Neutrophil Bleaching of GFP-Expressing
Staphylococci: Probing the Intraphagosomal
Fate of Individual Bacteria
This information is current as
of June 16, 2017.
Jamie Schwartz, Kevin G. Leidal, Jon K. Femling, Jerrold P.
Weiss and William M. Nauseef
J Immunol 2009; 183:2632-2641; Prepublished online 20
July 2009;
doi: 10.4049/jimmunol.0804110
http://www.jimmunol.org/content/183/4/2632
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References
The Journal of Immunology
Neutrophil Bleaching of GFP-Expressing Staphylococci:
Probing the Intraphagosomal Fate of Individual Bacteria1
Jamie Schwartz,* Kevin G. Leidal,* Jon K. Femling,*†‡ Jerrold P. Weiss,*‡
and William M. Nauseef2*‡
P
olymorphonuclear leukocytes (PMN)3 provide an important component of innate host defense against microbes
and are especially important in response to pyogenic bacterial infections, such as those caused by Staphylococcus aureus.
Beginning with phagocytosis, antimicrobial responses of PMN are
tightly coupled with activation of the phagocyte NADPH oxidase
and release of granule contents into the nascent phagosome (1).
Oxidants, generated de novo by the NADPH oxidase under aerobic
conditions, exert direct toxic effects on microbial targets, act as
substrates to support generation of other reactive species, and promote and modify the intrinsic activity of other agents present in the
phagosome. The fusion of preformed granules with the phagosome
delivers a wide array of biologically active agents that likewise
*Inflammation Program, Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Administration Medical Center,
Iowa City, IA 52240; †Medical Scientist Training Program, Roy J. and Lucille A.
Carver College of Medicine, University of Iowa, Iowa City, IA 52240; and ‡Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University
of Iowa, Iowa City, IA 52240
Received for publication December 8, 2008. Accepted for publication June 7, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The work was supported by National Institutes of Health Grants AI 18571 (to
J.P.W.) and AI 070958 (to W.M.N.), by a Merit Review Grant (to W.M.N.) from the
Veterans Administration, and by facilities and resources at the Veterans Administration in Iowa City, IA (to W.M.N.).
2
Address correspondence and reprint requests to Dr. William M. Nauseef, Inflammation Program and Department of Medicine, Roy J. and Lucille A. Carver College
of Medicine, University of Iowa, D160 MTF, 2501 Crosspark Road, Coralville, IA
52241. E-mail address: [email protected]
3
Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; HOCl, hypochlorous acid; MPO, myeloperoxidase; SA, Staphylococcus aureus; CFU, colony
forming unit; DPBS, Dulbecco’s PBS; TSB, tryptic soy broth; DPI, diphenylene
iodonium; MPO-D, MPO-deficient patient; GGO, glucose-glucose oxidase; MOI,
multiplicity of infection.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804110
exert both direct and indirect effects on the ingested microbe. As
such, the phagosome provides a specialized microenvironment
where many different cytotoxic compounds operate alone and synergistically to equip the PMN to respond to many different types of
microbes (2). The complex synergy in the phagosome is illustrated
by the capacity of the granule protein myeloperoxidase (MPO) in
the presence of H2O2 to catalyze the oxidation of chloride and
generate hypochlorous acid (HOCl), a potent microbicidal agent
(reviewed in Ref. 3).
Ironically, the same chemical and biochemical complexity that invests the PMN intraphagosomal microenvironment with such broad
and potent antimicrobial cytotoxicity has often created a challenge to
the identification of specific contributions of individual components to
antibacterial action. Where the principal indicator of antimicrobial
action is bacterial death, quantitated as diminished colony forming
units (CFU), the difficulty in distinguishing the contribution of specific components is especially difficult. Even in the case of PMN-S.
aureus interactions, where a functioning phagocyte NADPH oxidase
is prerequisite for optimal bacterial killing, debate persists as to the
nature of the potentiating effects of the oxidase and the specific nonoxidase components in the phagosome that contribute synergistically
to amplify bacterial damage.
In this report, we take advantage of the specific and selective
ability of the MPO-H2O2-chloride system to bleach GFP (4),
which can be expressed within the cytosol of a wide variety of
bacterial species, to study the fate of S. aureus in normal human
PMN. Considered in the context of clinical studies of interactions
between PMN and S. aureus that indicate that some ingested bacteria survive (5–7), the assessment of GFP bleaching during and
after phagocytosis directly demonstrates differences in the effects
of PMN cytotoxins on individual cocci under a variety of conditions. These studies provide novel insights into host and bacterial
factors that contribute to the fate of ingested staphylococci within
human PMN.
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Successful host defense against bacteria such as Staphylococcus aureus (SA) depends on a prompt response by circulating polymorphonuclear leukocytes (PMN). Stimulated PMN create in their phagosomes an environment inhospitable to most ingested
bacteria. Granules that fuse with the phagosome deliver an array of catalytic and noncatalytic antimicrobial peptides, while
activation of the NADPH oxidase at the phagosomal membrane generates reactive oxygen species within the phagosome, including
hypochlorous acid (HOCl), formed by the oxidation of chloride by the granule protein myeloperoxidase in the presence of H2O2.
In this study, we used SA-expressing cytosolic GFP to provide a novel probe of the fate of SA in human PMN. PMN bleaching of
GFP in SA required phagocytosis, active myeloperoxidase, H2O2 from the NADPH oxidase, and chloride. Not all ingested SA were
bleached, and the number of cocci within PMN-retaining fluorescent GFP closely correlated with the number of viable bacteria
remaining intracellularly. The percent of intracellular fluorescent and viable SA increased at higher multiplicity of infection and
when SA presented to PMN had been harvested from the stationary phase of growth. These studies demonstrate that the loss of
GFP fluorescence in ingested SA provides a sensitive experimental probe for monitoring biochemical events within individual
phagosomes and for identifying subpopulations of SA that resist intracellular PMN cytotoxicity. Defining the molecular basis of
SA survival within PMN should provide important insights into bacterial and host properties that limit PMN antistaphylococcal
action and thus contribute to the pathogenesis of staphylococcal infection. The Journal of Immunology, 2009, 183: 2632–2641.
The Journal of Immunology
Materials and Methods
2633
Staphylococcal culture
Bacterial killing
We used S. aureus ALC 1435, a derivative of RN6390 containing the
plasmid pALC1420 encoding GFP whose constitutive expression is controlled through the sar P1 promoter (9). S. aureus ALC 1435 was provided
by Dr. Ambrose Cheung, Department of Microbiology, Dartmouth Medical
School Hanover, NH.
