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The Journal of Immunology, 2008, 180, 500 -509
Copyright © 2008 by The American Association of Immunologists, Inc.
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Neutrophil Microbicides Induce a Pathogen Survival Response in Community-Associated Methicillin-Resistant Staphylococcus aureus1

Amy M. Palazzolo-Ballance*, Michelle L. Reniere{ddagger}, Kevin R. Braughton*, Daniel E. Sturdevant{dagger}, Michael Otto*, Barry N. Kreiswirth§, Eric P. Skaar{ddagger} and Frank R. DeLeo2,*

* Laboratory of Human Bacterial Pathogenesis and {dagger} Research Technologies Section, Genomics Unit, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; {ddagger} Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232; and § Public Health Research Institute Tuberculosis Center, International Center for Public Health, Newark, NJ 07103


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In recent years, there has been a dramatic increase in the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. MW2 (pulsed-field type USA400), the prototype CA-MRSA strain, is highly virulent and has enhanced ability to evade killing by neutrophils. Although progress has been made, the molecular basis for enhanced virulence of CA-MRSA remains incompletely defined. To that end, we studied resistance of MW2 to key microbicides of human neutrophils. Hydrogen peroxide (H2O2), hypochlorous acid, and azurophilic granule proteins had significant bacteriostatic but limited staphylocidal activity toward MW2 under the conditions tested. An MW2-specific microarray revealed common changes in S. aureus gene expression following exposure to each microbicide, such as up-regulation of transcripts involved in gene regulation (e.g., saeRS and kdpDE) and stress response. Azurophilic granule proteins elicited the greatest number of changes in MW2 transcripts, including up-regulation of mRNAs encoding multiple toxins and hemolysins (e.g., hlgA, hlgB, hlgC, hla, lukS-PV, lukF-PV, sec4, and set17–26). Notably, H2O2 triggered up-regulation of transcripts related to heme/iron uptake (e.g., isdA, isdB, and isdCDEFsrtBisdG), and an isogenic isdAB-negative strain of MW2 had increased susceptibility to H2O2 (p < 0.001) and human neutrophils (p < 0.05) compared with the wild-type parental strain. These findings reveal a S. aureus survival response wherein Iron-regulated surface determinant (Isd) proteins are important for resistance to innate host defense. Collectively, the data provide an enhanced view of the mechanisms used by S. aureus to circumvent destruction by the innate immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutrophils (polymorphonuclear leukocytes or PMNs3) are essential effector cells of the innate immune response and provide vital defense against infection. Phagocytosis of microbes elicits generation of reactive oxygen species (ROS) and triggers fusion of granules with forming phagocytic vacuoles, thereby enriching phagosomes with potent microbicides. Although most bacteria are killed by PMN microbicides, pathogens such as Staphylococcus aureus have devised means to circumvent destruction by neutrophils (reviewed in Ref. 1).

S. aureus is a leading cause of infections worldwide (2). Severity of infection ranges from mild skin diseases, such as impetigo, cellulitis, and skin abscesses, to serious invasive diseases, including sepsis, endocarditis, toxic shock syndrome, and necrotizing pneumonia (3, 4). Methicillin-resistant S. aureus (MRSA) remains a major problem in hospitals and healthcare facilities in developed countries, including the United States (5). Hospital-acquired MRSA infections usually occur in individuals with predisposing risk factors, such as surgery, or in patients with indwelling catheters or medical devices (4). In contrast, community-associated MRSA (CA-MRSA) infections typically occur in otherwise healthy individuals, and there has been a dramatic increase in the incidence and severity of CA-MRSA disease over the past several years (6, 7, 8, 9, 10). Strains known as USA300 (referred to here as Los Angeles County clone, LAC) and USA400 (referred to here as MW2, the prototype CA-MRSA strain) are the leading cause of CA-MRSA infections in the United States (10, 11, 12). Compared with prominent hospital-acquired MRSA, MW2 and LAC have significantly enhanced virulence, an attribute linked to increased ability to evade killing by human neutrophils (13).

Although progress has been made, there is limited understanding of the mechanisms used by S. aureus, especially CA-MRSA, to avoid killing by human neutrophils. To that end, we tested the ability of key neutrophil microbicide—hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and azurophilic granule proteins—to kill prominent CA-MRSA strains and evaluated global changes in MW2 gene expression following exposure to these cytotoxic molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial strains and culture

Staphylococcus aureus strains MW2 (USA400) (14), LAC (USA300) (13, 15), MRSA252 (USA200) (16), and COL (17) were grown overnight in tryptic soy broth (TSB; BD Biosciences). A Newman isdAB mutant strain was published previously (18) and is a deletion/insertion of isdAB using an ermC resistance marker. To generate an isogenic isdAB mutant strain in MW2 ({Delta}isdAB), {Delta}isdAB::ermC was transduced from strain Newman into MW2 by the transducing-phage {phi}-85 using a previously published protocol (19). S. aureus strains NCTC 12981 (ATCC 25923, originally isolated and used for quality control purposes at the University of Washington Clinical Laboratories in 1969) (20) and 502A (21) and serotype M1 group A Streptococcus (22) were published previously. For daily experiments, S. aureus cultures were seeded from overnight cultures using a dilution of 1/200 for MW2, LAC, 502A, and NCTC 12981; a 1/150 dilution for MRSA252; and a 1/100 dilution of COL. S. aureus were cultured at 37°C with shaking (250 rpm) to mid-exponential phase (OD600 = 0.75) of growth, at which time they were washed with Dulbecco’s PBS (DPBS; Sigma-Aldrich) or RPMI 1640 medium (Invitrogen Life Technologies) buffered with 10 mM HEPES (RPMI/H, pH 7.2) and resuspended in the same buffer (typically prechilled at 4°C) at a cell density of 108/ml. Group A Streptococcus was cultured in Todd-Hewitt broth containing 0.5% yeast extract and used as described above. Washed and resuspended cultures were used immediately in each experiment.

