Microbiology (2004), 150, 1315–1326
DOI 10.1099/mic.0.26822-0
Significant differences in type IV pilin allele
distribution among Pseudomonas aeruginosa
isolates from cystic fibrosis (CF) versus non-CF
patients
Julianne V. Kus,1,3 Elizabeth Tullis,2 Dennis G. Cvitkovitch3
and Lori L. Burrows1,3
1
Centre for Infection and Biomaterials Research, Hospital for Sick Children Research Institute
and Department of Surgery, University of Toronto, 7142A Elm Wing, 555 University Avenue,
Toronto, ON, Canada M5G 1X8
Correspondence
Lori L. Burrows
[email protected]
2
Adult Cystic Fibrosis Clinic, St Michael’s Hospital, Toronto, ON, Canada
3
Department of Microbiology, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada
Received 8 October 2003
Revised 2 December 2003
Accepted 4 December 2003
Type IV pili (TFP) are important colonization factors of the opportunistic pathogen Pseudomonas
aeruginosa, involved in biofilm formation and attachment to host cells. This study undertook a
comprehensive analysis of TFP alleles in more than 290 environmental, clinical, rectal and cystic
fibrosis (CF) isolates of P. aeruginosa. Based on the results, a new system of nomenclature is
proposed, in which P. aeruginosa TFP are divided into five distinct phylogenetic groups. Each
pilin allele is stringently associated with characteristic, distinct accessory genes that allow the
identification of the allele by specific PCR. The invariant association of the pilin and accessory
genes implies horizontal transfer of the entire locus. Analysis of pilin allele distribution among
isolates from various sources revealed a striking bias in the prevalence of isolates with group I pilin
genes from CF compared with non-CF human sources (P<0?0001), suggesting this particular
pilin type, which can be post-translationally modified by glycosylation via the action of TfpO
(PilO), may confer a colonization or persistence advantage in the CF host. This allele was also
predominant in paediatric CF isolates (29 of 43; 67?4 %), showing that this bias is apparent early
in colonization. Group I pilins were also the most common type found in environmental isolates
tested. To the authors’ knowledge, this is the first example of a P. aeruginosa virulence factor
allele that is strongly associated with CF isolates.
INTRODUCTION
Pseudomonas aeruginosa remains among the top opportunistic and nosocomial pathogens as well as the major cause
of morbidity and mortality among cystic fibrosis (CF)
patients. It elaborates a wide variety of virulence factors
including adhesins and toxins (Lyczak et al., 2000; Rahme
et al., 2000; Stanislavsky & Lam, 1997). One of the most
significant adhesins, polar type IV pili (TFP), is important in
bacteria–host cell interactions (Hahn, 1997), in formation
of biofilms (O’Toole & Kolter, 1998) and for twitching
Abbreviations: CF, cystic fibrosis; DSL, disulfide-bonded loop; TFP, type
IV pili.
Novel sequences from each of pilin groups I, II, IV and V described in
this study have been deposited in GenBank under accession numbers
AAM44066, AAM52055–AAM52061 and AAM62144–AAM62147.
Details of the Pseudomonas aeruginosa strains analysed for this study
are available in Microbiology Online.
0002-6822 G 2004 SGM
motility, a unique mechanism of surface propulsion
(Mattick, 2002; Semmler et al., 1999). Bacteria lacking
TFP have been shown to be significantly less able to bind
to eukaryotic cells (Farinha et al., 1994; Paranchych et al.,
1986) and to be unable to form microcolonies on artificial
surfaces, necessary for initiating formation of a biofilm
(O’Toole & Kolter, 1998). TFP are produced by many
Gram-negative bacteria in addition to Pseudomonas,
including Neisseria, Moraxella, Dichelobacter, Eikenella
and Myxococcus (Strom & Lory, 1993). The pilin structural
subunits (products of the pilA gene) are produced as
prepilins which are cleaved near the N terminus by a specific
prepilin peptidase (PilD/XcpA in P. aeruginosa) and Nmethylated prior to assembly into the mature pilus fibre
(Lu et al., 1997; Strom et al., 1993).
Previous small studies of P. aeruginosa pilin diversity
(Castric, 1995; Castric & Deal, 1994; Spangenberg et al.,
1995) have demonstrated that the N-terminal amino acid
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1315
J. V. Kus and others
region of mature pilins is highly conserved. This region
of the pilin is thought to mediate interaction between
subunits, permitting self-assembly of the helical quaternary
structure of the pilus fibre (Keizer et al., 2001). In contrast,
the primary amino acid sequence of the remainder of
the pilin protein is less conserved, particularly at the C
terminus which contains a disulfide-bonded loop (DSL)
structure (Castric & Deal, 1994; Spangenberg et al., 1995).
Despite this sequence diversity, however, P. aeruginosa pili
are thought to interact with the eukaryotic glycolipid
receptor asialoGM1 (aGM1) (Bryan et al., 1998; Saiman &
Prince, 1993) via the DSL, which is proposed to be exposed
only at the pilus tip (Doig et al., 1988; Irvin et al., 1989).
Unlike members of the Neisseriaceae, which can generate
pilin diversity through recombination of the expressed
pilin gene locus (pilE) with additional silent pilin gene
cassettes (pilS) present in the genome (Gibbs et al., 1989),
P. aeruginosa possesses only a single pilA structural gene
(Stover et al., 2000). Therefore, pilin diversity in P. aeruginosa is more likely to arise through random mutations
within a strain or by recombination with horizontally
acquired pilin genes from other strains or species.
Castric & Deal (1994) divided P. aeruginosa pilin sequences
into two related groups based on DNA sequence analysis
of pilin genes from nine strains. Group I pilin genes are
highly conserved, have a higher G+C content (approx.
51 mol%) and are longer than group II genes (approx.