Unless otherwise stated, we used mid-logarithmic growth staphylococci
in our studies. Bacteria were grown overnight at 37°C shaking at 180 rpm
in TSB supplemented with 10 ␮g/ml chloramphenicol to maintain the plasmid. Before use in the assay, the overnight culture was diluted to a density
of 0.05 at an OD550 in fresh TSB medium containing 0.01% HSA and 10
␮g/ml chloramphenicol. The bacteria were subsequently subcultured 2.5 h
to reach mid-logarithmic phase (OD550 ⬃0.1– 0.2) before harvesting by
centrifugation at 5,000 ⫻ g for 5 min in a tabletop microcentrifuge, and
resuspending in 20 mM HEPES buffered HBSS with divalent cations.
The collected cells were then used immediately or placed on ice until
opsonization.
In selected experiments, indicated in the text, staphylococci were serially subcultured to obtain a population of bacteria that was more uniformly
in the early exponential phase, using a modification of a previously described method (10). Initially, staphylococci in tryptic soy broth supplemented with 0.01% HSA and 10 ␮g/ml chloramphenicol were cultured at
37°C shaking at 180rpm for 7 h. Bacteria were pelleted (2,000⫻ g for 4
min at 4°C in Beckman Coulter J6-MI), washed twice with vortexing in
sterile endotoxin-free saline, and stored overnight on ice at 4°C in TSB
supplemented with 0.01% HSA and 10 ␮g/ml chloramphenicol. The following day, the stored bacteria were inoculated into fresh broth supplemented as above and the subculturing process was repeated three times
(once for 3 h, then twice for 2 h each), with each subculture starting with
a 1/100 dilution of the previous culture. After the final 2-h subculture, the
pelleted bacteria were washed twice in saline and resuspended in TSB
supplemented with 0.01% HSA, 10 ␮g/ml chloramphenicol, and 10% glycerol for storage at ⫺80°C. Either freshly prepared or thawed stored samples
were washed in DPBS, resuspended in 20 mM HEPES buffered HBSS with
divalent cations, and held on ice until opsonization.
After the 5 min incubation and resuspension (see above), aliquots were
removed to quantitate bacterial viability at the experimentally designated
zero time. Each sample was diluted 1/50 into a standard 96-well plate
containing 1% saponin prepared in H2O, incubated at room temperature for
15 min, mixed, and serially diluted with 0.9% saline. Several dilutions of
each sample were plated drop-wise on TSB agar and placed overnight at
37°C for enumeration of colonies. The CFU averaged from replicate plates
were calculated and data represented as a percent of viable organisms at
zero time.
Clinical grade dextran T500 (m.w. 500,000 daltons) was purchased from
Pharmacosmos A/S. Sterile endotoxin-free H2O and 0.9% sterile endotoxin-free sodium chloride for patient use were purchased from Baxter Healthcare. Ficoll-Paque PLUS was purchased from GE Healthcare (formally
Amersham Biosciences); HEPES, HBSS, and Dulbecco’s PBS (DPBS), with
or without divalent cations were obtained from Mediatech. PROTOCOL
HEMA-3 staining kit was purchased from Fisher Diagnostics, whereas human
serum albumin 25% was obtained from Talecris Biotherapeutics. Tryptic soy
broth (TSB) and bacto agar were purchased from BD Biosciences. Chloramphenicol, diphenylene iodonium (DPI), poly-L-lysine coated Poly-Prep slides,
and sodium gluconate were obtained from Sigma-Aldrich. Na125I (specific
activity 17.4 Ci/mg) was purchased from PerkinElmer, whereas unlabelled NaI
was purchased from Mallinckrodt. Saponin and glucose oxidase were obtained
from Fluka Biochemika. Affinity-purified rabbit polyclonal Ab against GFP
was purchased from Eusera. All other reagents were purchased from Fisher
Scientific.
Neutrophil isolation
Phagocytosis
Bacteria were suspended at 107/ml in 20 mM HEPES-buffered HBSS with
divalent cations, 1% HSA, and 10% pooled human serum and placed in a
37°C shaking water bath for 20 min to opsonize before being fed to PMN.
After opsonization, the bacteria were pelleted (2,000 ⫻ g, 5 min), washed,
and immediately used in phagocytosis assays (see below).
Before challenge with S. aureus, PMN were diluted to 107/ml and incubated at 37°C for 10 min with or without the addition of 10 ␮M DPI, or
0.5, 1, 2, or 5 mM NaN3 in HBSS containing divalent cations, 1% HSA,
and 10% pooled human serum. Subsequently, the opsonized S. aureus and
Fluorescence microscopy
At each time point, samples were vortexed, and ⬃7.5 ⫻ 104 PMN containing S. aureus were spun onto primed (20 mM HEPES buffered HBSS
supplemented with 0.2% HSA) poly-L-lysine coated poly-prep slides using
the cytocentrifuge (Shandon Cytospin 2; Shandon Southern Products) at
18 ⫻ g for 10 min. Each slide was fixed in 10% formalin for 15 min at
room temperature, washed once in DPBS, and covered with a coverslip
using mounting medium to preserve fluorescence. A Zeiss Axioplan 2 photomicroscope was used to analyze cocci:PMN and to visualize loss of GFP
fluorescence. For each sample, 50 –100 PMN were examined. Experiments
examining the effect of chloride concentration on loss of GFP fluorescence
by ingested staphylococci used a chloride-free buffer to which specified
amounts of a 122 mM chloride buffer (with identical cations) were added
to achieve the desired final concentration of chloride. The chloride-free
buffer had chloride salts in 10 mM PBS (pH 7.4) replaced with gluconate
salts.
Spectrofluorometric monitoring of bleaching of GFP-SA by
oxidants
GFP-SA (5 ⫻ 107/ml) were suspended in a 3 ml cuvette and scanned
(␭ex ⫽ 395 nm, ␭em ⫽ 480 – 540 nm) in a thermostated 37°C spectrofluorometer (Fluoromax 3, Jobin Yvon). Scanning was then performed immediately after each aliquot of H2O2, HOCl, NH2Cl (monochloramine)
over the indicated concentration ranges was added to the cuvette to assess
the effects of different concentrations of oxidants or the cell-free MPOH2O2-chloride system (see below) on GFP-SA fluorescence.