Isolation of human PMNs

PMNs were isolated from heparinized venous blood of healthy individuals using standard dextran sedimentation coupled with Hypaque-Ficoll gradient centrifugation as described (23). All work was performed according to a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Purity of PMN preparations and cell viability were assessed by flow cytometry (FACSCalibur; BD Biosciences), which showed that cell preparations contained ~98–99% granulocytes (mostly neutrophils with some eosinophils, ~2–5%).

Neutrophil azurophilic granule protein isolation and bactericidal activity assays

Subcellular fractionation of neutrophils was performed as described previously (24). In brief, neutrophils were resuspended in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, and 10 mM PIPES containing 1 mM ATP) and disrupted by nitrogen cavitation (400 psi for 20–30 min at 4°C). Neutrophil lysate was collected drop-wise into 100 mM EGTA such that the final concentration was 1.25 mM. Lysates were centrifuged at 200 x g for 6 min to remove unbroken cells and nuclei. Neutrophil lysates were overlaid atop Percoll step gradients and centrifuged at 48,300 x g for 15 min at 4°C. Fractions containing neutrophil primary/azurophilic granules and specific granules were collected as described, and Percoll was removed by ultracentrifugation at 100,000 x g for 90 min. Granule proteins were extracted by low pH (acid extraction) using a published protocol with modifications (25). In brief, glycine buffer (pH 2.0) was added to isolated granules for 30 min at 25°C (25). The mixture was centrifuged at 10,000 x g for 20 min at 25°C and supernatants were stored in aliquots at –80°C. The BCA Protein Assay (Pierce Biotechnology) was used to determine the concentration of solubilized protein in the supernatant as described in the kit protocol. To standardize bactericidal activity, aliquots of granule fractions were thawed once for use and then discarded. For daily experiments, the desired amount of solubilized granule protein (20–100 µg) was diluted in RPMI/H (pH 7.2) to 100 µl (pH remained at 7.2) and then added to an equal volume of 107 bacteria in RPMI/H. The suspension was rotated at 37°C for the desired times at which point the mixture was diluted in sterile saline and plated on tryptic soy agar (TSA). CFUs were counted the following day. Percent survival was determined using the equation (CFUtreated at t/CFUuntreated at t0) x 100.

Alternatively, specific granules were solubilized with 0.1% saponin in RPMI/H containing 1 x 107 bacteria. Specific granules isolated from 106, 5 x 106, and 107 PMNs (cell equivalents or CE) were added to the detergent/bacteria mixture and the suspension was rotated at 37°C for one hour. Bacteria were diluted in sterile saline and plated on TSA for overnight growth. Percent survival was determined as described above.

Hydrogen peroxide and hypochlorous acid killing assays

S. aureus (1 x 108 CFU/ml in RPMI/H) were exposed to varied concentrations of H2O2 (Sigma-Aldrich) or HOCl (prepared from Clorox, commercial source) for 60 or 30 min, respectively, at 37°C in sterile Eppendorf tubes. Inasmuch as Clorox is the sodium salt of HOCl (sodium hypochlorite, pKa = 7.48), the pH was adjusted to 7–8 to convert it to a mixture of HOCl and sodium hypochlorite and to mimic our assay conditions. The concentration of HOCl under these conditions was then determined by spectrophotometric analysis after dilution of pH 7–8 HOCl in sodium hydroxide (pH 12) using an absorbance maximum at 292 nm (pH 12, {epsilon}292 = 350 M–1 cm–1) (26). Bacteria were plated on TSA and allowed to grow overnight. Viable CFUs were counted the following day and percent survival was calculated as described above. Time response experiments were performed using either 5 mM H2O2 or 25 µM HOCl (the concentrations at which ~70% of MW2 remained viable) and plating samples at various time intervals following exposure to oxidant. Percent survival was determined as described above. The pH of the above reactions was measured using a pH meter to ensure a physiological pH (pH range was between 7.2 and 7.5).

Neutrophil phagocytosis and bactericidal activity assays

Neutrophil phagocytosis assays were performed as described previously (13). To measure neutrophil bactericidal activity, PMNs (106) were combined with MW2 or {Delta}isdAB (107) in 96-well tissue culture plates and S. aureus survival was determined as described previously (13). Alternatively, PMNs (106) were treated with 5 µM diphenyleneiodonium chloride (DPI; Sigma-Aldrich) for 30 min at 37°C before adding MW2 or {Delta}isdAB (each at 107 CFUs). Bactericidal activity was determined as described above. The assay measures total number of viable ingested and uningested bacteria.

Catalase activity assays

MW2 wild-type and isogenic {Delta}isdAB strains and strains 502A and NCTC 12981 were grown to mid-exponential phase as described above. A 1-ml aliquot of each culture was centrifuged at 10,000 x g for 3 min and catalase activity in the supernatant was measured using the Amplex Red Catalase Assay kit (Molecular Probes) as described by the manufacturer.