48 mol%). Strains with group II pilin genes include the
common laboratory strains PAO1, PA103 and PAK
(Johnson et al., 1986; Pasloske et al., 1985; Sastry et al.,
1985). Further analysis by Castric (1995) of the chromosome adjacent to the pilin structural gene showed that
group II pilin genes were immediately upstream of a
tRNAThr gene, while strains with group I pilin genes
contained an additional open reading frame (ORF), pilO,
between pilA and tRNAThr.
Another limited survey of 14 P. aeruginosa pilin gene
sequences (Spangenberg et al., 1995) identified, in addition
to group I and II pilins, a single unusual pilin sequence
from a CF isolate, G7, with only partial amino acid identity
(51 %) to the N-terminal 85 aa of group I pilins. The G7
pilA gene had a G+C content of 54?8 mol% and the
predicted amino acid sequence of the mature pilin was
larger (173 aa) than group I pilins. A recent representational difference analysis survey comparing the highly
virulent strain PA14 with PAO1 showed that the pilA gene
of PA14 was identical to that of G7 (Choi et al., 2002).
In addition to variations in pilin amino acid sequence, at
least one group I pilin, from strain 1244 (Castric, 1995),
has been shown to be post-translationally modified by
O-glycosylation on the C-terminal serine residue (Comer
et al., 2002). The glycosyl moiety was recently identified
as the O-antigen unit of the lipopolysaccharide of 1244,
which belongs to serotype O7 (Castric et al., 2001). The
carboxy terminus of the modified pilin was strongly
antigenic, eliciting antibodies that cross-reacted with the
1316
O antigen (Comer et al., 2002). A pilO knockout strain
continued to produce pili and to twitch, but its pilin had
altered migration on polyacrylamide and isoelectric focusing gels and lacked glycosylation by specific staining. PilO
appears to be the only protein required for pilin glycosylation, but the mechanism of PilO function and the
biological significance of pilin glycosylation in P. aeruginosa
are currently unknown.
The importance of TFP in the biology and virulence of
P. aeruginosa has made them attractive targets for vaccine
development (Hertle et al., 2001; Sheth et al., 1995). Saiman
et al. (1989) raised cross-reactive monoclonal antibodies
that recognized some pilin variants and reduced binding
to eukaryotic cells. However, to design a successful
P. aeruginosa vaccine based on TFP, it is important to be
aware of the scope of P. aeruginosa pilin diversity and the
prevalence of accessory genes such as pilO whose products
are capable of post-translationally modifying the pilin
subunit. In this study, we examined the pilin alleles of
a large panel of environmental, rectal, clinical and CF
isolates, and identified novel pilin and accessory genes.
These data provide the basis for a new system of
nomenclature for P. aeruginosa pilins. We show that the
distribution of pilin alleles amongst CF and non-CF
human isolates is sharply skewed, with the majority of
CF isolates (from both paediatric and adult populations)
belonging to pilin group I. This is the first example of
a P. aeruginosa virulence factor allele that is strongly
associated with isolates from CF patients.
METHODS
Bacterial strains and media. Patient-identity-blinded clinical and
rectal isolates of P. aeruginosa (Table I, supplementary data) were
prospectively collected from the microbiology laboratory at Mount
Sinai Hospital in Toronto, ON. Mount Sinai is a large tertiary
hospital which processes microbiology samples from several Greater
Toronto Area hospitals and long-term care facilities. Rectal colonizer
strains were isolated from routine rectal surveillance swabs (for resistant Enterobacteriaceae) plated on MacConkey agar supplemented
with 2 mg cefpodoxime ml21, to which P. aeruginosa is resistant.
Clinical isolates were obtained from various inpatient specimens
submitted from both sterile and non-sterile sites. CF isolates were
obtained with ethics approval from St Michael’s Hospital, Toronto,
ON, the Hospital for Sick Children, or as gifts from Dr David
Speert, University of British Columbia, and Dr John Govan,
University of Edinburgh. All CF strains were typed by PFGE to
eliminate clonal isolates. Environmental isolates were gifts from
Dr David Speert, Dr Stephen Lory of Harvard University, Dr Eric
Dezeil and Dr Richard Villemur of the INRS-Institut ArmandFrappier-Microbiologie et Biotechnologie, Dr Alan Hauser of
Northwestern University, and from Don Warburton of Health
Canada. All strains were maintained as glycerol stocks at 270 uC
after one passage on Luria–Bertani (LB) or Pseudomonas Isolation
agar. For cloning experiments, transformed Escherichia coli TOP10
(Invitrogen) were grown on LB agar supplemented with 50 mg
ampicillin ml21.
Twitching assay. Each strain was tested upon receipt for twitching motility by stabbing it with a sterile toothpick to the bottom
of a 3 mm thick 1 % LB agar plate (Semmler et al., 1999). After
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Microbiology 150
Restricted pilin diversity in CF isolates of P. aeruginosa
incubating overnight at 37 uC in a humidified chamber, the presence (or absence) of a hazy zone radiating from the point of inoculation at the agar–plate interface, indicating twitching motility, was
recorded. To improve visualization of twitching zones, agar was
removed from the plate and the twitching zones were stained with
crystal violet for 5 min then rinsed with tap water to remove
unbound dye.
PCR, cloning and DNA sequencing. PCR primers designed for
this study and used for amplification of pilA and adjacent sequences
from P. aeruginosa were: pilA (59-ATG AAA GCT CAA AAA GGC
TTT ACC TTG AT); pilB (59-TCC AGC AGC ATC TTG TTG ACG
AA); pilB2 (59-TGT TCA GGT CGC AAT AGG C); and tRNAThr
(59-CGA ATG AGC TGC TCT ACC GAC AGA GCT). Primers were
synthesized by ATGC, Toronto, ON. Chromosomal DNA templates
were prepared using Instagene (Bio-Rad) following the manufacturer’s instructions. Chromosomal DNA was isolated from mucoid
strains using a modified phenol/chloroform extraction procedure
(Goldberg & Ohman, 1984). The PCR mix consisted of 50 ml containing 1 ml template DNA, 3 ml 10 mM dNTPs, 50 pmol each
primer, 5 ml 106PCR buffer, 10 ml Q solution (Qiagen) and 0?5 ml
Hot StarTaq (Qiagen). PCR (using a Perkin Elmer 2400 thermocycler) consisted of a 15 min denaturation at 95 uC, followed by 30
cycles of 30 s at 95 uC, 45 s at 60 uC and 2 min at 72 uC, with a final
extension of 7 min at 72 uC, ending at 4 uC. Strains that were PCRnegative after the first round of amplification underwent a second
PCR amplification with a reduced annealing temperature of 55 uC
and an increased extension time of 2?5 min.