Spectrofluorometric monitoring of bleaching of GFP-SA by
cell-free MPO-H2O2-chloride system
In addition to assessing the impact of reagent-grade HOCl on fluorescence
of GFP-SA, the effect of a HOCl-generating system, composed of purified
MPO (2– 80 nM), H2O2 (100 – 450 ␮M), and chloride (130 mM), was
assessed by serial scanning. Purified MPO (Reinheit Zahl ⬃0.80) and H2O2
in PBS (pH 7.4) comprised the cell-free MPO-H2O2-chloride system. The
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PMN from normal healthy volunteers and myeloperoxidase-deficient individuals were purified from peripheral blood as described in Ref. 8. Donor
consent was obtained from each individual following the protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. Isolated PMN were suspended in sterile endotoxin-free
HBSS without divalent cations, counted, and diluted to a final density no
greater then 20 ⫻ 106/ml. From 95–98% of the isolated leukocytes were
PMN, as determined by microscopic examination of the cell suspension
after staining with the HEMA-3 kit. PMN were held on ice until use in
subsequent assays.
PMN samples were mixed at a ratio of one CFU of S. aureus (equivalent
to four cocci) to one PMN (i.e., multiplicity of infection (MOI) ⫽ 1:1, both
PMN and CFU bacteria at 5 ⫻ 106/ml) in 5 ml polypropylene roundbottom tubes, and incubated with shaking (180 rpm) in a 37°C water bath.
After 5 min, samples were gently spun (500 ⫻ g) for 5 min in a Savant high
speed tabletop centrifuge to pellet PMN and associated bacteria. The supernatant containing extracellular bacteria was discarded, and the pellet
resuspended in the original volume of HBSS containing divalent cations
supplemented with 1% HSA and 10% pooled human serum and returned to
the 37°C water bath for specified intervals. To confirm that PMN-associated GFP-SA were sequestered within PMN, DPI-PMN challenged with
opsonized GFP-SA were recovered after 10 min, stained with 0.1% crystal
violet for 15 min at room temperature, and examined by fluorescence
microscopy. Whereas fluorescence of GFP-SA directly exposed to crystal violet was immediately quenched, PMN-associated GFP-SA after 10
min of phagocytosis by DPI-PMN remained fluorescent (data not
shown). These data indicate that the GFP-SA were sequestered within
closed phagosomes by 10 min of phagocytosis under these experimental
conditions.
For experiments in which opsonized GFP-SA were coated with purified
MPO before being fed to PMN, the opsonized GFP-SA were pelleted and
resuspended in PBS ⫾ purified MPO for 1 h. Bacteria were pelleted, resuspended and fed to PMN as described above. It should be noted that
MPO bound to GFP-SA very inefficiently; exposure of GFP-SA to 1 ␮M
MPO resulted in retention of ⬃1.4 nM or 14 fmoles (0.2% of the initial
amount) on the bacterial pellet containing 108 cocci (n ⫽ 3). For experiments in which extracellular oxidants were provided by glucose-glucose
oxidase, the buffer was supplemented with 10 mM glucose and a final
concentration of 2 ␮g/ml glucose oxidase was added to the resuspended
DPI-treated samples.
2634
FATE OF STAPHYLOCOCCI IN HUMAN NEUTROPHILS
stock concentrations of MPO and H2O2 were determined spectrophotometrically, using ␧473 ⫽ 75 mM⫺1 cm⫺1 for reduced minus oxidized MPO
and ␧240 ⫽ 43.6 M⫺1 cm⫺1 for H2O2. GFP-SA (5 ⫻ 107) were suspended
in 3 ml of PBS containing the complete system, MPO alone, or H2O2 alone
and scanned serially every 2 min. In parallel, the chlorinating capacity of
the MPO-H2O2-chloride system was assessed using a previously described
method to quantitate taurine chlorination (11).
Quantitation of H2O2
In experiments in which exogenous H2O2 was generated by glucose-glucose oxidase, the amount of H2O2 was quantitated using a H2O2 electrode
(Apollo 4000, World Provision Instruments). The DPI-treated samples
were prepared as described in the phagocytosis assay and resuspended in
buffer supplemented with 10 mM glucose. The cell suspension was added
to the prewarmed 37°C chamber, stirred for 5 min until the signal stabilized, and glucose oxidase was added to a final concentration of 50 ␮g/ml.
The initial velocity of the glucose-glucose oxidase activity was calculated
from an H2O2 standard curve generated daily. Data are expressed as
nmoles/min.
Iodination
MPO-dependent iodination was quantitated as previously described (12,
13). PMN were suspended at 107/ml and incubated at 37°C for 10 min in
20 mM HEPES buffered HBSS with divalent cations, 1% HSA, and 10%
pooled human serum, with or without 100 ␮M sodium azide, in the presence of a mixed stock of radiolabeled Na125I and unlabeled iodide at a final
concentration of 10 ␮M. The pretreated cells were then mixed with the
opsonized S. aureus at an MOI of 1:1, 2:1, and 5:1, and placed into a 37°C
water bath for 30 min. The reaction was stopped using a mixture of sodium
thiosulfate and cold NaI, and the proteins and lipids precipitated with 20%
trichloroacetic acid. The samples were washed twice in 10% TCA, sedimented, and the final pellet was resuspended in 10% SDS and counted for
radioactivity.
Immunoblotting for GFP
The structural integrity of cytoplasmic GFP in GFP-SA after bleaching by
HOCl or 120 min after phagocytosis by PMN, both conditions under which
the fluorescence of GFP-SA was completely bleached, was assessed by
probing electroblots with a affinity-purified polyclonal rabbit Ab against
GFP. GFP-SA (5 ⫻ 107) treated with buffer or with reagent grade HOCl
were solubilized by vigorous vortexing in hot SDS sample buffer (14) for
5–10 min. Proteins were separated by SDS-PAGE in 5–20% gradient poly-
acrylamide gels and electroblotted to nitrocellulose filter. The electroblot
was blocked, probed with rabbit anti-GFP and a secondary donkey antirabbit Ab conjugated with HRP, and visualized with chemilumiscence reagents (SuperSignal West Detection Kit, Thermo Fisher Scientific). In experiments assessing the integrity of GFP in GFP-SA phagocytosed by
PMN, a subcellular fraction enriched for granules and phagosomes was
analyzed. PMN or PMN that had phagocytosed GFP-SA were pelleted,
resuspended in relaxation buffer (100 mM KCl, 3 mM NaCl, 1 mM Na2
ATP, 3.5 mM MgCl2 (pH 7.3)), and disrupted by N2 bomb cavitation, as
previously performed (15). Unbroken PMN and nuclei were removed by
low speed centrifugation (500 ⫻ g), and granules and phagosomes were
pelleted (20,000 ⫻ g, 20 min, 4°C). Pellets of PMN containing GFP-SA,
as well as a pellet of GFP-SA alone, were solubilized for SDS-PAGE and
immunoblotting as described above.