S. aureus microarray experiments

MW2 was cultured to mid-exponential phase (OD600 = 0.75) in TSB as described above, washed with cold RPMI/H (chilled on ice) and resuspended to a density of 2.5 x 108 CFU/ml in RPMI/H as described above. Bacteria were then cultured at 37°C for up to 120 min in the presence or absence of 5 mM H2O2, 25 µM HOCl, 20 µg (100 µg/ml final concentration) neutrophil azurophilic granule proteins as indicated. All assays and incubations were performed at 37°C. The bacteria were lysed using 700 µl of RLT buffer (Qiagen) and the lysate was homogenized using an FP120 FastPrep system (Qbiogene). Total RNA was isolated with RNeasy kits (Qiagen). Contaminating DNA was removed using DNase (on column DNase treatment, Qiagen; off column DNase treatment, Turbo DNase). Fragmented and biotin-dUTP-labeled cDNA was generated from purified RNA as described by the Affymetrix Target Preparation protocol (www. affymetrix.com/support/downloads/manuals/expression_s3_manual.pdf), except that cDNA synthesis was performed using 20 µg of total RNA instead of 10 µg. To synthesize cDNA, random primers at 25 ng/ml (Invitrogen), 10 mM DTT, 0.5 mM dNTPs, 0.5 U/µl SUPERase · In (Ambion), and 25 U/µl SuperScript II (Invitrogen) were added to ~20 µg RNA in 1x first-strand reaction buffer. The remaining RNA was hydrolyzed by adding 1 N NaOH at 65°C for 30 min after which 1 N HCl was added to neutralize the reaction. cDNA purification was performed using a QIAquick PCR purification kit (Qiagen) with nuclease-free water substituted for the elution buffer. cDNA (~5 µg/sample) was fragmented using 0.6 U of DNase I (GE Healthcare) per mg of cDNA in One-Phor-All Buffer (Amersham Biosciences). Labeling of the 3'termini of the fragmentation products was completed as described in the protocol using 7.5 mM GeneChip DNA Labeling Reagent (Affymetrix), terminal deoxynucleotidyl transferase (Promega) in 5x reaction buffer. The reaction was terminated using 0.5 M EDTA. Biotinylated S. aureus cDNA was hybridized to custom Affymetrix GeneChips (RMLChip 3) with 99.3% coverage of genes from MW2 (2613 probe sets of 2632 ORFs; the remaining 0.7% are represented by identical probe sets from other staphylococci) and scanned according to standard GeneChip protocols (Affymetrix) (13). Precise details for Affymetrix hybridization and scanning protocols can be found at the above internet address. Each experiment was replicated 3–9 times with microbicide treated MW2 samples time-matched with untreated MW2 samples. Affymetrix GeneChip Operating Software (GCOS v1.4; www.affymetrix.com) was used to perform the preliminary analysis of custom MW2 chips at the probe-set level. All *.cel files, representing individual biological replicates, were scaled to a trimmed mean of 500 using a scale mask consisting of only the MW2 probe-sets to produce the *.chp files. A pivot table with all samples was created, including calls, call p-value and signal intensities for each gene. The pivot table was imported into GeneSpring GX 7.3 (Agilent; www.chem.agilent.com/scripts/pds.asp?lpage = 27881), and hierarchical clustering (condition tree) using a Pearson correlation similarity measure with average linkage was used to determine similarity of biological replicates (data not shown). The pivot table was also imported into Partek software (Partek) to produce a Principal Component Analysis plot as a secondary check on similarity of biological replicates (data not shown). After data had passed these preliminary statistical tests, biological replicates were combined into a custom worksheet (Microsoft Excel 2003; Microsoft Corporation) used to correlate replicates of all test conditions and controls. Quality filters based upon combined calls and signal intensities were used in the worksheet to further evaluate individual gene comparisons. Present and marginal calls were treated as the same. Absent calls were negatively weighted for the filters and dropped completely from further calculations. All individual genes passing the above filters and combined from all usable replicates have the ratios of test (MW2 treated with microbicide)/control (untreated MW2) reported with associated probability of Student’s t test values. Significance Analysis of Microarrays (27) was also performed using the Excel sheet with a column added containing the results. p values obtained from ANOVA (Partek) were filtered using the false discovery rate. Gene lists were generated with emphasis placed upon the quality and statistical filters mentioned above. To be included in the final gene list, in addition to the above criteria, gene expression must have been changed at least 2-fold for one of the treatments. A complete set of microarray data are provided in Table I in the supplemental material4 and posted on the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/, platform accession number GPL4692, series accession number GSE6716).

TaqMan real-time RT-PCR analysis

Experiments and RNA preparation for TaqMan analysis were performed with procedures and conditions identical with those used for the microarray experiments. TaqMan real-time RT-PCR analysis of three separate experiments (each assayed in triplicate) using each of the oxidants was performed using an ABI 7500 thermocycler (Applied Biosystems). Relative quantification of S. aureus genes was determined by the change in expression of target transcripts relative to gyrB (a housekeeping gene commonly used for calibration) according to the manufacturer’s protocol (Applied Biosystems Relative Quantification Manual at http://docs.appliedbiosystems.com/ genindex.taf, doc. index no. 4347824).

The primer-probe sequences used for confirmation of microarray data were as follows: gyrB forward primer 5'-CAAATGATCACAGCATTTGGTACAG-3', gyrB probe 5'-AATCGGTGGCGACTTTGATCTAGCGAAAG-3', gyrB reverse primer 5'-CGGCATCAGTCATAATGACGAT-3'; hlgC forward primer 5'-CCCTCTTGCCAATCCGTTATT-3', hlgC probe 5'-AAATGCTAAAGCTGCCAACGATACTGAAGACAT-3', hlgC reverse primer 5'-TTGATAATTTCTATATCGCTTCCTTTACC-3'; isdB forward primer 5'-GGCGTTGCATCTGTAGCAATT-3', isdB probe 5'-ATTTGTACCACCTGTTTCTTCAGCTGCTGCT-3', isdB reverse primer 5'-TGGACTTGCAACTGCTTCAGTT-3'; fer forward primer 5'-CGAAGGTATTGCTTTCGTAATCCT-3', fer probe 5'-CCTCAGGTACTTCTGCAGTACCTTGGTTATCGT-3', fer reverse primer 5'-ATCAATTGCATCTTCCATATCTTCAT-3'; fur forward primer 5'-GGCGTCGCTCGTTTTGATT-3', fur probe 5'-AAGGCGCAAAACATTTCCACCAT-3', fur reverse primer 5'-CACGACCACATTCCATACATACTAAAT-3'; perR forward primer 5'-TGCGACAAGCAGGCGTAAG-3', perR probe 5'-ATTACACCTCAAAGACAAGCAATATTACGTT-3', perR reverse primer 5'-GCTGTTGGATGAGTATGTGAAGAAAT-3'; lukM forward primer 5'-TGCTACTGTTGAAAACCCTGAACT-3', lukM (MW1942) probe 5'-CTTTGCTTCAAAATATAGATACCCAGCAT-3', lukM (MW1942) reverse primer 5'-AAATTCTGGATTAAAGCCACTTCTTACT-3'; malE forward primer 5'-TCGTTCGAAAGAAGATATTGATAAAGC-3', malE probe 5'-AAAGATAATTCTAAAGACAAGCCTAACCAACTT-3', malE reverse primer 5'-TCGCCATCCACCCACATC-3'; dltA forward primer 5'-ACGAATTCTTCTTCTGTGGTGAAAT-3', dltA probe 5'-CTACCTCACAGAGCAGCAAAAGCGTTAG-3', dltA reverse primer 5'-CGCACTTGGGA AACGGTTT-3'.

