University of Iowa
Iowa Research Online
Theses and Dissertations
Fall 2009
Coping with stress: anaerobic respiratory and
oxidative stress tolerance mechanisms are critical
for Neisseria gonorrhoeae biofilm formation
Megan Lindsay Falsetta Wood
University of Iowa
Copyright 2009 Megan Lindsay Falsetta Wood
This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/450
Recommended Citation
Wood, Megan Lindsay Falsetta. "Coping with stress: anaerobic respiratory and oxidative stress tolerance mechanisms are critical for
Neisseria gonorrhoeae biofilm formation." PhD (Doctor of Philosophy) thesis, University of Iowa, 2009.
http://ir.uiowa.edu/etd/450.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Microbiology Commons
COPING WITH STRESS: ANAEROBIC RESPIRATORY AND OXIDATIVE
STRESS TOLERANCE MECHANISMS ARE CRITICAL FOR NEISSERIA
GONORRHOEAE BIOFILM FORMATION
by
Megan Lindsay Falsetta Wood
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Microbiology
in the Graduate College of
The University of Iowa
December 2009
Thesis Supervisor: Professor Michael A. Apicella
1
ABSTRACT
Many illnesses and infections are exacerbated and/or caused by biofilms.
Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently
asymptomatic in women, which can lead to persistent infection. Persistent
infection can result in pelvic inflammatory disease, tubo-ovarian abscesses,
infertility, and ectopic pregnancy. N. gonorrhoeae has been shown to form
biofilms over glass, primary and immortalized cervical cells, and during natural
cervical infection. Asymptomatic infection occurs in only 1% of infected males,
and the infection site is subject to periodic rapid fluid flow, which may limit biofilm
formation. Thus, biofilm formation may specifically play an important role in the
infection of women and could contribute to the infrequent occurrence of
symptoms.
Prior to work presented in this dissertation, little was known about biofilm
formation by N. gonorrhoeae. Therefore, we elected to compare the
transcriptional profiles of biofilms to their planktonic counterparts, and to identify
genetic pathways involved in biofilm formation and maintenance. We found that
3.8% of the genome was differentially regulated, and that genes involved in
anaerobic metabolism and oxidative stress tolerance were up-regulated in
biofilm, while genes involved in aerobic metabolism were down-regulated. We
determined that expression of aniA, ccp, and norB is required for robust biofilm
formation over glass and human cervical cells, and anaerobic respiration occurs
in the substratum of gonococcal biofilms. Disruption of the norB gene resulted in
severe attenuation of biofilm formation. We determined that the accumulation of
nitric oxide (NO) contributes to the phenotype of a norB mutant and can retard
biofilm formation when present at sublethal concentrations. However, higher
concentrations of NO can enhance biofilm formation in the absence of nitrite. NO
enhances biofilm formation in an aniA mutant, but cannot completely restore
2
biofilm formation, suggesting that NO can support anaerobic growth, although
nitrite is preferred. We determined that the majority of the genes involved in
gonococcal oxidative stress tolerance are required for normal biofilm formation,
as mutations in the following genes resulted in biofilm attenuation over cervical
cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm
formation may represent an adaptation for coping with the stresses present in the
female genitourinary tract.
Abstract Approved: ________________________________
Thesis Supervisor
________________________________
Title and Department
________________________________
Date
COPING WITH STRESS: ANAEROBIC RESPIRATORY AND OXIDATIVE
STRESS TOLERANCE MECHANISMS ARE CRITICAL FOR NEISSERIA
GONORRHOEAE BIOFILM FORMATION
by
Megan Lindsay Falsetta Wood
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Microbiology
in the Graduate College of
The University of Iowa
December 2009
Thesis Supervisor: Professor Michael A. Apicella
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Megan Lindsay Falsetta Wood
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Microbiology at the December 2009 graduation.
Thesis Committee: ______________________________
Michael A. Apicella, Thesis Supervisor
_______________________________
Lee-Ann Allen
_______________________________
Mary E. Wilson
_______________________________
Patricia L. Winokur
_______________________________
Timothy L. Yahr
To my biggest supporters, my loving family
ii
In happy homes he saw the light
Of household fires gleam warm and bright;
Above the spectral glaciers shone,
And from his lips escaped a groan,
Excelsior!
Henry Wadsworth Longfellow
An excerpt from Excelsior
iii
ACKNOWLEDGMENTS
I am indebted to my advisor, Michael Apicella, for his wisdom, guidance,
and encouragement. I am thankful for the opportunity to work with him, through
which experience I have grown tremendously as a scientist. My only regret is
that I will likely be the last graduate student to benefit from his tutelage. I also
thank the past and present members of the Apicella laboratory, and our
collaborators, who have helped me and renewed my confidence along the way.
I am grateful to the members of my thesis committee: Lee-Ann Allen,
Mary Wilson, Patricia Winokur, and Timothy Yahr. They have been extremely
supportive and have helped to steer my research in the right direction. It has
been a pleasure to work with them. I also owe a special thanks to Steven Clegg,
who served as the fifth member of my comprehensive exam committee.
I also would like to thank Tom Bair, Kevin Knudtson, and all of the
members of the University of Iowa DNA Facility. I could not have completed this
work without their help. I am also grateful for the staff of Central Microscopy.
I would never have pursued a career in science if were not for my mother,
Donna Falsetta, who has perpetually told me that I can do anything I set out to
do. She is, and always will be, my hero. I also owe my success to my father,
Albert Falsetta, who has coached from the field, the sidelines, and wherever I go.
I think he cheers for me the loudest. I give thanks to my “big” little brother,
Brandon Falsetta, who has often reminded me of who I am, and how much I love
science. I thank my husband, Zachary Wood, who has the almost magical ability
to help me relax when I am overwhelmed. I could not have gotten through the
stresses of graduate school without him. This work is for my son, Joshua Wood.
He reminds me that life is an awe-inspiring adventure, and although we do not
know where it will take us, getting there is at least half the fun.
iv
TABLE OF CONTENTS
LIST OF TABLES ................................................................................................ vii
LIST OF FIGURES ...............................................................................................ix
LIST OF ABBREVIATIONS ..................................................................................xi
CHAPTER
I.
INTRODUCTION................................................................................. 1
General Properties of Neisseria gonorrhoeae ..................................... 1
Gonorrhea Infection............................................................................. 2
Epidemiology ................................................................................ 2
Transmission................................................................................. 3
Disease ......................................................................................... 4
Antibiotic Resistance and Treatment ............................................ 7
Gonorrhea and Concurrent STD Infections................................... 8
Classicially Defined Virulence Factors ................................................ 9
Newly Recognized Virulence Factors................................................ 12
Proteins Involved in Anaerobic Metabolism ................................ 12
Proteins Involved in Oxidative Stress Tolerance......................... 17
Bacterial Biofilms and Chronic Infection ............................................ 23
What are Biofilms?...................................................................... 23
Biofilm Structure and Formation.................................................. 26
Advantages and Inherent Properties of Biofilm Formation .......... 30
Biofilm Formation by Neisserial Species ..................................... 36
Rationale for Research Conducted ................................................... 37
Hypothesis......................................................................................... 44
II.
TRANSCRIPTIONAL PROFILING REVEALS THE METABOLIC
PHENOTYPE OF GONOCOCCAL BIOFILM .................................... 45
Introduction........................................................................................ 45
Experimental Procedures .................................................................. 47
Bacteria....................................................................................... 47
RNA Isolation .............................................................................. 47
Microarray Analysis..................................................................... 48
Quantitative Real-Time PCR....................................................... 49
Mutant Construction.................................................................... 51
Biofilm Growth in Continuous-Flow Chambers Over Glass......... 52
Growth Curves Under Oxygen Tension Conditions Present
in Biofilm Medium........................................................................ 54
THCEC Culture ........................................................................... 54
Biofilm Growth in Continuous-Flow Chambers Over Cells .......... 55
Trypan Blue Viability Assays....................................................... 56
Complementation of aniA::kan, ccp::kan, and norB::kan
Mutants ....................................................................................... 57
Confocal Microscopy of Continuous-Flow Chambers ................. 57
COMSTAT Analysis of Confocal z-Series ................................... 58
Treatment of Biofilms with Nitric Oxide Donor and Inhibitor ........ 58
v
Statistical Analysis of COMSTAT Results ................................... 59
Results .............................................................................................. 59
Microarray Analysis of Genes Differentially Expressed
During Biofilm Growth ................................................................. 59
Validation of Microarray Results ................................................. 64
Expression Profiles of Highly Differentially Regulated Genes
in Biofilms Over Host Cells.......................................................... 64
Characterization of the Role of Anaerobic Metabolism Genes
in Biofilms Over Glass................................................................. 66
Characterization of the Role of Anaerobic Metaolism Genes
in Biofilms Over THCEC ............................................................. 73
Effect of Nitric Oxide on N. gonorrhoeae Biofilm Formation........ 80
Discussion ......................................................................................... 81
III.
ANAEROBIC METABOLISM OCCURS IN THE SUBSTRATUM
OF GONOCOCCAL BIOFILMS AND MAY BE SUSTAINED IN
PART BY NITRIC OXIDE .................................................................. 92
Introduction........................................................................................ 92
Experimental Procedures .................................................................. 95
Bacteria....................................................................................... 95
Construction of an aniA’-‘gfp Transcriptional Fusion................... 95
Biofilm Growth of the aniA’-‘gfp Fusion Over Glass .................... 97
Confocal Microscopy of Continuous-Flow Chambers ................. 98
Treatment of Biofilms with the NO Donors Sodium
Nitroprusside (SNP) and Diethylenetriamine/Nitric Oxide
Adduct (DETA/NO) ..................................................................... 98
THCEC Culture ........................................................................... 99
SNP Treatment of Biofilms Grown in Continuous-Flow
Chambers Over THCEC ........................................................... 100
Trypan Blue Viability Assays .................................................... 100
COMSTAT Analysis of Confocal z-Series ................................. 100
Statistical Analysis of COMSTAT Results ................................ 101
Results ............................................................................................ 101
Microscopic Examination of Anaerobic Respiration in Biofilm... 101
The Effect of High Concentrations of SNP on Biofilm
Formation.................................................................................. 102
The Effect of DETA/NO on Biofilm Formation ........................... 106
The Effect of DETA/NO on aniA::kan Insertion Mutant
Biofilms ..................................................................................... 109
The Impact of NO on Biofilms Grown Over THCEC.................. 114
Discussion ....................................................................................... 117
IV.
THE ABILITY TO TOLERATE OXIDATIVE STRESS IS
CRITICAL FOR GONOCOCCAL BIOFILM FORMATION ............... 127
Introduction...................................................................................... 127
Experimental Procedures ................................................................ 128
Bacteria..................................................................................... 128
Biofilm Growth in Continuous-Flow Chambers Over Glass....... 129
Confocal Microscopy of Continuous-Flow Chambers ............... 129
THCEC Culture and Biofilms over THCEC ............................... 130
COMSTAT Analysis of Confocal z-Series ................................. 130
Statistical Analysis of COMSTAT Results ................................ 130
vi
Results ............................................................................................ 131
Biofilm Formation by the OxyR Regulon Mutants oxyR::kan,
prx::kan, and gor::kan ............................................................... 131
Biofilm Formation by the PerR Regulon Mutants mntAB::kan
and mntC::kan........................................................................... 131
Biofilm Formation Over Glass by the NmlR Regulon Mutants
trxB::kan and estD::kan............................................................. 134
Biofilm Formation Over THCEC by the NmlR Regulon
Mutants trxB::kan and estD::kan ............................................... 134
Discussion ....................................................................................... 141
V.
DISCUSSION .................................................................................. 152
VI.
FUTURE DIRECTIONS AND IMPLICATIONS ................................ 170
REFERENCES ................................................................................................. 174
vii
LIST OF TABLES
Table
1.
Primers used in this study .......................................................................... 50
2.
Strains and plasmids used in this study ..................................................... 53
3.
Genes up-regulated during biofilm formation.............................................. 61
4.
Genes down-regulated during biofilm formation. ........................................ 62
5.
qRT-PCR validation of microarray results .................................................. 65
6.
Strains, plasmids, and primers used in this study....................................... 96
viii
LIST OF FIGURES
Figure
1.
Anaerobic metabolism in N. gonorrhoeae .................................................. 18
2.
Oxidative stress tolerance in N. gonorrhoeae............................................. 24
3.
A generalized model of bacterial biofilm architecture ................................. 28
4.
Mechanisms of biofilm tolerance. ............................................................... 34
5.
N. gonorrhoeae biofilms over glass ............................................................ 38
6.
N. gonorrhoeae biofilms in vivo .................................................................. 40
7.
Expression of aniA, ccp, norB, and nuoF in biofilms grown over cells........ 67
8.
Biofilm formation over glass. ...................................................................... 69
9.
COMSTAT analysis of biofilms grown over glass....................................... 71
10. Biofilm formation over THCEC ................................................................... 74
11. COMSTAT analysis of biofilms grown over THCEC................................... 76
12. Biofilm formation by complemented strains over THCEC........................... 78
13. NO halts biofilm formation in the wild type ................................................. 82
14. PTIO treatment enhances biofilm formation in the norB::kan mutant ......... 84
15. Biofilm formation by the aniA’-‘gfp fusion strain........................................ 103
16. NO enhances biofilm formation in biofilms that are undergoing
anaerobic respiration ................................................................................ 107
17. DETA/NO enhances biofilm formation in biofilms without nitrite............... 110
18. Biofilm formation by the aniA::kan mutant is enhanced in the presence
of DETA/NO ............................................................................................. 112
19. NO impedes biofilm formation over THCEC ............................................. 115
20. One possible model for the role of nitric oxide in biofilm formation........... 124
21. Biofilm formation by wild-type N. gonorrhoeae strain 1291 and the
oxyR::kan, prx::kan, and gor::kan mutant derivatives............................... 132
22. Biofilm formation by wild-type N. gonorrhoeae strain 1291 and the
mntAB::kan and mntC::kan mutants......................................................... 135
ix
23. Biofilm formation by wild-type N. gonorrhoeae strain 1291 and the
trxB::kan and estD::kan mutants over glass ............................................. 137
24. COMSTAT analysis of biofilm formation over glass by wild-type N.
gonorrhoeae strain 1291 and the trxB::kan and estD::kan mutants ......... 139
25. Biofilm formation by wild-type N. gonorrhoeae strain 1291 and the
trxB::kan and estD::kan mutants over THCEC ......................................... 142
26. COMSTAT analysis of biofilm formation over THCEC by wild-type N.
gonorrhoeae strain 1291 and the trxB::kan and estD::kan mutants.......... 144
x
LIST OF ABBREVIATIONS
ABC
ATP binding cassette
AHU-
Arginine, Hypozanthine, Uracil Auxotrophs
AdhC
Alcohol Dehydrogenase III
AniA (Pan1)
Nitrite Reductase
ATP
Adenosine Triphosphate
ASGP-R
Asialoglycoprotein Receptor
bc1
Cytochrome
bp
Base Pair
°C
Degrees Celsius
cbb3
Cytochrome Oxidase
Ccp
Cytochrome c Peroxidase
CDC
Centers for Disease Control
CEACAM
Carcinoembryonic Antigen-Related Cell Adhesion Molecules
CFU
Colony Forming Unit
CMP
Cytidine Monophosphate
CO2
Carbon Dioxide
CopA
Copper Transport Protein
CR3
Complement Receptor 3
DETA/NO
Diethylenetriamine/Nitric Oxide Adduct
DGI
Disseminated Gonococcal Infection
DNA
Deoxyribonucleic Acid
DnrN
Iron-Sulfur Cluster Repair Protein
EDTA
Ethylenediaminetetraacetic Acid
EstD
Esterase D
EPS
Exopolysaccharide
xi
Fe
Iron
FNR
Fumarate and Nitrate Regulator
Fur
Ferric Uptake Regulator
GC
Gonococcal
Gor
Glutathione Reductase
GFP
Green Fluorescent Protein
GSH
Glutathione
GSNO
S-nitrosoglutathione
Grx
Glutaredoxin
GRASP
Gonococcal Antimicrobial Resistance Surveillance Program
H2O
Water
H2O2
Hydrogen Peroxide
h
Hour
HIV
Human Immunodeficiency Virus
HSPG
Heparin Sulfate Proteoglycan
IM
Inner Membrane
JEM
Jenn Edwards Media
Kan
Kanamycin
KatA
Catalase
KEGG
Kyoto Encyclopedia of Genes and Genomes
K-SFM
Keratinocyte Serum-Free Media
LNnT
Lacto-N-neotetraose
LOS
Lipoligosaccharide
LysR
Diaminopimelate Decarboxylase Synthesis Regulator
µl
Microliter
µm
Micrometer
µM
Micromolar
xii
M
Molar
MerR
Mercury Resistance Regulator
min
Minute
ml
Milliliter
mm
Millimeter
Mn
Manganese
MntABC
Manganese ABC Transporter
MOI
Multiplicity of Infection
N2
Nitrogen Gas
N2O
Nitrous Oxide
NADH
Reduced Nicotinamide Adenine Dinucleotide
NADP+
Nicotinamide Adenine Dinucleotide Phosphate
NADPH
Reduced Nicotinamide Adenine Dinucleotide Phosphate
NarP/Q
Nitrite Responsive Two-component System
NarX/L
Nitrate Responsive Two-component System
ng
Nanogram
nM
Nanomolar
NmlR
Neisseria Mer-like Regulator
NO
Nitric Oxide
NorB
Nitric Oxide Reductase
NsrR
Nitric Oxide Sensitive Repressor
Nuo
NADH Dehydrogenase
O2-
Oxygen Gas
OM
Outer Membrane
OMP3
Outer Membrane Protein 3
Opa
Opacity-associated Protein
OxyR
Hydrogen Peroxide Regulator
xiii
P.I
Porin
P.II
Opacity-associated Protein
PerR
Peroxide Regulator
PCR
Polymerase Chain Reaction
PCR-SOE
PCR-Splicing by Overlap Extension
PMN
Polymorphonuclear Lymphocyte
ProB
Proline B
Prx
Peroxiredoxin
PTIO
2-Phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide
qRT-PCR
Quantitative RT-PCR
RNA
Ribonucleic Acid
RNS
Reactive Nitrogen Species
ROS
Reactive Oxygen Species
RT-PCR
Reverse Transcriptase PCR
S
Sulfur
SLCM
Scanning Laser Confocal Microscopy
Sod
Superoxide Dismutase
Spec
Spectinomycin
STD
Sexually Transmitted Disease
THCEC
Transformed Human Cervical Epithelial Cells
Trx(B)
Thioredoxin
UV
Ultraviolet
xiv
1
CHAPTER I
INTRODUCTION
General Properties of Neisseria gonorrhoeae
Neisseria gonorrhoeae (gonococcus) is the etiologic agent of gonorrhea
(Hansfield, 2005, Hook, 1999c). Gonorrhea, the infection caused by the
gonococcus, is the second most commonly reported notifiable disease in the
United States today (Centers for Disease Control and Prevention, 2007). Studies
indicate that the mechanisms of infection differ in men and women (Edwards &
Apicella, 2004). Women are susceptible to chronic complications from
undiagnosed gonorrhea infection, which frequently exhibits no noticeable
symptoms (Hook, 1999a, Hook, 1999b, Hook, 1999c). Up to 45 percent of
women with asymptomatic infection can develop upper genital infection and
pelvic inflammatory disease (PID) in (Hook, 1999b). PID increases the risk for
infertility, ectopic pregnancy, tuboovarian abscesses, and often results in chronic
pelvic pain, which can warrant surgical intervention (Hook, 1999b, Hook, 1999c).
Undiagnosed infection can even lead to death, as ectopic pregnancy is the
leading cause of first-trimester deaths among pregnant American women (1995).
The work presented in this thesis aims to offer insight into the complex biology of
gonococcal infection in women, proposing new explanations for the lack of
symptoms. The studies described here focus on the role of gonococcal biofilm in
the infection of cervical tissues, and its ability to resist the unique stresses
present in the cervical environment.
N. gonorrhoeae is an obligate human pathogen that is only infectious
through intimate physical contact with the mucosal surfaces of an infected
person. Gonococci are small gram-negative diplococci, which are nonmotile and
non-spore forming (Hook, 1999a). N. gonorrhoeae is a fastidious organism,
exhibiting complex growth requirements when cultured in vitro. All strains require
2
iron, several amino acids, vitamins, and other factors (Hansfield, 2005). N.
gonorrhoeae can utilize glucose, lactate, or pyruvate as sole carbon sources, but
cannot metabolize other carbohydrates (Catlin, 1973). Subsequently, carbon
utilization tests are the basis for distinguishing N. gonorrhoeae from other
Neisseria species (Hook, 1999a), such as Neisseria meningitidis, which can
metabolize both glucose and maltose, but not sucrose or lactose (Apicella,
2005). The following rich media are used in the clinical isolation of N.
gonorrhoeae: Thayer-Martin, modified Thayer-Martin, Lewis-Martin, and GCLect. These media have a chocolate agar base and contain antibiotics that
inhibit the growth of yeast and other bacteria (Spence et al., 2008). However,
most research laboratories use commercially prepared media bases (e.g. Difco
GC medium) that employ an enzymatic digest of meat and/or milk proteins to
provide nitrogen and amino acids (Spence et al., 2008). GC medium is typically
augmented with Kellogg’s supplement (Kellogg et al., 1963) or IsoVitalex
(Becton-Dickinson, Franklin Lakes, NJ) to enhance the growth of gonococci on
plates. N. gonorrhoeae requires 3-10 percent carbon dioxide (CO2) for growth.
This can be provided by growth on a solid medium in a CO2-incubator or candle
jar (Spence et al., 2008). N. gonorrhoeae is cultured at 37°C (body temperature),
and does not survive at temperatures below 25°C (Spence et al., 2008).
Colonies appear on plates within 24 to 48 hours of inoculation (Hansfield, 2005),
but viability is rapidly lost due to autolysis after depletion of glucose from the
medium (Spence et al., 2008).
Gonorrhea Infection
Epidemiology
On average 62 million new cases of gonorrhea are reported annually
worldwide with the greatest number of cases occurring in South and Southeast
3
Asia and Sub-Saharan Africa (Gerbase et al., 1998). However, the occurrence of
new cases is still prevalent in heavily industrialized regions including Eastern and
Central Europe, North America, and Western Europe (Gerbase et al., 1998).
Gonorrhea continues to be one of the most frequently reported communicable
diseases in the United States. In 2007 alone, 355,991 cases of gonorrhea were
reported (Centers for Disease Control and Prevention, 2008).
Gonorrhea rates declined in the United States in the 1940s and 1950s
with the advent of penicillin therapy (Fox et al., 1998), but began to climb
between 1966 and 1975 as a result of the changing sexual behavior patterns of
Americans and increasing resistance to penicillin G (Zaidi et al., 1983). In
response to this, public health officials and health care providers launched a
national campaign to control the spread of gonorrhea through the widespread
screening of asymptomatic women using newly developed selective culture
media (Thayer-Martin). This approach and the increasing use of barrier
contraception was largely successful, resulting in a decline in the number of
diagnosed gonorrhea cases (Zaidi et al., 1983). However, the number of cases
of gonorrhea increased between 2005 and 2007 in the United States (Centers for
Disease Control and Prevention, 2007), and overall the current rate of gonorrhea
transmission remains high (Centers for Disease Control and Prevention, 2008).
As evidence of this, one epidemiological study in Baltimore estimated that 5.3
percent of the (female) population between the ages of 18 and 35 had untreated
gonococcal infection (Turner et al., 2002).
Transmission
The efficiency of gonorrhea transmission depends on the anatomic sites
infected and exposed, and the total number of exposures. A man has an
estimated 20 percent risk for contracting gonorrhea from a single episode of
4
vaginal intercourse, which increases to 60 to 80 percent after four exposures
(Holmes et al., 1970, Hooper et al., 1978). A woman has an estimated 50 to 70
percent risk per contact, although no published studies have controlled for the
number of exposures to N. gonorrhoeae (Holmes et al., 1970, Hooper et al.,
1978, Lin et al., 1998). Transmission by anal intercourse is presumably efficient,
but has not been formally quantified (Hansfield, 2005, Hook, 1999a). Gonorrhea
may also be transmitted via fellatio, but transmission of pharyngeal infection to
the urethra is believed to be rare (Tice & Rodriguez, 1981, Wiesner et al., 1973).
The presence of and response to clinical symptoms of gonorrhea
dramatically impacts its transmission. Gonorrhea and other sexually transmitted
diseases (STDs) are typically transmitted by individuals who have no symptoms
or have symptoms that they ignore or discount (Handsfield et al., 1974).
Asymptomatic gonorrhea can occur in men and women, although asymptomatic
infection is more prevalent in women. Some studies estimate that 80 percent of
infected women have no noticeable symptoms, although these studies are based
on screening surveys of women referred to STD clinics because of prior sexual
contact (Pedersen & Bonin, 1971). Women attending acute care facilities are
more likely to present with symptoms. However, a recent study found that
approximately 40 percent of the women in the England Gonococcal Resistance
to Antimicrobials Surveillance Program (GRASP) have asymptomatic disease
(Bozicevic et al., 2006). Asymptomatic gonococcal infection occurs frequently in
women and contributes to the spread of gonorrhea by transmission to their
sexual partners.
Disease
N. gonorrhoeae is a well adapted human pathogen. Gonorrhea is one of
the oldest known human illnesses. The clinical manifestations of gonorrhea have
5
been described in several ancient texts, including the Old Testament (Leviticus,
1:19). The term gonorrhea, meaning “flow of semen,” was first termed by Galen
in 130 AD, who presumed that the urethral exudate of infected males was semen
(Hansfield, 2005, Hook, 1999c).
Men who become infected with N. gonorrhoeae usually present with acute
anterior urethritis, which is accompanied by symptoms of urethral discharge
and/or dysuria, typically without urgency or frequency (Hansfield, 2005, Hook,
1999c). The incubation period is normally 2 to 5 days (Hansfield, 2005, Hook,
1999c), but can range from 1 to 10 days, or even longer (Hansfield, 2005). The
initial discharge may be scant or mucoid, but becomes overtly purulent after 24
hours, which has been confirmed with experimental studies (Cohen & Cannon,
1999). However, approximately 1 percent of men can be asymptomatic for up to
6 weeks after exposure (Hansfield, 2005). This has been linked to infection by
particular N. gonorrhoeae strains, namely arginine, hypoxanthine, and uracil
auxotrophs referred to as AHU- strains (Brunham et al., 1985).
The endocervix is the primary locus of gonococcal infection in women, and
urethral colonization is present in 70 to 90 percent of those infected, although it is
uncommon in the absence of endocervical infection (Hook, 1999c). The
incubation period in women is likely more variable than in men. Those who
develop local symptoms usually do so within 10 days of infection (Platt et al.,
1983), however many women do not develop noticeable symptoms (Bozicevic et
al., 2006, Pedersen & Bonin, 1971). As with men, women infected with AHUstrains are more likely to have asymptomatic infection (Brunham et al., 1985).
Concurrent infection with Chlamydia trachomatis, Trichomonas vaginalis, and
bacterial vaginosis has eluded understanding of the natural course of gonococcal
infection in women (Hansfield, 2005). Symptoms may develop in most infected
women (Platt et al., 1983), but few seek medical attention because these
6
symptoms are typically minor or less noticeable than symptoms in men
(McCormack et al., 1977). Symptoms in women include increased vaginal
discharge, dysuria, intermenstrual bleeding, and menorrhagia, which can range
in intensity from minor to severe (Hansfield, 2005, Hook, 1999c). Many women
will exhibit cervical abnormalities that might not be detected during physical
examination, including mucopurulent cervical discharge, edema, and mucosal
bleeding (Hansfield, 2005, Hook, 1999c).
Women tend to be more susceptible to complicated gonococcal infection,
as a result of chronic undiagnosed infection. Acute salpingitis, or PID, is the
most frequent complication in women and occurs in an estimated 10 to 20
percent of those infected (Hook, 1999c). The most common symptoms of PID
are lower abdominal pain and genital tract infection (Hansfield, 2005). Some
women may also develop fever, chills, and nausea with or without vomiting, but
these symptoms are rare (Hansfield, 2005). Symptoms usually follow the onset
of menses by a few days (McCormack et al., 1977), and prior instances of PID
increase the risk for recurrent episodes (Hansfield, 2005).
Disseminated gonococcal infection (DGI) sometimes occurs in patients
with mucosal infection (Hansfield, 2005, Hook, 1999c). DGI is estimated to occur
in 0.5 to 3 percent of infected patients (Holmes et al., 1971), but the rate may be
lower due to the declining prevalence of the AHU- strains often associated with
DGI (Hansfield, 2005). The most common symptoms of DGI are joint pain and
skin lesions (Hansfield, 2005, Hook, 1999c), and an approximate 30 to 40
percent of DGI patients exhibit overt arthritis (Holmes et al., 1971). DGI-related
bacteremia is not continuous, sometimes making it difficult to positively identify
N. gonorrhoeae in patient blood cultures (Holmes et al., 1971). DGI is more
common in women than in men (Hook, 1999b), and several studies have
suggested that pregnancy may be a risk factor for DGI (Holmes et al., 1971).
7
The prevalence of AHU- strains associated with DGI also suggests that
asymptomatic infection may increase the risk for DGI, which occurs more
frequently in women (Bozicevic et al., 2006, Pedersen & Bonin, 1971).
Endocarditis is a rare, but serious complication of DGI, which occurred in 1 to 2
percent of infected patients in the pre-antibiotic era, and can result in death
(Holmes et al., 1971).
With the advent of antimicrobial therapy, fewer cases of gonorrhea result
in complications, such as urethral stricture (Hansfield, 2005, Hook, 1999c).
However, lasting damage, primarily infertility, can occur after infection with N.
gonorrhoeae, particularly in women who develop PID (1995, Hansfield, 2005,
Hook, 1999b, Hook, 1999c, McCormack et al., 1977). PID and DGI can be lifethreatening infections, especially when they cause ectopic pregnancy and
endocarditis, respectively (1995, Hansfield, 2005, Hook, 1999c). Asymptomatic
gonococcal infection likely puts patients at a higher risk for complicated infection
(Bozicevic et al., 2006, Pedersen & Bonin, 1971). Gonococcal infection in
women is not well understood due to confounding factors such as concurrent
infection with other STDs (Hansfield, 2005). This leaves no clear explanation for
the observed lack of symptoms in 60 to 80 percent of infected women (Pedersen
& Bonin, 1971). Subsequently, study is warranted to investigate the cause of
asymptomatic infection in women and the relationship of asymptomatic to
complicated infection (e.g. PID) in women.