Results
Time-dependent loss of GFP fluorescence and killing of
S. aureus within PMN
During our studies of the synergistic activity of the PMN NADPH
oxidase and extracellular Group IIA phospholipase A2 on ingested
S. aureus (16), we noted that GFP-expressing S. aureus (GFP-SA)
ingested by normal PMN lost fluorescence over time (Fig. 1A).
The loss of fluorescence was not strain-specific and was seen in
each of several different strains of S. aureus, including both
hospital-acquired and community-associated methicillin-resistant strains (data not shown). The loss of GFP fluorescence required ingestion of the bacteria by PMN, as extracellular SA remained fluorescent when incubated with PMN in which
phagocytosis was inhibited by treatment with dihydrocytochalasin
B (data not shown). Reasoning that the loss of GFP fluorescence
reflected damage to GFP-SA in the phagosome, we compared the
relative kinetics of the loss of fluorescence vs loss of viability of
ingested bacteria (Fig. 1B). There was a relative delay in the onset
of loss of GFP-SA fluorescence relative to the loss of viability in
PMN-associated bacteria (Fig. 1B). At early time points, PMN
killing of SA exceeded the loss of fluorescence, although values
were virtually identical at 120 min, with ⬃80% of the ingested
bacteria dead and nonfluorescent at that time. These data indicate
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FIGURE 1. Time-dependent loss
of GFP fluorescence and viability by
staphylococci within normal PMN. A,
Serum-opsonized GFP-SA were fed
to normal PMN at an MOI of 1 CFU
(4 cocci):1 PMN and fluorescence
monitored at 30 and 120 min after ingestion. Whereas ⬎90% of GFP-SA
remained fluorescent after 30 min,
most had lost fluorescence by 120 min
after phagocytosis. B, The kinetics of
killing, as assessed by quantitating
CFU, were more rapid than the loss of
GFP-SA fluorescence. However, the
percent of ingested bacteria that lost
viability and fluorescence was identical 120 min after phagocytosis (representative experiment of n ⬎ 20).
The Journal of Immunology
2635
that PMN cytotoxins significantly damaged or killed ingested SA
in the phagosome before GFP fluorescence was lost and, conversely, that ⬃20% of ingested GFP-SA resisted PMN cytotoxic
effects and remained fluorescent. Phagosomes containing GFP-SA
did not communicate with the extracellular space, as demonstrated
by the failure of crystal violet to quench intraphagosomal GFP-SA
at 120 min (data not shown), excluding leakage of cytotoxins from
the phagosome as an explanation for the subpopulation of surviving staphylococci.
Loss of fluorescence of GFP-SA: requirement for the phagocyte
NADPH oxidase
In contrast to the loss of GFP fluorescence in normal PMN (Fig.
1A), internalized GFP-SA remained fluorescent after ingestion by
PMN treated with diphenylene iodonium (DPI) (Fig. 2A), a potent
inhibitor of flavoproteins, including the phagocyte NADPH oxidase (17). Despite complete inhibition of NADPH oxidase activity,
phagocytosis is normal in DPI-treated PMN (16, 18). To determine
whether the dependence on an active NADPH oxidase reflected a
need for the oxidase as an oxidant source, we provided an exogenous H2O2-generating system to PMN with endogenous oxidase
activity inhibited by DPI. DPI-treated PMN were challenged with
GFP-SA in the absence or presence of glucose-glucose oxidase
(GGO), and fluorescence and viability of ingested GFP-SA were
assessed. The GGO system generated 90 –110 nmoles/ml H2O2, as
measured directly with a H2O2 electrode. Whereas GFP-SA within
DPI-treated PMN remained fluorescent at 120 min after ingestion
(Fig. 2B), significantly fewer organisms were persistently fluorescent in DPI-PMN exposed to the H2O2 source, although exogenous
H2O2 did not restore levels to that seen in normal PMN. In parallel,
DPI-PMN without supplemental H2O2 were less able to kill
GFP-SA than were control PMN, but the addition of GGO partially
restored bacterial killing to the DPI-treated PMN (data not shown).
The augmentation of activity in DPI-PMN in the presence of the
exogenous oxidant source required all components of the GGO
system and was inhibited by the addition of catalase (Fig. 2B).
These data indicate that the loss of GFP-SA fluorescence required
oxidants and that the NADPH oxidase was the source of ROS in
the phagosome.
Loss of fluorescence of GFP-SA: requirements for MPO and
chloride
To determine whether MPO contributed to or was required for the
loss of GFP-SA fluorescence, normal PMN were challenged with
GFP-SA in the presence of sodium azide (NaN3). Whereas NaN3
inhibits MPO activity in intact PMN (3), the NADPH oxidase is
NaN3-resistant (13, 19 –21), and NaN3-treated PMN generate more
oxidants than do normal PMN stimulated in the absence of NaN3
(13, 20, 22, 23). The fluorescence of GFP-SA persisted in NaN3treated PMN in a manner very similar to that seen in DPI-treated
PMN (Fig. 2C). In parallel, PMN from a MPO-deficient patient
(MPO-D) (24) were challenged with opsonized GFP-SA and ingested GFP-SA remained fluorescent in MPO-D PMN (Fig. 2C
and data not shown). Exposing GFP-SA to 50 nM MPO before
ingestion by MPO-D PMN partially reconstituted the PMN-mediated loss of GFP-SA fluorescence, but not to values seen with
normal PMN (Fig. 2C), perhaps a reflection of the inefficiency with
which MPO associated with GFP-SA during the exposure step
(vide supra). Taken together, these data confirmed that enzymatically active MPO was required for the loss of fluorescence by
GFP-SA phagocytosed by PMN.