Statistics (excluding microarray analyses)

Statistics were performed with a one- or two-way ANOVA, one-way repeated-measures ANOVA and/or Dunnett’s or Bonferroni’s posttest for multiple comparisons as appropriate (GraphPad Prism version 4.0 or 5.0 for Windows; GraphPad Software), or by using a paired Student’s t test (Microsoft Excel 2002; Microsoft). A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
S. aureus is resistant to major neutrophil microbicides

As a first step toward understanding the ability of CA-MRSA to evade killing by human neutrophils, we tested staphylocidal activity of H2O2, hypochlorous acid (HOCl), or neutrophil azurophilic granule proteins. Initially, we evaluated staphylocidal activity of these neutrophil microbicides toward several strains of S. aureus, including those that are prominent causes of community- and healthcare-associated infections (Fig. 1; Refs. 6, 7, 8, 9, 10, 11, 12, 13, 14). Although there was concentration-dependent bactericidal activity of H2O2 toward each strain, there was noteworthy survival after exposure to high concentrations of peroxide (Fig. 1A). For example, MW2 survival was 69.4 ± 5.4% after a 60-min exposure to 5 mM H2O2 and there was only a slight increase in bactericidal activity after exposure to 100 mM H2O2 (survival was 44.5 ± 7.6%; Fig. 1A). In addition, survival was similar among the strains tested, suggesting that known differences in strain virulence are not due to varied capacity of each to moderate effects of H2O2 (Fig. 1A). Results with H2O2 were similar using assays performed in TSB (data not shown).


Figure 1
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  		FIGURE 1. Prominent strains of MRSA are highly resistant to killing by neutrophil-generated oxidants. A, S. aureus were cultured for 60 min in RPMI/H or PBS (MW2 only, gray circles) with varied concentrations of H2O2 (percent survival, left). Right, data represented by CFU/ml. B, S. aureus were cultured for 30 min in RPMI/H or PBS (MW2 only, gray circles) with varied concentrations of HOCl (% survival, left). Right, data represented by CFU/ml. C, pH of RPMI/H with varied concentrations of HOCl was measured immediately following addition of HOCl (•) and again at 30 min ({circ}). D, RPMI/H supernatants of S. aureus cultured for 30 min with varied concentrations of HOCl were monitored for monochloramine generation (A284). E, S. aureus strains MW2, 502A, and NCTC 12981 (ATCC 25923) were cultured with the indicated concentrations of HOCl or H2O2 for 30 or 60 min, respectively. For (A) and (B) (left), the dotted lines indicate percent survival at t = 0 min (based upon the input number of CFUs), which is 100% by definition. For (A) and (B) (right), the symbols within the dotted circle indicate input CFUs. There is growth in RPMI/H during the incubation period of the assay, thereby accounting for survival greater than 100%.

 
Survival was also comparable among these S. aureus strains following exposure to varied concentrations of HOCl (12.5–500 µM) (Fig. 1B). However, there was unexpected bi-phasic bactericidal/bacteriostatic activity of HOCl in RPMI/H (Fig. 1B). For example, survival of MW2 was 80.9 ± 6.4% and 114.4 ± 8.0% after a 30-min exposure to 25 and 500 µM HOCl, respectively (Fig. 1B). This effect was specific to assays performed in RPMI/H, because bactericidal activity of HOCl was concentration-dependent in PBS (Fig. 1B, gray circles). Notably, pH remained relatively constant (pH ~ 7.5) throughout the range of concentrations tested (Fig. 1C). In as much as monochloramines, such as those generated from amino acids, are more reactive than secondary or tertiary chloramines that form with higher concentrations of HOCl (28, 29), it is possible that levels of these molecules varied in our assays with RPMI/H, thus accounting for the biphasic phenomenon. To test this notion, we measured monochloramines produced by the combination of HOCl and RPMI/H using our assay conditions (Fig. 1D). Production of monochloramines followed the general trend of HOCl-mediated staphylocidal activity, as peak production of primary chloramines occurred in the presence of 25 µM HOCl (Fig. 1D). This observation provides strong support to the idea that HOCl-derived monochloramines are acting as the major microbicide in RPMI/H. By comparison, there was little monochloramine production in assays with PBS (data not shown), and therefore HOCl is probably directly microbicidal in assays with PBS.

Previous studies (30, 31, 32) have evaluated S. aureus susceptibility to neutrophil microbicidal oxidants. However, it is difficult to compare the earlier results with our current data because studies were typically performed using phosphate buffer (rather than RPMI/H) and historical S. aureus strains such as 502A and NCTC 12981 (30, 31, 32). To that end, we compared survival of MW2 with S. aureus strains 502A and NCTC 12981 after exposure to HOCl and H2O2 using our assay conditions (Fig. 1E). Although survival varied among the strains, each was relatively resistant to killing by H2O2 and HOCl (e.g., survival was 51.8 ± 5.5%, 53.5 ± 2.9%, and 73.5 ± 6.3% for MW2, 502A, and NCTC 12981, respectively, after a 60 min exposure to 100 mM H2O2) (Fig. 1E).