Antibiotic Resistance and Treatment
Antibiotic resistance in N. gonorrhoeae is an increasing problem in the
treatment of gonococcal infection (2007, Centers for Disease Control and
Prevention, 2007, Hook, 1999a, Hook, 1999c, Wang et al., 2007a). Control
strategies for gonorrhea have traditionally relied on single-dose therapy to
8
promptly clear infection and prevent transmission to others. However,
antimicrobial resistance has often compromised these strategies (Wang et al.,
2007a). Currently, many strains of N. gonorrhoeae are resistant to sulfonamides,
penicillins, ciprofloxacin and tetracyclines (Centers for Disease Control and
Prevention, 2007, Hook, 1999a, Hook, 1999c, Wang et al., 2007a). In 2006, the
Centers for Disease Control and Prevention (CDC) updated their sexually
transmitted disease guidelines, recommending that fluoroquinolones no longer
be used to treat gonorrhea (2007). Attempts to design an effective vaccine for
the prevention of gonococcal infection have focused on the use of pilin and P.II
as an antigen. P.II is an opacity-associated protein that is expressed on the cell
surface (Hook, 1999a). However, pilin and opacity proteins undergo both phase
and antigenic variation so that they are not stably expressed, which is similar for
other gonococcal surface associated structures (Hook, 1999a). In 2006, the
number of cases of gonorrhea in the United States increased for the second
consecutive year (Centers for Disease Control and Prevention, 2007). Thus, it is
becoming increasingly important to pursue other treatment and/or prevention
strategies.
Gonorrhea and Concurrent STD Infections
Women diagnosed with gonorrhea are often concurrently infected with C.
trachomatis, T. vaginalis, and/or bacterial vaginosis (Hansfield, 2005).
Concurrent infection can confound the diagnosis of gonorrhea in women by
masking potentially minor symptoms, and it can lead to prolonged infection and
subsequent complications. A CDC study conducted in 2000 found that nearly
half of the women surveyed in the 15 to 19 age group were infected with both N.
gonorrhoeae and C. trachomatis. This was similar for the 15 to 24 age group,
and slightly lower (closer to 40 percent) for the 20 to 24 age group (Dicker et al.,
9
2003). A 2005 study examining the prevalence of gonorrhea, chlamydia, and
trichomonas in sexual partnerships found that a significant number of the
participating partners had concurrent gonorrhea and trichomonas, concurrent
gonorrhea and chlamydia, or concurrent gonorrhea, chlamydia, and trichomonas
infection (Khan et al., 2005). A recent study examining human immunodeficiency
virus (HIV) and STD coinfection found that 24 percent of the patients examined
presented with both gonorrhea and HIV, while two percent presented with
gonorrhea, chlamydia, and HIV (Huhn et al., 2008). This data supports the
finding that individuals infected with N. gonorrhoeae are at a higher risk for
contracting HIV (Centers for Disease Control and Prevention, 2007, Fleming &
Wasserheit, 1999). The presence of the gonococcus in the reproductive tract
has actually been shown to increase local expression of HIV viral RNA (Chen et
al., 2003).
Classically Defined Virulence Factors
Pilus is involved in the attachment of the gonococcus to host epithelial
cells (Pearce & Buchanan, 1978, Punsalang & Sawyer, 1973, Swanson, 1973).
N. gonorrhoeae expresses Type IV-A pili, which play important roles in the
virulence of pathogenic organisms, mediating important cellular functions, such
as surface motility, biofilm formation, host-cell adhesion, DNA uptake by natural
transformation, and cell signaling (Patel et al., 1991). In N. gonorrhoeae, Type
IV-A pili are required for the iC3b-mediated association of the gonococcus with
the CR3 receptor on primary cervical epithelial cells (Edwards et al., 2002). This
is critical for virulence, as CR3-mediated endocytosis is the primary mechanism
by which the gonococcus infects and invades the cervical epithelium of women
(Edwards et al., 2001). Novel regulatory mechanisms in the gonococcus allow
for both antigenic and phase variation of the pilus (Hansfield, 2005, Hook,
10
1999a). Thus, not all gonococci express pili at a given time, and rarely do two
cells express identical pilus epitopes. It has been postulated that N. gonorrhoeae
uses pilus phase and antigenic variation to cope with the immune responses of
the host mucosa (Seifert, 1996).
Opaque gonococcal colonies express one or more opacity-associated
outer membrane proteins (Opa, formerly P.II) that are not present in transparent
colonies, and the occurrence of Opa proteins is independent of pilus expression
(Swanson, 1978). A particular gonococcal strain may express multiple Opa
proteins simultaneously (Black et al., 1984), as a total of 11 or 12 loci occur
throughout the N. gonorrhoeae chromosome (Connell et al., 1990). All Opa
proteins share antigenic determinants, but the surface-exposed antigens of
various Opa proteins are different from one another (Swanson & Barrera, 1983).
Gonococci recovered after urogenital, cervical or rectal infection are typically
Opa+, including bacteria recovered from human volunteers infected with Opastrains (Jerse et al., 1994, Swanson et al., 1988). Initial attachment to the host
epithelium is mediated by pili, but Opa is thought to play a more intimate role in
cellular attachment (Dehio et al., 1998). Opa can bind two classes of receptors
on host cells: heparin sulfate proteoglycans (HSPGs), which are commonly
found on mammalian cells, and CD66 or carcinoembryonic antigen-related cell
adhesion molecules (CEACAM) (Dehio et al., 1998, Hansfield, 2005). Some Opa
proteins are capable of binding CEACAM receptors on B and T cells, which can
down-regulate the host immune defenses and may contribute to the poor
immune response during natural infection (Dehio et al., 1998). Antigenic and
phase variation of Opa can affect virulence by altering intercellular adhesion, the
ability to survive in human serum, and cytotoxicity (Black et al., 1984).
The outer membrane of N. gonorrhoeae is primarily comprised of
lipooligosaccharide (LOS), an amphipathic glycolipid that is composed of a lipid A
11
moiety, core polysaccharide, and oligosaccharide side (OS) chains (Griffiss et al.,
1988) to which sialic acid may be added as a terminal structure (Smith et al.,
1995). N. gonorrhoeae LOS is antigenically heterogeneous, and strains have
variable susceptibility to complement-mediated lysis (Schneider et al., 1984).
Common LOS components have the ability to convey serum resistance
(Schneider et al., 1985), and other mucosal pathogens share some of these
epitopes (Campagnari et al., 1990). Phase and antigenic variation allows for the
loss or gain of certain oligosaccharide moieties used in LOS assembly (Yang &
Gotschlich, 1996). N. gonorrhoeae and N. meningitidis also have LOS
components that are immunochemically similar to epitopes expressed on red
blood cells (Mandrell et al., 1988, Mandrell et al., 1990). The gonococcus cannot
synthesize or activate sialic acid, but it can acquire the activated nucleotide
sugar, CMP-sialic acid during natural infection and incorporate sialic acid onto
the LOS structure (Smith et al., 1995). When the LOS is sialylated, N.
gonorrhoeae cells are more resistant to bactericidal attack (Elkins et al., 1992)
and are less well engulfed by polymorphonuclear lymphocytes (PMNs) (Kim et
al., 1992). Sialylation also inhibits the ability of the gonococcus to invade urethral
epithelial cells (Harvey et al., 2001), but not cervical epithelial cells (Edwards &
Apicella, 2002). Expression of the lacto-N-neotetrose (LNnT) epitope mimics
human paragloboside and allows the gonococcus to invade urethral cells in men
by adherence to the asialoglycoprotein receptor (ASGP-R) (Harvey et al., 2001).
Adherence to ASGP-R on human sperm may also enhance transmission of N.
gonorrhoeae (Harvey et al., 2000). The LNnT epitope can be sialylated, which
contributes to serum resistance (Wetzler et al., 1992). Overall, LOS is extremely
important for infection by N. gonorrhoeae, as it can function as an adhesion
(Song et al., 2000), can aid in the invasion of host cells (Harvey et al., 2001),
may facilitate transmission of gonorrhea (Harvey et al., 2000), and contributes to
12
serum resistance and immune evasion (Griffiss et al., 1988, Mandrell et al., 1988,
Mandrell et al., 1990, Schneider et al., 1985, Wetzler et al., 1992).
Another important component of the gonococcal outer membrane is porin
(formerly P.I), which is closely associated with LOS (Hansfield, 2005, Hook,
1999a). Porin is a trimeric hydrophilic pore-forming protein that is necessary for
the survival of the gonococcus, as it allow nutrients to enter and waste products
to exit the cell (Young et al., 1983). Porin exists in two structurally related forms,
designated P.IA and P.IB (Knapp et al., 1984). Unlike other gonococcal outer
membrane components (pili, Opa, and LOS), porin does not undergo phase or
antigenic variation (Hansfield, 2005, Hook, 1999a) and is subsequently highly
immunogenic (Elkins et al., 1994). However, close association with LOS shields
porin from immunological attack (Elkins et al., 1992). Porin has been implicated
in a number of roles that are critical to pathogenesis of the gonococcus, including
invasion and apoptosis of host cells and inhibition of complement and immune
cell responses, which can occur when porin translocates into the membranes of
host cells (Massari et al., 2003). As with pili, porin is also required for association
of the gonococcus with CR3 on the host cell surface (Edwards et al., 2002).
Newly Recognized Virulence Factors
Proteins Involved in Anaerobic Metabolism
A number of gonococcal gene products are induced under conditions of
oxygen limitation (e.g. AniA, formerly Pan1) (Clark et al., 1987). Although the
role of these proteins in the pathogenesis of N. gonorrhoeae has not been
directly examined, it has been proposed that they may be important for virulence,
as it is presumed that the oxygen tension is low in some sites of gonococcal
infection, particularly the uterine cervix.
13
Aerobic respiration in N. gonorrhoeae is likely a significant source of
endogenous stress (Archibald & Duong, 1986, Jurtshuk & Milligan, 1974). In N.
gonorrhoeae, electron transfer from ubiquinol to oxygen is catalyzed by the
cytochrome bc1 complex and a single cbb3 type cytochrome oxidase (Seib et al.,
2006). cbb3 type cytochromes have a high affinity for oxygen, which indicates
that N. gonorrhoeae is likely well adapted for growth under oxygen-limited or
microaerophilic conditions (Pitcher et al., 2002). This may be a beneficial
adaptation for growth in the human host, as oxygen is thought to be limited in the
female genitourinary tract. Cytochrome oxidase is not a significant source of
reactive oxygen species (ROS) in Escherichia coli, like NADH dehydrogenase,
succinate dehydrogenase, and fumarate dehydrogenase (Seib et al., 2006).
Consequently, NADH hydrogenase (encoded by the nuo operon), succinate
dehydrogenase, and the bc1 complex are likely the most significant sources of
endogenous ROS generated by the gonococcus (Seib et al., 2006).
Due to an inability to culture N. gonorrhoeae under anaerobic conditions,
N. gonorrhoeae was initially classified as an obligate aerobe (James-Holmquest
et al., 1973). However, this conflicted with the observation that N. gonorrhoeae
was often isolated in the presence of obligate anaerobes (Smith, 1975). It
eventually became evident that N. gonorrhoeae can survive under anaerobic
conditions (Short et al., 1982), and previous attempts at anaerobic culture failed
because anaerobic respiration is coupled to nitrite reduction in the gonococcus
(Knapp & Clark, 1984). Thus, anaerobic growth may be facilitated by the
addition of a nitrite-containing disk to the center of a culture dish (Knapp & Clark,
1984). The requirement for nitrite is likely satisfied in vivo, as nitrite is present at
concentrations of roughly 28 µM in the cervical fluid of women (VaisanenTommiska et al., 2003). Nitrous oxide (N2O) is the end product of nitrite
reduction in the gonococcus (Overton et al., 2006). Therefore, N. gonorrhoeae
14
catalyzes partial denitrification by converting nitrite to nitrous oxide via nitric oxide
(Overton et al., 2006).
Anaerobic respiration in N. gonorrhoeae is catalyzed by the nitrite
reductase AniA, and the nitric oxide (NO) reductase NorB. AniA (formerly Pan 1)
is induced under anaerobic growth conditions and is one of three outer
membrane proteins whose expression is enhanced during anaerobic growth
(Clark et al., 1987). In patients that do make anti-gonococcal antibodies, there is
a strong antibody response to AniA, which indicates that AniA is expressed in
vivo (Clark et al., 1988). AniA is required for anaerobic growth, is tightly
regulated by oxygen availability, and is virtually undetectable in aerobically
cultured cells (Householder et al., 1999). Thus, expression of AniA in vivo
indicates that the gonococcus likely undergoes anaerobic respiration during
infection. AniA is a lipoprotein (Hoehn & Clark, 1992b) and a functional nitrite
reductase (Mellies et al., 1997). AniA is required for anaerobic growth, as an
aniA mutant can survive, but does not grow under anaerobic conditions
(Householder et al., 1999). In addition to its role in anaerobic metabolism, AniA
may be capable of modulating the immune response by binding complement
regulatory proteins (likely factor H), which can down-regulate expression of
immune factors (Cardinale & Clark, 2000). NorB is a heme protein that reduces
NO to N2O (Householder et al., 2000). NorB effectively reduces NO generated
by AniA, as well as NO that is present at the site of infection (Cardinale & Clark,
2005). NO is generated by the host immune defenses and other organisms that
colonize the human genitourinary tract (Seib et al., 2006). NO is also produced
by nitric oxide synthases of PMNs (Carreras et al., 1994, Fang, 1997,
MacMicking et al., 1997, McCall et al., 1989) and cervical endothelial and
epithelial cells (Ledingham et al., 2000, Tschugguel et al., 1999). NorB is
composed of a single functional subunit (Householder et al., 2000), unlike the
15
majority of NO reductases that are found in denitrifying organisms (Zumft, 1997),
including P. aeruginosa (Arai et al., 1995), N. gonorrhoeae is capable of
establishing a steady-state of NO during anaerobic growth and can rapidly (in
under 1 hour) reduce NO concentrations of 1 µm or greater (proinflammatory) in
the surrounding medium to 100 nM or less (anti-inflammatory) (Cardinale &
Clark, 2005). This implies that N. gonorrhoeae can effectively reduce
endogenously and environmentally produced NO.
High levels of NO (µM) stimulate the human immune response (Cardinale
& Clark, 2005, Davis et al., 2001, Stefano et al., 2000), and NO is toxic to some
bacterial cells (Davis et al., 2001, Fang, 1997, MacMicking et al., 1997, Zumft,
1997). Although controversial, it has been suggested that NO production is part
of the innate immune response to bacterial infection (Fang, 1997). As with an
aniA mutant, a norB mutant can survive anaerobic growth conditions, but does
not grow anaerobically (Householder et al., 2000). Thus, NO may not be as toxic
to N. gonorrhoeae, as it to some other organisms. This may result from the
presence of redundant mechanisms that reduce reactive nitrogen species (RNS)
in the gonococcus (Seib et al., 2006). Taken together, these findings indicate
that reduction of NO may be critical during gonococcal infection. Furthermore, N.
gonorrhoeae does not induce cytokine production during cervical infection, which
suggests that the gonococcus fails to induce the immune response or
suppresses the response via an unknown mechanism (Hedges et al., 1998,
Russell et al., 1999). In addition, only low levels of anti-gonococcal antibody are
detected during uncomplicated gonococcal infection in women (Hedges et al.,
1998). It is apparent that these low anti-gonococcal antibody levels cannot be
entirely attributed to antigenic and phase-variation (Hook, 1999a). Thus, rapid
reduction of proinflammatory to anti-inflammatory concentrations of NO may help
explain some of these observations (Cardinale & Clark, 2005).
16
Genes involved in anaerobic metabolism are tightly regulated, partly due
to the toxicity of NO, which is generated by AniA. In Escherichia coli, nitrite- and
nitrate- responsive genes are regulated by the fumarate and nitrate regulator
(FNR) and the two component regulatory systems NarP-NarQ and NarX-NarL
(Darwin et al., 1998, Rabin & Stewart, 1993). Homologues of narP, narQ, and fnr
exist in N. gonorrhoeae, and they are involved in regulation of anaerobic
respiration (Householder et al., 1999, Lissenden et al., 2000). However, N.
gonorrhoeae is incapable of reducing nitrate, and does not possess a narX or
narL homologue (Knapp & Clark, 1984). NarP and NarQ appear to be insensitive
to nitrate and nitrite (Overton et al., 2006), yet they contribute to the
transcriptional regulation of aniA. NarP is also autoregulatory and positively
regulates transcription of the narPQ operon (Overton et al., 2006). Transcription
of aniA is initiated by FNR when oxygen is limited and nitrite is present (Hoehn &
Clark, 1992a, Householder et al., 1999, Whitehead et al., 2007). FNR positively
regulates transcription of aniA under anaerobic growth conditions, as it is inactive
in the presence of oxygen (Crack et al., 2008). FNR is also inactivated by NO
(Crack et al., 2008, Cruz-Ramos et al., 2002). Another regulatory factor, referred
to as the nitric oxide-sensitive repressor (NsrR), also plays a role in
transcriptional regulation of aniA (Isabella et al., 2009, Overton et al., 2006).
NsrR is a negative regulatory factor that binds the promoter of aniA, effectively
blocking access to the promoter (Isabella et al., 2009, Overton et al., 2006).
When NO is present, NsrR dissociates from the promoter, allowing FNR to bind
and activate transcription of aniA (Isabella et al., 2009, Overton et al., 2006). At
present no signal has been detected for the NarP-NarQ two-component
regulatory system, and it has been proposed that NarP-NarQ is constituently
active and contributes to positive regulation of aniA once NsrR is removed from
the aniA promoter (Overton et al., 2006). However, it is possible that NarP-NarQ
17
senses a previously undetected molecule, which contributes to the positive
regulation of aniA transcription. Transcription of norB is also regulated by NsrR
in the same fashion (Isabella et al., 2009, Overton et al., 2006), although norB is
not regulated by NarP-NarQ or FNR (Householder et al., 2000, Isabella et al.,
2008). Another related protein DnrN, is similarly regulated by NsrR (Overton et
al., 2006). DnrN repairs iron-sulfur clusters that have been damaged by reactive
oxygen and nitrogen species, and is subsequently important for oxidative stress
tolerance in the gonococcus (Overton et al., 2008). The ferric uptake regulator
(Fur) has also been shown to positively regulate transcription of norB through a
novel mechanism that prevents binding of a second norB transcriptional
repressor whose binding site overlaps the Fur binding site (Isabella et al., 2009).
See Figure 1 for an illustration of these regulatory pathways.
Proteins Involved in Oxidative Stress Tolerance
As an obligate pathogen that colonizes the mucosal surfaces of the
human genitourinary tract, N. gonorrhoeae is continuously exposed to oxidative
stress. The gonococcus encounters oxidants that are typically generated in the
following ways: 1) as a by-product of gonococcal metabolism, 2) as a response
to infection by the human immune system, and 3) as a result of exposure to
oxidative stresses present in the mucosal milieu, which may be generated by
other inhabitants of the of human mucosa (Seib et al., 2006). Oxidants pose a
serious risk to bacterial survival and can cause damage to DNA, proteins, and
the cellular membrane by attacking protein iron-sulfur (Fe-S) clusters. This may
cause rapid mutagenesis when Fe is released from damaged clusters (Imlay,
2003). The potential for oxidative damage during growth in the human host, and
its often fatal effects has prompted investigation into the role(s) oxidative stress
defenses play in bacterial virulence. It has become increasingly clear that
18
Figure 1. Anaerobic metabolism in N. gonorrhoeae. The following image
depicts the regulatory pathways that control anaerobic metabolism in the
gonococcus. Genes are italicized, while the proteins they encode begin with a
capital letter and are not italicized. A line with an arrow indicates that a protein or
signal has a positive regulatory effect on the gene or protein to which the line is
pointing. Lines with a dash at the end indicate negative regulation. The largest
arrows denote the steps in the N. gonorrhoeae denitrification pathway, as nitrite
is reduced to nitrous oxide (N2O). The denitrification pathway in N. gonorrhoeae
is incomplete, and nitrogen gas (N2) is not evolved by the gonococcus. A
frameshift in the nos gene operon results in activation of the Nos proteins, which
would normally reduce N2O to N2. DnrN repairs oxidative damage that occurs to
protein iron-sulfur clusters and is negatively controlled by NsrR as denoted.
19
20
mechanisms used to avoid and/or cope with the oxidative stresses present in the
human body are important virulence determinants that are abundant in human
pathogens (Hassett & Cohen, 1989, Janssen et al., 2003, Seib et al., 2006).
Cytochrome c peroxidase (Ccp) is a lipoprotein that is encoded by the ccp gene
(Turner et al., 2003). Ccp is the sixth c-type cytochrome in N. gonorrhoeae and
is only present during anaerobic growth (Turner et al., 2003). Transcription of
ccp is controlled by FNR in a manner that is similar to aniA regulation (Lissenden
et al., 2000). This allows transcription of ccp to be selectively induced under
anaerobic conditions, as FNR senses both oxygen and NO (Crack et al., 2008,
Cruz-Ramos et al., 2002). Ccp defends against hydrogen peroxide-induced
killing by reducing hydrogen peroxide (H2O2) to water (H2O) (Seib et al., 2004,
Seib et al., 2006). Catalase, encoded by katA, is another potent detoxifier of
H2O2 in the gonococcus (Seib et al., 2006). The ccp gene is located downstream
of katA (Seib et al., 2004). Mutants in ccp or katA are sensitive to H2O2-induced
killing, while a ccp/katA double mutant is more sensitive to H2O2 in vitro (Seib et
al., 2004, Seib et al., 2006, Turner et al., 2003). Protection against H2O2 is also
important for survival of N. gonorrhoeae in vivo, as it is produced by PMNs
(Carreras et al., 1994) and Lactobacillus species present in the female
genitourinary tract (Eschenbach et al., 1989). Lactobacillus strains that produce
H2O2 can inhibit the growth of N. gonorrhoeae by acidification of the media,
H2O2-toxicity, and production of protein inhibitors (St Amant et al., 2002, Zheng et
al., 1994).
The peroxide stress response in N. gonorrhoeae is regulated by OxyR and
PerR (Seib et al., 2006). OxyR is a member of the LysR family of DNA-binding
transcriptional regulators and is activated when key cysteine residues are
oxidized to form a disulfide bond (Zheng et al., 1998). OxyR is common in other
gram-negative bacteria, and regulates three genes in N. gonorrhoeae: gor, prx,
21
and katA (Seib et al., 2006). Transcription of these genes is enhanced when the
gonococcus is exposed to peroxide stress (Seib et al., 2006, Stohl et al., 2005).
OxyR functions dually as a repressor and an activator by inhibiting katA
expression under low H2O2 concentrations and activating expression of gor and
prx under high H2O2 concentrations (Seib et al., 2006). The gor gene encodes
Gor, a glutathione reductase, while the prx gene encodes Prx, a peroxiredoxin
(Seib et al., 2006). Prx catalyses reduction of alkyl hydroperoxidases via reactive
cysteines (Poole, 2005). These cysteines are regenerated by thioredoxin (Trx) or
glutaredoxin (Grx), either of which can be reduced by NADPH, glutathione
(GSH), and Gor (Poole, 2005). Thus, Prx and Gor function coordinately, which
explains why they are transcriptionally linked through OxyR. Gor typically
maintains a reduced pool of GSH in the cell (Carmel-Harel & Storz, 2000), which
is critical because GSH is considered to be one of the first lines of defense
against oxidative stress (Pomposiello & Demple, 2001). PerR, a Fur paralogue,
regulates a larger operon, consisting of 12 genes (Seib et al., 2006, Wu et al.,
2006). The mntABC operon is among these genes, which encodes the MntABC
manganese (Mn) ATP binding cassette (ABC) transporter (Tseng et al., 2001,
Seib et al., 2006). Mn acts as an intracellular antioxidant and can protect N.
gonorrhoeae from H2O2 in the absence of catalase (Seib et al., 2004).
Accumulation of Mn also protects the gonococcus against damage caused by
superoxide (O2-), which is independent of the activity of superoxide dismutase
(SOD) activity (Tseng et al., 2001). A mntC mutant, which lacks the periplasmic
binding component of the MntABC Mn transporter, accumulates less Mn and is
significantly more sensitive to H2O2 than the wild type (Wu et al., 2006). A perR
mutant is also more resistant to H2O2 than the wild type, as PerR acts a
transcriptional repressor of mntABC and possibly ccp expression (Wu et al.,
2006). However, PerR does not regulate transcription of katA (catalase) (Wu et
22
al., 2006). PerR also regulates a couple of genes that may be involved in Fe
uptake (Wu et al., 2006). Thus, it has been proposed that PerR may monitor Fe
and Mn levels in gonococcal cells and respond to the free Mn/Fe ratio. This may
be critical for Fe acquisition in a host environment that likely contains high
concentrations of H2O2 (Wu et al., 2006).
NmlR, or the Neissieria mer-like regulator, is another transcriptional
regulator that plays an important role in oxidative stress tolerance in N.
gonorrhoeae (Kidd et al., 2005). NmlR possesses features that are distinctive of
the MerR family of prokaryotic transcriptional regulators (Kidd et al., 2005). The
archetype member of the MerR family is MerR, a metal ion responsive activator
and that senses the mercuric ion to convey resistance to mercury (Hobman et al.,
2005). NmlR regulates transcription of five genes: nmlR (autoregulation), trxB,
copA, estD, and adhC (Kidd et al., 2005, Potter et al., 2009). An nmlR mutant is
slightly less able to grow under microaerobic conditions and is more susceptible
to killing by cumene hydroperoxide and diamide than the wild type (Kidd et al.,
2005). However, an nmlR mutant is no more sensitive to H2O2 or NO, which
suggests that NmlR responds to a thiol-disulphide redox imbalance, as diamide
is an organic peroxide that can deplete the reduced thiol pool in gonococcal cells
(Kidd et al., 2005). NmlR represses transcription of copA, adhC, and trxB under
normal growth conditions, but NmlR also acts as an activator of expression, as
treatment with diamide results in a significant increase in the abundance of these
transcripts (Kidd et al., 2005). Thus, transcription of the NmlR operon is
inducible under conditions where disulfide stress is present. Members of this
operon are likely required for tolerance of thiol stress in N. gonorrhoeae. copA
encodes a CPx-type ATPase that is tentatively annotated as the copper transport
protein CopA (Kidd et al., 2005). adhC encodes a class III alcohol
dehydrogenase (Kidd et al., 2005, Potter et al., 2007). This enzyme is highly
23
conserved in both prokaryotes and eukaryotes and protects cells against
nitrosative stress by reducing S-nitrosoglutathione (GSNO) (Kidd et al., 2005,
Potter et al., 2007). However, the adhC gene of all gonococcal strains contains a
premature stop codon and does not encode a functional protein (Potter et al.,
2007). estD encodes esterase D, which also protects gonococcal cells from
GSNO toxicity by reducing GSNO to formate and GSH (Potter et al., 2009a).
estD is co-transcribed with adhC, and EstD is functional in N. gonorrhoeae,
contributing to oxidative stress tolerance and virulence (Potter et al., 2009a).
TrxB is a thioredoxin reductase that contributes to oxidative stress tolerance by
enhancing NO resistance in N. gonorrhoeae (Potter et al., 2009b). TrxB may
play a direct role in the reduction of reactive nitrogen species, as the thioredoxin
systems of E. coli are able to catalyze the denitrosation of GSNO and Snitrosothiols (Nikitovic & Holmgren, 1996, Sengupta et al., 2007). However, TrxB
may also contribute to NO resistance by affecting the regulation of the aniA and
norB denitrification genes, as a trxB mutant is defective in expression of these
transcripts (Potter et al., 2009b). Subsequently, lowered expression of aniA and
norB may account for the reduced capacity of an nmlR mutant to grow under
microaerobic conditions (Kidd et al., 2005, Potter et al., 2009b). See Figure 2 for
an illustration of the oxidative stress tolerance mechanisms of the gonococcus.
Bacterial Biofilms and Chronic Infection
What are Biofilms?
Since biofilms were first described (Zobell & Allen, 1935) and later
recognized as ubiquitous (Costerton et al., 1978), their significance in various
biological processes has gradually become apparent. Subsequently, the
understanding of the complex mechanisms that promote and govern biofilm
formation has steadily progressed. Although, the definition of a biofilm is often
24
Figure 2. Oxidative stress tolerance in N. gonorrhoeae. The following is an
illustration of the oxidative stress defenses present in a N. gonorrhoeae cell. The
outer membrane is designated OM, while the inner membrane is designated IM.
The space between the two membranes is the periplasm, and the space below
the IM is the cytoplasm. Reactive oxygen and nitrogen species and their
intermediates are designated as follows: O2-, superoxide; H2O2, hydrogen
peroxide; NO2-, nitrite; NO, nitric oxide; N2O, nitrous oxide; H2O, water; O2,
oxygen; GSSG, reduced glutathione; GSH, glutathione; GSNO, Snitrosoglutathione; NAPD+, nicotinamide adenine dinucleotide phosphate;
NADPH, reduced nicotinamide adenine dinucleotide phosphate. Proteins that
catalyze reactions in the oxidative stress pathways are represented as colored
ovals and are placed in the positions where they are located in the cell. The
arrows denote reactions, and the proteins that catalyze these reactions are
placed near or on the appropriate arrows. These proteins are designated as
follows: MntABC, manganese ABC transporter; KatA, catalase; SodB,
superoxide dismutase; Ccp, cytochrome c peroxidase; NorB, nitric oxide
reductase; AniA, nitrite reductase; EstD, esterase D; TrxB, thioredoxin oxidase;
Gor, glutathione reductase; Prx, peroxiredoxin.
25
26
debated and may change with research, a biofilm is generally defined as a group
of microorganisms that are enmeshed in an extracellular matrix working
cooperatively, while adhered to a surface (Costerton, 1999, Costerton et al.,
1995, Costerton et al., 1999, Dunne, 2002, Stoodley et al., 2002). This definition
includes aggregates or suspended flocs and bacteria attached to the pore
spaces of porous media (Costerton et al., 1995). Biofilms are predominant in
aquatic environments, are heterogeneous in their structure and composition, and
can be comprised of a variety of organisms that live and grow in these
environments (Costerton et al., 1995).
Biofilm Structure and Formation
Biofilm formation is a dynamic multi-step process. Biofilms have been
studied most extensively in Pseudomonas aeruginosa, a Gram-negative rodshaped bacterium that is an opportunistic pathogen and the predominant biofilm
colonizer in patients with cystic fibrosis (Davies & Bilton, 2009, Moreau-Marquis
et al., 2008, Wagner & Iglewski, 2008). Thus, P. aeruginosa often serves as a
model organism for the study of biofilm. Scanning laser confocal microscopy
(SLCM) has been used extensively to study the structure of P. aeruginosa
biofilms, which have the following generalized architecture: a basal layer of cells
is attached to a surface to which other cells are anchored, and differentiate into
microcolonies that are encased in an extracellular matrix. Microcolonies may
resemble mushroom or stalk structures and their matrix is comprised
predominantly of exopolysaccharides (EPS) in P. aeruginosa (Costerton, 1999,
Costerton et al., 1995, Costerton et al., 1999, Dunne, 2002, Hall-Stoodley &
Stoodley, 2002, Stoodley et al., 2002). Microcolonies have channels that allow
fluid to flow through the structure, and may also have void volumes were cells
are absent (Massol-Deya et al., 1995). Under laminar flow, portions of the biofilm
27
(streamers) may trail from the biofilm in the direction of flow, as the biofilm is
affected by fluid sheer (Costerton, 1999, Costerton et al., 1995, Costerton et al.,
1999, Dunne, 2002, Stoodley et al., 2002, Hall-Stoodley & Stoodley, 2002). See
Figure 3 for an illustration of this generalized structure.