Amplification products of interest were either sequenced directly or
cloned into pCR2.1 TOPO (Invitrogen) following the manufacturer’s
instructions, transformed into chemically competent E. coli TOP10
cells and plated on LB agar plates containing 50 mg ampicillin ml21
(L+A) and 2 % X-Gal (Bioshop, Mississauga, ON). White colonies
were screened for plasmid inserts using a rapid technique (Eckhardt,
1978). Recombinant clones were grown overnight on L+A plates
and plasmids isolated using the Qiagen plasmid mini-prep kit as
indicated by the manufacturer. The DNA sequence of cloned
amplicons was obtained using the dye-deoxy terminator method by
ACGT, Toronto, ON. Inserts were sequenced bidirectionally using
M13 forward and reverse primers and subsequently with internal
primers based on the first-round sequence.
Identification of novel accessory genes. For identification of
strains containing tfpOa, tfpOb, tfpY and tfpZ accessory genes downstream of pilA, specific PCR assays were performed. Primers tfpO up
(59-CGT ACT ATT CTA TTA TTG CTG A) and tfpO down (59CAA AGG ATG GGC TAC GAA), tfpO2 up (59-CTG ATG CTG
TTT TCC TTC), tfpO2 down (59-GCA TCT CGC CAC AAC ACG),
tfpY up (59-AAA TAG CAG GAG GAT GGC A) and tfpY down (59CTC CCC ACT ATC CGC TCA A), and tfpZ up (59-AAT ACA
CGG TGG CAG TTG G) and tfpZ down (59-AAA GCG AAG AGG
ACG GGA A) were used. Strain 1244 (gift of Dr Peter Castric) was
used as a positive control for tfpO, Pa4944 as the tfpY positivecontrol strain and Pa811061 as the tfpZ positive-control strain.
PCRs were identical to those described above except the extension
time was reduced to 30 s. PCR-negative strains were considered to
contain novel accessory DNA, which was then cloned and sequenced.
RFLP typing of tfpOb strains. Strains that were negative by speci-
fic PCR using primers tfpO up and down (which amplifies tfpOa but
not tfpOb) but positive using primers tfpO2 up and down (above)
were recorded as tfpOb-positive. To further distinguish variations
between tfpOb-positive strains, their pilB-tRNAThr gene product was
cloned into pCR2.1 and transformed into TOP10 chemically competent E. coli. Plasmids were isolated using QIAprep Spin Miniprep
Kit following the manufacturer’s instructions (Qiagen) and 2 ml
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plasmid DNA was digested with EcoRI, TaqI or RsaI for 1 h and
separated on a 1 % agarose gel. Samples that yielded dissimilar
restriction-fragment length patterns were sequenced.
PFGE. The relatedness of strains was assessed by PFGE as described
previously (Lightfoot & Lam, 1993; Romling & Tummler, 2000).
Briefly, cells from overnight cultures grown on Mueller–Hinton
plates were washed and resuspended in SE buffer (25 mM EDTA
pH 7?4, 75 mM NaCl) to a density of 8 McFarland and embedded
in an equal volume of 1?6 % low-melting-point agarose (Bio-Rad).
Embedded cells were incubated at 37 uC for 2 h with gentle shaking
in lysis buffer (10 mM Tris/HCl pH 7?2, 50 mM NaCl, 100 mM
EDTA pH 8?0, 0?2 % deoxycholate, 1 % Sarkosyl, 2 mg lysozyme
ml21). After a 15 min wash at 37 uC in wash buffer (20 mM Tris/
HCl pH 8?0, 50 mM EDTA pH 8?0), embedded cells were incubated overnight at 50 uC in proteinase K solution (100 mM EDTA
pH 8?0, 0?2 % deoxycholate, 1 % Sarkosyl, 50 ml proteinase K ml21).
Embedded cells were then washed 3630 min in wash buffer
(20 mM Tris/HCl pH 8?0, 50 mM EDTA pH 8?0). Agarose slices
2–3 mm thick were incubated overnight with SpeI in 16restriction
enzyme buffer (as supplied by the manufacturer; New England
Biolabs) at 37 uC. DNA fragments were separated in a 1 % PFGEgrade agarose (Bio-Rad) gel with 0?56Tris borate/EDTA running
buffer supplemented with 50 mM thiourea (Romling & Tummler,
2000) using a Bio-Rad CHEF II apparatus with the following programme: initial time 5 s; final time 35 s; run time 20 h; ratio 1;
voltage 200 V; temperature 12 uC. The gel was post-stained with
2 mg ml21 ethidium bromide in double-distilled H2O for 30 min
and de-stained for 1 h in double-distilled H2O. The bands were
visualized with UV light and photographed. Banding patterns were
interpreted based on the classification system outlined by Tenover
et al. (1997). Isolates are considered subtypes (differing by one or
two genetic events) when they differ by more than one band and
fewer than five bands; samples are considered different strains when
they differ by more than six bands (Arbeit, 1995; Tenover et al.,
1997).
Analysis of pilin proteins by SDS-PAGE. Pilin proteins were
isolated using the methods of Castric (1995) with modifications.