Based on the range of concentrations tested with RPMI/H, we selected 5 mM H2O2 and 25 µM HOCl for time-response experiments, because they represent sublethal concentrations of each microbicide (Fig. 2, A and B). Moreover, these concentrations have been estimated previously to exist within neutrophil phagosomes (31, 33, 34, 35, 36, 37); albeit, recent studies indicate the steady-state concentration of H2O2 is much lower (38). As with the concentration-dependent killing of S. aureus by either H2O2 or HOCl, there was limited time-dependent effect on viability (i.e., <100% survival) of MW2 following exposure to these microbicides (Fig. 2, A and B). In contrast, H2O2 and HOCl had significant bacteriostatic activity (i.e., inhibition of growth) toward MW2 for up to 180 min, the longest time point tested (p < 0.05) (Fig. 2, A and B). These observations are consistent with the previously reported bacteriostatic activity of neutrophils toward MW2 (13, 39).


Figure 2
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  		FIGURE 2. MW2 is resistant to killing by H2O2 or HOCl over time. A, MW2 was cultured in RPMI/H alone ({circ}) or with 5 mM H2O2 (•) at the indicated times. B, MW2 was cultured in RPMI/H alone ({circ}) or with 25 µM HOCl (•) for the indicated times.

 
We next tested susceptibility of the S. aureus strains to neutrophil azurophilic granule proteins, which confer much of the oxygen-independent killing of microbes by human neutrophils (Fig. 3A) (40). At 100 µg/ml, neutrophil azurophilic granule proteins were bacteriostatic toward S. aureus, but lacked significant staphylocidal activity (i.e., <100% survival) within 60 min (Fig. 3, A and B). By comparison, there was significant killing of Streptococcus pyogenes using the same concentration of azurophilic granule proteins (survival was reduced to 15.2 ± 4.9%, p < 0.001 vs input), confirming that acid extracted granule proteins retained antibacterial activity (Fig. 3A). There was significant bactericidal activity of azurophilic granule proteins toward MW2, but this required a much higher concentration of protein and longer periods of incubation (400 µg/ml after 180 min) (Fig. 3B). We note that the highest concentration of granule protein tested in this study is still less than that estimated recently to exist in neutrophil phagosomes under optimum conditions (31).


Figure 3
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  		FIGURE 3. Prominent strains of MRSA are resistant to killing by neutrophil azurophilic granule proteins (az-Grn). A, MRSA or Streptococcus pyogenes were cultured for 60 min in RPMI/H alone ({square}) or with 100 µg/ml neutrophil az-Grn ({blacksquare}) Left, Percent survival. Right, Average CFU/ml for the assays. B, MW2 was cultured for the indicated times for RPMI/H alone ({circ}) or with varied amounts of neutrophil az-Grn as indicated (•).

 
Solubilized proteins of neutrophil specific granules (up to 107 cell equivalents per assay) failed to reduce S. aureus viability (data not shown). Consistent with these findings, LL-37, the primary antimicrobial peptide of specific granules (41), had limited bactericidal activity toward MW2 (MIC50 was ~45 µM or 205 µg/ml; Ref. 42), a concentration much higher than required for S. pyogenes (MIC50 = 1.65 µM (43)). Therefore, we chose to evaluate effects of azurophilic rather than specific granule proteins for the remainder of the studies.

Because previous studies have shown that MW2 is resistant to killing by PMNs, causes rapid neutrophil lysis, and is highly virulent (13, 39), and because there were generally limited differences in susceptibility to microbicides among the strains, we used MW2 for the remainder of the studies.

Global changes in S. aureus gene expression after exposure to neutrophil components

As a step toward elucidating the molecular mechanisms used by S. aureus to evade killing by neutrophil components, we measured global changes in MW2 expression after exposure to 5 mM H2O2, 25 µM HOCl, or 100 µg/ml azurophilic granule proteins. We found that 33.6–45.9% of the genes in MW2 were differentially regulated at any given time following challenge with ROS or azurophilic granule proteins (e.g., 1195, 1199, 1140, and 853 genes at 15, 30, 60, and 120 min, respectively, were up- or down-regulated) (Fig. 4 and supplemental Table I). These results are consistent with previous studies using MW2 and intact human PMNs (13). Genes differentially expressed after exposure to microbicides were divided into categories based on function or annotation. The most robust changes in MW2 transcript levels were observed after culture with H2O2 and azurophilic granule proteins (Fig. 5 and supplemental Table I). Genes encoding proteins with unknown function and those involved in metabolism comprised the two largest categories of up- or down-regulated S. aureus transcripts (500 and 410 differentially regulated transcripts, respectively; supplemental Table I).


Figure 4
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  		FIGURE 4. Changes in patterns of (USA400) MW2 gene expression after exposure to neutrophil microbicides. Transcriptome analysis of MW2 was performed with custom Affymetrix microarrays containing probe sets encompassing 2613 of 2631 (99.3%) MW2 open reading frames (ORFs); the remaining 0.7% is represented by identical probe sets from other staphylococci. (A) Increases or (B) decreases in levels of MW2 transcripts after exposure to az-granule proteins (az-Grn, 100 µg/ml), H2O2 (5 mM) or HOCl (25 µM) in RPMI/H.

 

Figure 5
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  		FIGURE 5. MW2 transcripts are differentially expressed following challenge with neutrophil microbicides. Microarray results are presented as the mean fold-change of three separate experiments (see scale bar). White asterisks indicate genes called "absent" before (for up-regulated genes) or after (for down-regulated genes) exposure to microbicides. az-Grn, human neutrophil azurophilic granule proteins. Complete microarray data are provided in Table I in the supplemental material.