However, biofilm structure appears to be species-specific. For example,
EPS also has been shown to play a more significant role in biofilm formation by
P. aeruginosa than V. parahaemolyticus, subsequently influencing the biofilm
architecture (Lawrence et al., 1991). Other components including proteins, lipids,
nucleic acids (DNA), and LOS have been identified in the biofilm matrix, and are
important for biofilm formation (Karatan & Watnick, 2009, Ma et al., 2009, Mann
et al., 2009, Schooling et al., 2009, Steichen, 2008). Naturally, inclusion of
various combinations of these elements may affect the overall structure of a
biofilm. In addition, the architecture of a biofilm may be greatly affected by the
conditions under which it grows (Haase et al., 2006, Parsek & Greenberg, 2005,
Parsek & Tolker-Nielsen, 2008, Rodrigues et al., 2009). Thus, a single unifying
definition of biofilm has been elusive, although the aforementioned generalized
structure applies for a number of biofilm-forming organisms grown under laminar
flow (Costerton, 1999, Costerton et al., 1995, Costerton et al., 1999, Dunne,
2002, Hall-Stoodley & Stoodley, 2002, Karatan & Watnick, 2009, Stoodley et al.,
2002).
Biofilm formation in P. aeruginosa can be broken down into five discrete
development steps (Sauer et al., 2002, Southey-Pillig et al., 2005, Stoodley et al.,
2002). The first step in biofilm formation is reversible attachment, which occurs
within the first couple of hours following inoculation (Sauer et al., 2002). During
the reversible attachment phase, bacteria may transiently attach or begin to
make a more permanent association with the surface (Sauer et al., 2002). More
permanent association leads to irreversible attachment. During irreversible
28
Figure 3. A generalized model of bacterial biofilm architecture. In this model,
cell clusters are depicted as stalk and mushroom structures. The bulk fluid flows
above the cell clusters, and through channels in these clusters. Streamers trail
from the biofilm in the direction of the fluid flow. Void volumes, which do not
contain cells, are depicted within a mushroom cluster. Figure courtesy of Peg
Dirckx and the Center for Biofilm Engineering, Montana State University,
Bozeman, MT.
29
30
attachment, bacteria closely associate with and do not leave the surface of
attachment (Sauer et al., 2002). At this point, biofilm bacteria begin to exhibit
protein expression profiles that are different from planktonic cells (Sauer et al.,
2002, Southey-Pillig et al., 2005). Irreversibly attached cells undergo replication,
begin to produce biofilm matrix materials, and differentiate into microcolonies.
This step (maturation-1) typically occurs within 3 days and is defined as the point
at which cell clusters become thicker than 10 micrometers (µm) (Sauer et al.,
2002). P. aeruginosa biofilms progress into their penultimate stage (maturation2) as they attain a maximum thickness of approximately 100 µm (Davies et al.,
1998), which typically occurs within 9 days of inoculation (Sauer et al., 2002).
After 9 to 12 days, P. aeruginosa biofilms begin to actively disperse by upregulating cell motility in response to nutrient limitation (Klausen et al., 2006,
Romeo, 2006, Sauer et al., 2002, Southey-Pillig et al., 2005). Dispersal is
believed to contribute to the seeding of new biofilms downstream of the existing
biofilm (Romeo, 2006, Stoodley et al., 2002). These steps and the time in which
they are achieved may vary under different growth conditions in a speciesspecific manner.
Advantages and Inherent Properties
of Biofilm Formation
The ubiquity of biofilms in nature suggests that biofilm formation conveys
some advantage(s) over planktonic growth (Costerton et al., 1978). It was first
suggested that biofilm formation allows bacteria to obtain nutrients that are
available on surfaces (Zobell, 1943). While this is true, there are many other
established advantages of biofilm formation, which are briefly reviewed here.
The most frequently cited advantage is antibiotic resistance (Aparna & Yadav,
2008, Donlan, 2001, Dunne, 2002, Hall-Stoodley et al., 2004, Costerton et al.,
31
1999). Antibiotic resistance is a characteristic that is often used to distinguish
sessile bacteria from planktonic bacteria, as biofilms are inherently resistant to
antimicrobials. In order for bactericidal activity to be achieved, sometimes
antibiotic concentrations up to 4 orders of magnitude higher than those effective
against planktonic organisms are required to treat biofilm (Ceri et al., 1999).
It has become evident that there is no single explanation for antibiotic
resistance in biofilms and that resistance is conferred via a combination of the
following: 1) antibiotics fail to effectively diffuse through and penetrate the full
depth of the biofilm, 2) nutrient limitation within the biofilm leads to slow growing
or starved states that are less susceptible to antimicrobials, and 3) some cells in
a biofilm adopt a protected phenotype in response to growth on a surface
(Aparna & Yadav, 2008, Costerton et al., 1999, Donlan, 2001, Dunne, 2002, HallStoodley et al., 2004). The presence of EPS, a major component of the P.
aeruginosa biofilm matrix, has been shown to limit the diffusion of antibiotics
(Nichols et al., 1988, Stewart, 1998). EPS, microbial cells, inorganic materials,
and other matrix components (e.g. DNA) impede diffusion by reducing the
diffusion coefficient from that of pure water (Stewart, 1998). Most antibiotics
interfere with various aspects of bacterial cell growth to selectively target actively
growing cell populations. Portions of the biofilm population that are slow-growing
or dormant are likely wholly unaffected by antibiotics (Aparna & Yadav, 2008,
Costerton, 1999, Donlan, 2001, Dunne, 2002, Hall-Stoodley et al., 2004, Roberts
& Stewart, 2004). Studies have shown that biofilms are stratified and contain
zones of metabolically active and inactive cells, which was examined through the
immunofluorescent detection of pulse-labeled DNA (Rani et al., 2007, Werner et
al., 2004). Biofilms appear to be largely composed of anabolically inactive yet
viable cells, accounting for up to 70 percent of the total biomass, while
approximately 10 percent of the biofilm contains dead cells (Rani et al., 2007). In
32
P. aeruginosa, protein synthesis occurs in a narrow 30 to 60 µm band near the
biofilm-fluid interface, which correlates with the ability of oxygen to penetrate only
the first 50 µm of the biofilm (Werner et al., 2004). As a general rule, cells close
to the surface of attachment are mostly metabolically inactive, where oxygen and
nutrients are more limited (Rani et al., 2007, Werner et al., 2004, Roberts &
Stewart, 2004). Empiric evidence shows that metabolically inactive or slowgrowing biofilm cells are less susceptible to antibiotics than faster growing cells
(Duguid et al., 1992, Evans et al., 1990a, Evans et al., 1990b). P. aeruginosa
undergoes anaerobic respiration during growth as a biofilm, and this is thought to
be the primary mode of respiration during cystic fibrosis infection (Filiatrault et al.,
2006, Hassett et al., 2002, Van Alst et al., 2007, Yoon et al., 2002). Anaerobic
respiration occurs in areas of the biofilm near surface of attachment where cells
are less metabolically active. Dormancy or metabolic inactivity may contribute to
antimicrobial resistance (Werner et al., 2004). Biofilm formation is enhanced in
P. aeruginosa under anaerobic growth conditions, and strains with insertion
mutations in various anaerobic metabolism genes are impaired in their ability to
form biofilms (Yoon et al., 2002). Moraxella catarrhalis also up-regulates
anaerobic respiratory genes during biofilm formation. Although M. catarrahalis
cannot respire anaerobically, induction of these genes appears to contribute to
oxidative stress tolerance (Wang et al., 2007b). The idea that cells undergo
genetic and phenotypic changes in response to growth on a surface has
prompted the widespread study and comparison of the transcriptional and
proteomic profiles of planktonic and sessile cells (An & Parsek, 2007, Beloin &
Ghigo, 2005, Resch et al., 2006, Resch et al., 2005, Shemesh et al., 2007,
Wagner et al., 2003, Waite et al., 2006, Waite et al., 2005, Whiteley et al., 1999,
Wu et al., 2006). Although the transcriptional and proteomic profiles of various
bacterial populations examined at different times in their growth can vary
33
significantly, it is evident that biofilms have profiles that are distinctly different
from their planktonic counterparts. A variety of genes and proteins have
important roles in biofilm formation, some of which contribute to antimicrobial
resistance in biofilms, such as antibiotic efflux pumps (Zhang & Mah, 2008).
Relatively recently, a new mechanism for antibiotic tolerance in bacterial
biofilms has been proposed that involves the presence of a small population of
persister cells within the biofilm (Jayaraman, 2008, Lewis, 2005). Persisters are
rare non-growing cells (Balaban et al., 2004) that altruistically forfeit propagation
to ensure their survival in the presence of lethal factors (Lewis, 1998). Persisters
are able to survive antibiotic treatment and immune attack so they may
repopulate dwindling biofilms once the concentration of the antibiotic drops, or
antibiotic treatment is withdrawn (Jayaraman, 2008, Lewis, 2005). See Figure 4.
Biofilm formation conveys additional advantages to bacterial survival. Biofilms
are also inherently resistant to ultra-violet (UV) light exposure, acid exposure,
metal toxicity, dehydration, salinity, and phagocytosis (Hall-Stoodley et al., 2004).
Attachment to a surface can provide a source of nutrients that are often dilute in
aqueous solutions (Zobell, 1943, Hall-Stoodley et al., 2004). Attachment may
also offer a degree of stability in an ever changing growth environment and can
contribute to catalytic functions by bringing cells into close contact with one
another (Hall-Stoodley et al., 2004). Cells in close proximity to each other
achieve quorum, which allows for the amplification of chemical signals produced
by individual cells. These signals then dictate beneficial community behaviors
(Parsek & Greenberg, 2005). Attachment to surfaces can also prevent biofilm
organisms from being washed away in bodily secretions. Overall, biofilm
formation in the host can contribute to colonization, pathogenesis, transmission,
34
Figure 4. Mechanisms of biofilm tolerance. This cartoon depicts how the four
proposed mechanisms of antibiotic tolerance work in concert within a biofilm to
convey resistance to antimicrobials and host defenses. Areas of the biofilm
where the diffusion of antibiotics is slowed by the presence of EPS and other
matrix materials are highlighted in yellow. Areas of the biofilm where cells are
slow-growing or exist in an anabolically inactive state, located near the biofilm
substratum, are highlighted in pink. Cells that are undergo specific
transcriptional and proteomic changes in response to growth on a surface are
depicted in green. Persister cells are illustrated in purple.
35
36
and persistence (Davies & Bilton, 2009, Ehrlich et al., 2005, Hall-Stoodley &
Stoodley, 2005, Hall-Stoodley & Stoodley, 2009, Moreau-Marquis et al., 2008,
Wagner & Iglewski, 2008).
Many illnesses and infections in humans are exacerbated and/or caused
by biofilms, including dental caries, otitis media, osteomyelitis, native valve
endocarditis, and a number of nosocomial infections (Costerton et al., 1999).
The formation of a biofilm during infection typically allows these organisms to
persist within the host (Costerton et al., 1999). The ability of biofilms to persist
within the host has been attributed to the heightened antimicrobial resistance of
biofilm-forming organisms, which impedes the clearance of biofilm infections
(Ceri et al., 1999, Dunne, 2002, Schierholz et al., 1999). Therefore, it has been
postulated that nearly all chronic bacterial infections in humans persist as
biofilms (Costerton et al., 1999).
Biofilm Formation by Neisserial Species
N. gonorrhoeae is among those organisms that have recently been
investigated for the ability to form biofilms. N. gonorrhoeae develops structures
consistent with biofilm formation over abiotic surfaces (glass coverslips) under
continuous fluid flow conditions (Greiner et al., 2005). See Figure 5. N.
gonorrhoeae also forms biofilms over primary urethral epithelial and cervical
epithelial cells under static culture conditions (Greiner et al., 2005). N.
gonorrhoeae biofilms contain water channels and a continuous matrix, consistent
with the classically observed structures of P. aeruginosa biofilms (Greiner et al.,
2005). N. gonorrhoeae also forms biofilm over transformed human cervical
epithelial cells (THCEC) under continuous flow conditions, while biopsy evidence
from patients with culture-proven gonorrhea indicates that biofilms are present
during cervical infection (Steichen, 2008). See Figure 6. Altogether, these
37
observations indicate that N. gonorrhoeae forms biofilm during the natural
cervical infection of women. However, little is currently known about the
mechanisms that govern biofilm formation by N. gonorrhoeae.
Several case studies have also identified commensal Neisseria species as
the cause of infective endocarditis biofilms (Imperial et al., 1995, Kaplan & Fine,
2002, Kociuba et al., 1993, Struillou et al., 1993). In addition, N. meningitidis has
been shown to form biofilms over abiotic surfaces (Lappann et al., 2006, O'Dwyer
et al., 2009, Yi et al., 2004) and over human cells in tissue culture (Neil et al.,
2009, Neil & Apicella, 2009a, Neil & Apicella, 2009b). N. gonorrhoeae has also
been implicated in biofilm formation on intrauterine devices (Pruthi et al., 2003).
Overall, these observations strongly suggest that the Neisseria species are
capable of forming biofilms during infection, and that biofilm formation may play
an important role in Neisserial pathogenesis.
Rationale for Research Conducted
Many women do not develop noticeable symptoms of gonorrhea
(Bozicevic et al., 2006, Pedersen & Bonin, 1971). Men may also be
asymptomatic, but most develop readily identifiable symptoms and seek
immediate medical attention (Hansfield, 2005). Asymptomatic infection has been
linked to infection by AHU- strains, but these strains are no more prevalent in
women than men (Brunham et al., 1985). Antigenic variation of the gonococcal
pilus may limit the human immune response by adding to the extensive surface
variability of the gonococcus (Seifert, 1996). In addition, some Opa proteins
down-regulate host immune defenses by binding CEACAM receptors on B and T
cells (Dehio et al., 1998), while LOS may contribute to serum resistance and
38
Figure 5. N. gonorrhoeae biofilms over glass. Panel A shows a typical 4-day
biofilm formed in a continuous-flow chamber by wild-type N. gonorrhoeae strain
1291, and panel B shows the results of confocal analysis of a vertical
reconstruction of a z series using live/dead staining (Molecular Probes,
Invitrogen, Corp., Carlsbad, CA) of the biofilm. The majority of the organisms
nearest the stream of flow are viable. Panels C and E show confocal analyses of
a 4-day biofilm expressing plasmid-borne GFP. These biofilms were grown in
defined medium in the presence of 10 μM sodium nitrite. Panel C shows a
horizontal three-dimensional reconstruction of 60 images taken at 1-μm intervals
(a stacked z series), and panel E shows the vertical view of the same stacked z
series. Panels D and F show confocal analyses a 4-day biofilm that was grown
in defined medium in the absence of sodium nitrite. Panel D shows the stacked z
series, and panel F shows the vertical view of the same stacked z series. The
bars in panels C to F indicate 20 μm. Figure reprinted with permission from
Greiner et al., 2005.
39
40
Figure 6. N. gonorrhoeae biofilms in vivo. The following are electron
micrographs of cervical biopsy specimens from patients with gonococcal
cervicitis. The squamous epithelial cell layer can be seen in the lower portion of
panels A–C; above is a cluster of gonococci, which appear to be part of a biofilm
over this epithelial cell surface. The solid arrows in panels A and B indicate
blebs on the surface of the organisms, and dashed arrows indicate membranous
structures intercalated between the organisms. Panel C shows a control section
of a biopsy specimen where biofilm formation could not be identified. Scale bars
in panels A–C indicate 1 m. Panel D shows immunostaining of an Eponembedded cervical biopsy specimen from a woman with gonococcal cervicitis
demonstrating the edge of a cluster of gonococci, which appear to be part of a
biofilm on the cell surface. The section was stained with the anti-gonococcal H.8
monoclonal antibody 2C3 after an antigen retrieval process consisting of heat
and oxidation of the Epon with hydrogen peroxide. The top portion of panel D
shows the mass of organisms on the epithelial cell surface, and the bottom
portion shows a higher-magnification view demonstrating the colloidal gold
labeling. Figure reprinted with permission from Steichen et al., 2008.
41
42
immune evasion (Griffiss et al., 1988, Mandrell et al., 1988, Mandrell et al., 1990,
Schneider et al., 1985, Wetzler et al., 1992). However, the mechanisms for
phase and antigenic variation exist in all gonococcal strains, offering no
explanation for the apparent difference in symptoms among men and women.
Asymptomatic infection in women is problematic, as it appears to be associated
with an increased risk for serious and potentially life-threatening complications
(Hansfield, 2005, Hook, 1999c, Hook, 1999b).
Little is known about biofilm formation by N. gonorrhoeae, although this
organism has recently been demonstrated to form biofilm over primary cervical
cells and glass under continuous-flow conditions (Greiner et al., 2005). Biofilm
formation by N. gonorrhoeae is also evident during natural cervical infection
(Steichen, 2008). N. gonorrhoeae is one of a handful of human pathogens that
possesses a small genome specifically adapted to life within the host.
Subsequently, it is logical that biofilm formation may play a role in the
pathogenesis of N. gonorrhoeae, particularly in women.
Forming a biofilm offers numerous advantages, including inherent
resistance to antimicrobials and human immune defenses (Aparna & Yadav,
2008, Donlan, 2001, Dunne, 2002, Hall-Stoodley et al., 2004, Costerton et al.,
1999). Many illnesses and infections in humans are exacerbated and/or caused
by biofilms, and the formation of a biofilm during infection typically allows these
organisms to persist within the host (Costerton et al., 1999). The nature of N.
gonorrhoeae infection in men and women and the observation that biofilms form
over cervical tissues in vivo (Steichen, 2008), suggests that biofilm formation
may occur readily in women. In men, the site of N. gonorrhoeae infection is
subject to periodic rapid fluid flow, which may limit biofilm formation. Men also
frequently seek immediate treatment (within days of infection) (Hansfield, 2005,
Hook, 1999c), which can limit infection and likely impede mature biofilm
43
formation. Thus, biofilms may play an important role in the infection of the
female, but not male genitourinary tract and could contribute to the infrequent
occurrence of symptoms in women.
Biofilm cells undergo genetic and phenotypic changes in response to
growth on a surface, and microarrays have often served as a useful tool for
studying the transcriptional changes that occur during the formation of biofilm (An
& Parsek, 2007, Beloin & Ghigo, 2005, Resch et al., 2005, Shemesh et al., 2007,
Wagner et al., 2003, Waite et al., 2006, Waite et al., 2005, Whiteley et al., 1999,
Wu et al., 2006). Although the ability of N. gonorrhoeae to form a biofilm has
been established, little is currently known about the mechanism of biofilm
formation or the signals that regulate biofilm production, architecture, and
dispersal. Yet, biofilm formation may play an important role in the pathogenesis
of N. gonorrhoeae in women. Therefore, we elected to compare the
transcriptional profiles of N. gonorrhoeae biofilm to planktonic modes of growth in
order to identify genes that may be required for normal biofilm formation over
human cervical cells. We found that biofilm formation by the gonococcus may
aid in oxidative stress tolerance during host infection by positively regulating
factors, such as ccp and norB, which are required for the reduction of reactive
oxygen and nitrogen species, respectively. We determined that these genes and
others involved in anaerobic metabolism and/or oxidative stress tolerance are
critical for biofilm formation by N. gonorrhoeae. We also found that reactive
oxygen species, particularly NO, may dynamically affect biofilm formation by
promoting either biofilm dispersal or attachment in response to the environmental
concentration of NO. Overall, biofilm formation may be an important mechanism
by which the gonococcus copes with the inherent oxidative stresses that are
present during infection of human cervical tissues.
44
Hypothesis
N. gonorrhoeae forms biofilm during natural cervical infection, and biofilm
contributes to ability of the gonococcus to survive conditions where oxygen is
scarce and oxidative stresses are present, which occurs at the site of infection in
women. The biofilm of N. gonorrhoeae possesses a unique transcriptional profile
that distinguishes it from planktonic modes of growth. Genes that are involved in
anaerobic metabolism and oxidative stress tolerance are up-regulated during
biofilm formation and are required for normal biofilm formation over abiotic
surfaces (glass) and human cervical cells. The reduction of reactive oxygen and
nitrogen species, as well as the ability to grow under conditions of oxygen
limitation represent critical adaptations that facilitate the successful growth of the
gonococcus in the endocervix of women. The ability of biofilm to tolerate
oxidative stresses produced by the human immune system may also contribute
to the poor immune response that is observed in many women.
45
CHAPTER II
TRANSCRIPTIONAL PROFILING REVEALS THE METABOLIC PHENOTYPE
OF GONOCOCCAL BIOFILMS
Introduction
Neisseria gonorrhoeae is the etiologic agent of gonorrhea, the second
most commonly reported notifiable disease in the United States today (2007). On
average, 62 million new cases of gonorrhea are reported annually worldwide
(Gerbase et al., 1998). Individuals infected with gonorrhea are at higher risk for
contracting HIV (2007, Fleming & Wasserheit, 1999), as the presence of the
gonococcus in the reproductive tract has been shown to increase local
expression of HIV viral RNA (Chen et al., 2003). In addition, women are
susceptible to chronic complications from undiagnosed gonorrhea infection,
which is associated with the lack of noticeable symptoms in most women (Hook,
1999a, Hook, 1999b, Hook, 1999c). Women infected with N. gonorrhoeae
frequently develop upper genital tract infection, which leads to pelvic
inflammatory disease in up to 45 percent of women with asymptomatic infection
(Hook, 1999b).
Antibiotic resistance in N. gonorrhoeae is an increasing problem in the
treatment of gonococcal infection (2007, Centers for Disease Control and
Prevention, 2007, Hook, 1999c, Hook, 1999a, Wang et al., 2007a). Control
strategies for gonorrhea have traditionally relied on single-dose therapy to
promptly clear infection and prevent transmission to others. However,
antimicrobial resistance has often compromised these strategies (Wang et al.,
2007a). Attempts to design an effective vaccine for the prevention of gonococcal
infection have been universally unsuccessful (Hook, 1999a). In 2006, the
number of cases of gonorrhea in the United States increased for the second
46
consecutive year (2007). Thus, it is becoming increasingly important to identify
new strategies for treatment of this disease.
Many illnesses and infections in humans are exacerbated and/or caused
by biofilms, including dental caries, otitis media, osteomyelitis, native valve
endocarditis, and a number of nosocomial infections (Costerton, 1999). N.
gonorrhoeae is among those organisms that have recently been investigated for
the ability to form biofilms. N. gonorrhoeae forms structures consistent with
biofilms on abiotic surfaces (glass coverslips) under conditions of continuous fluid
flow and over primary urethral and cervical epithelial cells under static culture
conditions. These structures contain water channels and a continuous matrix,
consistent with classically observed biofilm structures (Greiner et al., 2005).
Results from our laboratory indicate that N. gonorrhoeae also forms biofilms over
THCEC under continuous-flow conditions, while biopsy evidence from patients
with culture-proven gonorrhea indicates that biofilms are present during cervical
infection (Steichen, 2008). Altogether, these observations suggest that N.
gonorrhoeae forms biofilm during natural cervical infection in women, which may
contribute to persistent infection and could be associated with the apparent
absence of symptoms.
Although the ability of N. gonorrhoeae to form biofilms has been
established, little is currently known about the mechanism of biofilm formation or
the signals that regulate biofilm production, architecture, and dispersal. Thus, we
elected to compare the transcriptional profiles of N. gonorrhoeae biofilms to those
of planktonic modes of growth in order to identify genetic pathways involved in
biofilm formation and maintenance. We found that when biofilm growth was
compared to planktonic growth, 3.8 percent of the genome was differentially
regulated. Our study provides new insights into gonococcal metabolism during
the interaction of the gonococcus with human epithelial cells.
47
Experimental Procedures
Bacteria
N. gonorrhoeae strain 1291, a piliated clinical isolate that expresses Opa
proteins, was used in this study. This strain was reconstituted from frozen stock
cultures and propagated at 37°C with 5% CO2 on GC agar (Becton Dickinson,
Franklin Lakes, NJ) supplemented with 1% IsoVitaleX (Becton Dickinson).
RNA Isolation
Overnight plate cultures were used to create cell suspensions of wild-type
N. gonorrhoeae strain 1291 for inoculation of biofilm flow chambers. N.
gonorrhoeae was grown in continuous-flow chambers in 1:10 GC broth (Kellogg
et al., 1963) diluted in phosphate-buffered saline with 1% IsoVitaleX and 100 µM
sodium nitrite. Nitrite was added to the biofilm medium, as biofilm formation is
significantly enhanced by the addition of nitrite, allowing mature biofilms to form
after 48 h of growth, although nitrite is not required for biofilm formation.
Subsequently, nitrite was added to all biofilm media, unless otherwise noted. Cell
suspensions (in biofilm medium) of 2 x 108 CFU/ml were used to inoculate 37- by
5- by 5-mm (approximately 1 ml volume) flow cell chamber wells. These
chambers were designed to reduce fluid shear on biofilms (versus that in typical
1-mm-deep wells). Flow chambers were incubated under static conditions at
37°C for 1 h postinoculation. Chambers were then incubated for another 48 h at
a flow rate of 150 µl/min. For the first 24 h of biofilm growth, the effluent was
passed through a sterile glass wool filter for the removal of detached biofilm flocs,
collected in a sterile waste flask, and cultured to assess culture purity. At this
time, the waste flask was replaced with a second sterile flask containing 10 ml of
RNAlater (Qiagen Corporation, Valencia, CA) and 100 µl of 10% sodium azide.
For the final 24 h of biofilm growth, the filtered planktonic effluent was collected in
48
this solution to preserve the RNA and to prevent transcriptional changes from
occurring. Planktonic RNA was extracted from the collected effluent, and biofilm
RNA was extracted directly from the biofilm chamber. Two flow chambers were
filtered into a single planktonic collection flask, and each biofilm RNA sample was
extracted from these two flow chambers, while each planktonic RNA sample was
extracted from the combined effluent of these two flow cells. RNA was extracted
using hot acid phenol extraction as follows. Samples were treated with 1 volume
of acid phenol (prepared by adding an equal volume of 2 M citric acid, pH 4.3, to
crystalline phenol and equilibrating it at room temperature for 24 h) with 0.1%
RNase-free sodium dodecyl sulfate, shaken vigorously, and incubated in a water
bath at 80°C for 10 min, with periodic shaking. The samples were cooled in an
ice water bath until cold and then spun at 7,000 rpm for 20 min at 4°C. The
supernatant was removed and extracted with 1 volume of phenol-chloroform (5:1;
Ambion/Applied Biosystems, Austin, TX) and centrifuged for 15 min. The
supernatant was then treated with 1 volume of chloroform and centrifuged for
another 15 min. The supernatant was precipitated with 1 volume of isopropanol
with 0.3 M sodium acetate overnight at –20°C. The RNA was pelleted and
washed with 80% ethanol, and the pellet was dried at room temperature. The
RNA was then digested with DNase I (New England Biolabs, Boston, MA) for 2 h
at 37°C and purified using a Qiagen RNeasy MinElute kit. RNA purity was
assessed on an Agilent 2100 bioanalyzer (Quantum Analytics, Foster City, CA),
and only samples with RNA integrity numbers of 7.5 or greater were used for
hybridization to microarrays.
Microarray Analysis
A total of two biofilm and two planktonic RNA samples were hybridized to
custom MPAUT1a520274F Affymetrix microarrays, which contain probes for N.
49
gonorrhoeae, N. meningitidis, and Haemophilus influenzae strains (Affymetrix
Inc., Santa Clara, CA). These RNAs were transcribed into labeled target cRNAs
as described in the Affymetrix GeneChip manual and were hybridized to the
MPAUT1a520274F arrays. Following hybridization, the chips were stained,
scanned, and analyzed by Affymetrix GeneChip operating software as described
by Affymetrix. ArrayAssist 5.5.1 software (Stratagene, an Agilent Technologies
Company, La Jolla, CA) was used to identify significantly differentially regulated
genes. All chips were normalized using the RMA algorithm. In comparing the
mean log2 ratios for biofilm versus planktonic samples, genes with absolute
changes of 2.0-fold or greater and P values of 0.05 or less were identified as
differentially regulated.
Quantitative Real-Time PCR
Array results were validated using SYBR green quantitative real-time PCR
(qRT-PCR). For each target, qRT-PCR primer sets (Table 1) were selected using
Primer Express software (Agilent Technologies) and obtained from Integrated
DNA Technologies (Coralville, IA). RNA samples were converted into cDNAs as
follows. Two µl of random hexamer primers (Invitrogen Corp., Carlsbad, CA) was
added to 2 µg total RNA in a total volume of 12 µl and incubated at room
temperature for 10 min to allow primers to anneal, then transitioned to 70°C to
relax RNA secondary structure, and finally cooled on ice for 2 min. Next, 2 µl 0.1
M dithiothreitol, 4 µl SuperScript II 10x reaction buffer, 1 µl of a mixture
containing a 10 mM concentration of each deoxynucleoside triphosphate, and 1
µl SuperScript II were added to each tube (all reagents were obtained from
Invitrogen Corp.) and incubated at 42°C for 4 h. At this point, RNA was degraded
with 3.5 µl of 0.5 M EDTA at 65°C for 15 min, and reactions were neutralized with
50
Table 1. Primers used in this study.