Overnight lawns of bacteria on Davis minimal agar plates were
resuspended in 2 ml sterile PBS. Pili were sheared by vigorous
vortexing for 5 min and cells removed by centrifugation. Supernatant proteins were precipitated by the addition of 1 M MgCl2 to a
final concentration of 0?1 M and incubation at 4 uC overnight.
Precipitated proteins were harvested by centrifugation and resuspended in 2 % SDS sample buffer. Pilin preparations were boiled
for 5 min, separated on 15 % SDS-PAGE gels with a pre-stained
Benchmark Protein Ladder (Invitrogen), and visualized by staining
with Coomassie brilliant blue.
Phylogenetic analysis. Amino acid sequences of PilA variants
from representative strains from each TFP group and the closest
non-P. aeruginosa homologues (GenBank, http://www.ncbi.nlm.nih.
gov) were aligned in CLUSTAL_X (Thompson et al., 1997) using
default parameters. Phylogenetic trees were generated using PHYLIP
3.5 using default parameters and the Jones–Taylor–Thornton substitution matrix for amino acids (Felsenstein, 1989; Jones et al.,
1992). One thousand bootstrap replicates were performed to generate the final consensus tree which was visualized in TREEVIEW v.
1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). For comparison, and with similar results, trees were also generated with
MEGA2 (Kumar et al., 2001).
Statistical analysis. The statistical significance of differences in
pilin gene distribution between sources was calculated using the chisquare test with Yates’ correction for continuity (http://www.unc.
edu/~preacher/chisq/chisq.htm). Distributions were considered
significantly different at P¡0?05.
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J. V. Kus and others
Accession numbers. Novel sequences from each of pilin groups I,
II, IV and V described in this study have been deposited in GenBank
and have been assigned the following accession numbers: strain
Pa302025, AAM62144 (PilAIa) and AAM62145 (TfpOa); strain Pa5658,
AAM62146 (PilAIb) and AAM62147 (TfpOb); strain Pa131533,
AMM52057 (PilAIb) and AMM52058 (TfpOb); strain Pa5235,
AAM44066 (PilAII); strain Pa5196, AAM52059 (PilAIV), AAM52060
(TfpW) and AAM52061 (TfpX); strain Pa110594, AAM52055 (PilAV)
and AAM52056 (TfpZ). Sequences of group III pilins from strains
G7 and PA14 are available at accession numbers AAC41410 and
AAL12242, respectively.
RESULTS
Twitching motility in CF versus non-CF strains
Macroscopic twitching motility was observed in 23 of 24
(95?8 %) environmental isolates (Table I, supplementary
data). Over 71 % (113 of 159) of non-CF rectal and clinical
isolates exhibited twitching in the agar stab assay. Loss of
twitching motility in CF isolates from chronically colonized
patients, as well as other phenotypic alterations such as
loss of swimming motility and increased alginate production, has been reported previously (Garrett et al., 1999;
Mahenthiralingam et al., 1994; Penketh et al., 1983). We
saw a similar phenomenon in this study: while 35 of 43
(81?4 %) paediatric CF isolates demonstrated twitching,
only 36 of 66 (54?5 %) adult CF isolates were positive for
twitching motility.
PCR amplification analysis of pilA and flanking
sequences
To amplify both pilA and any downstream sequences, an
upstream primer located in the divergently transcribed,
conserved pilB gene was used in conjunction with a downstream primer corresponding to the tRNAThr gene located
39 to pilA in PAO1 (Fig. 1). All strains tested produced a
single PCR product ranging in size from approximately
1?4 kb to greater than 4 kb. Based on product size from
agarose gel analysis, 97 of 159 non-CF human isolates
(61?0 %), 17 of 24 environmental isolates (70?8 %), and
89 of 109 CF strains (81?7 %) contained DNA between
pilA and tRNAThr (Table I, supplementary data). The
remaining strains generated PCR products predicted by
size to contain pilA alone, without accessory DNA. Selected
products from a range of sizes were cloned into pCR2.1 and
sequenced, or sequenced directly.
Fig. 1. Five distinct pilin alleles in P. aeruginosa. Each pilin
allele and associated accessory gene(s) are located in a
common chromosomal locus, between the conserved pilB and
tRNAThr genes. PCR primers used to amplify this locus are
indicated by small arrows. Diagram is not to scale.
Paslocke et al. (1988b). All group II PilA sequences have a
12 aa C-terminal DSL region related to that of common
laboratory strains PAO1 and PAK (Fig. 2). Pilins in this
group have been designated PilAII.
Unlike the other group II strains sequenced, strain Pa5235
possessed a novel pilin gene with a G+C content of
51 mol%, encoding a 154 aa prepilin with a 12 aa predicted DSL (Fig. 2). The Pa5235 pilin was 55 % identical
to FimZ (minor pilin precursor, GenBank accession no.
YQBZDZ) of Dichelobacter nodosus serogroup D, the aetiological agent of ovine footrot, whose TFP are important
colonization and virulence factors (Kennan et al., 2001).
The pilin of P. aeruginosa strain KB7 (Campbell et al., 1995;
Wong et al., 1995) appears to be closely related to that of
Pa5235, based on a short partial sequence corresponding
to the DSL region available in the databases for comparison (GenBank accession no. Q53391). Analysis of the
distribution of pilAII in CF versus non-CF human strains
(clinical and rectal isolates) showed that this pilin allele
was significantly less likely (P=0?0005) to be present in CF
strains (18?3 % vs 37?7 %) (Table 1). This allele was present
in 29?2 % of environmental strains (Table 1).