 
Genes encoding proteins that facilitate virulence and defense mechanisms, toxins, hemolysins, and proteases are up-regulated by exposure to azurophilic granule proteins

Within 15 min after exposure to microbicides, at least 71 transcripts encoding virulence molecules and those involved in defense mechanisms were up-regulated (Fig. 5 and supplemental Table I). Notably, the greatest number of genes encoding key virulence determinants was up-regulated by exposure to azurophilic granule proteins (Fig. 5 and supplemental Table I). For example, transcripts encoding extracellular matrix binding protein (ssp), fibrinogen- and fibronectin-binding proteins (e.g., fnbB), IgG-binding protein (sbi), von Willebrand factor-binding protein (VWbp), {alpha}-hemolysin (hla), {delta}-hemolysin (hld), {gamma}-hemolysin (hlgA, hlgB, hlgC), Panton-Valentine leukocidin (lukS-PV, lukF-PV), leukotoxin M (MW1941, MW1942), staphylococcal enterotoxin C4 (sec4), and eleven putative staphylococcal exotoxins (set17–set26), were up-regulated after exposure to azurophilic granule proteins (Fig. 5). By comparison, H2O2 or HOCl either failed to trigger changes in these transcripts or changes were typically delayed or muted.

S. aureus genes involved in transcriptional regulation and cell envelope biosynthesis are differentially regulated by neutrophil components

Transcripts encoding S. aureus molecules involved in gene regulation, including two-component gene regulatory systems SaeRS, KdpDE, and LytRS, were up-regulated after exposure to each microbicide (Fig. 5 and supplemental Table I). These findings indicate that the observed increases in these transcripts following PMN phagocytosis (13) are a general response to microbicides, rather than due to binding or phagocytosis per se. By comparison, vraR and vraS (vraRS) were up-regulated primarily after exposure to azurophilic granule proteins (Fig. 5). VraRS coordinates key steps in cell wall biosynthesis and is induced after cell wall injury (44), consistent with our findings using azurophilic granule proteins and peptides (Fig. 5), some of which are known to disrupt the bacterial cell envelope (45, 46). Transcripts encoding accessory gene regulator (Agr) subunits B and D (agrB and agrD) were up-regulated by at least one time point with two of the three treatments, and there was a uniform decrease in sarA transcript within 30 min (Fig. 5 and supplemental Table I). Because these results are at variance with those following phagocytosis by intact PMNs (13), other factors within intact PMN phagosomes contribute to altered levels of these molecules.

Transcripts encoding DltA, DltB, and DltD (dltA, dltB, and dltD), multiple peptide resistance factor MprF (mprF, annotated as fmtC) and VraFG (vraF and vraG) were up-regulated within 15 min by exposure of MW2 to neutrophil azurophilic granule proteins (Fig. 5). Resistance of S. aureus to neutrophil {alpha}-defensin, an azurophilic granule protein component, has been attributed to the dlt operon, which mediates incorporation of D-alanine into teichoic acids of the staphylococcal cell envelope and regulates cell envelope-dependent processes, such as activity of autolytic enzymes and binding of divalent cations (47, 48).

Genes encoding stress response proteins are up-regulated

Transcripts encoding proteins associated with a response to ROS were up-regulated by each of the microbicides, including thioredoxin and thioredoxin reductase (trxA and trxB), general stress protein (dps), superoxide dismutase A and M (sodA and sodM), glutathione peroxidase (bsaA), and alkyl hydroperoxide reductase subunits C and F (ahpC and ahpF) (Fig. 5 and supplemental Table I). It is possible that these transcripts are up-regulated by neutrophil azurophilic granule proteins because they comprise a general response to stress triggered by a gene regulatory molecule, as suggested above.

Hydrogen peroxide challenge up-regulates S. aureus genes encoding proteins that are associated with metal response elements

Forty-five MW2 transcripts related to metal uptake or use were increased following exposure to H2O2 (Fig. 5 and supplemental Table I). By comparison, only a limited number of these molecules were up-regulated after treatment with azurophilic granule proteins and HOCl (10 and 19 transcripts, respectively) (Fig. 5 and supplemental Table I). Transcripts associated with the iron and/or heme acquisition, including those encoding a ferric siderophore transport system (fhuA, fhuB, fhuD, and fhuG), iron-regulated surface determinants (isdA, isdB, and isdCDEFsrtBisdG), ferritin (fer), and staphylococcal iron-regulated proteins (sirA, sirB, and sirC), were among those up-regulated after exposure to H2O2 (Fig. 5 and supplemental Table I). In contrast, these changes in gene expression failed to occur after treatment with azurophilic granule proteins (some of the transcripts were down-regulated) and levels of transcripts were changed only slightly and/or at the latest time point after exposure to HOCl (Fig. 5). Moreover, perR and fur, transcripts encoding ferric uptake regulators, were increased following treatment with H2O2 (Fig. 5 and supplemental Table I). These results were confirmed by TaqMan real-time RT-PCR, which were generally consistent with the microarray data (Fig. 6). Consistent with these collective findings, previous studies suggest PerR functions as a sensor for peroxide, responds to stress imparted by HOCl, and directly regulates expression of Fur (49).


Figure 6
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  		FIGURE 6. TaqMan real-time RT-PCR confirmation of microarray results. Genes (n = 8) identified as differentially expressed by microarray analysis were selected for confirmation by TaqMan real-time RT-PCR using strains MW2, MRSA252 and COL. Transcripts were measured at 15, 30, or 60 min after exposure to az-Grn (100 µg/ml, red bars), H2O2 (5 mM, green bars) or HOCl (25 µM, blue bars) as indicated. There was general consistency (79.2%) between MW2 TaqMan and microarray results as previously described (13 ).

 
As a step toward determining whether the changes in transcript levels following exposure to specific neutrophil microbicides comprised a common S. aureus response, we compared levels of selected transcripts in MW2 with MRSA252 and COL using TaqMan real-time RT-PCR (Fig. 6). Although there were some strain-to-strain differences (e.g., malE in strain COL after exposure to azurophilic granule proteins), the responses were relatively similar among the strains tested (Fig. 6). The results are consistent with the observed comparable resistance of these strains to neutrophil microbicides (Figs. 1–3).