Primer
Target
Sequence
qRT-PCR primers:
MFRTF8
MFRTR8
MFRTF27
MFRTR27
MFRTF28
MFRTR28
MFRTF29
MFRTR29
MFRTF30
MFRTR30
MFRTF31
MFRTR31
MFRTF32
MFRTR32
MFRTF33
MFRTR33
MFRTF35
MFRTR35
MFRTF36
MFRTR36
MFRTF37
MFRTR37
MFRTF38
MFRTR38
MFRTF39
MFRTR39
MFRTF40
MFRTR40
MFRTF41
MFRTR41
MFRTF42
MFRTR42
MFRTF43
MFRTR43
omp3
omp3
secD
secD
aniA
aniA
ccp
ccp
fimT
fimT
nifU
nifU
nuoF
nuoF
opaB
opaB
tatC
tatC
ngo0023
ngo0023
modA13
modA13
norB
norB
secF
secF
tatB
tatB
narP
narP
narQ
narQ
mucD
mucD
5’-CGTCGGCATCGCTTTTG-3’
5’-CAGGCTGTTCATGCGGTAGTC-3’
5’-ACCAACCGACAAGCCATCAT-3’
5’-CAATCGACCGCGACTATCCT-3’
5’-CAATCGACCGCGACTATCCT-3’
5’-GGTTTTTTCGACGGTTTCCA-3’
5’-GCGCAAGGTGTATTCCAACCT-3’
5’-AACGGACGGATTTTCTGCAT-3’
5’-GACAAAAACGGCAATAAGGAATATG-3’
5’-CGCTGCGGAGGAAAACAT-3’
5’-TTCCGCCATCGCTTCGT-3’
5’-TCATCCAGACTTTTGCCTTTG-3’
5’-GCATTGCTTGAATCGTTGGAA-3’
5’-GGGAACGGCGGTTTGAA-3’
5’-CAACCGCTCCAGGCAAAA-3’
5’-TGCGTACGGATGTTTCTGAAAT-3’
5’-AGACCCTGCTTGCCATTCC-3’
5’-AGCGTCCGAACCAAATGC-3’
5’-TTGAGCCCGATTGCGAAT-3’
5’-CCGCCGGTAATGACAAACTG-3’
5’-CGAGCAAGGCGGTATCTCAA-3’
5’-GGAAAGTTCGGCATAAACAAACTC-3’
5’-CGAAAGCATCCTGCCTTACTATC-3’
5’-GCGGGTGGTTTGCAACTT-3’
5’-GCAGGGTGCGGATGTCA-3’
5’-CACCCATTTTCAGCGTATCGA-3’
5’-TTTGGGCGAGCTGATTTTTG-3’
5’-GGCGTTCTGGACCAAGGA-3’
5’-CCGCAGCAGGCAGTCATT-3’
5’-CGGTAAGGTCGTCGCTGTCT-3’
5’-TGCAGGAGCGTGCCAAA-3’
5’-TTGTGCTTGGGAACGGATTT-3’
5’-GCCGGTATGGGCAGTATCAA-3’
5’-CCGATGAGTTTGGCGGTATATT-3’
Cloning primers:
aniA_F
aniA_R
aniA_5'Xho1F
aniA_5'Xho1R
aniA_upstm
aniA_downstm
NorBF
NorBRev
aniAcomp_F
aniAcomp_R
ccpcomp_F
ccpcomp_R
norBcomp_F
norBcomp_R
5’-ATGAAACGCCAAGCCTTAG
5’-TAAACGCTTTTTTCGGATGCAG
5’-ACCGCTCGAGGCCGTACTTCCACATTCA
5’-TGAATGTGGAAGTACGGCCTCGAGCGGT
5’-AACTACCCTGCCTTTGCCTGATT
5’-CCGAGGAAAAATAACCGGACATAC
5’-CAGAGCAGGCAAAGGCAG
5’-GAACAGCCCTACCGCATC
5’-GTGTATACATAAATTGCCCTGCCTTTGCC
5’-GTCTTAAGGCGCCGTCTGAAAAATCAATCTTCC
5’-GTCCCGGGGTGGTTTGATTCAATGA
5’-CCCTTAAGTTCAAACGCATCATGGCTTA
5’-TTCCCGGGTTGCAAAGGCAGGGCAATTTC
5’-GGCTTAAGAGGGTGCGGTTTCCATTTTCAG
*Primers for qRT-PCR with their targets and sequences are listed above. An F in the last letter position of
the name denotes the forward primer, while an R denotes the reverse primer. Primers for cloning are listed
below the qRT-PCR primers. Here the primer name denotes the target.
51
5 µl 1 M Tris and 21.5 µl Tris-EDTA (all reagents were obtained from
Ambion/Applied Biosystems). cDNAs were purified using a Qiagen PCR cleanup
kit, quantitated, diluted to 10 ng/µl, and used as templates for qRT-PCR.
Relative RNA quantities were determined with a standard curve (five-fold
dilutions of purified genomic DNA ranging from 100 ng/µl to 0.00032 ng/µl), and
all values were normalized to the amount of outer membrane protein 3 (OMP3)
RNA in each sample. Fifty-microliter reaction mixtures were set up in triplicate,
which contained 1x SYBR Green master mix (Ambion/Applied Biosystems) with
3.5 mM magnesium chloride, 10 ng of template or genomic DNA standard, and a
1 µM final concentration of each primer. RT-PCR was performed on an ABI
Prism 7000 sequence detection system (Quantum Analytics). Each RT-PCR
assay was performed a minimum of two times, and genes were considered
validated if results were consistent for both assays and absolute changes were
equivalent to or greater than the corresponding array changes.
Mutant Construction
An insertion mutant construct of the aniA gene was made via introduction
of a kanamycin resistance cassette (pUC4Kan; Amersham Biosciences,
Piscataway, NJ) into a suitable unique restriction site in the coding region of the
aniA gene. The aniA gene from N. gonorrhoeae strain 1291 was amplified in two
fragments, A and B, using primer set A (aniA_F and aniA5'XhoI_R) and primer
set B (aniA_R and aniA5'XhoI_F), which contains the restriction site XhoI. The
aniA gene containing the XhoI site (at bp 454 in the coding region) was amplified
using fragments A and B and the primers aniA_F and aniA_R, and then cloned
into pGEM-T Easy (Promega, Madison, WI), generating pGEM-aniA(XhoI). The
aniA insertional mutations were created by digesting pGEM-aniA(XhoI) with XhoI
and ligating it with the isolated SalI kanamycin resistance cassette-containing
52
fragment derived from pUC4Kan. The mutant construct was linearized with
SacII, and N. gonorrhoeae strain 1291 was transformed with aniA::kan as
described previously (Jennings et al., 1995). Multiple independent mutant strains
were isolated and confirmed by sequencing and PCR, using the primers
aniA_upstm and aniA_downstm. An insertion mutant construct of the norB gene
was also made via introduction of the kanamycin resistance cassette from
pUC4Kan (Amersham) into a suitable unique restriction site in the coding region
of the norB gene. The norB gene from N. gonorrhoeae strain 1291 was amplified
using primers NorBF and NorBRev, and the resulting product was cloned into
pGEM-T Easy (Promega). The norB insertional mutation was created by
digesting this plasmid with BsaBI and ligating it with the isolated HincII kanamycin
resistance cassette-containing fragment derived from pUC4Kan (Amersham).
The mutant construct was linearized with NotI and transformed into N.
gonorrhoeae strain 1291 as described previously (Jennings et al., 1995). For
each mutant construct, multiple independent mutant strains were isolated and
confirmed by sequencing and PCR, using the same primers. See Table 1 for
cloning primers and Table 2 for plasmids and strains.
Biofilm Growth in Continuous-Flow Chambers Over Glass
Wild-type N. gonorrhoeae strain 1291 and aniA::kan, ccp::kan, and
norB::kan insertional mutants were assayed for the ability to form biofilms. A
ccp::kan mutant was described in an earlier publication (Seib et al., 2004) and
was transformed with pGFP for confocal microscopy. Strains were propagated
from frozen stock cultures on GC agar with 1% IsoVitaleX (Becton Dickinson,
Franklin Lakes, NJ) and incubated at 37°C and 5% CO2. Overnight plate cultures
were used to create cell suspensions for the inoculation of biofilm chambers.
53
Table 2. Strains and plasmids used in this study
Strain or plasmid
Relevant genotype or properties
Strains:
N. gonorrhoeae 1291
aniA::kan
norB::kan
ccp::kan
wild type
aniA::kan ∆aniA
norB::kan ∆norB
ccp::kan ∆ccp
Plasmids:
pGEM-Teasy
pUC4Kan
pUC4/kanamycinR
pGEM-aniA(XhoI)
pGEM-aniA:kan
pGEM-norB
pGEM-norB:kan
pGFP
pGEM-Teasy/aniA
pGEM-Teasy/aniA::kan ∆aniA
pGEM-Teasy/norB
pGEM-Teasy/norB::kan ∆norB
pLES98/GFP/chloramphenicalR
pCTS32
pGEM-Teasy/proB::spec/ampicillinR
pCTS32-aniAcomp
pCTS32-ccpcomp
pCTS32-norBcomp
pCTS32/aniA
pCTS32/ccp
pCTS32/norB
Source or
reference
ATCC
This study
This study
(Seib et al.,
2004)
Promega
Amersham
Biosciences
This study
This study
This study
This study
(Edwards and
Apicella, 2005
(Steichen et al.,
2008)
This study
This study
This study
*Strains and plasmids used during this study are listed above with a description of their relevant
genotypes or properties. The source of each strain or plasmid is also denoted. This study refers
to any strains or plasmids constructed during the course of this study, while a citation refers to the
appropriate reference listed in the references section.
54
N. gonorrhoeae was grown in continuous-flow chambers over glass as described
previously. Chloramphenicol was added to the medium at a final concentration of
5 µg/µl to maintain pGFP. After 48 h, the biofilm effluent was cultured to assure
culture purity, and biofilm formation was assessed via confocal microscopy.
Growth Curves Under Oxygen Tension Conditions
Present in Biofilm Medium
A GEM Premier 3000 blood gas meter (Instrumentation Laboratory
Company, Bedford, MA) was used to measure the dissolved oxygen content
present in the biofilm medium collected from the medium reservoir and the biofilm
outflow at 0, 24, and 48 h of biofilm growth. The medium was collected under
mineral oil to prevent gas exchange during collection. Approximately 0.2 ml of
each sample was run on the blood gas meter immediately following collection.
Static growth curves for N. gonorrhoeae aniA::kan, ccp::kan, and norB::kan
mutants and the wild-type parent strain were then performed under similar
oxygen tension conditions in GC broth with 1% IsoVitalex and 100 µM sodium
nitrite and assessed by measuring the dissolved oxygen content of the culture
medium. Growth was monitored for 48 h.
THCEC Culture
Primary cervical cells were obtained from cervical biopsies performed at
the University of Iowa Hospitals and Clinics, Iowa City, IA, and immortalized by
the methodology developed by Klingelhutz et al. (Klingelhutz et al., 1994). The
resulting cell line was designated THCEC. Our studies have confirmed the
presence of complement receptor type III on the surfaces of these transformed
cells and their ability to secrete complement components. These factors are
important in gonococcal attachment to and invasion of cervical epithelial cells
55
(Edwards et al., 2001). THCEC were cultured in 100-mm tissue culture plates in
serum-free keratinocyte growth medium (K-SFM) supplemented with 12.5 mg
bovine pituitary extract, 0.08 µg epidermal growth factor per 500-ml bottle, and a
1:500 dilution of penicillin-streptomycin liquid (10,000 units penicillin and 10,000
µg streptomycin in 0.85% saline;Gibco Cell Culture) at 37°C and 5% CO2. Once
confluent, the cells were split onto collagen-coated coverslips, which were
prepared by autoclaving 22- by 50-mm no. 1 coverslips in bovine tendon collagen
type I (Worthington Biochemical Corp., Lakewood, NY), removing the coverslips
from solution, and allowing them to dry for 30 min in 100-mm tissue culture plates
at room temperature. Cells were split as follows. Two ml of 0.25% tryspin-1 mM
EDTA (Gibco) was applied to a confluent 100-mm tissue culture plate for 4 min at
room temperature and then aspirated, and the plate was incubated for an
additional 5 min at 37°C and 5% CO2. Cells were collected in 5 ml K-SFM with
5% fetal bovine serum (Gibco) to neutralize trypsin and then spun down and
resuspended in a final volume of K-SFM to result in a 1:8 dilution per glass
coverslip. A total of 0.5 ml of this cell suspension was applied to each collagencoated coverslip, covering the entire surface. Coverslips were then incubated for
3 to 4 h at 37°C and 5% CO2 to allow cells to adhere, and then plates were
flooded with K-SFM (10 ml). Cells were grown until confluent at 37°C and 5%
CO2 (2 days) and were stained with Cell Tracker Orange (Molecular Probes,
Invitrogen Corp., Carlsbad, CA) just prior to infection.
Biofilm Growth in Continuous-Flow Chambers
Over Cells
N. gonorrhoeae was grown in continuous-flow chambers adapted for
tissue culture in 1:5 JEM medium (2 parts serum-free hybridoma medium, 1 part
McCoy's 5A medium, and 1 part defined K-SFM; all available from Gibco) diluted
56
in phosphate-buffered saline with 1% IsoVitaleX, 100 µM sodium nitrite, 0.5 g/liter
sodium bicarbonate to buffer the medium, and 5 µg/µl chloramphenicol to
maintain pGFP. Fifty- by 22- by 5-mm tissue culture biofilm chambers
(approximately 3 ml volume) were assembled with confluent THCEC coverslips,
which were placed between the top and bottom portions of the chamber and
sealed using a rubber gasket and screws that fasten the top and bottom portions
together. Chambers were inoculated at a multiplicity of infection (MOI) of 100:1,
using cell suspensions prepared in biofilm medium. Flow chambers were
incubated under static conditions at 37°C and 5% CO2 for 1 h postinfection to
allow attachment to THCEC. Chambers were then incubated for 48 h at 37°C at
a flow rate of 180 µl/min. After 48 h, the biofilm effluent was cultured to assure
culture purity, and biofilm formation was assessed via confocal microscopy.
Trypan Blue Viability Assays
To assess the impact of infection with the various strains used in this
study, THCEC were treated with 0.25% trypsin-EDTA (Gibco), collected in PBS,
and stained with trypan blue (Invitrogen) after 48 hours of biofilm formation.
THCEC were infected with wild-type N. gonorrhoeae strain 1291 and aniA::kan,
ccp::kan, and norB::kan insertional mutants. Uninfected chambers were used as
a control. Viable counts were made using a hemocytometer, and the percentage
of adherent cells remaining after 48 h was calculated by comparing the number
of viable cells after 48 h to the initial number of adherent cells in each chamber.
57
Complementation of
aniA::kan, ccp::kan, and norB::kan Mutants
The ccp and norB genes and their nascent promoters were PCR amplified
from N. gonorrhoeae strain 1291 genomic DNA, using primers that generated a
XmaI site at the 5′ end and an AflII site at the 3′ end. The aniA gene and its
promoter was amplified as a single fragment from N. gonorrhoeae strain 1291
genomic DNA, using primers that generated a BstZI7I site at the 5′ end and an
AflII site at the 3′ end. The resulting fragments were cloned into pGEM-T easy
(Promega). These fragments were then subcloned into pCTS32 (Steichen,
2008) for recombination into the proline B (proB) gene of N. gonorrhoeae. The
restriction sites generated by PCR amplification were selected for ligation into the
pCTS32 in the opposite orientation of the SpecR promoter to allow transcription
to be initiated from the nascent promoters of these genes. The resulting
constructs were sequenced and used to transform N. gonorrhoeae 1291
ccp::kan, norB::kan, and aniA::kan accordingly. Transformants were selected on
GC agar containing spectinomycin and supplemented with proline. Integration of
these constructs was confirmed via PCR and Southern blot. To assess
complementation, biofilms were grown over THCEC as previously described with
the following modification: proline was added to the media at a concentration of
0.2 g/ml.
Confocal Microscopy of Continuous-Flow Chambers
z-Series photomicrographs of flow chamber biofilms were taken with a
Nikon PCM-2000 confocal microscope scanning system (Nikon, Melville, NY),
using a modified stage for flow cell microscopy. Green fluorescent protein (GFP)
was excited at 450 to 490 nm and Cell Tracker Orange dye (Molecular Probes)
was excited at 540 to 580 nm for biofilm imaging. Three-dimensional images of
58
the biofilms were created from each z series, using Volocity high-performance
three-dimensional imaging software (Improvision Inc., Lexington, MA). The
images were adjusted to incorporate the pixel sizes for the x, y, and z axes of
each image stack.
COMSTAT Analysis of Confocal z-Series
Quantitative analysis of each z series was performed using COMSTAT
(Heydorn et al., 2000), available from http://www.im.dtu.dk/comstat/. COMSTAT
is a mathematical script written for MATLAB 5.3 (The Mathworks, Inc., Natick,
MA) that quantifies three-dimensional biofilm structures by evaluating confocal
image stacks so that pixels may be converted to relevant measurements of
biofilm, including total biomass and average thickness. To complete COMSTAT
analysis, an information file was created for each z series to adjust for the pixel
sizes of the x, y, and z axes and the number of images in each z series.
COMSTAT was then used to obtain threshold images to reduce the background.
Biomass and the average thicknesses in each z series were calculated by
COMSTAT, using the threshold images.
Treatment of Biofilms with Nitric Oxide Donor
and Inhibitor
N. gonorrhoeae 1291 wild-type biofilms were treated with the nitric oxide
donor sodium nitroprusside (SNP), either at the start of the biofilm or after 24 h of
biofilm formation. When treating cells at the start of the biofilm, a final
concentration of 500 nM SNP was added to the biofilm medium prior to initiating
flow, and this medium was used to create suspensions for inoculation of the
biofilm. When treating cells after 24 h of biofilm formation, a final concentration of
500 nM SNP was added to the medium reservoir after the biofilm had been
59
allowed 24 h of growth time. To demonstrate that the effects of SNP were due to
nitric oxide release, wild-type biofilms were also treated with the nitric oxide
quencher 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). These
biofilms were treated at the start of the biofilm with final concentrations of 500 nM
SNP and 1 µM PTIO. All treated samples and untreated controls were run in
quadruplicate for a minimum of four experiments. N. gonorrhoeae 1291
norB::kan mutant biofilms were also treated with PTIO (0.1 mM) and compared to
untreated norB::kan mutant biofilms, which were run at the same time. Biofilm
formation was evaluated by confocal microscopy and COMSTAT analysis as
previously described.
Statistical Analysis of COMSTAT Results
Statistical analysis was performed with Prism 4 software (GraphPad
Software, Inc., La Jolla, CA). Paired Student’s t tests and nonparametric oneway analysis of variance with Dunn's multiple comparison posttest were used to
compare the biomass and average thickness of the aniA::kan, ccp::kan, and
norB::kan insertional mutant biofilms to those of the wild-type biofilm, or treated
samples to the 48-h untreated biofilm sample in the case of the NO studies.
Values that met a P value cutoff of 0.05 were considered statistically different.
Results
Microarray Analysis of Genes
Differentially Expressed During Biofilm Growth
Our working hypothesis was that physiological differences between biofilm
and planktonic cells would be reflected in their transcriptional profiles. We
compared the transcriptional profiles of cells grown as a biofilm to those of
planktonic cells collected from the biofilm outflow, allowing for the identification of
60
gene expression patterns that are specific to biofilm growth and development.
Genes with an absolute change of 2.0-fold or greater and a P value of 0.05 or
less were identified as differentially regulated. Under these analysis conditions,
83 genes were identified as differentially regulated, when we compared biofilm to
planktonic growth (see Tables 3 and 4). This represents approximately 3.8
percent of the genome.
Forty-eight hypothetical genes were identified, which accounts for 57.8
percent of the differentially regulated genes. However, the majority of the upregulated genes (11 of 16 genes) do have identified functions. Five of these
genes had changes that were greater than 2.5-fold, including three genes, aniA,
ccp, and norB, which play critical roles in the anaerobic respiratory pathways of
N. gonorrhoeae (Seib et al., 2006). AniA is an inducible nitrite reductase (Mellies
et al., 1997) that is required for anaerobic growth (Householder et al., 1999) with
nitrite as an electron acceptor (Knapp & Clark, 1984). NorB is a nitric oxide
reductase that is also required for oxygen-limited growth and has been shown to
play a key role in the protection of cells against NO toxicity (Householder et al.,
2000). Ccp is a cytochrome c peroxidase that is expressed only during anaerobic
growth (Lissenden et al., 2000) and is responsible for protecting the gonococcus
from H2O2-mediated killing (Turner et al., 2003). The majority of the downregulated genes did not have identified functions, as 43 of the 67 genes are
hypotheticals. Of the 24 genes with identified functions, 6 belong to the nuo
operon, which is an NADH dehydrogenase (ND-1) involved in respiratory electron
transfer (Yagi, 2004). All genes in the nuo operon (nuoA to nuoN) were found to
be down-regulated via microarray, although the remaining eight genes met only a
1.5-fold cutoff.
61
Table 3. Genes up-regulated during biofilm formation
Fold Change
(B/P)
Gene
p value
Number
5.40
3.29
2.84
2.75
2.64
2.62
2.60
2.54
2.31
2.26
2.19
0.0042
0.0130
0.0031
0.0072
0.0045
0.0119
0.0390
0.0104
0.0481
0.0370
0.0028
NGO1276
NGO0893
NGO0080
NGO1275
NGO1769
NGO0562
NGO0492
NGO1473
NGO2052
NGO0641
NGO0340
2.14
2.04
2.04
2.01
2.00
0.0212
0.0135
0.0165
0.0192
0.0115
NGO0895
NGO0474
NGO1289
NGO1277
NGO0754
Name
Function
nitrite reductase
putative oxidoreductase
opaB
outer membrane opacity protein
norB
nitric oxide reductase
ccp
cytochrome c peroxidase
dihydrolipoamide dehydrogenase
hypothetical phage associated protein
mdaB
modulator of drug activity B
PhnA protein
modA13 mod methylase
cysK
cysteine synthase/cystathionine betasynthase
hypothetical protein
hypothetical phage associated protein
hypothetical protein
hypothetical protein
molybdopterin-guanine dinucleotide
biosynthesis
aniA
*This table lists genes up-regulated during biofilm versus planktonic growth meeting a foldchange cutoff of 2.0 and a p-value cutoff of 0.05. Gene numbers refer to the annotations
available from the Kyoto Encyclopedia of Genes and Genomes (KEGG), which are available at
http://www.kegg.com. A gene name and function is identified where possible.
62
Table 4. Genes down-regulated during biofilm formation
Fold Change
(P/B)
p value
Gene
Number
9.74
5.89
5.61
5.55
5.14
0.0050
0.0184
0.0056
0.0141
0.0288
NGO0015
NGO1777
NGO1348
NGO1419
NGO0023
3.74
3.47
3.34
3.09
3.05
3.05
2.86
2.85
2.70
2.70
2.67
2.66
2.61
2.60
2.59
2.58
2.57
2.56
2.52
2.51
2.50
0.0263
0.0436
0.0178
0.0040
0.0215
0.0112
0.0348
0.0002
0.0285
0.0028
0.0025
0.0026
0.0075
0.0482
0.0487
0.0094
0.0371
0.0267
0.0161
0.0157
0.0142
NGO1051
NGO1760
NGO0630
NGO1293
NGO1563
NGO0179
NGO0054
NGO0518
NGO0967
NGO2096
NGO1720
NGO2162
NGO1361
NGO1953
NGO1106
NGO0378
NGO1230
NGO1929
NGO0983
NGO0667
NGO1537
2.50
2.38
2.37
2.37
0.0039
0.0053
0.0130
0.0040
NGO0784
NGO0644
NGO0752
NGO1494
2.37
2.35
2.34
2.32
2.31
2.29
2.29
2.28
2.27
2.20
2.20
2.18
2.18
0.0132
0.0101
0.0100
0.0024
0.0207
0.0102
0.0041
0.0177
0.0059
0.0107
0.0291
0.0022
0.0398
NGO1744
NGO2003
NGO1741
NGO0843
NGO1960
NGO0141
NGO1747
NGO0927
NGO1418
NGO0182
NGO1147
NGO0651
NGO2091
nuoH
2.16
0.0156
NGO0656
oxlT
Name
lip
Function
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
ABC transporter, periplasmic binding
protein
hypothetical protein
hypothetical protein
putative chaperone protein HscB
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical phage associated protein
hypothetical protein
putative sodium-dependent transporter
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical phage associated protein
hypothetical protein
hypothetical protein
hypothetical protein
outer membrane lipoprotein
hypothetical protein
phospho-N-acetylmuramoyl-pentapeptidetransferase
narL
nuoK
nuoE
nqrF
tatB
hypothetical protein
ribosomal-binding factor A
two-component system
ABC transporter, periplasmic binding
protein
NADH dehydrogenase I
hypothetical protein
NADH dehydrogenase I
hypothetical protein
hypothetical protein
hypothetical protein
NADH dehydrogenase I
hypothetical protein
NADH:ubiquinone oxidoreductase
sec-independent protein translocase
hypothetical protein
hypothetical protein
ABC transporter, permease protein,
enterobactin
membrane transporter
63
Table 4 Continued.
Fold Change
(P/B)
p value
Gene
Number
2.15
2.14
2.12
2.11
2.10
2.10
2.09
2.07
2.06
2.06
2.06
2.05
2.05
0.0386
0.0190
0.0242
0.0080
0.0436
0.0117
0.0295
0.0092
0.0450
0.0220
0.0006
0.0370
0.0245
NGO0181
NGO1740
NGO1746
NGO0171
NGO0876
NGO0622
NGO1536
NGO1742
NGO1752
NGO0028
NGO18781
NGO1298
NGO0713
2.04
2.04
0.0012
0.0306
NGO2142
NGO0606
2.04
0.0114
NGO1535
2.03
2.03
2.02
2.01
2.01
2.01
2.01
0.0122
0.0022
0.0134
0.0000
0.0017
0.0029
0.0454
NGO0785
NGO2172
NGO0703
NGO1917
NGO0083
NGO2136
NGO0905
Name
Function
tatC
nuoL
nuoF
sec-independent protein translocase
NADH dehydrogenase I
NADH dehydrogenase I
50S ribosomal protein L19
hypothetical protein
hypothetical protein
hypothetical protein
NADH dehydrogenase I
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
2-deydro-3-deoxyphosphogluconate
aldolase
hypothetical protein
putative sodium-dependent transport
protein
nuoJ
murD
UDP-N-acetylmuramoyl-L-alanyl-D-glutamate
synthase
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
pilin glycosylation protein
hypothetical protein
hypothetical protein
*This table lists genes down-regulated during biofilm versus planktonic growth meeting a foldchange cutoff of 2.0 and a p-value cutoff of 0.05. Gene numbers refer to the annotations
available from the Kyoto Encyclopedia of Genes and Genomes (KEGG), which are available at
http://www.kegg.com. A gene name and function is identified where possible.
64
Validation of Microarray Results
To confirm our microarray results, we performed SYBR Green qRT-PCR.
We selected a variety of highly up- and down-regulated genes, including aniA,
ccp, norB, and a representative member of the nuo operon, nuoF. In addition, we
selected some genes with changes that were below our 2-fold cutoff (those that
met a 1.5-fold cutoff). Relative RNA quantities were determined by a standard
curve, and all values were normalized to the amount of OMP3 RNA in each
sample. Reactions were performed in triplicate, and each qRT-PCR assay was
performed a minimum of two times. Expression profiles were validated for
differentially regulated genes (absolute change of 2.0-fold or greater) when
results were consistent for both assays and absolute change values were
equivalent to or greater than the corresponding microarray change values.
Thirteen of 16 selected targets were validated via qRT-PCR (Table 5).
The three genes that did not meet our criteria for validation are known or
suspected to be phase variable, which might account for their inconsistent qRTPCR profiles. The unverified genes included ngo0023 (encoding a periplasmic
binding protein of an ABC transporter), ngo0641 (modA13 methylase gene), and
ngo0080 (opaB).
Expression Profiles of Highly Differentially Regulated
Genes in Biofilms Grown Over Host Cells
Microarray and qRT-PCR results indicated that aniA, ccp, norB, and the
nuo operon are among the most highly differentially expressed genes between
biofilm and planktonic growth states. This observation and the identified roles of
aniA, ccp, and norB in anaerobic respiration made them attractive candidates for
further study. However, before characterizing the roles of these genes in
65
Table 5. qRT-PCR validation of microarray results
Gene Number
Name
Array Fold Change
(B/P)
qRT-PCR Fold Change
(B/P)
Up-regulated genes meeting a 2.0 fold cutoff:
NGO0641
NGO1769
NGO0070
NGO1275
NGO1276
modA13
ccp
opaB
norB
aniA
2.3
2.6
2.8
2.8
5.4
1.4 ± 0.2
6.6 ± 1.6
1.7 ± 1.3
6.7 ± 2.0
17.9 ± 4.2
Up-regulated genes not meeting a 2.0 fold cutoff:
NGO0452
fimT
1.8
1.3 ± 0.3
Down-regulated genes meeting a 2.0 fold cutoff:
NGO1746
NGO0182
NGO0752
NGO0023
nuoF
tatB
narL
2.1
2.2
2.4
5.1
3.5 ± 0.8
2.6 ± 0.6
3.1 ± 0.6
1.3 ± 0.3
Down-regulated genes not meeting a 2.0 fold cutoff:
NGO0189
NGO0190
NGO0138
NGO0181
NGO0633
NGO0753
secD
secF
mucD
tatC
nifU
narX
1.5
1.5
1.6
1.7
1.7
1.9
2.6 ± 1.3
3.0 ± 1.4
3.5 ± 1.2
1.9 ± 0.3
4.7 ± 0.8
2.5 ± 0.3
*This table depicts the expression profiles of genes identified as differentially regulated, which we
attempted to validate with qRT-PCR. The original microarray fold-change and qRT-PCR foldchange values plus or minus one standard deviation from the mean are listed, along with gene
functions and KEGG accession numbers. The bolded genes did not meet our criteria for
validation.
66
biofilms, and in view of the fact that N. gonorrhoeae is an obligate human
pathogen (Hook, 1999a), we elected to study the expression profiles of these
genes in biofilms grown over THCEC. We did not perform microarray studies for
biofilms grown over THCEC in order to avoid the problem of contaminating
THCEC RNAs. In order to study the expression profiles of aniA, ccp, norB, and
nuoF in biofilms over cells, we grew biofilms over THCEC in flow cells for 48 h,
confirmed the presence of a biofilm by confocal microscopy, and collected the
planktonic effluent as described for the microarray studies. We collected
duplicate planktonic and biofilm RNA samples from two independent experiments
and used these RNAs as templates for qRT-PCR. We compared aniA, ccp,
norB, and nuoF qRT-PCR expression profiles from this experiment to the qRTPCR profiles for biofilms on glass (Figure 7). We found that aniA, ccp, and norB
were up-regulated in biofilms grown over THCEC, similar to their expression in
biofilms grown on glass. The nuoF gene was also down-regulated in biofilms
over THCEC, similar to the case for biofilms on glass. The similar expression of
these genes in biofilms grown over cells and glass warranted pursuit of the role of
anaerobic respiration in N. gonorrhoeae biofilms.
Characterization of the Role of Anaerobic
Metabolism Genes in Biofilms Over Glass
Wild-type N. gonorrhoeae clinical strain 1291 and isogenic aniA::kan,
ccp::kan, and norB::kan mutants were assayed for the ability to form biofilms in
flow cells over glass. These mutants were transformed with pGFP for
examination of biofilms via confocal microscopy (Figure 8). We found that there
was no significant difference in the biomass of the aniA::kan or ccp::kan mutant
when either was compared to the wild type. However, the aniA::kan and ccp::kan
mutants did have significantly smaller average thicknesses (P ≤ 0.001).
67
Figure 7. Expression of aniA, ccp, norB, and nuoF in biofilms grown over cells.
Relative RNA quantities were determined by a standard curve, and all values
were normalized to the amount of OMP3 RNA in each sample. Reactions were
performed in triplicate, and each qRT-PCR assay was performed a minimum of
two times. qRT-PCR values for biofilms grown over THCEC are displayed by
gray bars, and qRT-PCR values for biofilms grown over glass are displayed by
white bars. The error bars represent 1 standard deviation from the mean for the
averaged qRT-PCR data from duplicate assays.