Group II pilins
Strains predicted to contain pilA alone were assigned to
group II, as previously established by Castric & Deal (1994)
(Fig. 1). The predicted amino acid sequences of PilA
from two randomly selected group II strains (Pa260611,
Pa270176) were similar to P. aeruginosa strain K122-4
(Pasloske et al., 1988a). Both strains had an identical R43A
substitution compared with K122-4. Strains Pa281298,
Pa270958, Pa100443 and Pa100683 had pilins most closely
related to that of strain CD, a CF isolate identified by
1318
Group III pilins
Of strains predicted to contain both pilA and additional
DNA by amplification product size, several (Pa4494,
Pa5024, Pa5112, Pa5122, Pa5223 and Pa141123) encoded
pilins closely related to PilA of strain G7, a CF isolate
previously found by Spangenberg et al. (1995) to have an
unusually large pilin sequence, and strain PA14, a human
clinical strain (Choi et al., 2002) (Table I, supplementary
data). These strains were assigned to group III, and the
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Restricted pilin diversity in CF isolates of P. aeruginosa
Fig. 2. Amino acid sequence alignment of
the pilins’ C-terminal DSL regions. Strains
sequenced for this study are underlined.
PCR and sequencing revealed two subgroups of group I: Ia and Ib. Invariant hydrophobic (F/Y) and proline residues are
bolded. Asterisks indicate residues conserved in each group. The length of the
region between the cysteine residues is indicated to the right.
pilins designated PilAIII. The PilAIII proteins from these
strains were 95–100 % identical to the G7 pilin (Table I,
Fig. 2). Immediately downstream of the pilin gene was an
ORF that we named tfpY (type four pilin gene Y), which
corresponds to the previously reported gene called ORF1
(Spangenberg et al., 1995). The protein encoded by tfpY
is essentially identical in all strains carrying PilAIII (not
shown) and is most homologous to FimB, a pilin accessory
protein of unknown function from D. nodosus (Kennan
et al., 2001). PCR with tfpY-specific primers showed that
41 of 159 human non-CF isolates (25?8 %) and three of
24 environmental strains (12?5 %) contained tfpY, while
only nine of 109 CF strains (8?2 %) contained this gene
(Table 1).
Table 1. Distribution of pilin alleles among isolates from various sources
Pilin allele
Accessory gene(s)
Number of strains (n=292)
Clinical (n=95)
Rectal (n=64)
Clinical+rectal (n=159)
Environmental (n=24)
CF adult (n=66)*
CF paediatric (n=43)D
CF total (n=109)
Clinical vs rectal P value
Clinical+rectal vs environmental P value
Clinical+rectal vs CF total P value
CF total vs environmental P value
Adult CF vs paediatric CF P value
I
tfpO
(46?9 %)
(30?5 %)
(28?1 %)
(29?6 %)
(58?3 %)
(71?2 %)
(67?4 %)
(69?7 %)
0?882
0?011d
<0?0001
0?401
0?838
137
29
18
47
14
47
29
76
II
89
36
26
62
7
13
7
20
None
(30?5 %)
(37?9 %)
(40?6 %)
(39?0 %)
(29?2 %)
(19?7 %)
(16?3 %)
(18?3 %)
0?856
0?484
0?0005
0?361
0?843
III
53
26
15
41
3
4
5
9
tfpY
(18?2 %)
(27?4 %)
(23?4 %)
(25?8 %)
(12?5 %)
(6?1 %)
(11?6 %)
(8?2 %)
0?710
0?244
0?0005
0?793
0?499
IV
1
0
1
1
0
0
0
0
tfpWX
(0?3 %)
(0?0 %)
(1?6 %)
(0?6 %)
(0?0 %)
(0?0 %)
(0?0 %)
(0?0 %)
ND
V
12
4
4
8
0
2
2
4
tfpZ
(4?1 %)
(4?2 %)
(6?3 %)
(5?0 %)
(0?0 %)
(3?0 %)
(4?6 %)
(3?7 %)
ND
ND
ND
ND
0?470
ND
ND
ND
ND
ND, Not done; numbers in these groups were too small for meaningful statistics.
*Combined adult CF strains from Toronto and Vancouver; there were no significant differences between these two groups.
DCombined paediatric CF strains from Toronto and Edinburgh; there were no significant differences between these two groups.
dNumbers in bold are statistically significant.
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Group IV pilin
Group IV currently contains a single isolate, Pa5196. The
pilin gene of strain Pa5196 was novel, with a G+C content
of 54?8 mol%, encoding a 155 aa prepilin protein with a
23 aa DSL (Fig. 2). The closest homologue of the Pa5196
pilin (40 % identical, 55 % similar) was PilE, a Neisseria
meningitidis fimbrial protein precursor (GenBank accession
no. S55496). Immediately downstream of pilA in Pa5196
were two novel ORFs. The first, tfpW, encodes a large
hydrophobic hypothetical transmembrane protein, while
the second, tfpX, encodes a putative pilin accessory protein most similar to TfpZ (below; 33 % identical, 58 %
similar) and to PilB and FimB of Eikenella corrodens and
D. nodosus, respectively. E. corrodens is a member of the
HACEK group of bacteria (Haemophilus, Actinobacillus,
Cardiobacterium, Eikenella, Kingella) which cause various
human infections including infectious endocarditis.
Group V pilins
Strains Pa271457, Pa081061 and Pa110594 had novel pilA
genes with a G+C content of 52?3–52?5 mol%. The mature
pilins are one residue shorter than that of G7, with a
predicted DSL of 29 aa (Fig. 2). These pilins were assigned
to group V. A database search using BLASTP (Altschul et al.,
1997) showed the prepilins were 53–54 % identical (67–
68 % similar) to the G7 prepilin, and the next closest
homologues were TFP-like proteins from the plant pathogen Ralstonia solanacearum (GenBank accession no.
NP_518679). Adjacent to the pilA gene in each of these
strains was a novel accessory gene tfpZ, which encodes a
putative pilin accessory protein that is most homologous
(31 % identity, 53 % similarity) to the PilB protein of
E. corrodens strain VA1 (Villar et al., 1999). PCR with
tfpZ-specific primers showed that eight of 159 non-CF
human samples (5?0 %) and four of 109 CF strains (3?7 %)
contained tfpZ DNA. These distributions are not significantly different, but the numbers in each group are low.