Iron-regulated surface determinants (Isd) are important for resistance to killing by human neutrophils and H2O2

Although molecules involved in iron and/or heme uptake and use are associated with the oxidative stress response in S. aureus (50, 51, 52), the contribution of individual components of this response to survival is incompletely characterized. The Isd proteins participate in the shuttling of heme-iron across the staphylococcal cell envelope from host iron sources and is likely the predominant heme-iron uptake system at the surface of S. aureus (18, 53, 54, 55). The increase in isdA, isdB, and isdCDEFsrtBisdG after exposure of MW2 to H2O2 suggests that these transcripts are important for resistance to peroxides and thus may contribute to survival after interaction with phagocytic leukocytes. To test this notion, we evaluated survival of MW2 wild-type and isogenic isdAB-negative ({Delta}isdAB) strains after phagocytosis by human neutrophils and following exposure to H2O2 (Fig. 7). Compared with the wild-type strain, survival of {Delta}isdAB was decreased significantly by 15 min after interaction with human neutrophils (survival was 63.3 ± 5.8% for MW2 and 47.5 ± 5.1% for {Delta}isdAB, p < 0.05) and there was a trend for reduced survival of the mutant strain at all later time points (Fig. 7C). Reduced survival of {Delta}isdAB was restored completely by pretreating human neutrophils with DPI, an inhibitor of NADPH oxidase (Fig. 7D). There were no significant differences in phagocytosis, consistent with the notion that reduced survival of {Delta}isdAB compared with MW2 was due to resistance to NADPH-derived oxidants (Fig. 7E). In accordance with those findings, survival of {Delta}isdAB was reduced 22.6% compared with the wild-type strain following exposure to 5 mM H2O2 (survival was 84.8 ± 2.7% for MW2 vs 65.6 ± 1.8% for {Delta}isdAB, p = 0.001; Fig. 7F). Susceptibility of the {Delta}isdAB mutant strain to H2O2 was not a function of catalase activity, as the enzyme activity level of {Delta}isdAB was comparable to that of the wild-type strain (data not shown). Taken together, these results indicate IsdA and/or IsdB promote resistance to H2O2 and killing by neutrophils (Fig. 7, C–F). Moreover, the data suggest heme-uptake is important for resistance to H2O2 and/or that absence of IsdA and IsdB, major protein components of the cell envelope (56, 57), alter properties of the cell envelope such that the bacterium has increased sensitivity to H2O2 and/or components of innate host defense. Consistent with this idea, recent studies by Clarke et al. (58) demonstrated that IsdA reduces surface hydrophobicity of S. aureus, thereby, rendering the pathogen more resistant to bactericidal fatty acids and peptides.


Figure 7
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  		FIGURE 7. The isdA and isdB genes are important for resistance to H2O2 and evasion of killing by neutrophils. A, Growth curves of MW2 wild-type and isogenic isdAB mutant ({Delta}isdAB) S. aureus strains were performed in TSB. Left panel, OD600. Right panel, CFU/ml. Evaluation of MW2 and {Delta}isdAB by microscopy revealed no differential clumping between the strains. B, DNA was isolated from overnight cultures of MW2 and {Delta}isdAB, and PCR was performed using isdB primers (forward primer 5'-GGCGTTGCATCTGTAGCAATT-3', reverse primer 5'-TGGACTTGCAACTGCTTCAGTT-3') to generate a 150 bp product. C, Killing of MW2 and {Delta}isdAB by human PMNs. D, DPI inhibits increased susceptibility of {Delta}isdAB to killing by human PMNs. E, Phagocytosis of MW2 and {Delta}isdAB by human neutrophils. F, MW2 and {Delta}isdAB were cultured for 60 min in RPMI/H with 5 mM H2O2. Percent S. aureus survival during interaction with neutrophils was determined using the equation (CFU+PMN at t/CFU+PMN at t0).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Neutrophils are the first line of cellular defense against bacterial pathogens. As such, individuals with neutropenia or defects in neutrophil function are highly susceptible to severe bacterial infections, especially those caused by S. aureus (59, 60). In contrast, CA-MRSA causes disease in otherwise healthy individuals with no obvious risk factors for infection (3, 6, 7, 8, 9, 61). CA-MRSA strains that represent the leading cause of community-associated infections in the United States (i.e., USA300-0114 (LAC) and USA400 (MW2)) have enhanced ability to avoid destruction by human PMNs and cause rapid host cell lysis (13, 39). Herein, we demonstrate that prominent S. aureus strains are highly resistant to key neutrophil microbicides (Figs. 1–3). The apparent lack of staphylocidal activity by individual neutrophil microbicides may be due to the simplicity of our in vitro assay system, which does not represent fully the complex milieu of microbicidal components in the phagosome. In preliminary experiments, we tested the microbicidal activity of combinations of azurophilic granule proteins, H2O2, and HOCl toward MW2, but viability remained unchanged compared with exposure to any of the three microbicidal components alone (data not shown). As a step toward understanding this resistance, we evaluated MW2 gene expression following exposure of the pathogen to H2O2, HOCl, and neutrophil azurophilic granule proteins, and discovered patterns of gene expression that explain in part the ability of S. aureus to circumvent destruction by neutrophils.

Neutrophil azurophilic granule proteins, H2O2, and HOCl triggered changes in MW2 gene expression common to all three treatments (Fig. 5 and supplemental Table I), thereby constituting a general "stress response" in S. aureus that promotes survival. Our data reveal mechanisms that likely contribute to the resistance of MW2 to neutrophil azurophilic granule proteins. Proteins encoded by dltABCD, mprF, and vraFG promote resistance to cationic antimicrobial peptides (62, 63) and each of these transcripts was up-regulated within 15 min after exposure to azurophilic granule proteins (Fig. 5 and supplemental Table I). These findings are consistent with induction of the antimicrobial peptide sensor (aps) (64). In addition, transcripts encoding teichoic acid biosynthesis proteins B and D (tagB and tagD) and VraRS (vraRS) were increased after treatment with azurophilic granule proteins (Fig. 5 and supplemental Table I).