68
69
Figure 8. Biofilm formation over glass. Biofilm masses over 2 days of growth for
wild-type N. gonorrhoeae strain 1291 (1) and aniA::kan (2), ccp::kan (3), and
norB::kan (4) insertion mutants. All four strains were visualized by GFP plasmid
expression. The images are three-dimensional reconstructions of stacked z
series taken at a magnification of x200 and rendered by Volocity. These
experiments were performed a minimum of three times, and representative
results are shown.
70
71
Figure 9. COMSTAT analysis of biofilm formation over glass. COMSTAT and
statistical analyses of biomass (A) and average thickness (B) for wild-type
(checkered bars) and aniA::kan (black bars), ccp::kan (gray bars), and norB::kan
(white bars) insertion mutant biofilms grown over glass for 2 days.
Representative images of these biofilms are depicted in Figure 8. All strains
were run in duplicate in a minimum of three experiments, and at least four
images of each biofilm chamber were used for COMSTAT analysis. The error
bars represent 1 standard deviation from the mean. Statistical differences
between mutants and the wild type, determined by Student's t test, are denoted
by asterisks above the error bars. ***, P value of 0.001 or less.
72
73
The norB::kan mutant had the most dramatic biofilm-deficient phenotype, with
significantly less biomass and significantly smaller average thicknesses than the
wild type (P ≤ 0.001) (Figure 9). To confirm that the defect in biofilm formation
was not due to a defect in growth under the oxygen tension conditions present in
the medium in our biofilm system, we measured the dissolved oxygen content of
medium entering and exiting the flow chambers at 0, 24, and 48 hours. We found
that the oxygen concentration (150 to 200 mm Hg) was much higher than
concentrations that are typically considered to be anaerobic or microaerophilic (5
to 27 mm Hg) (Dunn et al., 1979, Loesche et al., 1983). When we performed 48hour growth curves under similar oxygen tension conditions, we found that there
was no defect in the growth of aniA::kan, norB::kan, and ccp::kan insertion
mutants compared to the wild-type strain. This finding was the same for growth
curves conducted under aerobic culture conditions (data not shown).
Characterization of the Role of Anaerobic
Metabolism Genes in Biofilms Over THCEC
Wild-type N. gonorrhoeae clinical strain 1291 and aniA::kan, ccp::kan, and
norB::kan insertional mutants were also assayed for the ability to form biofilms
over THCEC, as all three anaerobic mutants were deficient in some aspect of
biofilm formation over glass. We used confocal microscopy to evaluate whether
the phenotypes of biofilms grown on glass were similar to or more exaggerated
than those of biofilms grown over cells (Figure 10). As the qRT-PCR results
suggested, based on at least equivalent expression of these genes in biofilms
grown over THCEC, these biofilms were more severely attenuated over THCEC
than on glass. All three mutants had significantly less biomass and significantly
smaller average thicknesses than the wild type did (P ≤ 0.001) (Figure 11).
Infection reduced the viability of THCEC, but there was no difference in the
74
Figure 10. Biofilm formation over THCEC. Biofilm masses over 2 days of
growth for wild-type N. gonorrhoeae strain 1291 (1) and aniA::kan (2), ccp::kan
(3), and norB::kan (4) insertion mutants. All four strains were visualized by GFP
plasmid expression, while THCEC were visualized by Cell Tracker Orange
(Molecular Probes, Invitrogen Corp., Carlsbad, CA) staining. The images are
three-dimensional reconstructions of stacked z series taken at a magnification of
x200 and rendered by Volocity. These experiments were performed a minimum
of three times, and representative results are shown.
75
76
Figure 11. COMSTAT analysis of biofilms grown over THCEC. COMSTAT and
statistical analyses of biomass (A) and average thickness (B) for wild-type
(checkered bars) and aniA::kan (black bars), ccp::kan (gray bars), and norB::kan
(white bars) insertion mutant biofilms grown over THCEC for 2 days.
Representative images of these biofilms are depicted in Figure 10. All strains
were run in duplicate in a minimum of three experiments, and at least four
images of each biofilm chamber were used for COMSTAT analysis. The error
bars represent 1 standard deviation from the mean. Statistical differences
between mutants and the wild type, determined by Student's t test, are denoted
by asterisks above the error bars. *, P value of 0.05 or less; **, P value of 0.01 or
less.
77
78
Figure 12. Biofilm formation by complemented strains over THCEC. Biofilm
masses over 2 days of growth for wild-type N. gonorrhoeae strain 1291 (1) and
complemented aniA::kan (2), complemented ccp::kan (3), and complemented
norB::kan (4) insertion mutants. All four strains were visualized by GFP plasmid
expression, while THCEC were visualized by Cell Tracker Orange (Molecular
Probes) staining. The images are three-dimensional reconstructions of stacked z
series taken at a magnification of x200 and rendered by Volocity. These
experiments were performed a minimum of three times, and representative
results are shown.
79
80
viability of THCEC infected with aniA::kan, ccp::kan, norB::Kan, or wild type.
Complementation of these mutations restored biofilm formation to wild type levels
(Figure 12). There was no significant difference in the biomass or average
thicknesses of the complemented mutants when compared to the wild type (P ≥
0.4).
Effect of Nitric Oxide on N. gonorrhoeae
Biofilm Formation
We observed that the norB mutant had a more pronounced biofilmdeficient phenotype than an aniA mutant when N. gonorrhoeae biofilms were
grown over glass. One explanation for this phenotype is that NO accumulates in
the norB mutant, and not in the aniA mutant, since AniA produces NO and this
radical species is reduced by NorB to nitrous oxide. Findings for Pseudomonas
aeruginosa (Barraud et al., 2006) and Staphylococcus aureus (Schlag et al.,
2007) suggest that NO may be an important signaling molecule that causes
biofilm detachment when present at sublethal concentrations within the biofilm.
This prompted us to investigate the effect of NO on wild-type N. gonorrhoeae
1291 biofilms grown over glass. Wild-type biofilms treated with a sublethal
concentration of NO (500 nM) either at the start of the biofilm or after 24 hours of
biofilm growth did have significantly less biomass and smaller average
thicknesses than untreated biofilms. There was no apparent difference in
biomass and average thickness for biofilms grown for 24 hours without treatment
and biofilms grown for 24 hours and then treated with SNP for another 24 hours.
This suggests that the addition of SNP to a 24-hour biofilm halts development of
the biofilm. Furthermore, biofilm formation was restored to wild type levels when
biofilms were simultaneously treated with NO and an NO quencher, suggesting
that defects in biofilm formation were due to the accumulation of NO (Figure 13).
81
Treatment with the NO quencher (PTIO) alone did not alter biofilm formation
(data not shown).
In order to directly assess the effect of NO accumulation on norB mutant
biofilms, we also treated N. gonorrhoeae 1291 norB:kan biofilms with the NO
quencher and compared these biofilms to untreated norB:kan biofilms. We found
that treatment with the NO quencher enhanced norB mutant biofilm formation
compared to untreated biofilm formation (Figure 14). Moreover, we found that a
higher concentration of NO quencher was required to restore the defect in biofilm
formation by the norB mutant than that for wild-type biofilms artificially treated
with an NO donor. This finding suggests that NO accumulates at concentrations
in excess of 500 nM in norB mutant biofilms.
Discussion
Microarray analysis indicated that biofilms of N. gonorrhoeae possess a
unique transcriptional profile that distinguishes them from planktonic modes of
growth. Specifically, genes required for anaerobic respiration (aniA, ccp, and
norB) were more highly expressed during biofilm growth, while genes involved in
respiration with NADH as an electron donor (nuo operon) were more highly
expressed during planktonic growth. Mutants in which the aniA, ccp, or norB
gene was interrupted by the insertion of a kanamycin cassette were attenuated
for biofilm formation over glass and THCEC. Overall, these biofilms were less
cohesive, were structurally unstable, and often contained significantly less
biomass or were thinner than wild-type biofilms. N. gonorrhoeae is frequently
isolated from the genitourinary tract in the presence of obligate anaerobes
(Smith, 1975), and AniA is a major antigen recognized in sera from patients with
gonococcal disease (Clark et al., 1988). Thus, anaerobic respiration probably
occurs naturally during infection, and since it also seems likely that
82
Figure 13. NO halts biofilm formation in the wild type. (A) Biofilm masses over 2
days of growth for wild-type N. gonorrhoeae strain 1291, either untreated (1),
treated with SNP at the start of the biofilm (t = 0) (2), grown as a biofilm for only
24 h and left untreated (3), treated with SNP after 24 h of biofilm growth (t = 24 h)
(4), or treated simultaneously with SNP and PTIO (5). All biofilms were
visualized by GFP plasmid expression. The images are three-dimensional
reconstructions of stacked z series taken at a magnification of x200 and rendered
by Volocity. These experiments were performed a minimum of four times, and
representative results are shown. (B) Table showing results of COMSTAT
analysis of biomass and average thickness. Statistical differences between
treated samples and the untreated control (48-h biofilm) were determined via
paired Student's t tests, and the corresponding P values are listed.
83
84
Figure 14. PTIO treatment enhances biofilm formation in the norB::kan mutant.
(A) Biofilm masses over 2 days of growth for N. gonorrhoeae strain 1291
norB::kan, either untreated (1) or treated with PTIO (2). Biofilms were visualized
by GFP plasmid expression. The images are three-dimensional reconstructions
of stacked z series taken at a magnification of x200 and rendered by Volocity.
These experiments were performed a minimum of three times, and
representative results are shown. (B) Table showing results of COMSTAT
analysis of biomass and average thicknesses. Statistical differences between
treated samples and the untreated control were determined via paired Student's t
tests.
85
86
N. gonorrhoeae forms biofilms during cervical infection (Steichen, 2008), this
suggests that biofilm formation is critical for successful infection of the female
genitourinary tract. N. gonorrhoeae was initially considered incapable of
anaerobic growth (James-Holmquest et al., 1973), despite being isolated in the
presence of obligate anaerobes (Smith, 1975). However, Short and coworkers
demonstrated that N. gonorrhoeae is capable of survival under anaerobic
conditions (Short et al., 1982). Knapp and Clark later determined that anaerobic
growth in N. gonorrhoeae is coupled to nitrite reduction and that supplementation
with nitrite is required for growth under these conditions, explaining previous
unsuccessful attempts to grow the organism anaerobically (Knapp & Clark,
1984). Overton and coworkers went on to demonstrate that nitrous oxide is the
end product of nitrite reduction, indicating that N. gonorrhoeae catalyzes partial
denitrification, converting nitrite to nitrous oxide via nitric oxide (Overton et al.,
2006). AniA (formerly called Pan 1) was identified in a screen for anaerobically
regulated genes, as one of three outer membrane proteins whose expression is
induced under anaerobic growth conditions (Clark et al., 1987). Examination of
sera from patients with complicated or uncomplicated gonococcal infection
indicated that there is a strong antibody response to AniA, demonstrating that
AniA is expressed in vivo and that anaerobic growth likely occurs during infection
(Clark et al., 1988). AniA was later shown to be a lipoprotein (Hoehn & Clark,
1992b) that functions as a nitrite reductase (Mellies et al., 1997) and is
subsequently required for anaerobic growth (Householder et al., 1999).
AniA is tightly regulated by oxygen availability and is virtually undetectable
in aerobically cultured cells (Householder et al., 1999). Thus, elevated AniA
expression in biofilms is a strong indicator that gonococcal biofilms grow
anaerobically or microaerobically. Isolation of N. gonorrhoeae in the presence of
obligate anaerobes suggests that oxygen is limited in the female genitourinary
87
tract (Smith, 1975), and therefore biofilm formation may aid in the ability to
respire under conditions of oxygen limitation in the host environment. With N.
meningitidis, Rock and Moir have shown that the addition of nitrite significantly
enhances growth of this bacterium under oxygen-limited conditions (Rock & Moir,
2005), and thus it is possible that a combination of (micro)aerobic respiration and
partial denitrification is optimal in gonococcal biofilms. Furthermore, if AniA is
capable of modulating the immune response by binding complement regulatory
proteins that can down-regulate the immune response, as suggested by
Cardinale and Clark (Cardinale & Clark, 2000), then biofilm growth may also
convey an advantage to growth by aiding in immune evasion.
NO, the product of nitrite reduction by AniA in N. gonorrhoeae, has been
shown to be an important signaling molecule and a modulator of cellular events
(Davis et al., 2001, Liaudet et al., 2000, MacMicking et al., 1997, Ortega Mateo &
Amaya Aleixandre de, 2000, Stefano et al., 2000, Tschugguel et al., 1999). Low
basal levels of NO (nM scale) have been determined to be anti-inflammatory,
while high levels of NO (µM scale) have been shown to be proinflammatory,
stimulating the immune response (Cardinale & Clark, 2005, Davis et al., 2001,
Stefano et al., 2000). NO is also known to be toxic to some bacterial cells (Davis
et al., 2001, Fang, 1997, MacMicking et al., 1997, Zumft, 1997) and is produced
by nitric oxide synthases of polymorphonuclear neutrophils (Carreras et al., 1994,
Fang, 1997, MacMicking et al., 1997, McCall et al., 1989). Thus, it has been
postulated that NO production may be part of the innate immune response to
bacterial infection, although this is currently a controversial topic (Fang, 1997).
NorB is a heme protein responsible for reducing AniA-generated NO to
nitrous oxide (Householder et al., 2000). Unlike most NO reductases that are
found in denitrifying organisms (Zumft, 1997), such as Pseudomonas aeruginosa
(Arai et al., 1995), NorB is composed of a single functional subunit (Householder
88
et al., 2000). N. gonorrhoeae is capable of establishing a steady-state NO level
during anaerobic growth and can reduce NO concentrations of greater than 1 µM
(proinflammatory) in the surrounding medium to approximately 100 nM (antiinflammatory) in less than an hour (Cardinale & Clark, 2005). This suggests that
N. gonorrhoeae is capable of rapidly reducing not only endogenously produced
NO that is generated during anaerobic respiration but also environmentally
produced NO from phagocytic cells. Cervical endothelial and epithelial cells have
also been found to produce NO (Ledingham et al., 2000, Tschugguel et al.,
1999), again implying that reduction of NO by the gonococcus may be critical
during infection. During cervical infection, the gonococcus does not induce
cytokine production in the host, suggesting that the organism either fails to induce
the immune response or suppresses the response via an unknown mechanism
(Hedges et al., 1998, Russell et al., 1999). Additionally, only low levels of
antigonococcal antibody can be detected during uncomplicated infection in
women (Hedges et al., 1998), which cannot be attributed completely to the
antigenic and phase variation of many surface-exposed gonococcal structures
(Hook, 1999a). The ability of the gonococcus to rapidly reduce host-produced
NO from proinflammatory to anti-inflammatory levels may then help to account for
the lack of a strong immune response in women with uncomplicated gonorrhea.
Suppression of the immune response would likely result in the inability to clear
the organism, as well as in down-regulation of the inflammatory response,
resulting in the absence of noticeable symptoms. Thus, biofilm formation may be
a special adaptation to growth in the cervical environment, enabling the
gonococcus to persist in a chronic infection state.
N. gonorrhoeae is not the only organism that appears to catalyze
anaerobic respiration during biofilm growth. P. aeruginosa, a well-studied biofilmforming organism, has been shown to undergo anaerobic respiration during
89
growth as a biofilm, and this is thought to be the primary mode of respiration
during cystic fibrosis infection (Filiatrault et al., 2006, Hassett et al., 2002, Van
Alst et al., 2007, Yoon et al., 2002). P. aeruginosa has been shown to form
better biofilms under anaerobic than under aerobic growth conditions, and
mutants with insertions in many of the anaerobic metabolism genes do not form
biofilms as well as the wild type does (Yoon et al., 2002). A recent study of
Moraxella catarrhalis also determined that genes involved in anaerobic
metabolism are highly up-regulated during biofilm formation versus planktonic
growth. Interestingly, M. catarrhalis is believed to be incapable of anaerobic
growth. However, it is suggested that the ability to reduce NO may aid in the
survival of M. catarrhalis in the nasopharynx (Wang et al., 2007b). The
observation that other organisms catalyze anaerobic respiration during biofilm
growth suggests that biofilm formation could contribute to the ability to respire
under oxygen-limited conditions. Our observation that a norB mutant had a more
pronounced biofilm-deficient phenotype than an aniA mutant when biofilms were
grown over glass bears similarity to findings for P. aeruginosa. In P. aeruginosa,
a norBC (NO reductase) insertion mutant forms virtually no biofilm under
anaerobic growth conditions, while a nir (nitrite reductase) mutant forms biofilms
similar to those of the wild type (Yoon et al., 2002). One explanation for this
observation could be that NO accumulates under anaerobic growth conditions in
the absence of an NO reductase, resulting in slow growth or cell death. An
alternative explanation is that NO is an important signaling molecule in N.
gonorrhoeae biofilms that elicits biofilm dispersal, which has been shown to be
true for P. aeruginosa (Barraud et al., 2006) and Staphylococcus aureus biofilms
(Schlag et al., 2007). Inherent antimicrobial resistance in biofilms results in the
inability to clear biofilm infection even with high doses of antibiotics (Ceri et al.,
1999, Dunne, 2002, Schierholz et al., 1999), which makes biofilm dispersal an
90
appealing target for the treatment of biofilm infection. NO treatment has been
shown to augment the effects of antimicrobial treatment of P. aeruginosa biofilms
(Barraud et al., 2006). In agreement with these results, we found that treatment
of N. gonorrhoeae biofilms with an NO donor at the start of the biofilm growth or
after 24 hours of growth either hindered biofilm formation or elicited biofilm
dispersal. Respectively, addition of an NO quencher to norB mutant biofilms
enhanced biofilm formation, suggesting that NO accumulation accounts for the
severe biofilm-deficient phenotype observed with this mutant. We considered the
possibility that production of NO in the norB::kan mutant could result in a growth
defect by inhibition of cytochrome oxidase, subsequently impairing aerobic
respiration. However, it has been shown that cytochrome cbb3 of Pseudomonas
stutzeri, an isoenzyme in the family of cytochrome c oxidases, actually possesses
nitric oxide reductase capability (Forte et al., 2001). The nitric oxide reductase
activity of cytochrome cbb3, although considerably lower than the nitric oxide
reductase activity in P. stutzeri, is the highest within the heme-copper oxidase
superfamily (Forte et al., 2001). Furthermore, the norB::kan mutant showed no
growth defect when grown under oxygen tension conditions that mimic those
present in the medium of our biofilm apparatus, and cytochrome cbb3 is the only
oxidase present in N. gonorrhoeae.
Cytochrome c peroxide, encoded by ccp, is a lipoprotein and is the sixth ctype cytochrome induced during anaerobic growth (Turner et al., 2003).
Transcription of ccp is controlled by FNR, which controls the expression of other
important anaerobic metabolism genes in N. gonorrhoeae, such as aniA
(Lissenden et al., 2000). Ccp plays an important role in oxidative stress defense
by reducing H2O2 to water (Seib et al., 2004). H2O2 is produced by
polymorphonuclear neutrophils (Carreras et al., 1994) and Lactobacillus species
present in the female genitourinary tract (Eschenbach et al., 1989). Specifically,
91
H2O2-producing Lactobacillus strains are capable of inhibiting the growth of N.
gonorrhoeae by acidification of the medium, H2O2 toxicity, and production of
protein inhibitors (St Amant et al., 2002, Zheng et al., 1994). Thus, up-regulation
of ccp during biofilm formation by N. gonorrhoeae may represent a critical
adaptation for survival in the female genitourinary tract. It is established from
studies of the closely related mitochondrial complex 1 that NADH
dehydrogenases of this type are highly susceptible to inactivation by nitric oxide,
peroxynitrite, and S-nitrosothiols (Brown & Borutaite, 2004). The lower level of
expression of nuo in biofilms suggests that the gonococcus is also able to alter
expression of primary dehydrogenases in the respiratory chain.
Biofilm formation by the gonococcus may aid in oxidative stress tolerance
during host infection by positively regulating factors, such as ccp and norB, which
are required for the reduction of reactive oxygen and nitrogen species,
respectively. Overall, anaerobic growth may be an important mechanism by
which the gonococcus copes with the inherent oxidative stresses that are present
during infection of human cervical tissues.
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CHAPTER III
ANAEROBIC METABOLISM OCCURS IN THE SUBSTRATUM OF
GONOCOCCAL BIOFILMS AND MAY BE SUSTAINED IN PART BY NITRIC
OXIDE
Introduction
Sixty-two million new cases of gonorrhea are reported annually worldwide
(Gerbase et al., 1998), which are caused by the bacterium Neisseria
gonorrhoeae (Hansfield, 2005, Hook, 1999a, Hook, 1999c). Gonorrhea is one of
the oldest known human illnesses (Hansfield, 2005, Hook, 1999c), and it remains
prevalent, as gonorrhea is among the most frequently reported communicable
diseases in the United States today (Centers for Disease Control and Prevention,
2007, Centers for Disease Control and Prevention, 2008). Both men and
women may become infected with N. gonorrhoeae, although the mechanism of
infection differs between men and women (Edwards & Apicella, 2004). Men who
become infected with N. gonorrhoeae typically develop acute anterior urethritis
with urethral discharge and/or dysuria (Hansfield, 2005, Hook, 1999c), while up
to 80 percent of infected women do not develop any noticeable symptoms
(Bozicevic et al., 2006, Hansfield, 2005, Hook, 1999c, McCormack et al., 1977,
Pedersen & Bonin, 1971). Asymptomatic infection may occur in men, but is rare,
occurring in only 1 percent of those infected (Hansfield, 2005, Handsfield et al.,
1974). Undiagnosed infection in women can lead to prolonged or persistent
infection (Hansfield, 2005, Hook, 1999b, Hook, 1999c). Women with persistent
infection may develop PID, ectopic pregnancy, chronic pain, infertility, and/or DGI
(1995, Hansfield, 2005, Hook, 1999b, Hook, 1999c). In addition, gonorrhea
increases the risk for contracting other sexually transmitted diseases, including
HIV (Centers for Disease Control and Prevention, 2007, Chen et al., 2003,
Fleming & Wasserheit, 1999).
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It has been recently acknowledged that biofilm formation frequently
contributes to infection by pathogenic and opportunistic bacterial species
(Costerton, 1999, Costerton et al., 1995, Costerton et al., 1999, Dunne, 2002,
Hall-Stoodley et al., 2004, Hall-Stoodley & Stoodley, 2009, Stoodley et al., 2002).
Biofilm formation can facilitate persistence within the human host, as the majority
of biofilms are inherently resistant to antimicrobials and host immune defenses
(Aparna & Yadav, 2008, Donlan, 2001, Dunne, 2002, Hall-Stoodley et al., 2004,
Costerton et al., 1999). N. gonorrhoeae is capable of forming biofilms over glass
and over primary and transformed human cervical epithelial cells (Greiner et al.,
2005). Microscopic examination of human cervical biopsies also indicates that N.
gonorrhoeae forms biofilms during natural cervical infection (Steichen, 2008). It
has been demonstrated that the ability of N. gonorrhoeae to perform anaerobic
respiration and tolerate oxidative stress is critical for normal biofilm formation
(Falsetta et al., 2009, Lim et al., 2008, Potter et al., 2009a, Potter et al., 2009b,
Seib et al., 2007). Mutations in a number of oxidative stress tolerance genes,
including trxB, estD, gor, oxyR, prx, mntABC, ccp, and norB impair the ability of
the gonococcus to form biofilm over THCEC and/or glass surfaces (Seib et al.,
2007, Potter et al., 2009a, Potter et al., 2009b, Falsetta et al., 2009, Lim et al.,
2008).
The capacity to tolerate the oxidative and nitrosative stress associated
with the innate immune system in the human body can be an important virulence
determinant, and mechanisms for oxidative stress tolerance are abundant in
human pathogens (Hassett & Cohen, 1989, Janssen et al., 2003, Seib et al.,
2006). N. gonorrhoeae can neutralize reactive oxygen and nitrogen species,
such as H2O2 and NO (Seib et al., 2004, Seib et al., 2006). NO is toxic to many
bacterial species (Davis et al., 2001, Fang, 1997, MacMicking et al., 1997, Zumft,
1997), and the production of NO may be a strategy used by the innate immune
94
system to respond to bacterial infection (Fang, 1997), as it is produced by
polymorphonuclear lymphocytes (PMNs) (Carreras et al., 1994, Fang, 1997,
MacMicking et al., 1997, McCall et al., 1989) and cervical endothelial and
epithelial cells (Tschugguel et al., 1999, Ledingham et al., 2000). N.
gonorrhoeae is inherently resistant to NO-mediated killing and can rapidly
achieve non-inflammatory steady-state levels of NO (Cardinale & Clark, 2005).
N. gonorrhoeae uses NorB, a respiratory nitric oxide reductase to reduce NO
(Householder et al., 2000). NorB is also responsible for the reduction of NO
produced by the respiratory nitrite reductase AniA, and together NorB and AniA
facilitate anaerobic growth of the gonococcus (Householder et al., 1999).
Although norB and aniA insertion mutants are both defective in some aspect of
biofilm formation, a norB insertion mutant is more severely attenuated for biofilm
formation (Falsetta et al., 2009). This appears to be due to the accumulation of
NO in the norB mutant, as nanomolar concentrations of NO can impair biofilm
formation and/or elicit biofilm dispersal in N. gonorrhoeae (Falsetta et al., 2009).
This has also been observed in Pseudomonas aeruginosa (Barraud et al., 2006)
and Staphylococcal (Schlag et al., 2007) biofilms. However, higher
concentrations of NO (µM-mM) can also enhance biofilm formation in P.
aeruginosa (Barraud et al., 2006, Zaitseva et al., 2009).
We elected to investigate the anaerobic respiratory profiles of N.
gonorrhoeae biofilms using a GFP transcriptional fusion to aniA, as mutations in
aniA and norB impair but do not entirely prevent biofilm formation (Falsetta et al.,
2009). We hypothesized that N. gonorrhoeae uses a combination of
anaerobic/microaerobic and aerobic metabolism to support biofilm growth. The
observation that biofilm formation is more severely attenuated in a norB mutant
and that NO can affect P. aeruginosa biofilm in a concentration-dependent
manner (Barraud et al., 2006, Zaitseva et al., 2009) prompted us to investigate
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the effect of higher concentrations of NO on gonococcal biofilm formation. We
found that anaerobic metabolism occurs primarily at the biofilm-surface interface
or substratum of gonococcal biofilms, and that higher NO concentrations can
enhance biofilm formation, especially in the absence of nitrite. NO may also help
to sustain anaerobic growth, as addition of NO enhances biofilm formation in an
aniA::kan insertion mutant, which cannot utilize nitrite.
Experimental Procedures
Bacteria
N. gonorrhoeae strain 1291, a piliated clinical isolate that expresses Opa
proteins, was used in this study. This strain was reconstituted from frozen stock
cultures and propagated at 37°C with 5% CO2 on GC agar (Becton Dickinson)
supplemented with 1% IsoVitaleX (Becton Dickinson). See Table 1.
Construction of an aniA’-’gfp Transcriptional Fusion
The approximately 190 base pair (bp) promoter region of the aniA gene was PCR
amplified from N. gonorrhoeae strain 1291 genomic DNA, using primers that
generated a XmaI site at the 5′ end. The coding sequence of GFP was amplified
from the pGFP plasmid (Edwards & Apicella, 2005) using primers that generated
an AflII site at the 3′ end. These fragments were then used as a template for
PCR splicing by overlap extension (PCR-SOE) to fuse the GFP coding sequence
(beginning at the ATG start codon) to the aniA promoter from N. gonorrhoeae
1291. The resulting fragment was cloned into pGEM-T easy (Promega). This
fragment was then subcloned into pCTS32 (Steichen, 2008) for recombination
into the proline B synthesis gene (proB) of N. gonorrhoeae.
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Table 6. Strains, plasmids, and primers used in this study.
Strain, plasmid, or primer
Genotype or Sequence
Reference
Strains:
N. gonorrhoeae 1291
aniA::kan
wild type
aniA::kan ∆aniA pGFP
aniA’-‘gfp
aniA’-‘gfp
ATCC
(Falsetta et al.
2009)
This study
Plasmids:
pCTS32
pGEM-T easy/specR/∆proB
pGFP
pLES98/GFP/chloramphenicolR
paniA’-‘gfp
pCTS32/aniA’-‘gfp
(Steichen et
al. 2008)
(Edwards and
Apicella
2005)
This study
Primers:
aniA promoter For
aniA promoter Rev
GFP For
GFP Rev
5’-TACCCGGGAACTGCCTTTGCCTGCTCTG
5’-TCTTCTCTTTTACTCATAATGTTTCCTTTT
5’-AAAAGGAAAACATTATGAGTAAAAGGAGAAGA
5’-GTCTTAAGTTCTGCAGGAGGTCTGGACATT
*Strains, plasmids, and primers used during this study are listed above with a description
of their relevant genotypes or sequences (primers only). The source of each strain or
plasmid is also denoted by a citation. This study refers to any strains or plasmids
constructed during the course of this study. Primers are named for the region they
amplify.
97
The restriction sites generated by PCR amplification were selected for ligation
into pCTS32 in the opposite orientation of the SpecR promoter to allow
transcription to be initiated from the aniA promoter only. The resulting construct
was sequenced and used to transform wild-type N. gonorrhoeae strain 1291.
Transformants were selected on GC agar containing spectinomycin and
supplemented with proline. Integration of these constructs was confirmed via
PCR and Southern blot. See Table 1.
Biofilm Growth of the aniA’-’gfp Fusion Over Glass
The N. gonorrhoeae 1291 aniA’-‘gfp fusion was assayed for its ability to
form biofilm. This strain was propagated from frozen stock culture on GC agar
with 1% IsoVitaleX (Becton Dickinson) and 0.2 g/L proline, and incubated at 37°C
and 5% CO2. An overnight plate culture was used to create a cell suspension for
inoculation of biofilm flow chambers. N. gonorrhoeae was grown in continuousflow chambers over glass as described previously (Falsetta et al., 2009). The
media was supplemented with 0.2 g/L of proline to facilitate growth of the aniA’‘gfp strain. After 43 h of biofilm formation, chambers were stained with the 2C3
antibody to gonococcal H.8. H.8 is present in the outer membrane of the
gonococcus and was used to visualize all cells within the biofilm for overlay with
cells expressing GFP from the aniA’-‘gfp fusion. Staining was performed as
follows: a 1:100 dilution of 2C3 (in biofilm media) was pumped through the
chambers for 2 h, after which the media was replaced and pumped through the
chambers for another 30 min to remove unbound 2C3. Finally, a 1:500 dilution of
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the secondary antibody AlexaFluor 568 goat anti-mouse IgG (Molecular Probes)
was pumped through the chambers for 2 h in the dark. Biofilm formation was
then examined via confocal microscopy.
Confocal Microscopy of Continuous-Flow Chambers
z-Series photomicrographs of flow chamber biofilms were taken with a
Nikon PCM-2000 confocal microscope scanning system using a modified stage
for flow cell microscopy. GFP was excited at 450 to 490 nm and AlexaFluor 568
dye (Molecular Probes) was excited at 540 to 580 nm for biofilm imaging. Threedimensional images of the biofilms were created from each z series, using
Volocity high-performance three-dimensional imaging software (Improvision Inc.).