Dependence of intraphagosomal GFP-SA fluorescence decay on
oxidants and on active MPO implicated a likely role for the PMN
MPO-H2O2-Cl system in mediating the loss of fluorescence by
ingested bacteria (3). Consistent with this prediction, loss of intraphagosomal GFP-SA fluorescence correlated inversely with the
concentration of chloride in the incubation buffer (Fig. 2D). In the
absence of chloride, ⬃90% ingested GFP-SA remained fluorescent
at 120 min, in contrast to the ⬃25% value at 122 mM chloride. As
phagocytosis, degranulation, and NADPH oxidase activity in PMN
were normal at the varied concentrations of chloride (data not
shown) and gluconate (up to 150 mM) did not inhibit chlorination
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FIGURE 2. Loss of GFP fluorescence required an intact MPO-H2O2chloride system. Serum-opsonized
GFP-SA were fed to DPI-treated PMN
at an MOI of 1:1 and fluorescence monitored at 30 and 120 min after ingestion
(A). The effect of added glucose-glucose oxidase (GGO) ⫾ catalase on the
loss of fluorescence was examined to
determine the contribution of oxidants
to the observed changes in GFP-SA (B).
To assess the contributions of MPO to
GFP-SA fluorescence, the effect of
added NaN3 on loss of fluorescence of
GFP-SA in normal PMN as well as the
fate of GFP-SA in MPO-deficient PMN
(MPO-D) were examined (C). Pretreating GFP-SA with 50 nM MPO partially
restored the loss of fluorescence of
GFP-SA in MPO-D PMN (C). To assess the contribution of chloride to the
loss of GFP-SA fluorescence in PMN,
the fluorescence of PMN-associated
GFP-SA 120 min after phagocytosis
was examined at varied concentrations
of chloride (D) (n ⫽ 3 of experiments
done in duplicate).
2636
FATE OF STAPHYLOCOCCI IN HUMAN NEUTROPHILS
by the cell-free MPO-H2O2 system (data not shown), the persistence of fluorescence by ingested GFP-SA in the absence of chloride reflected the consequences of an incomplete HOCl-generating
system.
HOCl, but neither H2O2 nor monochloramine, mediated loss of
GFP-SA fluorescence
To assess the susceptibility of GFP-SA to the direct effects of
HOCl, GFP-SA were incubated in varied concentrations of reagent
grade HOCl while GFP fluorescence was simultaneously moni-
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FIGURE 3. Selective loss of GFPSA fluorescence by HOCl. A, The fluorescence of a suspension of GFP-SA
(5 ⫻ 107) was monitored continuously (␭ex ⫽ 395 nm, ␭em ⫽ 480 –
540 nm) and exposed to reagent grade
HOCl, from) to 100 ␮M. At concentrations ⱖ20 ␮M HOCl, fluorescence
of GFP-SA decreased in a dose-response fashion and was eliminated
completely ⬃100 ␮M HOCl (representative experiment). In five independent experiments, each done in
triplicate, 46.2 ⫾ 9.9 ␮M HOCl decreased fluorescence of GFP-SA
50%. B, GFP-SA (5 ⫻ 107) lost fluorescence as well when incubated
with a cell-free HOCl-generating system. In the presence of 50 nM MPO,
150 ␮M H2O2, and 130 mM NaCl,
fluorescence was monitored spectrophotometrically over time. Fluorescence of GFP-SA decreased 50%
within 12.3 min (representative experiment shown of n ⫽ 5). Despite
the loss of fluorescence, the GFP protein remained intact, as judged by immunoblotting, after exposure to reagent millimolar HOCl (C) or in
phagosomes recovered 120 min after
ingestion by PMN (D). Similar treatment of GFP-SA with H2O2 (up to 10
mM) or NH2Cl (up to 7 mM) (E) had
no effect on GFP fluorescence (representative of n ⫽ 3 experiments).
tored in a spectrofluorometer (Fig. 3A). Fluorescence of GFP-SA
decayed in the presence of HOCl in a dose-dependent fashion, with
fluorescence of 5 ⫻ 107 GFP-SA decreased to 50% of control in
the presence of 46.2 ⫾ 9.9 ␮M (mean ⫾ SEM, n ⫽ 5 separate
experiments, each in triplicate). The HOCl-generating system was
likewise effective; in the presence of 50 nM purified MPO, 150
␮M H2O2, and 130 mM chloride, fluorescence of 5 ⫻ 107 GFP-SA
decreased 50% within 12.3 min at room temperature (n ⫽ 5) (Fig.
3B). For both HOCl and the MPO-H2O2– chloride system, there
was an initial delay in loss of GFP-SA fluorescence; at low
The Journal of Immunology
Effect of growth phase of GFP-SA on its bleaching in PMN
phagosome
HOCl is highly reactive, with a clearly defined hierarchy in its
propensity to react with biological substrates (28, 29). Although
HOCl can rapidly diffuse across membranes, successful diffusion
depends on the presence and relative susceptibility of potentially
competing substrates that might consume HOCl before it could
enter a target cell. We reasoned that the susceptibility of GFP to
bleaching would depend on the presence and relative susceptibility
of competing substrates on the surface and in the cytoplasm of
GFP-SA that would compete with GFP for reaction with HOCl.
The composition and properties of the staphylococcal envelope
change extensively during the progression of bacteria from logarithmic to stationary phase (30). Accordingly, the array of competing substrates available to react with HOCl would likewise vary
with the growth-phase of the GFP-SA. We therefore anticipated
that the susceptibility of ingested GFP-SA to bleaching within the
FIGURE 4. The bleaching of GFP-SA within PMN depended on the
growth phase of the bacteria. GFP-SA grown to mid-log, enriched mid-log,
and stationary phase (see Materials and Methods) were fed to normal or
DPI-treated PMN at an MOI 1CFU (4 cocci):1 PMN for 120 min, at which
time the percent of fluorescent GFP-SA was enumerated microscopically.
A higher percentage of the more rapidly growing GFP-SA from enriched
mid-log phase were more susceptible to bleaching of GFP, relative to log
or stationary bacteria.
PMN would depend on the growth stage of the bacteria at the time
that they were ingested. To test this hypothesis, we compared
PMN bleaching of GFP-SA recovered from mid-logarithmic
phase, our standard condition, stationary phase, or from cultures in
which rapidly growing SA were selectively enriched (see Materials and Methods). The population that was enriched in rapidly
growing GFP-SA (early exponential phase) was more susceptible
to PMN killing and bleaching than were our standard mid-log
grown bacteria (Fig. 4). Conversely, a significantly greater fraction
of GFP-SA harvested after overnight culture (stationary phase)
was more resistant to bleaching. In light of the relative kinetics of
GFP-SA bleaching and killing (Fig. 1B), the data suggest that
growth phase-related changes in the composition of surface structures, dictated the potential substrates available to compete with
GFP for reacting with HOCl.