Previous studies (65) used a microarray-based approach to investigate S. aureus (strain NCTC 8325) responses to H2O2 and identified changes in transcripts associated with metabolism and DNA repair. In contrast to our work, they reported limited changes in molecules associated with a general stress response or those known to be involved in the defense against ROS (e.g., ahpC, ahpF, bsaA, perR, sodM, trxA, and trxB) or heme uptake (e.g., isdAB, isdC, isdD, isdE, isdF, and isdG) (65). The basis for the noted variance in patterns of gene expression is unclear, but might be related to differences in assay culture conditions (Luria-Bertani broth vs RPMI/H).

Although patterns of gene expression were in general similar between MW2 treated with H2O2 or HOCl, there were key differences (Fig. 5 and supplemental Table I). For example, some of the changes in transcripts triggered by HOCl were muted compared with those elicited by H2O2. More notably, HOCl typically failed to cause changes in transcripts encoding proteins associated with metal and/or heme-iron uptake, or the magnitude of change was reduced by comparison (Fig. 5 and supplemental Table I). This finding is unexpected because changes in transcripts encoding gene regulators and stress response molecules following exposure of MW2 to HOCl and H2O2 are similar (Fig. 5). There are several possible explanations for this observation. First, transition metals (Fe2+ and Cu+) are known to undergo Fenton chemistry with H2O2 to generate other ROS, such as highly reactive hydroxyl radical (·OH). Sequestration of transition metals by S. aureus would potentially restrict generation of secondary ROS within phagosomes, thereby limiting oxidative damage caused by these molecules. Second, many redox proteins have metal centers (e.g., catalase and superoxide dismutase) or are regulated by metal-sensitive mechanisms (alkyl hydroperoxide reductase) (66), and previous studies have highlighted the importance of iron in the peroxide stress response by S. aureus (50, 51, 52). In the present studies, we used MW2 wild-type and isogenic isdAB-negative S. aureus strains to discover a previously unrecognized role for the IsdA and/or IsdB in the defense against H2O2 and evasion of killing by human neutrophils (Fig. 7). This phenomenon may be related to altered heme-uptake or modification of the surface characteristics of staphylococci (58), thereby conferring increased susceptibility to peroxides and possibly other components of innate host defense.

We reported previously global changes in S. aureus gene expression, including those in strain MW2, that occur following PMN phagocytosis (13). The present study was directed in part to identify the neutrophil-derived effectors of that S. aureus response as means to better understand how the pathogen circumvents killing by neutrophils. Compared with postphagocytosis gene expression, there were many similar changes in S. aureus transcripts following exposure of MW2 to individual neutrophil microbicides (compare Fig. 5 and supplemental Table I with data in Refs. 13 , 39). For example, transcripts encoding numerous toxins and hemolysins, such as {alpha}- and {gamma}-hemolysin (hla and hlgABC), Panton-Valentine leukocidin (lukS-PV and lukF-PV) (13, 39), enterotoxin C (sec4; hybridization was to sec3 in Refs. 13 , 39), aerolysin/leukocidin (MW1941, MW1942), and a number of exotoxins (e.g., set26, set17, and set25; named differently in Ref. (13), were up-regulated after phagocytosis and following exposure to neutrophil microbicidal components (Fig. 5 and Ref. 13). Transcripts involved in stress response, gene regulation, and virulence/defense mechanisms, including ahpC, ahpF, bsaA, clpC, dps, groEL, groES, sodA, trxA, trxB, saeRS, vraRS, kdpDE, sbi, ssp, lrgA, lrgB, and psm, were up-regulated after phagocytosis (13, 39) and by one or more of the individual neutrophil microbicides tested (Fig. 5). There were also transcripts similarly down-regulated after phagocytosis and following exposure to at least one of the neutrophil microbicides, including clfB, sdrC, rsbV, and sigB (Fig. 5). These data provide an enhanced understanding of the postphagocytosis transcriptome reported for S. aureus (13, 39) (e.g., up-regulation of lukS/F-PV after phagocytosis is due to effects of azurophilic granule proteins).

In contrast, some of the changes in gene expression after exposure to microbicides, such as differential regulation of agrB, agrD, hld, isdB, rot, sarA, rsbU, and hrcA, were at variance with the changes observed after phagocytosis (compare Fig. 5 with data in Refs. 13 , 39). Moreover, levels of some transcripts changed only after phagocytosis or exposure to microbicides, such as spa, epiEF, katA, splA, splB, splC, splF, sspA and sspC. The difference between the postphagocytosis S. aureus transcriptome and that following exposure to microbicides might be explained by the complexity of the neutrophil phagosome, because S. aureus is exposed to multiple microbicides concurrently after phagocytosis and many were not tested in our studies.

In summary, we used a microarray-based approach to generate a comprehensive view of the CA-MRSA response to neutrophil microbicides, thereby providing a better understanding of how the pathogen avoids killing by the human innate immune system. An enhanced understanding of the host-pathogen interface at both the cell and molecular levels is critical for treatment and control of infections caused by this emerging human pathogen.


   Acknowledgments
 
We thank Dr. Mark T. Quinn for critical review of the manuscript and Dr. Jovanka M. Voyich for technical advice.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This research was supported by the Intramural Research Program of the National Institutes of Health, National Institutes of Allergy and Infectious Diseases (to F.R.D.), a research grant from Johnson & Johnson Pharmaceutical Research and Development (to B.N.K.), and a United States Public Health Service Grant (AI69233) from the National Institute of Allergy and Infectious Diseases (to E.P.S). M.L.R. is supported by a National Institutes of Health Training Grant in Mechanisms of Vascular Disease T32 HL07751. Back

2 Address correspondence and reprint requests to Dr. Frank R. DeLeo, Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840. E-mail address: fdeleo{at}niaid.nih.gov Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; ROS, reactive oxygen species; CA, community associated; MRSA, methicillin-resistant S. aureus; TSB, tryptic soy broth; TSA, tryptic soy agar; HOCl, hypochlorous acid; DPI, diphenyleneiodonium chloride; Isd, Iron-regulated surface determinant. Back

4 The online version of this article contains supplemental material. Back

Received for publication July 24, 2007. Accepted for publication October 28, 2007.


   References
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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