The images were adjusted to incorporate the pixel sizes for the x, y, and z axes
of each image stack.
Treatment of Biofilms with the NO Donors
Sodium Nitroprusside (SNP) and
Diethylenetriamine/Nitric Oxide Adduct (DETA/NO)
Wild-type N. gonorrhoeae strain 1291 biofilms were treated with SNP, a
rapid release NO donor, at concentrations ranging from 1 mM to 20 µM at the
start of the biofilm formation. For most experiments, SNP was added to the
biofilm medium prior to initiating flow, but not to the medium that was used to
create suspensions for inoculation of the biofilm. Nitrite was added to the biofilm
media at a concentration of 100 µM. Biofilms were also treated with a gradual
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release NO donor DETA/NO in the absence of nitrite. Wild-type N. gonorrhoeae
strain 1291 biofilms were treated with 10 µM and 20 µM concentrations of
DETA/NO and assessed for biofilm formation compared to biofilms grown in the
absence of nitrite and NO. The impact of SNP treatment following growth as a
biofilm in the presence of nitrite for 24 h was also investigated. Wild-type N.
gonorrhoeae strain 1291 biofilms were grown for 24 h in the presence of nitrite
and then transitioned to media with 20 µM SNP or media without nitrite and NO.
Biofilm formation was evaluated after another 24 h of growth under these
conditions. Biofilm assays were run in quadruplicate in a minimum of two
experiments for each described condition. Biofilm formation was evaluated by
confocal microscopy and COMSTAT analysis.
THCEC Culture
THCEC were cultured in 100-mm tissue culture plates in K-SFM
supplemented with 12.5 mg bovine pituitary extract, 0.08 µg epidermal growth
factor per 500-ml bottle, and a final concentration of 1% penicillin-streptomycin
(Gibco Cell Culture) at 37°C and 5% CO2. THCEC were split to bovine collagen
coated coverslips for biofilm assays, as previously described (Falsetta et al.,
2009).
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SNP Treatment of Biofilms Grown in
Continuous-Flow Chambers over THCEC
Wild-type N. gonorrhoeae strain 1291 biofilms were grown over THCEC in
the presence of 100 µM nitrite, as previously described (Falsetta et al., 2009).
Biofilms were treated with 500 nM and 1 µM SNP and assessed for the ability to
form biofilm compared to untreated biofilms. Biofilm formation was evaluated by
confocal microscopy and COMSTAT analysis.
Trypan Blue Viability Assays
To assess the impact of SNP and DETA/NO treatment on THCEC, cells
were treated with 0.25% trypsin-EDTA (Gibco), collected in PBS, and stained
with trypan blue (Invitrogen) after 48 hours of biofilm formation in the presence
and absence of SNP, with and without infection by wild-type N. gonorrhoeae
strain 1291. Viable counts were made using a hemocytometer, and the
percentage of adherent cells remaining after 48 h of infection was calculated by
comparing the number of viable cells after 48 h to the initial number of adherent
cells in each chamber.
COMSTAT Analysis of Confocal z-Series
Quantitative analysis of each z series was performed using COMSTAT
(Heydorn et al., 2000), available from http://www.im.dtu.dk/comstat/. COMSTAT
is a mathematical script written for MATLAB 5.3 (The Mathworks, Inc.) that
101
quantifies three-dimensional biofilm structures by evaluating confocal image
stacks so that pixels may be converted to relevant measurements of biofilm,
including total biomass and average thickness. To complete COMSTAT analysis,
an information file was created for each z series to adjust for the pixel sizes of the
x, y, and z axes and the number of images in each z series. COMSTAT was then
used to obtain threshold images to reduce the background. Biomass and the
average and maximum thicknesses in each z series were calculated by
COMSTAT, using the threshold images.
Statistical Analysis of COMSTAT Results
Statistical analysis was performed with Prism 4 software (GraphPad
Software, Inc.). Student’s t tests were used to compare the biomass and
average thicknesses of untreated biofilms to those treated with the NO donors
SNP and DETA/NO. Values that met a P value cutoff of 0.05 were considered
statistically different.
Results
Microscopic Examination of Anaerobic Respiration
in Biofilm
Confocal imaging of the aniA’-‘gfp transcriptional fusion indicated that
transcription of aniA occurs in the majority of cells in the substratum of N.
gonorrhoeae biofilms, while few cells express the fusion in other regions of the
biofilm (Figure 15). Anaerobic respiration occurs most readily near the surface of
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attachment in gonococcal biofilms. aniA is induced during anaerobic growth and
is tightly repressed under aerobic growth conditions (Householder et al., 1999).
Thus, it can be concluded that anaerobic respiration occurs most readily near the
surface of attachment in gonococcal biofilms. Since expression of aniA’-‘gfp
does not occur in all regions of the biofilm it can be concluded that the oxygen
partial pressure is sufficient to repress anaerobic gene expression. Although the
majority of GFP positive cells can be found in the substratum, they are also
present in other areas of the biofilm. GFP expression appeared to be in areas
where water channels were less abundant. Water channels can facilitate the
diffusion of oxygen into the biofilm (Costerton, 1999, Costerton et al., 1995,
Dunne, 2002), and this may lead to inhibition of anaerobic gene expression.
Almost no GFP expression can be detected at the surface-liquid interface of the
biofilm where oxygen would be most abundant in the bulk fluid. See Figure 15.
The Effect of High Concentrations of SNP
on Biofilm Formation
In a previous report, we determined that low concentrations of the NO
donor SNP (500 nM) inhibited biofilm formation when administered at the start of
biofilm or after 24 hours of biofilm formation (Falsetta et al., 2009). However,
reports in P. aeruginosa indicate that SNP could also enhance biofilm formation
when administered at higher doses (25-100 µM) (Barraud et al., 2006, Zaitseva
et al., 2009). This observation prompted us to investigate the effect of NO
concentration on gonococcal biofilm formation. We first treated N. gonorrhoeae
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Figure 15.
Biofilm formation by the aniA’-‘gfp fusion strain. Panel A shows the
biomass mass over 2 days of growth for N. gonorrhoeae strain 1291 aniA’-’gfp.
This image is a three-dimensional reconstruction of a representative stacked z
series taken at a magnification of x200 and rendered by Volocity. Cells
expressing GFP appear green or yellow (co-localization of GFP and 2C3), while
cells stained with 2C3 that are not expressing GFP appear red. Panel B is a
series of side views of the biofilm depicted in panel A. These images are
oriented so the bottom of each image is the substratum of the biofilm, or surface
of attachment. The top of each image is the portion of the biofilm that is exposed
to the fluid flow. The highest point of biofilm formation in these images is
approximately 130 µM. Image 1 in panel B is the merged image where the red
and green channels are overlaid. Image 2 is the green channel alone (aniA-gfp),
while image 3 is the red channel alone (2C3).
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105
biofilms with 1 mM SNP at the start of biofilm formation and then examined
biofilms after 48 hours of growth using confocal microscopy. We found that 1
mM SNP completely prevented biofilm formation in N. gonorrhoeae, as no cells
were associated with the glass surface in continuous-flow chambers. This was
an unexpected result, because N. gonorrhoeae is thought to be inherently
resistant to NO, at least more so than P. aeruginosa (Seib et al., 2004, Seib et
al., 2006). To eliminate the possibility that the presence of SNP was interfering
with the initial adherence of biofilm cells, we used media without SNP to
inoculate flow chambers. We administered SNP to these biofilms when the flow
was initiated through the addition of SNP to the medium reservoir. However, this
did not significantly improve biofilm formation. We then tested lower
concentrations of SNP (500, 250, 50 and 20 µM), but all concentrations
completely prevented biofilm formation. SNP is a rapid release NO donor.
Therefore, high concentrations of SNP administered at the start of biofilm may
overwhelm the NO defenses of the gonococcus, prior to the initiation of
anaerobic respiration. Expression of norB is induced under anaerobic conditions,
and the norB transcript is virtually undetectable under aerobic growth conditions
(Householder et al., 2000). This expression pattern is similar to that of aniA
(Householder et al., 1999). To assess the induction of anaerobic respiration in
gonococcal biofilms, we used confocal microscopy to examine GFP expression
in N. gonorrhoeae strain 1291 aniA’-’gfp in overnight plate cultures, the biofilm
inoculum, and after 24 hours of biofilm growth. GFP expression could not be
detected in cells from overnight plate cultures or the biofilm inoculum, after
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spotting these cells on glass slides. However, GFP could be detected in biofilms
grown for 24 hours in the presence of nitrite (data not shown). Therefore, we
elected to examine the effect of SNP on biofilms that were first grown for 24
hours in the presence of nitrite. Biofilms that were transitioned to media with 20
µM SNP and no nitrite, formed biofilms with significantly increased biomasses
and average thicknesses compared to biofilms that were transitioned to media
without SNP or nitrite (Figure 16). Biofilms grown in the presence of SNP for the
final 24 hours of growth formed biofilms similar to the biofilms grown for 48 hours
in the presence of nitrite, as described in an earlier publication (Falsetta et al.,
2009). This suggests that NO may be able to enhance growth in biofilms
undergoing anaerobic respiration, similar to nitrite, which enhances but is not
required for biofilm formation (Falsetta et al., 2009).
The Effect of DETA/NO on Biofilm Formation
We decided to examine the effect of NO treatment on biofilms that were
not previously treated with nitrite, because SNP treatment enhanced biofilm
formation after nitrite was removed from the media. Our aim was to determine if
NO could be substituted for nitrite, as nitrite and NO are both consumed during
anaerobic respiration. This line of study was of interest because anaerobic
respiration plays an important role in biofilm formation (Falsetta et al., 2009). To
test this, we treated biofilms with DETA/NO, a more stable, slow-release NO
donor. SNP proved unsuitable for these experiments, as treatment with
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Figure 16. NO enhances biofilm formation in biofilms that are undergoing
anaerobic respiration. Panel A shows the biofilm mass over 2 days of growth for
wild-type N. gonorrhoeae strain 1291 in the absence of nitrite (1) and the
presence of 20 µM SNP (2). Biofilms were grown for 24 hours in the presence of
nitrite and then transitioned to media without nitrite that either did or did not
contain SNP. Experiments were performed a minimum of two times and a
representative result is depicted in panel A. N. gonorrhoeae was visualized by
GFP expression, and these images are three-dimensional reconstructions of
stacked z series taken at a magnification of x200 and rendered by Volocity.
Panel B shows graphs of the COMSTAT analysis of biomass and the average
thickness. There was a significant difference between biofilms with and those
without nitrite, as determined by Student’s t test (P < of 0.001).
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109
concentrations of 20 µM or greater at the start of biofilm completely blocked
biofilm formation. Therefore, we treated biofilms with 20 µM DETA/NO and
compared this to biofilms grown in the absence of nitrite. We found that biofilm
formation was enhanced when wild-type N. gonorrhoeae strain 1291 biofilms
were grown in the presence of DETA/NO for 48 hours. Biofilms treated with 20
µM DETA/NO at the start of and throughout biofilm growth formed thicker biofilms
with significantly more biomass than biofilms grown in the absence of nitrite and
NO (Figure 17). Biofilms grown with DETA/NO also formed biofilms with
biomasses and average thicknesses that were similar to wild type biofilms grown
in the presence of nitrite (Falsetta et al., 2009).
The Effect of DETA/NO on
aniA::kan Insertion Mutant Biofilms
NO can be reduced by NorB, or it may be sufficient to regenerate nitrite
(Rodionov et al., 2005). If nitrite was regenerated, AniA could then use it as a
substrate for anaerobic/microaerobic growth. Therefore, we elected to examine
the effect of DETA/NO treatment on an aniA::kan insertion mutant, because this
mutant cannot reduce nitrite. Biofilm formation was enhanced in N. gonorrhoeae
1291 aniA::kan after treatment with 20 µM DETA/NO in the absence of nitrite.
Biofilms grown in the presence of DETA/NO had significantly increased
biomasses and average thicknesses compared to aniA::kan biofilms grown in the
absence of NO or nitrite (Figure 18).
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Figure 17. DETA/NO enhances biofilm formation in biofilms without nitrite.
Panel A shows the biofilm mass over 2 days of growth for wild-type N.
gonorrhoeae strain 1291 in the absence of nitrite (1) and the presence of 20 µM
DETA/NO (2). Experiments were performed a minimum of three times and a
representative result is depicted in panel A. N. gonorrhoeae was visualized by
GFP expression, and these images are three-dimensional reconstructions of
stacked z series taken at a magnification of x200 and rendered by Volocity.
Panel B shows graphs of the COMSTAT analysis of biomass and the average
and maximum thickness. Statistical differences between biofilms with and
without nitrite were determined via Student’s t test (P < of 0.001).
111
112
Figure 18. Biofilm formation by the aniA::kan mutant is enhanced in the
presence of DETA/NO. Panel A shows the biofilm mass over 2 days of growth
for N. gonorrhoeae strain 1291 aniA::kan in the absence of nitrite (1) and in the
presence of 20 µM DETA/NO (2). Experiments were performed a minimum of
three times and a representative result is depicted in panel A. N. gonorrhoeae
was visualized by GFP expression, and these images are three-dimensional
reconstructions of stacked z series taken at a magnification of x200 and rendered
by Volocity. Panel B shows graphs of the COMSTAT analysis of biomass and
the average thickness. Statistical differences between biofilms with and without
nitrite were determined via Student’s t test (P < of 0.001).
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114
However, treatment with DETA/NO did not fully restore biofilm formation to
levels similar to the wild type grown in the presence of nitrite. Thus, NO can
enhance biofilm formation when nitrite is unavailable or cannot be consumed, but
NO does not compensate for the inability to utilize nitrite. This indicates that N.
gonorrhoeae is capable of using NO to partially sustain anaerobic respiration in
biofilm, yet nitrite is preferred. It is also possible that we were unable to achieve
the optimal level of NO, which supports anaerobic growth in these biofilms.
Overall, these data indicates that N. gonorrhoeae biofilms are capable of utilizing
both nitrite and NO to enhance biofilm formation, depending on the relative
concentration of these substrates.
The Impact of NO on Biofilms Grown over THCEC
N. gonorrhoeae is an obligate human pathogen (Hansfield, 2005, Hook,
1999c, Hook, 1999a). Thus, we elected to use a more relevant model to
examine the impact of NO on biofilms by growing biofilms over THCEC.
Analogous to previous studies, we treated wild-type biofilms with 500 nM SNP at
the start of biofilm and assessed biofilm formation after 48 hours of treatment.
However, we found that there was no significant difference between untreated
and SNP-treated biofilms (data not shown). Therefore, we considered the
possibility that growth over cervical cells provided added protection against NO
toxicity, which might occur through the cervical cell-mediated reduction of NO.
Accordingly, we increased the concentration of SNP to 1 µM. We found that
115
Figure 19.
NO impeded biofilm formation over THCEC. Panel A shows the
biofilm mass over 2 days of growth for wild-type N. gonorrhoeae strain 1291 over
THCEC in the absence of SNP (1) and the presence of 1 µM SNP (2).
Experiments were performed a minimum of three times and a representative
result is depicted in panel A. THCEC were visualized by Cell Tracker Orange
(Molecular Probes) staining, while N. gonorrhoeae was visualized by GFP
expression. These images are three-dimensional reconstructions of stacked z
series taken at a magnification of x200 and rendered by Volocity. Panel B shows
graphs of the COMSTAT analysis of biomass and the average and maximum
thickness. Statistical differences between biofilms with and without nitrite were
determined via Student’s t test (P < of 0.001).
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117
wild-type N. gonorrhoeae strain 1291 biofilms had significantly less biomass and
decreased average thicknesses after treatment with 1 µM SNP (Figure 19). We
performed trypan blue viability assays to assess the effect of SNP treatment on
THCEC. We found that treatment with 1M SNP did not decrease the viability of
THCEC compared to untreated cells (data not shown). We also set out to
examine the effect of higher concentrations of SNP on biofilms grown over
cervical cells. However, the media used in our tissue culture system is
proprietary. Therefore, we could not control the amount of nitrite or deplete nitrite
from the media after 24 hours biofilm growth, which would have been analogous
to our studies over glass. We also could not assess the effect of DETA/NO on
biofilms over THCEC, because DETA/NO is toxic to cervical cells at the
concentration used to treat biofilms grown over glass.
Discussion
N. gonorrhoeae biofilms can use a combination of anaerobic and aerobic
metabolism to support growth. Genes that are anaerobically induced and/or
encode proteins involved in anaerobic respiration are required for normal biofilm
formation, while the transcripts of some genes involved in aerobic respiration are
less abundant in biofilm (Falsetta et al., 2009). We found that the addition of NO
can improve biofilm formation in the absence of nitrite and help to restore biofilm
formation in an aniA::kan mutant that is incapable of reducing nitrite. However,
NO does not fully restore biofilm formation in this mutant, which may indicate that
nitrite is the preferred substrate for anaerobic respiration. Biofilm formation may
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also be inhibited by NO when it is present at sublethal concentrations within the
biofilm, even when nitrite is available (Falsetta et al., 2009). This suggests that
the gonococcus is capable of sensing and responding to the concentration of NO
in the surrounding media, as well as the availability of nitrite.
The gonococcus was initially considered to be incapable of anaerobic
growth (James-Holmquest et al., 1973), although N. gonorrhoeae was often
isolated in the presence of obligate anaerobes (Smith, 1975). It was
subsequently determined that nitrite is required for the anaerobic growth of N.
gonorrhoeae on plates (Knapp & Clark, 1984). AniA (nitrite reductase) reduces
nitrite to NO (Mellies et al., 1997), which is then reduced to N2O by NorB (NO
reductase) (Householder et al., 2000). N. gonorrhoeae does not evolve nitrogen
gas, as there is a frameshift mutation in the nos genes that would normally
encode proteins that reduce N2O (Overton et al., 2006). Therefore, nitrite and
NO could be reduced during anaerobic growth, both of which are present at the
site of infection in women (Ledingham et al., 2000, Tschugguel et al., 1999,
Vaisanen-Tommiska et al., 2003). Previous studies demonstrated that an
aniA::kan insertion mutant cannot respire anaerobically, but can survive
incubation under anaerobic conditions (Householder et al., 1999). However, NO
was not present under these growth conditions. We found that exogenouslysupplied NO can partially rescue biofilm formation in an aniA::kan mutant. This
suggests that strains with an impaired aniA function may be capable of anaerobic
respiration in the presence of NO, if it is present at concentrations that enhance
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biofilm formation. Naturally, this may be difficult to achieve outside of a biofilm or
other chemostatic system.
NorB establishes a NO steady-state that rapidly reduces NO from
proinflammatory (1µM) to anti-inflammatory levels (100 nM) (Cardinale & Clark,
2005). This indicates that NorB is efficient at reducing endogenously- and
exogenously-produced NO, which is produced by PMNs (Carreras et al., 1994,
Fang, 1997, MacMicking et al., 1997, McCall et al., 1989) and cervical
endothelial and epithelial cells in the host (Tschugguel et al., 1999, Ledingham et
al., 2000). This poses a potentially serious threat to the survival of bacterial cells
that cannot reduce NO. NorB plays an important role in the tolerance of reactive
nitrogen species, and it is important for gonococcal survival in the cervical
environment (Seib et al., 2004, Seib et al., 2006). NorB not only reduces
nitrosative stress, but it also participates in anaerobic respiration (Householder et
al., 2000). We previously demonstrated that normal biofilm formation in N.
gonorrhoeae is dependent on the ability to grow anaerobically (Falsetta et al.,
2009). In this study, we determined that NO can partially restore biofilm
formation in an aniA::kan mutant that is incapable of anaerobic growth using
nitrite (Householder et al., 1999). Therefore, our results suggest that NorB may
be sufficient to support anaerobic growth when NO is present at concentrations
that enhance biofilm formation. Thus, reducing the oxidative state of
endogenous NO may contribute to anaerobic respiration, especially if nitrite is
unavailable or the function of AniA is impaired. It has been demonstrated that N.
meningitidis can respire anaerobically (Deeudom et al., 2006, Rock et al., 2005,
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Rock & Moir, 2005, Rock et al., 2007). However, AniA is only functional in a few
strains of N. meningitidis, and it is not required for pathogenesis (Stefanelli et al.,
2008). If an aniA::kan insertion mutant can respire anaerobically through the
NorB-mediated reduction of NO, this may also help to explain why biofilm
formation is more severely attenuated in the norB::kan mutant (Falsetta et al.,
2009). Although unlikely, we acknowledge that NO could enhance biofilm
formation through a novel mechanism, which is independent of anaerobic
respiration. We considered the construction of a norB’-‘gfp transcriptional fusion,
which we might use to examine anaerobic respiration in an aniA::kan
background, as norB is also repressed under aerobic conditions (Householder et
al., 2000). However, transcription of norB is induced in the presence of NO via
derepression of NsrR-dependent inhibition of transcription (Isabella et al., 2009,
Overton et al., 2006). Thus, treatment with DETA/NO could induce expression of
norB irrespective of oxygen availability.
NorB is the simplest form of a respiratory nitric oxide reductase. It uses
ubiquinol as electron donor and reduces NO at the outer face of the cytoplasmic
membrane (de Vries & Schroder, 2002). As a consequence, this enzyme does
not conserve energy. In some cases its sole functional role appears to be to
detoxify NO, as in the cyanobacterium Synechocystis (Busch et al., 2002).
However, provided that NO respiration was coupled to the activity of protontranslocating NADH dehydrogenase, the respiratory pathway to NO would
generate a proton motive force. The AniA nitrite reductase is located in the outer
membrane of N. gonorrhoeae (Clark et al., 1987). Although the respiratory
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proteins that shuttle electrons across the periplasm to AniA have not been
definitively identified, the sensitivity of nitrite respiration to the inhibitor
myxothiazol is a clear indication that the cytochrome bc1 complex is involved
(Deeudom et al., 2006). This means that electron transfer from NADH to nitrite
involves two energy-conserving steps. This may explain why nitrite is a superior
electron acceptor for respiration compared to NO. Neisseria species possess a
single electrogenic cytochrome oxidase (cytochrome cbb3). Thus, respiration
from NADH to oxygen involves three energy-conserving steps. It is notable that
cytochrome cbb3 has a very high affinity for oxygen, with a Km in the nM range
(Pitcher & Watmough, 2004). This means that oxygen consumption and energy
conservation via aerobic respiration probably takes place under very low
concentrations of oxygen, which would induce expression of aniA and norB.
Under these conditions, NorB may have an additional role in detoxification. It
has been established that NADH dehydrogenase is susceptible to inhibition by
NO, and thus NorB may play a key role in preventing inhibition of this key
energy-conserving enzyme.
We determined that N. gonorrhoeae uses a combination of aerobic and
anaerobic/microaerobic metabolism to support its growth as a biofilm. This
agrees with our previous findings that biofilm formation is impaired, but not
prevented entirely in the aniA::kan and norB::kan insertion mutants (Falsetta et
al., 2009). P. aeruginosa, the paradigm organism for the study of biofilm
formation, also undergoes a combination of anaerobic and aerobic respiration
during growth as a biofilm. However, anaerobic respiration is believed to be the
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primary mode of respiration during cystic fibrosis infection (Filiatrault et al., 2006,
Hassett et al., 2002, Van Alst et al., 2007, Yoon et al., 2002). This appears to be
similar for N. gonorrhoeae, as a large proportion of the biofilm expresses aniA,
although many of these cells are concentrated in the substratum of the biofilm.
Normal biofilm formation is dependent on expression of anaerobic respiratory
genes in P. aeruginosa (Yoon et al., 2002), which is similar for N. gonorrhoeae
(Falsetta et al., 2009). We found that anaerobic respiration occurs most
prevalently in the substratum of N. gonorrhoeae biofilms. Our images also
demonstrate that anaerobic respiration does not occur in the upper portions of
the biofilm, which are directly exposed to the fluid flow. Oxygen can penetrate
approximately the first 50 µM of P. aeruginosa biofilms, while the first 30-60 µM
of these biofilms contain actively respiring cells that synthesize protein (Rani et
al., 2007, Werner et al., 2004). Overall, oxygen and nutrients become limited
near the surface of attachment in biofilms, and this portion of the biofilm largely
contains metabolically inactive cells (Rani et al., 2007, Werner et al., 2004,
Roberts & Stewart, 2004). Metabolically inactive or slow-growing cells are less
susceptible to antibiotics, which appears to contribute to the inherent
antimicrobial resistance of biofilms (Duguid et al., 1992, Evans et al., 1990a,
Evans et al., 1990b). This suggests that anaerobic respiration could have some
relationship to antibiotic resistance and persistence during gonococcal infection
of cervical tissues.
N. gonorrhoeae biofilms do not immediately catalyze anaerobic
respiration. Overnight plate cultures and biofilm inocula do not produce
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detectable levels of GFP in the N. gonorrhoeae 1291 aniA’-’gfp transcriptional
fusion strain. However, GFP can be detected after 24 hours of growth as a
biofilm, which indicates that N. gonorrhoeae biofilms do become
anaerobic/microaerobic over time. This likely explains why high doses of SNP
prevent biofilm formation when administered at the start of biofilm, but can
enhance growth in biofilms that have been primed for anaerobic respiration by
growing for 24 hours in the presence of nitrite. Biofilms undergoing anaerobic
respiration can reduce NO, which contributes to nitrosative stress tolerance and
anaerobic respiration in the gonococcus. However, nitrite appears to be the
preferred substrate for anaerobic metabolism in N. gonorrhoeae. DETA/NOtreated biofilms are indistinguishable from nitrite-treated biofilms, while those
grown in the absence of nitrite and NO are severely attenuated. DETA/NO
partially restores biofilm formation in the aniA::kan mutant, but these biofilms do
not achieve the same level of biofilm production as wild type biofilms grown in the
presence of nitrite. Thus, wild type biofilms that can utilize both NO and nitrite
would have a distinct advantage over aniA::kan biofilms that can only use NO.
Respiration is heterogeneous in N. gonorrhoeae biofilms, which employ a
combination of aerobic and anaerobic or microaerobic metabolism. Aerobic
respiration likely plays an important role during initial biofilm formation, which
allows time for these biofilms to induce transcription of genes in the anaerobic
respiratory chain.
N. gonorrhoeae is an obligate human pathogen (Hansfield, 2005, Hook,
1999c, Hook, 1999a). Therefore, it is essential to study N. gonorrhoeae in the
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context of human infection. We found that low doses of SNP prevent biofilm
formation over human cervical cells in vitro, which is similar to the effect of low
doses of SNP on biofilms over glass (Falsetta et al., 2009). We observed that
higher doses of SNP were required to elicit a response over cervical cells (1 µM
versus 500 nM over glass), which was not entirely surprising. The need for
higher doses of SNP, indicates that growth over cervical cells may offer added
protection against NO-toxicity or the NO-mediated dispersal of gonococcal
biofilm. We found that viability of the cervical cells was not affected by SNP
treatment, which suggests that these cells have a mechanism(s) for coping with
or reducing NO. If THCEC reduce NO, this could allow N. gonorrhoeae to
withstand higher concentrations of NO during an infection. High doses of NO,
donated from SNP or DETA/NO, appear to enhance biofilm formation in the
absence of nitrite. Inherent protection from NO by human cervical cells could
promote gonococcal association with the cervical epithelium when NO levels are
high, in turn stimulating biofilm formation. Biofilm growth also induces expression
of several factors involved in anaerobic respiration and oxidative stress
tolerance, which would convey protection against NO (Falsetta et al., 2009). The
gonococcus can adhere to and/or invade human cervical cells (Edwards &
Apicella, 2002, Edwards & Apicella, 2004, Edwards et al., 2001, Edwards et al.,
2002), although it is not clear what regulates the transition between adherent and
invasive growth. The ability to sense the concentration of NO in the surrounding
media appears to influence biofilm formation, which in turn may regulate the
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Figure 20. One possible model for the role of nitric oxide in biofilm formation.
During step 1, N. gonorrhoeae cells (depicted in pink) recognize high levels of
NO and bind to the surface of the cervical cells (depicted in red). During step 2,
these cells differentiate into a biofilm, producing a biofilm matrix. In step 3 biofilm
formation turns on transcription of aniA and norB, which reduces the
concentration of NO in the surrounding media. In step 4, low levels of NO signal
dispersal of the biofilm, which likely occurs through degradation of the biofilm
matrix (step 5). In step 6, cells that are released from the biofilm may be swept
away in bodily secretions, allowing these cells to potentially colonize new areas
of cervical tissue.
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gonococcal lifestyle, governing the switch between attached and intracellular
modes of growth. See Figure 20 for our proposed model, which illustrates the
potential role of NO in biofilm formation and detachment. We acknowledge that
further investigation is necessary to support or refute this model.
N. gonorrhoeae biofilms use a combination of aerobic and anaerobic respiration.
Although aerobic respiration may largely support the initial growth of
biofilms, anaerobic respiration uniquely confers protection against the oxidative
stresses that are present in the natural cervical environment. The ability to sense
and respond to NO also appears to be critical for biofilm formation by N.
gonorrhoeae, as NO may contribute to anaerobic metabolism as well as
influence the mechanisms that govern biofilm detachment and formation.
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CHAPTER IV
THE ABILITY TO TOLERATE OXIDATIVE STRESS IS CRITICAL FOR
GONOCOCCAL BIOFILM FORMATION
Introduction
During colonization of the cervical epithelium, N. gonorrhoeae is
continuously exposed to oxidative stress. In the host, oxidants are produced as
a by-product of gonococcal metabolism, by the human immune system, and by
other inhabitants of the human mucosa (Seib et al., 2006). Oxidative stress can
cause damage to DNA, proteins, and the cellular membrane (Imlay, 2003).
Mechanisms used to avoid and/or cope with the oxidative stresses present in the
human body are abundant in human pathogens and are increasingly being
recognized as important virulence factors (Hassett & Cohen, 1989, Janssen et
al., 2003, Seib et al., 2006).
There are several regulons in N. gonorrhoeae that govern the response to
the oxidative stresses present in the mucosal milieu. The gonococcus has
redundant mechanisms to cope with reactive oxygen and nitrogen species (Seib
et al., 2004, Seib et al., 2006). The peroxide stress response in N. gonorrhoeae
is regulated by OxyR and PerR (Seib et al., 2006). OxyR is common in other
Gram-negative bacteria, and regulates three genes in N. gonorrhoeae: gor
(glutathione), prx (peroxiredoxin), and katA (catalase) (Seib et al., 2006).
Transcription of these genes is enhanced when the gonococcus is exposed to
peroxide stress (Seib et al., 2006, Stohl et al., 2005). PerR is a Fur paralogue
that regulates expression of 12 genes (Seib et al., 2006, Wu et al., 2006).