To examine in a different way the impact of competing substrates on bleaching of GFP-SA, we challenged PMN with
GFP-SA at varied MOI, comparing our standard condition (1
CFU:1 PMN, equivalent to 4 cocci: 1 PMN) with higher ratios of
2 CFU:1 PMN and 5 CFU:1 PMN (equivalent to 8 cocci: 1 PMN
and 20 cocci: 1 PMN, respectively). The number of cocci per PMN
was enumerated and the fraction of intracellular organisms that
remained fluorescent determined 120 min after ingestion by PMN
(Fig. 5A). As the multiplicity of infection increased, PMN ingested
more bacteria, as reflected by an increase in the average number of
cocci per PMN. In parallel, the fraction of GFP-SA that remained
fluorescent (Fig. 5A) and resisted killing increased at higher MOI.
The viability of intracellular GFP-SA 120 min after phagocytosis
was 30.9 ⫾ 6.0, 89.0 ⫾ 17.4, and 101.8 ⫾ 8.8 (mean ⫾ SEM, n ⫽
6) at MOI of 1 CFU: 1 PMN, 2 CFU: 1 PMN, and 5 CFU: 1 PMN,
respectively. Thus, survival of ingested GFP-SA paralleled the increases in the bacterial challenge presented to PMN, suggesting
that the organism load at higher ratios of infection exceeded the
capacity of intraphagosomal antimicrobial activity, the H2O2MPO-chloride system, or both. To assess specifically the capacity
of MPO-mediated biochemistry independent of GFP bleaching, we
quantitated protein radioiodination by PMN stimulated by
GFP-SA at varied MOI (Fig. 5B). As more bacteria were ingested
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concentrations of HOCl (Fig. 3A) or at early time points in MPOdependent generation of HOCl (Fig. 3B), there was little change in
fluorescence, followed by more rapid loss of fluorescence. For example, the initial additions of HOCl had little, if any, influence on
fluorescence of GFP-SA; once 40 ␮M HOCl was added, loss of
GFP-SA fluorescence was dose-dependent (Fig. 3A). Likewise, in
the first 6 min, the HOCl-generating system had little effect on
GFP-SA fluorescence, whereas subsequently the loss of fluorescence became nearly linear (Fig. 3B). Despite loss of fluorescence,
the GFP in GFP-SA remained structurally intact. Immunoblots of
GFP-SA exposed to millimolar concentrations of HOCl (Fig. 3C),
⬃300-fold greater than those mediating loss of fluorescence, or of
GFP-SA after 120 min in PMN (Fig. 3D), a time when ⬎90% of
the organisms have lost fluorescence, demonstrated no decrease in
size or amount of GFP Thus, the MPO-dependent HOCl-generating system, both in vitro and within human PMN, reduced the
fluorescence of GFP-SA without degrading GFP.
H2O2 accounts for nearly all the oxygen consumed by the
NADPH oxidase of activated PMN (25, 26) and serves as the
substrate for the production of HOCl in the phagosome. To assess
the susceptibility of GFP-SA fluorescence to more proximal oxidants generated by activated PMN, GFP-SA were exposed to varied amounts of reagent-grade H2O2 while simultaneously monitoring fluorescence spectrofluorometrically (Fig. 3E). There was
no loss of fluorescence at concentrations of H2O2 as high as 10
mM. This failure of H2O2 treatment to compromise GFP-SA fluorescence is consistent with the data from MPO-deficient PMN,
where relatively larger amounts of oxidants from the NADPH oxidase in the absence of MPO (13, 20, 22, 23) did not diminish the
fluorescence of ingested GFP-SA (Fig. 2C and data not shown).
These data demonstrate that only a subset of oxidants modified by
reaction with MPO abrogated the fluorescence of GFP-SA.
In addition to HOCl, the MPO-H2O2-Cl system reacts with both
bacterial and host proteins in the phagosome to generate an array
of biologically active derivatives, including long-lived chloramines (reviewed in Ref. 3). Reasoning that chloramines might also
contribute to the loss of GFP-SA fluorescence, we investigated
whether monochloramine treatment altered the fluorescence of
GFP-SA. However, GFP-SA fluorescence was unaffected by concentrations of monochloramine (NH2Cl) as high as 7 mM (Fig.
3E). Taken together, these data strongly implicate HOCl produced
in the phagosome by the MPO-H2O2-chloride system as the agent
responsible for the loss of GFP-SA fluorescence. Given that peroxidase-dependent chlorination of tyrosine 66 in GFP renders GFP
no longer fluorescent (27), the loss of fluorescence by GFP-SA
likely reflects bleaching of GFP by HOCl in the phagosome.
2637
2638
FATE OF STAPHYLOCOCCI IN HUMAN NEUTROPHILS
at higher MOI (Fig. 5C), there were commensurate increases in
protein radioiodinated and in the fraction of bacteria that survived.
To determine whether the changes in bleaching and killing of
GFP-SA at higher MOI were due to limiting amounts of the components of the MPO-H2O2-chloride system, we supplemented
PMN with MPO or with oxidants. Pre-exposing GFP-SA to 50 nM
purified MPO before incubation with PMN did not significantly
increase either radioiodination (Fig. 5B) or bacterial killing (data
not shown) that occurred during and after phagocytosis. Even
when GFP-SA were exposed to 1 ␮M MPO before feeding to
PMN, conditions in which 1.4 ⫾ 0.3 nM (n ⫽ 3) enzymatically
MPO could be detected after incubation; neither killing nor bleaching of GFP-SA was increased. Thus, the ability of some ingested
GFP-SA to escape killing and bleaching at the MOI tested did not
reflect insufficient mobilization of MPO from granules during
phagocytosis. However, supplemental H2O2, achieved by coincubating the PMN-GFP-SA with glucose-glucose oxidase, increased
the percentage of ingested GFP-SA that were bleached, but the
increase was statistically significant only at the highest MOI examined (Fig. 5C). Thus, insufficient production of oxidants, but not
the release of MPO, accounted, at least in part, for the ability of
GFP-SA in PMN phagosomes to escape killing and bleaching at
the each of the MOI tested.