The group V CF isolates were distributed equally among
Toronto (one patient), Vancouver (one patient) and
Edinburgh (two patients). This rare allele was not detected
in the environmental samples tested.
Because this pilin type had not been reported previously,
and we identified a number of strains with this unusual
allele, strain typing by PFGE was performed to determine
if the strains were clonal. PFGE of SpeI-digested chromosomal DNA showed that two of 13 group V strains were
identical (Pa110594, Pa290177) and two others were closely
related to these (Pa281457, Pa5517) (Fig. 3). Epidemiological analysis showed that identical strains Pa110594 and
Pa290177 were collected from endotracheal tubes from
different individuals in the same hospital, but the samples
were collected over a month apart, and patients were in
separate rooms. The closely related strains (Pa281457,
Pa5517) were from two other area hospitals. The remaining
tfpZ-bearing strains, including the four CF isolates, were
unrelated.
1320
Fig. 3. PFGE typing of group V strains. The relatedness of
strains bearing group V pilins was assessed using PFGE of
SpeI-digested chromosomal DNA, shown as a negative image
of the ethidium bromide-stained gel. Strains Pa110594 and
Pa290177 are identical (bold and underlined) and Pa281457
and Pa5517 (underlined) are closely related (Tenover et al.,
1997). The remaining strains are considered unrelated.
Group I pilin genes and their predominance in
CF isolates
Forty-seven of 159 (29?6 %) human non-CF isolates, 14 of
24 (58?3 %) environmental strains and 76 of 109 (69?7 %)
CF strains yielded PCR products of approximately 3 kb,
the predicted size for pilAI plus the glycosylation gene pilO.
Of note, the P. aeruginosa PAO1 genome sequence (Stover
et al., 2000) contains a completely unrelated gene also
named ‘pilO’ (ORF PA5042), which is not involved in
pilin glycosylation. Therefore, to reduce future confusion,
and to harmonize the nomenclature with that of the other
pilA accessory genes described above, we have renamed the
glycosylation gene pilO (adjacent to pilA) as tfpO.
PCR with primers based on the published sequence of
tfpO yielded amplification products for approximately half
these strains, 28 of 159 human non-CF strains (17?6 %),
seven of 24 environmental strains (29?2 %) and 45 of 109
CF strains (41?3 %). The pilAI and tfpO genes from the
PCR-positive strains were highly homologous to those of
the prototype strain 1244 (not shown). The original 3 kb
PCR products from the remaining strains were digested
with EcoRI, TaqI and RsaI to distinguish RFLPs. Three
different RFLP patterns were obtained (not shown) and a
representative product from each pattern was sequenced.
All three strains encoded PilAI proteins with 70–74 % amino
acid identity to PilAI from tfpO-containing strain 1244.
These strains contained tfpO homologues encoding proteins with 87–92 % identity with the 1244 TfpO (PilO)
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Microbiology 150
Restricted pilin diversity in CF isolates of P. aeruginosa
protein. Sequence differences occurred mainly in the 59
region of the tfpO gene, proximal to pilAI. Based on the
differences in the pilin and accessory genes, the group I
pilin-containing strains could be divided into subgroups:
TfpOa (Ia) strains, including the original strain 1244, and
TfpOb (Ib) strains [Tables I (supplementary data) and 1]. All
group Ia and Ib pilins sequenced in this study contained
the terminal serine residue recently shown to be the site of
glycosylation in strain 1244 (Comer et al., 2002).
Analysis of novel pilin proteins
The potential role of TfpOb and other novel accessory
proteins in pilin modification was examined by separation
of sheared pilin preparations from representative strains
on SDS-PAGE. In strain 1244, glycosylation by TfpOa of
the C-terminal serine residue of PilAI with a lipopolysaccharide O-antigen unit was previously shown to slow pilin
migration on SDS-PAGE (Comer et al., 2002). Pilins from
strains PAK (group II), 1244 (group I) and a tfpOa : : GmR
insertion mutant of strain 1244 were used as controls
(Fig. 4a). TfpOb strain Pa131533 pilin migrated more
slowly than its predicted mass of 15?6 kDa, similar to the
glycosylated 1244 pilin (Fig. 4a). Group I pilin proteins
from other strains also showed reduced migration (not
shown). These results show that strains carrying either
version of TfpO are capable of producing modified pilins.
Pilins from strains carrying tfpY or tfpZ migrated more
rapidly than their predicted sizes of 17?4 and 17?7 kDa,
Fig. 4. SDS-PAGE of sheared pilins. Sheared pilin preparations were separated on a 15 % SDS-PAGE gel and stained
with Coomassie blue. Pilins from strains Pa100683 and
Pa131533 (group Ia and Ib) and Pa5196 (group IV) migrate
more slowly than their predicted molecular mass, similar to
those of strain 1244, suggesting that they may be post-translationally modified. When glycosylation of the 1244 pilin is abrogated by a mutation of tfpO, the migration of the pilin is
increased (fourth lane). Low-molecular-mass markers are shown
on the left.
http://mic.sgmjournals.org
respectively (Fig. 4a), suggesting that they may be processed differently from other pilins, generating a shorter
form, or post-translationally modified. These possibilities
are currently under investigation.
Interestingly, the pilin from group IV strain Pa5196, which
contains tfpW–tfpX downstream of pilAIV, migrated more
slowly than predicted by its molecular mass (Fig. 4b),
similar to the glycosylated form of group I pilin. The pilin
from Pa5196 does not contain the terminal serine residue
that is glycosylated in group I pilins, suggesting that posttranslational modification, if present, is likely to be at a
position different from that of PilAI. The pilin of this
strain is related to those of Neisseria (see below), which
are modified on internal serine residues (Forest et al., 1999;
Marceau & Nassif, 1999). Based on protein structure and
hydropathy analyses (Fig. 5), TfpW is predicted to have
multiple transmembrane domains, similar to TfpO (Castric,
1995), although there is no primary amino acid sequence
homology between the proteins. We are in the process of
generating Pa5196 mutants lacking TfpW to demonstrate
whether this protein is involved in modification of the
pilin.