Among these genes, PerR regulates the mntABC operon, which encodes the
Mn2+ MntABC transporter (Seib et al., 2006, Tseng et al., 2001). Mn2+ acts as
an intracellular antioxidant and can protect N. gonorrhoeae from H2O2 (Seib et
al., 2004). NmlR, or the Neissieria mer-like regulator, is another transcriptional
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regulator that plays an important role in oxidative stress tolerance in N.
gonorrhoeae (Kidd et al., 2005). NmlR regulates transcription of five genes:
nmlR (autoregulation), trxB (thioredoxin), copA (CPx-type ATPase), estD
(esterase D), and adhC (alcohol dehydrogenase) (Kidd et al., 2005, Potter et al.,
2009a, Potter et al., 2007). An nmlR mutant is slightly less able to grow under
microaerobic conditions and is more susceptible to killing by cumene
hydroperoxide and diamide than the wild type (Kidd et al., 2005).
Anaerobic respiration also contributes to the oxidative stress response in
the gonococcus, as NorB, a major anaerobically-induced protein, reduces the
reactive oxygen species NO (Householder et al., 2000). Although it is not
involved in anaerobic respiration, cytochrome c peroxidase (Ccp) is also induced
under anaerobic conditions and plays a role in oxidative stress defense by
reducing H2O2 (Turner et al., 2003). Expression of both norB and ccp is critical
for normal gonococcal biofilm, which indicates that both anaerobic respiration
and oxidative stress tolerance mechanisms are important for biofilm formation
(Falsetta et al., 2009). Thus, we elected to examine the importance of other
oxidative stress tolerance mechanisms in biofilm formation by N. gonorrhoeae.
We determined that members of the OxyR, PerR, and NmlR regulons are
required for normal biofilm formation over transformed human cervical cells
and/or glass surfaces.
Experimental Procedures
Bacteria
N. gonorrhoeae strain 1291, a piliated clinical isolate that expresses Opa
proteins, was used in this study. This strain was reconstituted from frozen stock
cultures and propagated at 37°C with 5% CO2 on GC agar (Becton Dickinson,
Franklin Lakes, NJ) supplemented with 1% IsoVitaleX (Becton Dickinson). The
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following insertion mutation strains were also used: oxyR::kan (Tseng et al.,
2003), prx::kan (Seib et al., 2007), gor::kan (Seib et al., 2007), mntAB::kan (Wu
et al., 2006), mntC::kan (Tseng et al., 2001), trxB::kan (Potter et al., 2009b), and
estD::kan (Potter et al., 2009a).
Biofilm Growth in Continuous-Flow Chambers
Over Glass
N. gonorrhoeae strain 1291 wild-type and the oxyR::kan, prx::kan,
gor::kan, mntAB::kan, mntC::kan, trxB::kan, and estD::kan insertional mutants
were assayed for the ability to form biofilms. Strains were propagated from
frozen stock cultures on GC agar with 1% IsoVitaleX (Becton Dickinson) and
incubated at 37°C and 5% CO2. Overnight plate cultures were used to create cell
suspensions for inoculation of biofilm flow chambers. N. gonorrhoeae was grown
in continuous-flow chambers over glass as previously described (Falsetta et al.,
2009). Chloramphenicol was added to the medium at a final concentration of 5
µg/µl to maintain pGFP. After 48 h, the biofilm effluent was cultured to assure
culture purity, and biofilm formation was assessed via confocal microscopy.
Confocal Microscopy of Continuous-Flow Chambers
z-Series photomicrographs of flow chamber biofilms were taken with a
Nikon PCM-2000 confocal microscope scanning system using a modified stage
for flow cell microscopy. GFP was excited at 450 to 490 nm and Cell Tracker
Orange dye (Molecular Probes) was excited at 540 to 580 nm for biofilm imaging.
Three-dimensional images of the biofilms were created from each z series, using
Volocity high-performance three-dimensional imaging software (Improvision Inc.).
The images were adjusted to incorporate the pixel sizes for the x, y, and z axes
of each image stack.
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THCEC Culture and Biofilms over THCEC
THCEC were cultured in 100-mm tissue culture plates in K-SFM
supplemented with 12.5 mg bovine pituitary extract, 0.08 µg epidermal growth
factor per 500-ml bottle, and a final concentration of 1% penicillin-streptomycin
(Gibco Cell Culture) at 37°C and 5% CO2. THCEC were split to bovine collagen
coated coverslips and run in biofilm assays, as previously described (Falsetta et
al., 2009).
COMSTAT Analysis of Confocal z-Series
Quantitative analysis of each z series was performed using COMSTAT
(Heydorn et al., 2000), available from http://www.im.dtu.dk/comstat/. COMSTAT
is a mathematical script written for MATLAB 5.3 (The Mathworks, Inc.) that
quantifies three-dimensional biofilm structures by evaluating confocal image
stacks so that pixels may be converted to relevant measurements of biofilm,
including total biomass and average thickness. To complete COMSTAT analysis,
an information file was created for each z series to adjust for the pixel sizes of the
x, y, and z axes and the number of images in each z series. COMSTAT was then
used to obtain threshold images to reduce the background. Biomass and the
average and maximum thicknesses in each z series were calculated by
COMSTAT, using the threshold images.
Statistical Analysis of COMSTAT Results
Statistical analysis was performed with Prism 4 software (GraphPad
Software, Inc.). Student’s t tests and were used to compare the biomass and
average thicknesses of oxidative stress response mutant biofilms to the wild
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type. Values that met a P value cutoff of 0.05 were considered statistically
different.
Results
Biofilm Formation by the OxyR Regulon Mutants
oxyR::kan, prx::kan, and gor::kan
We determined that the oxyR::kan, prx::kan, and gor::kan insertion
mutants were attenuated for biofilm formation over glass. All three mutants
formed biofilms with reduced biomasses and lower average thicknesses than the
wild type, which was not due to a general defect in the growth of this strains (data
not shown). These mutant biofilms had less than 10 percent of the biomass of
wild type biofilms. Overall, the mutant biofilms were very thin, lacked secondary
structure, and consisted of small clusters of cells sparsely distributed over the
surface of the glass coverslip. This is in stark contrast to the wild type, which
formed thick robust biofilms with almost no gaps between biofilm clusters. See
Figure 21.
Biofilm Formation by the PerR Regulon Mutants
mntAB::kan and mntC::kan
We determined that the mntAB::kan and mntC::kan mutant strains were
also deficient in biofilm formation over glass. COMSTAT analysis showed that
both the mntAB::kan and mntC::kan mutants have reduced biomasses and
average thicknesses compared to the wild type strain (P< 0.0001). Specifically,
the mntAB::kan mutant had 20 percent of the biomass of the wild type, while the
mntC::kan mutant had 7 percent of the biomass of the wild type.
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Figure 21. Biofilm formation by wild-type N. gonorrhoeae strain 1291 and the
oxyR::kan, prx::kan, and gor::kan mutant derivatives. Panel A shows the biofilm
mass over 2 days of growth for the N. gonorrhoeae 1291 parent strain (1), and
the oxyR::kan (2), prx::kan (3), and gor::kan (4) mutant strains. These images
are three-dimensional reconstructions of representative stacked z series taken at
a magnification of x200 and rendered by Volocity. Panel B shows a COMSTAT
analysis of the stack biofilm, analyzing the sections for biomass and the average
thickness of the biofilm. The error bars represent 1 standard deviation from the
mean. These experiments were performed in duplicate on two different
occasions. There is a statistically significant difference in the mean biomass of
the oxyR::kan, prx::kan, and gor::kan mutant strains relative to the wild-type
strain (P < 0.05). There is also a statistically significant difference in the average
thickness of the biofilm of the oxyR::kan, prx::kan, and gor::kan mutant strains
relative to wild-type strain (P < 0.05).
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135
The mntAB::kan and mntC::kan mutants possessed 10 percent and 12 percent
the average thickness of the wild-type, respectively. Three-dimensional images
of these biofilms show that overall both mutants form thinner and more diffuse
biofilms with large gaps between biofilm clusters compared to wild type. See
Figure 22.
Biofilm Formation Over Glass by the NmlR
Regulon Mutants trxB::kan and estD::kan
The trxB::kan mutant was deficient in biofilm formation compared to the
wild-type strain after 48 hours of biofilm growth over glass. Surprisingly, the
estD::kan mutant formed biofilm equally as well as wild type under these growth
conditions. Three dimensional images of the biofilms indicated that the trxB::kan
mutant formed diffuse biofilms over glass with large gaps between biofilm
clusters, while the estD::kan and wild-type strains formed robust biofilms with few
gaps between clusters. See Figure 23. COMSTAT analysis of stacked z series
indicated that the trxB::kan mutant formed biofilms with significantly less biomass
and lower average thicknesses than the wild-type strain. However, the estD::kan
mutant, as indicated by the appearance of the biofilms images, showed no
difference in biomass or average thickness compared to the wild-type. See
Figure 24.
Biofilm Formation Over THCEC by the NmlR
Regulon Mutants trxB::kan and estD::kan
There was no difference in biofilm formation between the wild type and
estD::kan mutant when these strains were grown as biofilms over glass.
Therefore we elected to examine biofilm formation over THCEC, as N.
gonorrhoeae is an obligate pathogen and this is a more relevant model system.
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Figure 22. Biofilm formation by wild-type N. gonorrhoeae 1291 and the
mntAB::kan and mntC::kan mutants. Panel A shows the biofilm mass over 2
days of growth for N. gonorrhoeae 1291 wild-type (1) and the mntAB::kan (2) and
mntC::kan (3) mutants. These images are three-dimensional reconstructions of
representative stacked z series taken at a magnification of x200 and rendered by
Volocity. Panel B shows a COMSTAT analysis of the stack biofilm, analyzing the
sections for biomass and the average thickness of the biofilm. The error bars
represent 1 standard deviation from the mean. Asterisks denote statistically
significant differences in biomass and average thickness compared to the wild
type. These experiments were repeated on three different occasions, and a
representative result is shown. There is a statistically significant difference in the
mean biomasses of the mntAB::kan and mntC::kan mutants relative to wild type
(P < 0.0001). There is also a statistically significant difference in the average
thicknesses of the biofilm of the mntAB::kan and mntC::kan mutants relative to
wild type (P < 0.0001).
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138
Figure 23. Biofilm formation by wild-type N. gonorrhoeae 1291 and the trxB::kan
and estD::kan mutants over glass. Biofilm masses over 2 days of growth for
wild-type N. gonorrhoeae strain 1291 (1) and the trxB::kan (2) and estD::kan (3)
insertion mutants. All three strains were visualized by GFP plasmid expression.
The images are three-dimensional reconstructions of stacked z series taken at a
magnification of x200 and rendered by Volocity. These experiments were
performed a minimum of three times, and representative results are shown.
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140
Figure 24. COMSTAT analysis of biofilm formation over glass by wild-type N.
gonorrhoeae 1291 and the trxB::kan and estD::kan mutants. COMSTAT and
statistical analyses of biomass (A) and average thickness (B) for wild type (black
bars), trxB::kan (white bars), and estD::kan (gray bars) insertion mutant biofilms
grown over glass for 2 days. Representative images of these biofilms are
depicted in Figure 23. All strains were run in duplicate in a minimum of three
experiments, and at least four images of each biofilm chamber were used for
COMSTAT analysis. The error bars represent 1 standard deviation from the
mean. Statistical differences between mutants and the wild-type, determined by
Student's t test, are denoted by asterisks above the error bars. *, P value of 0.05
or less.
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Previous studies indicated that biofilm-deficient phenotypes may be
equally or more pronounced over THCEC compared to phenotypes over glass
surfaces (Falsetta et al., 2009). The gaps present between biofilm clusters in the
trxB::kan mutant appeared to be larger when the biofilm was grown over THCEC
(Figure 23), suggesting that this mutant is more severely attenuated for biofilm
formation over host cells. Accordingly, the estD::kan showed a defect in biofilm
formation that was not detected when these biofilms were grown over glass. The
estD::kan mutant formed very little biofilm over THCEC with large gaps between
sparsely- populated biofilm clusters. The estD::kan and trxB::kan mutant biofilms
were indistinguishable in their rendered three-dimensional images over THCEC.
However, the wild-type strain formed biofilms equally well on glass and THCEC.
Overall, the wild type formed compact biofilms with almost no gaps between
biofilm clusters, while the mutants formed sparse patches of loosely packed
biofilm. See Figure 25. COMSTAT analysis of stacked z series indicated that
the trxB::kan and estD::kan mutants formed biofilms with significantly less
biomass and lower average thicknesses than wild type. See Figure 26.
Discussion
We determined that members of the OxyR, PerR, and NmlR regulons are
required for normal biofilm formation, as interruptions in the oxyR, gor, prx,
mntAB, mntC, trxB, and estD genes resulted in strains that were unable to form
biofilms as well as the wild type in at least one growth condition tested. We also
determined that some phenotypes were only apparent or were more pronounced
when these strains were grown over THCEC (e.g. estD::kan). This suggests that
the ability to tolerate oxidative stress is important for normal biofilm formation,
particularly in the presence of host cells, which may produce oxidants in
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Figure 25. Biofilm formation by wild-type N. gonorrhoeae 1291 and the trxB::kan
and estD::kan mutants over THCEC. Biofilm masses over 2 days of growth for
wild-type N. gonorrhoeae strain 1291 (1) and the trxB::kan (2) and estD::kan (3)
insertion mutants. All three strains were visualized by GFP plasmid expression,
while THCEC were visualized by Cell Tracker Orange (Molecular Probes,
Invitrogen Corp., Carlsbad, CA) staining. The images are three-dimensional
reconstructions of stacked z series taken at a magnification of x200 and rendered
by Volocity. These experiments were performed a minimum of three times, and
representative results are shown.
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Figure 26. COMSTAT analysis of biofilm formation over THCEC by wild-type N.
gonorrhoeae 1291 and the trxB::kan and estD::kan mutants. COMSTAT and
statistical analyses of biomass (A) and average thickness (B) for the wild-type
(black bars), trxB::kan (white bars), and estD::kan (gray bars) insertion mutant
biofilms grown over THCEC for 2 days. Representative images of these biofilms
are depicted in Figure 25. All strains were run in duplicate in a minimum of three
experiments, and at least four images of each biofilm chamber were used for
COMSTAT analysis. The error bars represent 1 standard deviation from the
mean. Statistical differences between mutants and the wild type, determined by
Student's t test, are denoted by asterisks above the error bars. *, P value of
0.0001 or less.
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response to infection (Ledingham et al., 2000, Tschugguel et al., 1999,
Vaisanen-Tommiska et al., 2003). There is precedence for these findings, as
genes involved in anaerobic respiration are also required for normal biofilm
formation (Falsetta et al., 2009). AniA and NorB, members of the anaerobic
respiratory pathway in N. gonorrhoeae, must be functional to achieve wild type
levels of biofilm formation. AniA and NorB contribute to oxidative stress
tolerance by reducing nitrite and NO, respectively (Householder et al., 1999,
Householder et al., 2000). A norB::kan insertion mutant is more severely
attenuated for biofilm formation than an aniA::kan mutant, which likely
corresponds to NO accumulation in a norB::kan mutant, as this mutant cannot
reduce AniA-generated NO (Falsetta et al., 2009). This strongly suggests that
oxidative stress tolerance is critical for biofilm formation.
By definition biofilms are inherently resistant to a variety of biological and
environmental stresses including antimicrobials, UV light, acids, metals,
dehydration, salinity, and phagocytosis (Hall-Stoodley et al., 2004). Thus, it is
logical that biofilms would possess mechanisms necessary for the tolerance of
oxidative stresses, which abound in the environment and the human host (Seib
et al., 2006). N. gonorrhoeae biofilms up-regulate transcript levels of the
anaerobic respiratory/oxidative stress tolerance genes aniA, norB, and ccp, as
compared to planktonic or batch culture cells (Falsetta et al., 2009). N.
gonorrhoeae forms biofilms over cervical tissue in vivo (Steichen, 2008), which
indicates that biofilm formation is a physiologically relevant occurrence that could
contribute to the ability of the gonococcus to resist the myriad of oxidative
stresses present in the human genitourinary tract. We determined that other
members of the major oxidative stress tolerance regulons are also required for
biofilm formation, in addition to AniA, Ccp, and NorB. These findings support the
hypothesis that biofilm contributes to the ability of the gonococcus to resist
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oxidative stress in vivo. Although antibiotic resistance has increased in this
organism, N. gonorrhoeae has not been shown to be recalcitrant to antibiotic
treatment like other biofilm-forming organisms (2007, Centers for Disease
Control and Prevention, 2007). Perhaps then, the main function of biofilm
formation in N. gonorrhoeae is to convey resistance to oxidants, not
antimicrobials. Women can become chronically infected with N. gonorrhoeae,
which is often the result of asymptomatic infection (Hansfield, 2005, Hook,
1999c, Hook, 1999b). Thus, women may not receive immediate medical
treatment that typically employs the use of antimicrobials. However, the
gonococcus is exposed to oxidative stress immediately upon infection of the
cervical epithelium (Seib et al., 2004, Seib et al., 2006). Genes involved in
antibiotic efflux, which can be up-regulated during biofilm formation in other
species, have not been detected as differentially expressed in N. gonorrheoae
biofilm. However, genes involved in oxidative stress tolerance are significantly
up-regulated (Falsetta et al., 2009). Thus, the need to combat oxidative stress is
likely more immediate. If this is true, biofilm formation may represent an
adaptation for coping with the oxidative stresses that arise in vivo.
OxyR is a member of the LysR family of DNA-binding transcriptional
regulators (Zheng et al., 1998), which regulates expression of gor, prx, and katA
in N. gonorrhoeae (Seib et al., 2006). OxyR inhibits katA (catalase) expression
when the H2O2 concentration is low and activates gor and prx expression when
the H2O2 concentration is high (Seib et al., 2006). Prx catalyses reduction of
alkyl hydroperoxidases (Poole, 2005), while Gor maintains a reduced pool of
GSH in the cell (Carmel-Harel & Storz, 2000). The function of Gor is critical,
because GSH is one of the first lines of defense against oxidative stress in the
cell (Pomposiello & Demple, 2001). Interruption of the gor and prx genes
resulted in strains that were severely attenuated for biofilm formation. These
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phenotypes were indistinguishable from the phenotype of the oxyR::kan mutant,
which cannot induce transcription of gor or prx. This suggests that a mutation in
either of these genes is sufficient to cause the oxyR::kan phenotype, and that the
effect of these mutations is not cumulative. Either mutation is sufficient to impair
biofilm formation. All three mutations nearly abolish biofilm formation, which
indicates that OxyR and members of its regulon (gor and prx) are likely equally
important for normal biofilm formation. We did not examine the effect of a
katA::kan mutation on biofilm formation, because the function of catalase is
redundant in N. gonorrhoeae (Seib et al., 2006). We previously demonstrated
that a ccp::kan mutant is also deficient in biofim formation (Falsetta et al., 2009).
Like catalase, Ccp reduces H2O2 in the gonococcus (Seib et al., 2004, Seib et al.,
2006).
PerR is a Fur paralogue that regulates transcription of 12 genes (Seib et
al., 2006, Wu et al., 2006). Of these, the mntABC operon is one of a few genes
in this regulon that have identified functions (Seib et al., 2006). The mntABC
operon encodes the MntABC transporter, which imports Mn2+ into the cells
(Tseng et al., 2001, Seib et al., 2006). Mn2+ acts as an intracellular antioxidant
and can also protect N. gonorrhoeae from H2O2 in strains lacking catalase activity
(Seib et al., 2004). In addition, Mn2+ protects N. gonorrhoeae from O2- (Tseng et
al., 2001). A mntC mutant lacking the periplasmic binding component of the
Mn2+ transporter accumulates less Mn2+ and is sensitive to H2O2 (Wu et al.,
2006). The mntAB::kan and mntC::kan mutants are both severely attenuated for
biofilm formation over glass compared to the wild type, and there is no
appreciable difference in the ability of these mutants to form biofilm. This
indicates that the accumulation of Mn2+ is critical for biofilm formation. Although it
has not been directly demonstrated, these results suggest that the mntAB::kan
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mutant is equally impaired in its ability to accumulate Mn2+, as both form poor
biofilms with similar phenotypes.
NmlR shares homology with the MerR family of prokaryotic transcriptional
regulators (Kidd et al., 2005). MerR senses the mercuric ion and regulates
genes that confer resistance to mercury (Hobman et al., 2005). In N.
gonorrhoeae NmlR regulates five genes: nmlR, trxB, copA, estD, and adhC
(Kidd et al., 2005, Potter et al., 2009a). An nmlR mutant is more susceptible to
killing by cumene hydroperoxide and diamide than the wild type and is somewhat
impaired in its ability to grow anaerobically (Kidd et al., 2005). Transcription of
the NmlR operon is normally repressed by NmlR, but is induced in the presence
of disulfide stress (Kidd et al., 2005). The function of copA is not known at this
time, although CopA is tentatively annotated as a copper transport protein CopA
(Kidd et al., 2005). Thus, we did examine the effect of a copA mutation on
biofilm formation. adhC encodes a class III alcohol dehydrogenase, but this
gene contains a premature stop codon in all N. gonorrhoeae strains and does not
encode a functional protein (Kidd et al., 2005, Potter et al., 2007). Therefore, we
did not pursue study of this gene either. We did examine biofilm formation in an
estD::kan mutant, as estD encodes the esterase D protein, which protects
gonococcal cells from GSNO toxicity (Potter et al., 2009a). We also examined
the role of TrxB in biofilm. TrxB is a thioredoxin reductase that contributes to
oxidative stress tolerance by enhancing NO resistance in N. gonorrhoeae (Potter
et al., 2009b). TrxB may also contribute to NO resistance in the gonococcus by
affecting regulation of the aniA and norB denitrification genes, as a trxB mutant is
defective in expression of these transcripts (Potter et al., 2009b).
We found that an estD::kan mutant is not defective in its ability to form
biofim over glass, yet it is severely attenuated in its ability to form biofilm over
THCEC. This suggests that GSNO may be more abundant during infection.
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Cervical epithelial and endothelial cells produce NO (Ledingham et al., 2000,
Tschugguel et al., 1999), which is reduced to GSNO in the gonococcus (Seib et
al., 2006). Thus, a greater abundance of NO in our THCEC culture system may
explain why an estD::kan is severely attenuated over cells, but not attenuated
over glass. In our glass flow cell system, the most abundant source of NO would
be anaerobic metabolism in the gonococcus (Seib et al., 2006). It has been
demonstrated that N. gonorrhoeae rapidly achieves a NO steady-state under
anaerobic growth conditions (Cardinale & Clark, 2005). Thus, anaerobic
respiration is likely not an abundant source of NO. A trxB::kan mutant is
attenuated for biofilm formation over glass and THCEC. Attenuation appears to
be more severe over cervical cells, although trxB::kan forms biofilms with
significantly less biomass and lower average thickness than wild type under both
growth conditions. At first glance these findings appear to conflict, as TrxB is
also involved in NO tolerance. Thus, one would expect that the trxB::kan mutant
would also be attenuated over THCEC, but not glass. However, a trxB::kan
mutant is impaired in its ability to undergo anaerobic respiration, and transcription
of both aniA and norB is reduced in this mutant compared to wild type (Potter et
al., 2009b). We previously demonstrated that AniA and NorB are required for
wild type levels of biofilm formation (Falsetta et al., 2009). Thus, the deficit in
biofilm formation over glass is likely attributable to the reduced expression of
aniA and norB in the trxB::kan mutant. This phenotype may be more severe over
cervical cells, because the concentration of NO is likely higher under these
conditions, and trxB::kan lacks the ability to efficiently reduce NO when norB
transcription is impaired.
Overall, these findings suggest that the ability to tolerate oxidative stress
is critical for normal biofilm formation. Some genes involved in oxidative stress
tolerance are more highly expressed in biofilm (Falsetta et al., 2009), suggesting
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that biofilm may represent an adaptation that enhances oxidative stress tolerance
during infection of the human host.
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CHAPTER V
DISCUSSION
Human understanding of the behaviors of microorganisms has
significantly advanced since the 1670s when Antonie van Leeuwenhoek looked
through one of the first compound light microscopes and saw what he termed
“animalcules.” For centuries following this discovery, microorganisms were
viewed as free-floating or planktonic, unicellular organisms that acted
independently of each other. However, in the 1930s, researchers began to
recognize that bacteria, particularly marine or aquatic bacteria, had a tendency to
associate with one another in a microbial community (Zobell & Allen, 1935).
These communities were later termed biofilms, and have received significant
attention in the field of microbiology over the last 30 to 40 years (Costerton,
1999, Costerton et al., 1995, Costerton et al., 1999, Dunne, 2002, Stoodley et al.,
2002). Biofilms are ubiquitous (Costerton et al., 1978) and play significant roles
in various biological processes with both positive and negative outcomes.
Biofilms have received the most attention for the detrimental roles that they play
in human disease and other processes, such as biofouling (Costerton, 1999,
Costerton et al., 1995, Costerton et al., 1999, Dunne, 2002, Stoodley et al.,
2002). Biofilms significantly contribute to the morbidity and mortality of several
human diseases, including cystic fibrosis and nosocomial infections. Biofilms are
inherently resistant to antimicrobials and the human immune defenses, which
facilitates persistent infection by biofilm-forming organisms (Ceri et al., 1999).
Recently it has been postulated that nearly all pathogens that cause persistent
infection grow as biofilms (Costerton et al., 1999). Therefore, more organisms
are being evaluated for their ability to form a biofilm.
The mechanism of infection by N. gonorrhoeae differs in men and women
(Edwards & Apicella, 2004). Men typically suffer from acute infection with overt
154
symptoms (Hansfield, 2005, Hook, 1999c), while up to 80 percent of infected
women exhibit no symptoms (Bozicevic et al., 2006, Pedersen & Bonin, 1971).
Therefore, infected males typically seek immediate medical attention, are treated
with antibiotics, and rapidly clear the infection (Hansfield, 2005, Hook, 1999c).
Women, however, can become chronically infected, leading to the potentially
serious complications of PID, infertility, ectopic pregnancy, and DGI (Hansfield,
2005, Hook, 1999c). Women frequently do not seek medical attention until there
is a complication that results in more severe and noticeable symptoms (Platt et
al., 1983). At this point, irreparable damage has often occurred, as PID can lead
to the chronic re-infection by a woman’s own normal flora (Hansfield, 2005,
Hook, 1999c). Gonorrhea rates declined in the United States once penicillin
therapy became popular (Fox et al., 1998), but later rose as the sexual behavior
patterns of Americans began to change in the 1960s (Zaidi et al., 1983).
Compulsory screening was implemented to detect asymptomatic infection in
women, which was initially largely successful (Zaidi et al., 1983). However, the
number of cases of gonorrhea has gradually increased over the last several
years in the United States, along with the rates of antibiotic resistance (Centers
for Disease Control and Prevention, 2007, 2007). Women in poor communities
without access to regular screening are more likely to develop complications from
gonorrhea infection. No single unifying explanation exists for the apparent
difference in the occurrence of symptoms in men and women.
The observation that women are susceptible to chronic gonococcal
infection prompted our laboratory to evaluate the ability of N. gonorrhoeae to
form biofilms over abiotic surfaces (glass), primary and immortalized cervical
tissues, and over cervical tissues in vivo (Steichen, 2008, Greiner et al., 2005).
N. gonorrhoeae readily forms biofilm in continuous-flow systems over glass and
cervical tissues (Greiner et al., 2005). Patient biopsies indicate that gonococcal
155
biofilm structures are present during natural cervical infection (Steichen, 2008).
N. gonorrhoeae is a highly adapted human pathogen, which strongly suggests
that biofilm formation plays a role in infection in vivo and that biofilm formation is
not a novel occurrence. In contrast to women, the site of infection in men is
subject to periodic rapid fluid flow, and the incubation period is generally short.
Thus, the conditions in men are likely not conducive to biofilm formation, as are
the conditions in women.
Prior to the studies presented in this dissertation, little was understood
about the mechanisms that contribute to or govern biofilm formation in N.
gonorrhoeae. Much of the current biofilm literature suggests that biofilms exhibit
unique patterns of gene expression that dictate community behaviors, including
resistance to antimicrobials and host immune defenses (An & Parsek, 2007,
Beloin & Ghigo, 2005, Resch et al., 2005, Shemesh et al., 2007, Wagner et al.,
2003, Waite et al., 2006, Waite et al., 2005, Whiteley et al., 1999, Wu et al.,
2006). In order to better understand gonococcal biofilm, we used microarrays to
examine the transcriptional profiles of N. gonorrhoeae biofilms for comparison to
their planktonic counterparts (Chapter II). We identified 83 genes that met our
criteria for differential expression (fold-change ≥ 2.0 and P ≤ 0.05) when we
compared biofilms grown over glass to planktonic cells collected from the biofilm
effluent. The vast majority of these genes were hypothetical (57.8%), although
many of the proteins encoded by these genes could play important roles in
biofilm formation. We elected not to pursue these genes for the purposes of this
study, as a significant pattern of expression emerged in some of the genes with
identified functions. However, the hypothetical genes we identified could be
pursued in future investigations. We found that genes involved in anaerobic
respiration (aniA, ccp, and norB) were highly up-regulated during biofilm
formation, while genes involved in aerobic respiration were down-regulated (nuo
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operon). We used qRT-PCR to confirm expression of these genes over glass
and to examine expression in a more relevant model system (growth over
THCEC). We found that the pattern of regulation was similar over glass and over
THCEC. This finding indicates that expression of these genes is relevant to
infection. This data also suggests that our continuous-flow system for biofilm
growth over glass may be a suitable model for biofilm formation when the
experimental design does not permit the use of cervical cells. For example, this
system may be a useful tool for modeling biofilm behaviors during treatment with
compounds that are toxic to or impair the growth of cervical cells. However, we
prefer to use the tissue culture system to model biofilm growth when possible, as
N. gonorrhoeae is an obligate pathogen.
Transcripts of both aniA and norB are virtually undetectable under aerobic
growth conditions (Householder et al., 1999, Householder et al., 2000). Thus,
high levels of expression of aniA and norB during growth as a biofilm indicates
that anaerobic respiration occurs in gonococcal biofilms. N. gonorrhoeae is often
isolated in the presence of obligate anaerobes and the cervical environment is
presumed to be oxygen limited (Smith, 1975). Thus, anaerobic growth may be
important for survival in vivo. Anaerobic respiration also contributes to the ability
of the gonococcus to tolerate oxidative stress, as NorB can rapidly achieve a NO
steady-state that reduces proinflammatory concentrations of NO to
concentrations that are not inflammatory (Cardinale & Clark, 2005). Although
Ccp does not play a role in anaerobic respiration, expression of ccp is only
induced under anaerobic conditions (Turner et al., 2003). Like NorB, Ccp also
reduces oxidants present in the cervical environment. Ccp effectively reduces
the H2O2 produced by host PMNs and the Lactobacillus species that also inhabit
the female genitourinary tract (Carreras et al., 1994, Eschenbach et al., 1989,
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Turner et al., 2003). Therefore, elevated expression of these genes in biofilm
indicates that biofilms may be suited for survival in the female host.
