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* Laboratory of Human Bacterial Pathogenesis,
Rocky Mountain Veterinary Branch, and
Rocky Mountain Microscopy Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;
Public Health Research Institute Tuberculosis Center, International Center for Public Health, Newark, NJ 07103; and
¶ Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor University College of Medicine, Houston, TX 70030
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen causes a variety of diseases including impetigo, cellulitis, food poisoning, toxic shock syndrome, necrotizing pneumonia, endocarditis, and sepsis (1). Risk of S. aureus infection is increased during hospitalization, after surgery or dialysis, and in patients with indwelling percutaneous medical devices and/or catheters (1). Notably, there is an alarming increase in the incidence of community-acquired (CA) S. aureus infections in seemingly healthy individuals, a problem which underscores the need to better understand virulence mechanisms (2). A number of factors are known to contribute to pathogenesis (1, 2, 3, 4, 5, 6); however, a comprehensive analysis of changes in S. aureus gene expression that occur during interaction with the human innate immune system has not been performed. Furthermore, our understanding of the molecular mechanisms used by pathogens to avoid destruction by innate host defense is limited. To that end, we studied S. aureus pathogenesis and global gene expression during phagocytic interaction with human PMNs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Staphylococcus aureus strains MW2 (5, 7), MnCop (8), LAC (9, 10, 11, 12), MRSA252 (13), and COL (14) were selected carefully for this study based on several criteria, including the type of human disease caused by each (e.g., MW2 as the prototype for CA methicillin-resistant S. aureus (MRSA), and MRSA252 for its high prevalence in hospitals in the United States and United Kingdom), available genome sequence information (MW2, MRSA252, and COL), historical significance (COL), available genotype information (e.g., MLST, spa type, agr type, and SCCmec type), and methicillin susceptibility (MnCop). Specifically, MW2 (pulsed-field type USA400) was originally isolated from a healthy 16-mo-old girl in North Dakota who died of septicemia and septic arthritis in 1998 (7, 15). LAC (pulsed-field type USA300) has caused recent S. aureus outbreaks in athletes, many of which are skin infections (10, 11, 16). Recent reports also describe USA300 as the causative agent of staphylococcal necrotizing fasciitis (9) and severe community-onset pneumonia in healthy adults (12). MnCop was originally isolated from an 8-mo-old infant who had S. aureus toxic shock syndrome (8). Thus, each of the CA strains used in this study, i.e., MW2, MnCop, and LAC, has been linked to infections in individuals with no known risk factors. In contrast, MRSA252 (pulsed-field type identical to EMRSA16), is the second leading cause of healthcare-associated S. aureus infections in the United States (16). Descriptions of each strain are summarized in Table I.
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Mouse model of S. aureus disease
All animal studies conformed to National Institutes of Health guidelines and were approved by the Animal Use Committee at Rocky Mountain Laboratories. Female CD1 Swiss mice were purchased from Charles River. Animals were between 10 and 12 wk of age, housed in microisolator cages, and received food and water ad libitum. S. aureus strains were grown to E1, washed twice with sterile DPBS, and resuspended to 108/100 µl in DPBS. Mice were inoculated with 108 S. aureus or with sterile DPBS via the tail vein. The experiment was performed once with 10 mice per S. aureus strain and 5 mice for DPBS control. Criteria for determining morbidity/sickness in mice included hunched posture, decreased activity, ruffled fur, and labored breathing. Animals were euthanized if unable to eat or drink, or if they became immobile. All mice were euthanized by 48 h. Survival statistics were performed with a log-rank test (GraphPad Prism version 4.0 for Windows; GraphPad Software).