Phylogenetic analysis of PilA proteins from
P. aeruginosa confirms that there are five
distinct groups
Representative amino acid sequences from each pilin group
(I–V) and homologous proteins with the highest degree
of similarity (based on BLASTP analysis) from species
other than P. aeruginosa were aligned using CLUSTAL_X
(Thompson et al., 1997). Phylogenetic trees were generated
with PHYLIP 3.5 using the neighbour-joining method
(Fig. 6). The pilins clustered unequivocally into the same
groups to which they were previously assigned based on
the length of the C-terminal DSL and the presence (or
absence) and identity of characteristic accessory genes.
High bootstrap confidence values confirm the clades within
the tree are statistically significant. Use of only the highly
conserved N-terminal regions of the proteins generated
similar trees (not shown), demonstrating that the clades
are not reflective only of sequence differences in the
divergent C termini of the proteins.
Of interest, Pa5196 PilAIV groups more closely with E.
corrodens PilA1, Neisseria gonorrhoeae PilE and N. meningitidis PilE than other P. aeruginosa pilins within this data
set. PilAII from the anomalous group II strain Pa5325
groups more closely with D. nodosus FimZ than other
P. aeruginosa PilAII proteins, but its relationship to P.
aeruginosa group II pilins is still statistically significant
(bootstrap confidence value of 78 %).
DISCUSSION
P. aeruginosa is an opportunistic pathogen that lives in a
broad range of environments and infects hosts across the
evolutionary spectrum. Common virulence factors have
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1321
J. V. Kus and others
Fig. 5. Kyte–Doolittle hydropathy plots of
TfpO and TfpW proteins. The TfpOa and
TfpOb proteins show almost identical hydropathy plots with some variation at the N and
C termini. Although TfpW has no sequence
similarity to TfpO, its hydropathy plot has
similar predicted transmembrane domains.
The plots were generated in GENE RUNNER
3.00 (Hastings Software) using the predicted amino acid sequences. The scales
are equivalent for comparisons and positions
of the amino acid residues are shown on
the x-axis.
been shown to be required for pathogenesis in various
hosts ranging from amoebae, yeast and nematodes through
plants and mammals (Cosson et al., 2002; D’Argenio et al.,
2001; Hogan & Kolter, 2002; Pukatzki et al., 2002; Yorgey
et al., 2001). Also, no obvious differences have been detected
with respect to pathogenicity between clinical and environmental strains (Alonso et al., 1999), suggesting any strain
is equally capable of colonization or infection given the
appropriate circumstances. Because P. aeruginosa virulence
factors, including TFP, have been well studied, we were
surprised when our initial analysis of pilin diversity in
non-CF P. aeruginosa clinical isolates revealed previously
unrecognized pilin types and accessory genes. To explain
this observation, we hypothesized that CF isolates, upon
which many P. aeruginosa researchers have traditionally
focused, may carry a restricted range of pilin types, leading
to an underestimation of the diversity of pilins in this
species. We then analysed the pilin types of a large set of
CF strains to test this supposition.
Comparison of the distribution of pilin alleles between
CF and non-CF human isolates supports our hypothesis.
Group I pilins, which can be modified by glycosylation
through the activity of TfpO, were identified in 69?7 % of
genetically distinct CF isolates but only 29?6 % of non-CF
human isolates, a highly significant difference (P<0?0001;
Table 1). This distribution pattern was apparent regardless
of whether CF strains were collected in Toronto, Vancouver
1322
or Edinburgh CF clinics (P=0?298), showing the high
prevalence of group I TFP in the CF population is not due
to a regional bias. Interestingly, 29 of 43 paediatric CF
isolates (67?4 %) belong to pilin group I (Table 1), similar
to adult CF isolates (71?2 %; P=0?838), demonstrating
that the bias in pilin type is already evident in patients
early in the progression of CF disease. Correspondingly,
group II and III pilins were more likely to be present in
non-CF human strains compared with CF isolates (P=
0?0005 and P=0?0005, respectively). The remaining types
are currently too rare for meaningful analyses.
Whether strains carrying group I pilins are better at
colonizing the unique CF lung environment than strains
with other pilin types, or have associated characteristics
that make them more likely to persist in the CF lung,
remains to be determined experimentally. It is intriguing
that the percentage of group I pilins within the environmental strains tested for this study was similar to that
within CF isolates (58?3 % vs 69?7 %, P=0?401). The
environment has been suggested to be the source of
P. aeruginosa in CF patients (Speert et al., 2002), and the
similarity in pilin distribution supports this theory. In
contrast, other human isolates, whether they are rectal
colonizing strains or clinical isolates, appear to have
approximately equal distributions of strains within pilin
groups I, II and III. This remarkably parallel distribution
pattern may imply that opportunistic, non-CF P. aeruginosa
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Restricted pilin diversity in CF isolates of P. aeruginosa
Fig. 6. Phylogenetic relationships between PilA and related proteins. A neighbour-joining tree was produced in PHYLIP 3.5
using the Jones–Taylor–Thornton matrix for amino acid substitutions and visualized using MEGA2.1. The pilin groups that were
originally generated based on presence of accessory gene(s) and length of the C-terminal DSL sequence are strongly
supported by this analysis. Bootstrap replicates are indicated on branches as percentages, and indicate confidence for the
relationship. GenBank accession numbers are indicated and strains from this study are underlined. Pa, P. aeruginosa (bolded);
Mb, Mycobacterium bovis; Dn, D. nodosus; Nm, N. meningitidis; Ng, N. gonorrhoeae; Ec, E. corrodens; Rs, R. solanacearum.
infections, which are often associated with use of medical
devices, may arise from the patient’s own transient flora. We
showed previously (Gardam et al., 2002) that even severely
immunocompromised transplant patients are most frequently infected by their own rectal flora.