Sera from patients infected with N. gonorrhoeae exhibits a strong antibody
response to AniA (Clark et al., 1988), which is further evidence that anaerobic
respiration occurs in vivo. Biofilm formation can be detected in patient biopsies
and would likely confer distinct growth advantages through the enhanced
expression of oxidative stress tolerance genes (Steichen, 2008). Thus, it is
plausible that this is the preferred mode of growth in the cervical environment. In
the past, the development of a vaccine against N. gonorrhoeae has focused on
P.II as a potential antigen (Hook, 1999c). However, the development of a
vaccine has been largely unsuccessful, as most gonococcal surface structures
are phase and/or antigenically variable (Hansfield, 2005, Hook, 1999c). Some
groups have proposed using AniA as a vaccine antigen, as AniA is a stably
expressed outer membrane protein that is likely directed outside the cell
(Boulanger & Murphy, 2002). AniA is highly expressed in biofilms grown over
cervical cells, which represents a greater than 20-fold induction versus planktonic
cells. Our data indicate that biofilms are the preferred mode of growth in vivo,
and it has not been established that an anti-AniA antibody could penetrate the full
depth of the biofilm to access AniA in the substratum of these biofilms.
Furthermore, the fact that patients with active disease display robust anti-AniA
antibody responses suggests that these antibodies are indeed not protective
against disease. For these reasons we suggest that AniA is not a good vaccine
target.
To further investigate the role of anaerobic respiratory/anaerobicallyinduced genes in biofilm, we created or obtained strains with kanamycin
resistance cassette insertions in the coding sequences of the aniA, ccp, and norB
genes (Chapter II). We found that all three mutant strains were attenuated in at
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least one aspect of biofilm formation over glass and over THCEC. We
determined that the aniA::kan and ccp::kan mutants formed biofilms with
equivalent biomasses over glass, but these biofilms were much more compact
and had significantly reduced average thicknesses compared to the wild type.
However, the norB::kan mutant formed very little biofilm, which was dramatically
reduced in both biomass and average thickness. To evaluate biofilm formation in
a more relevant system, we infected THCEC with our mutant and wild type
strains. We found that all three mutants had significantly reduced biomasses and
average thicknesses compared to the wild type when grown over THCEC for 48
hours. This suggests that the role of these genes is more critical in an infection
model, which better resembles the conditions present in the host environment.
The role of Ccp is redundant, as catalase also reduces H2O2 (Seib et al.,
2006). If mutations are made in either the ccp or katA (catalase) gene, the
resulting strains become sensitive to H2O2-mediated killing (Seib et al., 2006).
However, a ccp/katA double mutant is more sensitive to H2O2 than either single
mutant (Seib et al., 2006). This may help to account for the observation that the
ccp::kan mutant is only partially impaired in its ability to form biofilms over glass.
Thus, we would expect that a ccp/katA mutant may be further impaired in its
ability to form biofilm. This would be a reasonable objective for future studies.
However, the role of AniA is not functionally redundant and yet a similar
phenotype is observed in the aniA::kan mutant. Other organisms also catalyze
anaerobic respiration during growth as a biofilm (Filiatrault et al., 2006, Hassett et
al., 2002, Van Alst et al., 2007, Wang et al., 2007b, Yoon et al., 2002). It has
become increasingly evident that anaerobic respiration is critical for biofilm
formation in a number of organisms, including the biofilm paradigm organism; P.
aeruginosa. Evidence suggests that anaerobic respiration is the primary mode of
growth for P. aeruginosa during cystic fibrosis infection (Filiatrault et al., 2006,
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Hassett et al., 2002, Van Alst et al., 2007, Yoon et al., 2002). This mode of
growth may contribute to the antimicrobial resistance of P. aeruginosa biofilms,
as cells that undergo anaerobic respiration are concentrated near the biofilm
substratum, are less metabolically active, and are likely less susceptible to
antimicrobials that target active cellular processes (Rani et al., 2007, Werner et
al., 2004). Oxygen only penetrates approximately the first 50 µM of P.
aeruginosa biofilms (Werner et al., 2004), although these biofilms typically
achieve thicknesses of approximately 100 µM or more (Davies et al., 1998,
Sauer et al., 2002). This observation may help to explain why the aniA::kan
mutant forms biofilms with biomasses that are similar to wild type, yet these
biofilms are significantly thinner than wild type. If oxygen cannot penetrate the
entire depth of the biofilm, the thicknesses of the biofilm may be limited by the
availability of oxygen in mutants that cannot respire anaerobically. The thickness
of an aniA::kan mutant biofilm does exceed 60 µM, which appears to correlate
with the findings in P. aeruginosa.
Biofilm formation was more severely attenuated in the norB::kan mutant,
as this mutant had significantly less biomass and lower average thicknesses than
the wild type. This was initially puzzling, as norB and aniA are members of the
same metabolic pathway and previous studies demonstrated that mutations in
either norB or aniA resulted in strains that were unable to respire anaerobically
(Householder et al., 1999, Householder et al., 2000). However, there was one
obvious difference in these two mutant strains. AniA reduces nitrite to NO
(Mellies et al., 1997), which is often toxic to many bacterial species (Davis et al.,
2001, Fang, 1997, MacMicking et al., 1997, Zumft, 1997). NorB then reduces
NO to nitrous oxide (Householder et al., 2000), which is not generally considered
to be toxic (Seib et al., 2006). A mutation in norB would render the gonococcus
unable to reduce AniA-generated NO. We considered the possibility that the
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accumulation of NO may be toxic or could impair biofilm formation in the
norB::kan mutant through another mechanism, as NO is an important signaling
molecule in eukaryotes (Davis et al., 2001, Liaudet et al., 2000, MacMicking et
al., 1997, Ortega Mateo & Amaya Aleixandre de, 2000, Stefano et al., 2000,
Tschugguel et al., 1999). N. gonorrhoeae is presumed to be inherently resistant
to NO, and a norB::kan mutant survives incubation under anaerobic conditions,
which should result in the accumulation of AniA-generated NO (Householder et
al., 2000). Thus, we hypothesized that NO affected biofilm formation through
another mechanism. The current P. aeruginosa biofilm literature suggested that
sublethal concentrations of NO may prevent biofilm formation or facilitate biofilm
dispersal (Barraud et al., 2006). Therefore, we attempted to rescue biofilm
formation in the norB::kan mutant by treating these biofilms with an NO quencher
(Chapter II). We found that treatment with PTIO improved biofilm formation in
this mutant. Although PTIO improved biofilm formation, biofilms were not
completely restored to wild type levels in the presence of PTIO. This result was
expected, as a norB::kan mutant cannot respire anaerobically (Householder et
al., 2000). We treated wild type biofilms with the NO donor SNP at the beginning
of biofilm formation and after 24 hours of biofilm formation to determine if NO
could also impair biofilm formation in the wild type. We found that treatment at
the start of biofilm impaired biofilm formation, but did not completely inhibit biofilm
formation. Treatment with SNP after 24 hours of biofilm formation resulted in
biofilms with less biomass and lower average thicknesses than untreated
biofilms. This indicates that the introduction of NO after 24 hours of biofilm either
halts biofilm development or causes the biofilm to disperse. We also considered
the possibility that NO could inhibit cytochrome oxidase, thus impairing aerobic
respiration in the norB::kan mutant. However, we found that there was no defect
161
in the growth of the norB::kan mutant when it was cultured under oxygen tension
conditions that were similar to those present in our biofilm system.
Treatment with NO did not completely inhibit N. gonorrhoeae biofilm
formation. The effective dose of NO is likely considerably lower than the
concentration of NO donor supplied. Thus, a higher dose of NO may be required
to completely block biofilm formation and/or other mechanisms that govern
gonococcal biofilm formation and dispersal. Interestingly, higher concentrations
of NO appear to enhance biofilm formation in P. aeruginosa (Barraud et al.,
2006). Concentrations that were considerably higher than the typical lethal dose
increased the ratio of adherent to planktonic cells in one P. aeruginosa biofilm
study (Barraud et al., 2006). This suggests that NO may have a dynamic effect
on biofilm, encouraging detachment at low concentrations and encouraging
biofilm formation at high concentrations. Biofilms are inherently resistant to
antimicrobial treatment, UV light, salinity, host defenses, acid exposure,
dehydration, and metal toxicity (Hall-Stoodley et al., 2004). Thus, it is plausible
that biofilm formation may also convey resistance to oxidants, such as NO. This
may explain why P. aeruginosa biofilm formation is elevated in the presence of
lethal NO concentrations, as biofilm formation may enhance the ability of these
cells to tolerate NO. Subsequently, we elected to examine the affect of higher
doses of SNP on N. gonorrhoeae biofilm formation (Chapter III). We found that
treatment with 20 µM concentrations and higher (up to 1 mM) completely
prevented biofilm formation, if SNP was administered at the start of the biofilm.
Removing SNP from the media used to inoculate the biofilm chambers did not
improve biofilm formation. These findings indicate that higher concentrations of
NO could completely block biofilm formation in N. gonorrhoeae. However, P.
aeruginosa, which is inherently more sensitive to NO, can form better biofilms in
the presence of high concentrations of NO (Barraud et al., 2006). When we
162
considered this, it occurred to us that anaerobic respiration would not be
immediately induced in gonococcal biofilms, as aniA and norB are repressed
under aerobic growth conditions (Barraud et al., 2006, Householder et al., 1999,
Householder et al., 2000). Thus, we constructed a fluorescent transcriptional
fusion to the aniA gene (aniA’-‘gfp) to monitor the induction of anaerobic
respiration in biofilms (Chapter III). We used light microscopy to monitor
induction in overnight plate cultures, the biofilm inoculum, and biofilms grown for
24 and 48 hours. We were unable to detect fluorescence in plate cultures or the
biofilm inoculum. However, we could readily detect GFP in biofilms grown for 24
and 48 hours. These data indicate that N. gonorrhoeae biofilms become
anaerobic over time, as aniA expression was induced between 0 and 24 hours of
biofilm formation. Gradual induction of anaerobic respiration in biofilms could
explain why immediate treatment with low concentrations of NO was partially
inhibitory, while high concentrations completely inhibited biofilm formation.
However, we observed that low doses of SNP halted biofilm formation in 24-hour
biofilms that were likely expressing aniA and undergoing anaerobic respiration.
This suggests that the effect of NO on N. gonorrhoeae biofilms is not dictated
solely by NO toxicity. This agrees with our data that indicates that the norB::kan
mutant is not impaired in its ability to grow under microaerophilic conditions.
We also used the aniA’-‘gfp fusion strain to evaluate the profile of
anaerobic metabolism in biofilm. P. aeruginosa biofilms are stratified and use a
combination of anaerobic and aerobic metabolism (Rani et al., 2007, Werner et
al., 2004). Protein synthesis occurs in the first 30-60 µm of biofilm, while
anaerobic/metabolically inactive cells comprise the majority of the biofilm and are
localized near the substratum (Werner et al., 2004). To determine if this was
similar for N. gonorrhoeae, we grew the aniA’-‘gfp fusion strain for 48 hours and
then stained the biofilm with 2C3 (H.8 antibody) to visualize all cells in the biofilm.
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We found that the majority of the biofilm cells expressed aniA, and that cells
expressing aniA were located near the surface of attachment in the substratum of
biofilm. The uppermost region of the biofilm was almost entirely comprised of
cells that did not express aniA. This indicates that cells near the fluid flow
interface use aerobic metabolism. This pattern is similar to the patterns
observed in P. aeruginosa biofilms (Rani et al., 2007, Werner et al., 2004). aniA
expression also did not localize to areas where there were gaps in the biofilm,
indicating the presence of water channels. The expression of aniA and norB is
repressed when oxygen is abundant (Householder et al., 1999, Householder et
al., 2000, Whitehead et al., 2007), as it would be in the bulk fluid. In Chapter II,
we calculated the dissolved oxygen concentrations of media entering and exiting
the biofilm chamber and found that the concentration of oxygen was well above
anaerobic or microaerophilic concentrations. Although mutations in anaerobic
respiratory genes can cause severe impairment of biofilm formation, especially
when cultured in the presence of host cells, these mutations do not completely
prevent biofilm formation. This finding reflects the metabolic profile of N.
gonorrheoae biofilms, which catalyze both anaerobic and aerobic respiration.
Expression of aniA is not induced in the biofilm inoculum, which suggests that
biofilms may rely on aerobic respiration early during an infection, prior to the
establishment of a biofilm and transcription of the anaerobic respiratory genes.
To further examine the effect of NO on biofilm formation, we devised a
method to induce anaerobic respiration in biofilms before evaluating the effect of
NO. We grew biofilms for 24 hours in the presence of nitrite, then transitioned
these biofilms to media without nitrite, which either was or was not supplemented
with 20 µM SNP. We previously determined that growing biofilms in the
presence of nitrite for 24 hours induces expression of aniA (Chapter III). Biofilm
formation was enhanced by the addition of SNP under these conditions. It
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appears that treatment with high doses of SNP can enhance biofilm formation, if
anaerobic respiration has been initiated. Anaerobic respiration likely allows norB
to be transcribed at the levels necessary to effectively reduce NO to
concentrations that are not lethal to the gonococcus. This observation further
supports the hypothesis that N. gonorrhoeae biofilms are important for and
contribute to oxidative stress tolerance during infection. In our aim to develop a
better model for studying the impact of NO on biofilm, we selected another NO
donor for the treatment of biofilms. We chose to treat biofilms with DETA/NO, as
it is a slower release NO donor that may donate NO at a rate that is not
overwhelming during the initial stages of biofilm development. We found that 20
µM DETA/NO did not prevent biofilm formation, but rather enhanced biofilm
formation in the absence of nitrite. This finding suggests that DETA/NO donates
NO at a rate that is easily tolerated during the early stages of biofilm
development, before the induction of anaerobic respiration. Overall, it appears
that the concentration of NO, the rate at which it is donated, and the time at
which it is administered determines the effect on biofilm formation. Low
concentrations of NO prevent biofilm formation, even in the presence of nitrite,
while high concentrations enhance biofilm formation if a slow-release NO donor
is used or anaerobic respiration is occurring in biofilm. We have proposed a
model that illustrates the potential effects of NO on biofilm formation (Figure 20).
However, additional evidence is needed to either support or refute this model,
and it is not clear what protein(s) may be sensing the concentration of NO in
biofilm. We would propose that the NsrR regulator may play a role in sensing the
concentration of NO in biofilm, as NsrR takes its name from its sensitivity to NO
(NO-sensitive repressor) (Whitehead et al., 2007). When NO is present, NsrR is
unable to function as a negative regulator of aniA and norB, which results in the
de-repression of aniA and norB expression (Whitehead et al., 2007). It may be
165
possible that NsrR regulates other previously unidentified targets that could play
a role in the NO response. Further study is warranted to investigate this
hypothesis. Microarrays could be used as tools to screen for genes that are
differentially regulated after treatment with SNP or DETA/NO. This may be
useful in the identification of genes involved the NO response. This screen might
also identify genes that are involved in biofilm detachment, which could represent
new therapeutic targets for the treatment of gonorrhea.
Biofilms treated with DETA/NO from the start of biofilm formation or
treated with SNP after 24 hours of growth resembled those grown in the
presence of nitrite for 48 hours. Prior to determining that N. gonorrhoeae biofilms
catalyze anaerobic respiration, we observed that nitrite enhanced biofilm
formation, although biofilms could form in the absence of nitrite (Greiner et al.,
2005). Biofilms grown in the absence of nitrite develop at slower rate than those
grown in the presence of nitrite. Thus, we began supplementing our biofilms with
nitrite, which allowed mature biofilms to form by 48 hours postinoculation. In light
of this observation, it was not surprising that N. gonorrhoeae utilizes anaerobic
respiration during growth as a biofilm. N. gonorrhoeae was initially considered to
be incapable of anaerobic growth, due to the inability to culture cells under
anaerobic conditions (James-Holmquest et al., 1973). This led to the finding that
N. gonorrhoeae is unique in that it uses nitrite as a terminal electron acceptor for
anaerobic growth, and it is incapable of using nitrate (Knapp & Clark, 1984).
However, we found that biofilm growth can be partially restored in an aniA::kan
mutant by adding NO to the biofilm media (Chapter III). This mutant cannot
reduce nitrite and similar mutants were considered incapable of anaerobic
growth. Previous mutants survived, but did not grow under anaerobic conditions
(Householder et al., 1999). However, our results suggest that this mutant may
be able to grow under anaerobic conditions using NO. NO was not used to
166
supplement the culture media in earlier studies that concluded that an aniA
insertion mutant is unable to respire anaerobically (Householder et al., 1999).
Although oxygen is abundant in the media of our biofilm system, the majority of
the cells in these biofilms catalyze anaerobic respiration. Anaerobic respiration
occurs in more than two-thirds of the total thickness of N. gonorrhoeae biofilms,
as visualized by aniA expression. Thus, the partial restoration of biofilm
formation in the aniA::kan mutant strongly suggests that these biofilms are able
to undergo anaerobic respiration, as the thickness of these biofilm exceeds the
thickness of the aerobic portion of gonococcal biofilms. However,
supplementation with NO does not fully restore biofilm formation in the aniA::kan
mutant. This indicates that we may not have supplemented our media with the
optimal concentration of NO, or that nitrite may be the preferred substrate for
anaerobic metabolism. The ability to use both nitrite and NO to support
anaerobic growth would be of advantage to gonococcal biofilms. If this were the
case, NO could be used to support anaerobic growth if the function of AniA was
impaired. In support of this hypothesis, some N. meningitidis strains possess a
frameshift mutation in aniA, but are still able to respire anaerobically (Deeudom
et al., 2006, Rock et al., 2005, Rock & Moir, 2005, Rock et al., 2007, Stefanelli et
al., 2008). N. gonorrhoeae biofilms are metabolically heterogeneous and may be
able to use a variety of substrates to catalyze anaerobic and aerobic respiration.
Biofilm heterogeneity confers advantages for biofilm survival, including the ability
of metabolically inactive cells to resist antimicrobial treatment and the host
immune response (Barraud et al., 2006, Costerton et al., 1999, Werner et al.,
2004).
If oxygen is abundant in our biofilm system, why do N. gonorrhoeae
biofilms predominantly use anaerobic metabolism? The cbb3 family of
cytochrome oxidases have a high affinity for oxygen (Pitcher et al., 2002), and it
167
has been speculated that the oxygen concentration in vivo would have to be
considerably lower than the predicted concentration in order to hinder aerobic
growth of the gonococcus. Studies that examined the oxygen profiles of P.
aeruginosa biofilms determined that oxygen is limited in its ability to diffuse into
the biofilm (Barraud et al., 2006, Werner et al., 2004). This may be the simplest
explanation for the metabolic profile of N. gonorrhoeae biofilms, which catalyze
aerobic respiration at the fluid-flow interface and anaerobic respiration in the
depths of the biofilm. However, the matrix of N. gonorrhoeae biofilm is
dramatically different than P. aeruginosa biofilm, and it is not clear as to whether
the diffusion of oxygen is limited in gonococcal biofilms (Costerton, 1999,
Steichen, 2008). Our results suggest that diffusion of oxygen into N.
gonorrhoeae biofilms is limited, as aniA expression is typically repressed when
oxygen is abundant (Householder et al., 1999). We suggest that direct
measurement of the dissolved oxygen content at different depths of the biofilm
would be the best way to study the kinetics of oxygen diffusion in gonococcal
biofilms.
We also considered the possibility that the induction of anaerobic genes in
biofilm plays a more important role in oxidative stress tolerance. NorB functions
dually in anaerobic respiration and oxidative stress tolerance by reducing NO
(Mellies et al., 1997), while Ccp contributes to oxidative stress tolerance by
reducing H2O2 (Turner et al., 2003). Induction of these genes in biofilm suggests
that biofilm formation may enhance oxidative stress tolerance in N. gonorrhoeae.
Although we did not directly challenge gonococcal biofilms with oxidative
stressors (other than NO), we determined that a variety of oxidative stress
tolerance genes are required for biofilm formation including trxB, estD, mntABC,
oxyR, prx, and gor (Chapter IV). A reasonable future objective would be to
assess the ability of biofilm to withstand oxidative stress as compared to
168
planktonic cells. Ideal oxidants to test would be NO and H2O2. Despite the
varied function of the proteins encoded by these genes, all are required for
normal biofilm formation. Inhibiting the function of the periplasmic binding portion
(MntC) or intermembrane domain (MntAB) of the MntABC transporter severely
attenuates biofilm formation over glass. Mutations in either portion of the
transporter should impair the ability of the gonococcus to take up Mn. These
findings indicate that the antioxidant properties of Mn help to protect gonococcal
biofilms from oxidative stress. However, the mntABC mutants are not as
severely impaired as other oxidative stress tolerance mutants, which includes
members of the OxyR regulon. This may reflect the relative importance of
different oxidative stress tolerance mechanisms in N. gonorrhoeae. The OxyR
regulator and members of its operon (prx and gor) are also required for robust
biofilm formation. oxyR::kan mutant biofilms are indistinguishable from prx::kan
or gor::kan mutant biofilms, which suggests that disruption of either member of
this operon is sufficient to hinder biofilm formation. The effects of the gor and prx
mutations do not appear to be cumulative, although they are not functionally
redundant (Seib et al., 2006). However, they do play complementary roles in the
reduction of H2O2 (Seib et al., 2006). Although the gonococcus has several
mechanisms for coping with H2O2 stress, disrupting a single gene involved in
H2O2 tolerance (gor, prx, or ccp) can impair biofilm formation. This suggests that
protection against H2O2 is paramount in biofilms, which may correspond to the
prevalence of H2O2-producing Lactobacillus species in the female genitourinary
tract and the use of H2O2 by the host immune system (Carreras et al., 1994,
Eschenbach et al., 1989). Of all the mutants tested, the estD::kan mutant
displayed the most unique phenotype. This mutant is not defective in its ability to
form biofilm over glass, yet it is severely attenuated in its ability to form biofilm
over THCEC. Although aniA::kan and ccp::kan are more severely attenuated
169
over THCEC, both mutants have reduced average thicknesses compared to wild
type when cultured over glass. Cervical epithelial and endothelial cells produce
NO (Ledingham et al., 2000, Tschugguel et al., 1999), which is reduced to GSNO
in the gonococcus (Seib et al., 2006). NO and subsequently GSNO would likely
be more abundant in the THCEC culture system. GSNO is toxic and would
normally be reduced by EstD in the cell (Seib et al., 2006). This may explain why
an estD::kan mutant is severely attenuated over cells, but is not attenuated over
glass. In our glass flow cell system, the most abundant source of NO would be
anaerobic respiration in the gonococcus. However, NO is rapidly reduced by
NorB in vitro (Cardinale & Clark, 2005). In contrast to the estD::kan mutant, a
trxB::kan mutant is attenuated for biofilm formation over glass and THCEC. TrxB
is also involved in NO tolerance, but NO is likely not abundant in our continuousflow system over glass, as previously discussed. However, the trxB::kan mutant
is likely attenuated in both systems, because it is impaired in its ability to undergo
anaerobic respiration. Transcription of both aniA and norB is reduced in this
mutant compared to the wild type (Potter et al., 2009b). Thus, deficient biofilm
formation is likely attributable to the reduced expression of aniA and norB. The
phenotype may be more severe over cells where the NO concentration is higher,
as the trxB::kan mutant would lack the ability to efficiently reduce NO. Overall,
our findings in Chapter II and IV clearly demonstrate that the ability to tolerate
oxidative stress is necessary for robust levels of biofilm formation.
Biofilm formation by N. gonorrhoeae may aid in oxidative tolerance during
the cervical infection of women by positively regulating factors, such as ccp and
norB, which reduce reactive oxygen and nitrogen species, respectively.
Anaerobic respiration in biofilm may also represent an adaptation to oxygen
limitation within the biofilm or the host environment. However, biofilm formation
may play a more prominent role in oxidative stress defense, as we determined
170
that the majority of the genes involved in these pathways are required for robust
biofilm formation. The propensity for biofilms to form during natural cervical
infection, the conditions present during male infection that are likely not conducive
to biofilm formation, and the lack of evidence of biofilm formation in men, suggests
that biofilm formation may be specific to the infection of women. Biofilm formation
may confer properties to the gonococcus through the induction of the anaerobic
metabolism and oxidative stresses defense pathways, enhancing the ability to
cope with or evade the host immune response. The ability to do so likely
contributes to the occurrence of persistent infection in women and may help to
account for the greater likelihood of asymptomatic infection in women.
Mechanisms that govern biofilm formation might be manipulated to improve
treatment or diagnosis of N. gonorrhoeae infection in women. Our findings and
others suggest that AniA may be a suitable candidate for vaccine development.
171
CHAPTER VI
FUTURE DIRECTIONS AND IMPLICATIONS
N. gonorrhoeae biofilms are present during natural cervical infection
(Steichen, 2008), and we have determined that biofilm formation likely
contributes to the ability of the gonococcus to adapt to and withstand the unique
stresses present in the cervical environment. Thus, biofilm-specific properties
could serve as new targets for the prevention, diagnosis, and treatment of
gonorrhea in women. Patients with gonorrhea have low anti-gonococcal
antibody levels, which are partially attributable to the phase and antigenic
variation of the major surface-exposed structures of the gonococcus (pili, LOS,
Opa) (Hook, 1999c, Hedges et al., 1998). However, many patients show a
strong antibody response to AniA, which does not phase or antigenically vary
(Clark et al., 1988). Attempts to make a vaccine against P.II (Opa) have been
largely unsuccessful (Hook, 1999c), leading some researchers to suggest that
AniA should be investigated as a potential vaccine target.
Our results demonstrate that transcription of aniA is induced over 20-fold
in biofilm versus planktonic modes of growth and that aniA is expressed in the
majority of the cells within the biofilm with the exception of those immediately
exposed to oxygen that is abundant in the bulk fluid. The abundance of AniA in
biofilm suggests that AniA may be a reasonable target for the development of a
vaccine or new anti-gonococcal therapies. However, an anti-AniA antibody may
not be able to penetrate the full depth of gonococcal biofilms to access the
populations of AniA that are most abundant in the biofilm substratum. Therefore,
we would suggest trying to identify small molecules that inhibit the function of
AniA or NorB. This would likely be a more valuable line of study, and may be
useful the creation of new therapies directed against gonorrhea. AniA and NorB
are critical for biofilm formation over cervical cells (Falsetta et al., 2009).
172
Therefore, inhibiting the function of either AniA or NorB could help to combat
cervical infection. NCBI BLAST searches (http://blast.ncbi.nlm.nih.gov/Blast)
indicate that the nucleotide sequences of aniA and norB only have significant
homology to sequences that are present within the genomes of other Neisserial
species. Although the amino acids sequences of these genes share homology
with other bacterial species, none of the species identified are known to inhabit
the female genitourinary tract. However, these sequences do not have
significant homology to human proteins. Thus, AniA and NorB appear to be
unique to the cervical environment. If this is true, they might be selectively
targeted to impede gonococcal infection with little to no detrimental effect on
human cervical cells or inhabitants of the normal flora.
Researchers have focused on trying to elucidate the complex signaling
mechanisms that govern biofilm formation and dispersal in hopes that these
mechanisms could be manipulated to impair biofilm formation or dissociate
existing biofilms. Biofilms are inherently resistant to antibiotics and a wide range
of environmental and host-related stresses, which makes them recalcitrant to
treatments that are typically effective against “normal” planktonic bacteria (Ceri et
al., 1999, Hall-Stoodley et al., 2004). Some studies have shown that dispersed
biofilm cells or flocs are more susceptible to antimicrobial treatment than their
biofilm counterparts (Barraud et al., 2006). This suggests that manipulating the
typical biofilm life cycle may be advantageous for the treatment of biofilm
infections. Our data indicate that NO may be an important signaling molecule in
N. gonorrhoeae biofilms. NO appears to be involved in regulation of the
phenotypic switches that occur in the life cycle of the gonococcus, which
alternates among suspended (planktonic), adherent (biofilm), and invasive
modes of growth (Edwards & Apicella, 2004). The ability to manipulate the
gonococcal life cycle could be useful in the treatment or diagnosis of gonorrhea.
173
Further investigation is needed to define the mechanism(s) that governs
the gonococcal response to NO. We would propose using microarrays to identify
genes that are induced or repressed by the addition of NO. We would expect
this screen to identify genes that may be involved in the degradation or synthesis
of the biofilm matrix. N. gonorrhoeae is non-motile (Hook, 1999a) and would
have to rely on mechanisms that build or degrade the biofilm matrix to control
biofilm formation and dispersal. If our model is accurate (Figure 20), sublethal
concentrations of NO would likely up-regulate factors involved in degradation and
down-regulate factors involved in synthesis of the biofilm matrix. Conversely,
high or potentially lethal concentrations of NO should encourage biofilm
formation by up-regulating matrix production and down-regulating degradation.
We would also suggest examining the effect of NO during an infection by
quantifying the number of cells that are suspended, adherent, or invasive
following NO treatment. A more directed approach might focus on the role of
NsrR in the NO-sensing response. NsrR is a key regulator of the gonococcal
anaerobic respiratory genes (aniA and norB), which is given its name for its
sensitivity to NO (Overton et al., 2006). Thus, NsrR may sense the concentration
of NO in the cell and regulate the transcription of genes involved in this response.
Although NsrR has not been shown to regulate such genes, studies that have
examined the NsrR regulon have not looked at its regulation in the presence of
NO (Overton et al., 2006). Unpublished studies from our laboratory have
identified a DNA nuclease that is involved in degrading foreign and gonococcal
DNA. These studies have also determined that DNA appears to be a major
component of the N. gonorrhoeae biofilm matrix. Exogenous addition of this
purified nuclease causes N. gonorrhoeae biofilms to disperse. Thus, this
nuclease may be up-regulated in response to sublethal concentrations of NO.
174
We would propose examining transcription of this gene in response to NO
treatment and/or evaluating what role, if any, NsrR might have in its regulation.
Overall our findings suggest that biofilm formation is advantageous to
gonococcal growth in the human cervical environment. Specifically, biofilm
formation appears to represent an adaptation to oxygen limitation and the
presence of oxidative stressors. However, we did not perform any studies to
directly assess the ability of N. gonorrhoeae biofilms to withstand oxidative
stress. Thus, we would also propose experiments that would examine the impact
of NO or H2O2 treatment on gonococcal biofilms. By comparing the survival of
biofilm to planktonic cells, we could assess the relative fitness of biofilm in
response to oxidative stress. We would expect biofilms to have enhanced fitness
based on our observations that oxidative stress tolerance mechanisms are
critical for biofilm formation and biofilms appear to be relatively resistant to high
concentrations of NO if NorB is functional and expressed at the time of treatment.
If biofilms have enhanced survival in the presence of oxidants, this may help to
explain how N. gonorrhoeae successfully maintains infection over long periods of
time in the female host. The ability to tolerate and subvert some of the host
immune defenses may also help to account for the lack of a strong immune
response and the absence of symptoms in many women.
Understanding the life cycle of N. gonorrhoeae, specifically the biofilm
phase of growth, may enhance the overall understanding of gonococcal infection.
Only then may more effective treatment and prevention strategies be developed
to minimize the complications that are common in infected women.
175
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