To determine whether S. aureus had disseminated to large organs during bacteremia, liver and lung tissues were harvested at time of animal death, homogenized, and aliquots of each homogenate were plated on tryptic soy agar. Colonies were enumerated the following day. Alternatively, parts of the liver, lung, kidney, heart, and brain were collected in parallel for histopathology. Tissues were fixed in 10% neutral-buffered formalin, processed for 12 h in an Excelsior Tissue Processor (ThermoShandon), and dehydrated using a graded series of ethanol. Tissues were then cleared with Histosolve (ThermoShandon) and infiltrated and embedded in paraffin. Paraffin blocks were sectioned at 5 microns and slides were stained with H&E. A trained veterinary pathologist (D. J. Gardner) examined all of the tissue samples. Statistics for dissemination of S. aureus to the liver and lung were performed with a Kruskal-Wallis analysis using Dunn’s posttest for multiple comparisons. Images of organ sections were sharpened and adjusted equally for brightness and contrast with Photoshop CS (Adobe Systems).
Isolation of human PMNs
PMNs were isolated from heparinized venous blood of healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Briefly, blood was incubated for 20 min at room temperature at a 1:1 ratio with 0.9% sodium chloride (Irrigation USP; Baxter Healthcare) containing 3.0% Dextran T-500 (Amersham Biosciences) to sediment erythrocytes. The leukocyte-containing supernatant was centrifuged at 670 x g for 10 min and resuspended in 35 ml of 0.9% sodium chloride. The cell suspension was underlaid with 10 ml of Ficoll-PaquePLUS (1.077 g/L; Amersham Biosciences) and centrifuged for 25 min to separate PMNs from PBMCs. PBMCs were removed by aspiration and erythrocytes were lysed with water (Irrigation USP; Baxter Healthcare) for 15–30 s followed by immediate mixing with 1.7% sodium chloride. Purified PMNs were centrifuged at 380 x g, resuspended in RPMI/H, and enumerated by microscopy. Purity of PMN preparations and cell viability were assessed by flow cytometry (FACSCalibur; BD Biosciences). Cell preparations contained 
99% PMNs and all reagents used contained <25.0 pg/ml endotoxin (Limulus Amebocyte Lysate assay; Fisher Scientific).
Phagocytosis of S. aureus by human PMNs
Phagocytosis of S. aureus by human PMNs was determined with fluorescence microscopy as previously described (17). Bacteria were grown to E1, opsonized with 50% normal human serum for 30 min at 37°C, and washed in DPBS. After opsonization, bacteria were labeled with AlexaFluor 488 (Molecular Probes) in DPBS for 20 min at 37°C, and unlabeled fluorochrome was removed by two washes with DPBS. PMNs (3 x 105) were added to serum-coated glass coverslips in 24-well tissue culture plates and allowed to adhere at room temperature for 15 min. Cells were chilled on ice for 10 min and AlexaFluor 488-labeled bacteria were added at a ratio of 10 bacteria:PMN. Plates were then centrifuged at 380 x g for 8 min at 4°C to synchronize phagocytosis (18). Samples were incubated at 37°C in a CO2 incubator for the indicated times (t = 0 was processed immediately after centrifugation), medium was removed from the wells by aspiration, and cells were fixed on ice for 30 min with 4% paraformaldehyde. Fixative was removed by aspiration, and uningested bacteria were counterstained with AlexaFluor594 conjugated-Ab specific for AlexaFluor 488 for 15 min at room temperature. Samples were visualized with a Zeiss Axioskop 2 Plus fluorescence microscope (Carl Zeiss) and imaged with a Zeiss Axiocam digital camera. The total number of S. aureus bound and/or ingested was evaluated in 50 neutrophils per assay from at least five separate fields of view. Percent phagocytosis was calculated with the equation: (number of ingested bacteria per cell/total number of PMN-associated bacteria per cell, bound or ingested) x 100. Statistics were performed with a repeated-measures ANOVA and Tukey’s posttest for multiple comparisons (GraphPad Prism version 4.0 for Windows; GraphPad Software). Images were adjusted for brightness and contrast with Photoshop CS (Adobe Systems).