Discussion
Innate host defense against infecting staphylococci depends in
large part on optimal PMN function, an association most dramatically demonstrated by the frequency and severity of such infections in patients with neutropenia or with inherited PMN disorders
such as chronic granulomatous disease (31). In addition, both oxidants and MPO figure prominently in efficient killing of staphylococci, as PMN from patients with chronic granulomatous disease
or inherited MPO deficiency exhibit defective antimicrobial activ-
ity (32). However, it has long been recognized that a significant
fraction of ingested S. aureus can survive in the phagosome of
normal PMN, thereby creating a potential reservoir for protracted
infection and inflammation (6). The recent international emergence
of community-associated methicillin-resistant S. aureus as a serious infectious disease problem has reinvigorated efforts to unravel
both the strategies of the organism and the PMN (33– 41) to influence the outcome of the host-pathogen interaction. In this context, our observations of the fate of GFP-SA provide several important insights into events within the phagosome of normal PMN
and identify variables in SA-PMN interactions that may contribute
to the eventual fate of ingested staphylococci.
Our data extend previously reported observations of the susceptibility of purified GFP or GFP expressed in Escherichia coli to
HOCl or the MPO-H2O2-Cl system of PMN (4). The loss of GFP
fluorescence in the ingested SA depended on the presence of an
intact MPO-H2O2-chloride system (Fig. 2), with partial reconstitution of GFP bleaching by DPI-treated PMN when an exogenous
source of H2O2 was supplied (Fig. 2B), indicating an essential role
for HOCl in this setting as well. PMN-mediated killing of SA
exhibited the same features and prerequisites, thus demonstrating
that the phagocyte oxidase provides oxidants that are essential for
efficient killing of staphylococci and that operate synergistically
with MPO to support the antimicrobial HOCl-generating system.
The loss of GFP-SA fluorescence was produced only by HOCl and
was not seen with H2O2 or NH2Cl, reactive species also generated
in the PMN phagosome (42), even at ⬃1000-fold higher concentrations those that were effective when using HOCl. Furthermore,
HOCl and PMN-mediated loss of GFP-SA fluorescence occurred
without detectable degradation of GFP (Fig. 3, C and D). Thus, the
modification that disrupts the chromophore of GFP reflects very
specific HOCl-mediated chemistry within the PMN phagosome.
Although either chlorination or nitration of tyrosine 66 in GFP
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FIGURE 5. The bleaching of GFPSA depended on the organism-load per
PMN. A, PMN fed GFP-SA at MOI of
1 CFU (4 cocci), 2 CFU (8 cocci), and
5 CFU (20 cocci) to 1 PMN were examined after 120 min for the number of
cocci per PMN and for the fraction of
the intracellular organisms that remained fluorescent. The number of
cocci per PMN increased in parallel
with the MOI. In parallel, the percent of
intracellular cocci that remained fluorescent increased as the MOI increased.
B, Effect of MOI on the extent of radioiodination by PMN challenged with
GFP-SA and by precoating of GFP-SA
with 5 or 50 nM MPO before engaging
PMN. C, Effect of added glucose-glucose oxidase system on bleaching of
GFP-SA fed to PMN at MOI of 2 CFU
(8 cocci) or 5 CFU (20 cocci) to 1
PMN. p values (two tailed Students t
test) at MOI 1:1, 2:1, and 5:1 were
0.050, 0.173, and 0.036 respectively
(n ⫽ 3).
The Journal of Immunology
HOCl on microbial targets. Vulnerable but nonessential surface
sites undergo oxidation and N-chlorination first, consuming HOCl
but not compromising microbial viability. Subsequently, HOCl attacks and inactivates membrane proteins essential for energy generation, thereby culminating in critical damage and microbial death
(reviewed in Ref. 54). Ultimately, HOCl will react with susceptible
cytoplasmic elements, as seen with the GFP in our GFP-SA; the
close correlation between the percent of SA that retained GFP
fluorescence and the number of surviving intracellular SA recovered as CFU strongly suggest that the bleaching of GFP after 120
min corresponded to dying or dead bacteria.
One of the most important features of assessing GFP bleaching
during and after phagocytosis is the insight it provides on the fate
of individual cocci within PMN. Our results provide compelling
evidence for differences in the fate of individual bacteria, in some
circumstances even within a single neutrophil (Fig. 1A). Several
previous studies have strongly suggested that the killing of intraphagosomal staphylococci by PMN is incomplete (5–7, 33, 35,
55, 56) and clinical observations demonstrate the survival of a
subpopulation of ingested staphylococci that persist in infected
patients (57). The very close correlation between the number of SA
surviving within PMN and the fraction of intraphagosomal bacteria retaining GFP fluorescence supports the finding that some ingested SA can survive within PMN and raises the possibility that
the differential retention of GFP fluorescence could be used to sort
and separately monitor the subsequent fates of living and dead SA
within PMN.
The host and microbial variables that contribute to the eventual
fate of ingested SA in PMN are likely extremely complex, but our
studies point to three important factors: 1) the growth phase of the
staphylococci, 2) the MOI and the resultant number of organisms
ingested by each PMN, and 3) the amount of H2O2 generated in an
individual phagosome. The relative resistance of ingested GFP-SA
to killing and bleaching paralleled the duration bacteria were in
culture before being fed to PMN. The rank order of resistance to
GFP bleaching, overnight cultures ⬎ mid-log phase cultures ⬎
repetitive consecutive early mid-log cultures (Fig. 4), suggests that
slowly or non growing SA were more resistant to PMN cytotoxicity than were those growing more rapidly. This differential susceptibility of the array of potential competitive substrates to cytotoxins in the phagosome due to growth rate-dependent changes in
the bacterial envelope the profile of secreted microbial products, or
both. Independent of other bacterial variables, higher MOI and the
associated greater intracellular bacterial loads also compromised
the effectiveness of PMN antimicrobial action. By monitoring GFP
bleaching as a function of MOI and the corresponding number of
ingested and bleached bacteria within individual PMN, we clearly
demonstrated that it is the number of SA per PMN that determined
the likelihood of GFP bleaching and microbial killing. This relationship was the same at each MOI tested (Fig. 5A); the higher
MOI simply increased the probability of greater bacterial loads
within individual PMN and thus favored conditions in which intraphagosomal cytotoxins were insufficient to more extensively
damaged SA. Under these taxing conditions, it is possible that one
or more antimicrobial component in the phagosome becomes limiting. The increase in iodination proportional to the more robust
bacterial uptake at higher MOI (Fig. 5B) indicates the presence of
sufficient MPO to support MPO-dependent cytotoxicity, a conclusion supported by the failure of added MPO to augment iodination,
bleaching, or killing. In contrast, supplementation with exogenous
H2O2, especially at higher MOI (Fig. 5C), reduced the percent of
ingested GFP-SA that resisted bleaching, suggesting that the increased phagocytic burden taxed the capacity of the NADPH oxidase to contribute, directly or indirectly, to antimicrobial action in
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ablates its fluorescence (27), nitration does not occur in the phagosome of human PMN (43, 44). Consequently the bleaching of ingested GFP-SA by PMN likely reflects chlorination of GFP and
thus provides a probe of MPO-dependent chlorination in an individual phagosome in real time.