It is interesting that strains capable of expressing the type
of pilin that can be glycosylated are most prevalent in CF
patients. The role of pilin glycosylation in host–bacterium
interactions is currently unknown. Castric (1995) showed
that inactivation of tfpO (pilO) had no apparent functional
consequences with respect to pilus expression and twitching
motility, but did not determine the effect of the mutation
on adherence to host cells. The position of the glycosyl
moiety at the pilin’s C terminus, immediately adjacent to
the putative receptor-binding domain, was suggested to
play a role either in modulating cell–cell binding or in
protecting the adhesive portion of the pilus from proteolytic
degradation (Comer et al., 2002). Further investigation is
necessary to determine whether pilin glycosylation confers
a colonization advantage in the CF lung.
http://mic.sgmjournals.org
While all strains tested possessed the pilin structural gene,
some strains lacked twitching motility by the agar stab
assay. Biosynthesis, function and regulation of TFP remain
complex and poorly defined processes involving more than
50 different genes, including some of unknown function
(Jacobs et al., 2003; Mattick, 2002). Therefore, strains that
lack macroscopic twitching motility may still produce
adhesive pili but be unable to twitch due to defects in pilus
motor function or chemotaxis, or may lack pili altogether
due to mutations in a variety of genes. In two cases (strains
Pa5226 and Pa5289), we found that the pilin locus was
disrupted with different insertion sequence elements
(Table I, supplementary data). In general, strains isolated
from patients at a single time point may represent nontwitching derivatives of a colonizing strain that originally
had twitching motility. Loss of twitching motility has
previously been reported in serial isolates of strains from
chronically colonized CF patients (Mahenthiralingam et al.,
1994). This observation is supported by differences in twitching motility seen in our paediatric versus adult CF isolates
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1323
J. V. Kus and others
(81?4 % vs 54?5 %; P=0?008), even though both populations are colonized predominantly with group I strains.
Variation at the pilA locus in P. aeruginosa is interesting
from the perspective of bacterial evolution. Since P. aeruginosa contains only a single pilin locus, it must either
undergo mutations within the existing pilA gene or acquire
foreign pilA genes through horizontal genetic transfer to
generate novel pilin types. Both scenarios were evident in
this study. We identified five distinct groups of pilins in
P. aeruginosa, including some with homology to pilins
from other mammalian (D. nodosus and E. corrodens)
(Kennan et al., 2001; Villar et al., 2001) or plant (R. solanacearum, Xanthomonas campestris) (da Silva et al., 2002;
Liu et al., 2001) pathogens, suggesting possible horizontal
transfer from organisms sharing common environments.
There was also evidence of sequence variation within pilin
groups, ranging from single to multiple amino acid changes
(Fig. 2). Notably, each pilin type is stringently associated
with specific accessory gene(s), i.e. pilAIII is always found
with tfpY and never with the group V gene tfpZ. This
invariant relationship implies that the pilin and its accessory gene(s) were acquired together, and allows the diagnostic use of accessory-gene-specific PCR to determine the
pilin type of the strain.
The presence immediately adjacent to the pilA locus of a
tRNAThr gene suggests that bacteriophage-mediated transduction may be involved in generation of pilin diversity in
P. aeruginosa, since tRNA genes represent preferred sites
for bacteriophage integration (Reiter et al., 1989). Recent
DNA sequence analysis of the highly infectious P. aeruginosa bacteriophage phiKZ showed it carries a tRNAThr gene
in its genome (Mesyanzhinov et al., 2002). Despite linkage
of TFP to genetic competence in other species, P. aeruginosa
has not been found to be competent for natural transformation, making transduction a more likely mechanism of pilin
variation in this species.
Among the most striking and significant amino acid
sequence variations we noted in this study were differences
in the pilin C-terminal DSL region (Fig. 2) that has been
implicated as the binding domain for the eukaryotic
glycolipid asialoGM1 (aGM1). Previous studies of receptor
binding using peptide mimics of the C-terminal DSLs of
group II pilins from strains PAK, PAO1 and KB7 and the
group I pilin from strain P1 showed that all peptides
bound to aGM1 despite differences in amino acid composition (Campbell et al., 1997). In contrast, a recent study
(Schroeder et al., 2001) using whole, piliated cells showed
that a number of clinical isolates (of undefined pilin type),
as well as laboratory strains including PAO1 and PAK, did
not demonstrate differences in binding to host cells in the
presence or absence of exogenously added aGM1 receptor.
Therefore, the effect on host cell adhesion of the major
differences in the length and amino acid sequence of the
putative receptor binding domain in these novel pilins, and
the effect of their presentation as peptides compared to
intact pili or on whole bacterial cells, require further
1324
studies. Similarly, the possibility that there may be other
potential host receptor(s) for these novel pilins requires
investigation.
In conclusion, our analyses of the type IV pilin gene locus in
P. aeruginosa have led to the identification of novel pilins
and accessory genes, expanding the known diversity of
type IV pilins within this species and providing the basis
for a new system of pilin nomenclature. This information
will be useful for the design of comprehensive pilin-based
vaccines. Our intriguing finding that the preponderance of
CF isolates have group I pilin genes is, to our knowledge,
the first example of a P. aeruginosa virulence factor allele
that is strongly linked with CF, and will be the focus of
future research.
ACKNOWLEDGEMENTS
We thank the many generous individuals listed in Methods for
providing strains for this study. We also thank Dr Dave Guttman, John
Stavrinides, Dr J.-M. Moncalvo, and the Bioinfomatics groups at HSC
for helpful discussions about phylogenetics. Winston Wong and
Raynah Fernandes provided excellent technical assistance. This work
was supported by grant MOP-49577 from the Canadian Institutes of
Health Research to L. L. B. J. V. K. is a Harron Scholar and a CIHR
Strategic Training Fellow in Cell Signalling in Mucosal Inflammation
and Pain (STP-53877).
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