The application of GFP bleaching as a probe of the antimicrobial activity of PMN-derived HOCl requires recognition of HOCl
chemical reactivity within the complex biological context of the
neutrophil phagosome. In general, HOCl-mediated modifications
of biological targets depend on 1) sufficient production of HOCl,
2) access of HOCl to potential targets, and 3) the relative susceptibility of substrates to modification. When exposed to HOCl or the
MPO-H2O2-chloride system, recombinant GFP in solution is rapidly bleached in a dose-dependent fashion, with ⬍20 ␮M HOCl
causing complete loss of GFP fluorescence by direct oxidative
bleaching of the chromophore (4). In contrast, our data (Fig. 3, A
and B) and previous studies demonstrate that bleaching of GFP
when expressed in bacterial cytoplasm required higher concentrations of HOCl or more time, when HOCl is provided by the MPOH2O2-chloride system. Because phagocytosis rapidly triggers production of HOCl by human PMN (3) and bleaching of GFP-SA
occurred in PMN, we concluded that sufficient HOCl was generated in phagosomes to support bleaching of GFP and that the observed delay in GFP bleaching more likely reflected the reaction
properties of HOCl rather than insufficient HOCl.
In contrast to the situation with recombinant GFP, GFP-SA
present a more complex and varied substrates for reaction with
HOCl. HOCl readily reacts with surface structures when bacteria
are exposed to HOCl extracellularly, as discussed in detail by
Hurst (reviewed in Ref. 45). The initial focus of microbial attack
by HOCl is on the bacterial envelope. Consequently, HOCl will
encounter and react with many susceptible targets on the surface of
GFP-SA, thus consuming some of the HOCl before it can breach
the bacterial surface. Furthermore, any biomolecules in the cytoplasm that are intrinsically very susceptible to HOCl-mediated oxidation are affected later and only after concentrations of HOCl in
great excess of that required for killing are added (45– 47). The
competition with GFP for reacting with HOCl is even more complex in the phagosomes of neutrophils, where the granule proteins
dominate and provide ample substrate for reactions with HOCl and
other oxidants. In fact, most of chlorotyrosine residues recovered
from neutrophils that ingest S. aureus are of host origin (44).
Even if HOCl rapidly accessed the bacterial cytoplasm, biomolecules in the cytoplasm would compete with GFP for reacting for
HOCl, depending on their relative intrinsic vulnerability for oxidation by HOCl. Sulfur-containing amino acids (notably, cysteine
and methionine) are roughly a million-fold more susceptible to
reaction with HOCl than is tyrosine (⬃3 ⫻ 107 M⫺1 s⫺1 for methionine or cysteine vs 44 M⫺1 s⫺1 for tyrosine) (28, 29) and it is
thus likely that HOCl would react with such targets in S. aureus
before bleaching GFP. As a consequence of these complexities and
the competition between GFP and more vulnerable substrates, the
relative kinetics of bleaching and killing of GFP-SA differed (Fig.
1B). Analogous findings were reported by Palazzo et al. (4), where
a similar delay in the bleaching of GFP-E. coli after additions of
HOCl were noted and where ⬃10-fold more HOCl was required to
bleach cytosolic GFP than was needed to kill the GFP-E. coli (4).
Very similar observations regarding the relative rates of bacterial
killing vs oxidative damage by HOCl have been reported for several bacterial targets, including a variety of sulfhydryl groups, bcytochromes, ␤-carotene, iron-sulfur centers in succinate dehydrogenase, ␤-galactosidase, aldolase, and F1-ATPase (45, 47–53).
Collectively, the survival curves of bacteria exposed to HOCl or to
the MPO-H2O2-chloride system reflect the sequential attack of
2639
2640
Acknowledgments
We thank Sally McCormick for technical assistance with some aspects of
these studies.
Disclosures
The authors have no financial conflict of interest.
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the phagosome independent of the MPO system. Furthermore, decreased bleaching of GFP-SA at higher MOI even in the presence
of supplemental H2O2 suggests that additional factors in the
phagosome may be somewhat limiting in the face of increased
substrate load. Among the possible contributing factors are antibacterial peptides and proteins whose mobilization from preformed granules to individual phagosomes may be limiting at high
bacterial loads. However, the failure of GFP-SA to lose fluorescence in the absence of oxidants, as in DPI-PMN, or active MPO,
as in NaN3-PMN or MPO-deficient PMN, indicates that granule
proteins alone were unable to kill and bleach GFP-SA. The presence of bleached and fluorescent GFP-SA in neighboring phagosomes within the same neutrophil underscores the variability in the
potential fate of ingested SA, whether as a reflection of the individual organism’s metabolic state or due to stochastic differences
in mobilization of granule proteins and oxidase assembly at a
given phagosome. An important clinical implication of these
findings is that the bacterial metabolic state and relatively small
differences in bacterial inoculum in relation to the number of
PMN recruited could dictate significant differences in the outcome of interactions between SA and PMN, possibly helping to
explain the practical challenges often seen in resolving infections with S. aureus.
In summary, the specific bleaching of GFP expressed within
staphylococci demonstrates that oxidants generated by the
NADPH oxidase and that chlorination mediated by MPO collectively contribute to the potent antimicrobial activity within the
PMN phagosome. The loss of GFP fluorescence by ingested SA
provides a sensitive experimental probe for monitoring biochemical events within individual phagosomes and for identifying the
subpopulation of organisms that resist PMN cytotoxins. Defining
the molecular basis of SA survival within PMN will provide important insights into the pathophysiology of staphylococcal infection, the microbial attributes that contribute to the clinical challenges that staphylococci pose, and the development of novel
therapeutic agents (58).
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