PMN ROS production
PMN ROS production was measured using a previously described method (18) with minor modifications. PMNs were incubated with 25 µM 2', 7'-dihydrodiclorofluorescein diacetate (DCF; Molecular Probes) for 20–30 min at room temperature in RPMI/H. DCF-containing PMNs (106) and opsonized S. aureus were combined in wells of a 96-well microtiter plate at 4°C (ratio of 10 bacteria:PMN), centrifuged for 8 min at 380 x g, and transferred to a microplate fluorometer (Spectramax Gemini; Molecular Devices). ROS production was measured continuously at 1-min intervals for up to 180 min at 37°C using excitation and emission wavelengths of 485 and 538 nm, respectively. The rate of PMN ROS production over time (second-order kinetics) were determined from the Vmax (max increase in fluorescence) within each 10-min time period.
PMN bactericidal activity
Killing of S. aureus by human PMNs was determined as described (17) with some modification. Briefly, PMNs (106) were combined with 107 opsonized S. aureus in 24-well tissue culture plates, centrifuged at 380 x g for 8 min and incubated at 37°C for up to 6 h. At indicated times, PMNs were lysed with 0.1% saponin (20 min on ice) and S. aureus were plated on tryptic soy agar. Colonies were enumerated the following day, and percent S. aureus survival was calculated with the equation (CFU+PMN at t/CFU+PMN at t0) x 100. The assay measures total number of viable ingested and uningested bacteria. Statistics were performed with a repeated-measures ANOVA and Tukey’s posttest for multiple comparisons (GraphPad Prism).
S. aureus-induced PMN lysis
Following phagocytosis of S. aureus, PMN lysis was determined with a standard assay for release of lactate dehydrogenase (LDH) as described by the manufacturer (Cytotoxicity Detection kit; Roche Applied Sciences). Alternatively, PMN lysis was determined by counting intact cells on a hemacytometer. Statistics were performed with a repeated-measures ANOVA (LDH release) or one-way ANOVA (hemacytometer counts) and Tukey’s posttest for multiple comparisons (GraphPad Prism).
Electron microscopy
For transmission electron microscopy (TEM), phagocytosis assays were performed as described above, but with the following modifications. PMNs (3 x 105) were combined with 3 x 106 serum-opsonized S. aureus in wells of a 24-well tissue culture plate containing serum-coated Thermanox coverslips (Nalge Nunc International). Cells were incubated at 37°C with 5% CO2 for up to 180 min. At the indicated times, cells were fixed in Karnovsky’s fixative containing 4% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2 (Electron Microscopy Sciences). Samples were washed twice in 0.1 M phosphate, and postfixed in 1% osmium/0.8% potassium ferrocyanide in 0.1 M phosphate. Following two additional washes in water, samples were stained in-block with 1% uranyl acetate in water, washed again, and then dehydrated in ethanol, and embedded in Spurr resin (Ted Pella), as described (19). Thin sections were examined on a model H7500 TEM (Hitachi High Technologies) at 80 kV. Images were captured with an Advantage HR digital camera system (Advanced Microscopy Techniques) and adjusted for brightness and contrast with Photoshop CS (Adobe Systems). For scanning electron microscopy, phagocytosis assays were performed as described above, but with the following modifications. Cells were fixed in 0.15 M cacodylate buffer, pH 7.2, containing 2.5% glutaraldehyde and washed twice in cacodylate buffer. Samples were then postfixed with 1.0% osmium tetroxide in cacodylate buffer, washed twice with water, dehydrated with ethanol, and critical-point dried through carbon dioxide. After mounting on stubs, the samples were lightly coated with iridium using an ion beam sputterer (South Bay Technology), and examined with a Hitachi S4500 field emission scanning electron microscope (Hitachi High Technologies America). Digital images were collected with an Orion system (Focused Resolutions), and adjusted for brightness and contrast with Photoshop CS (Adobe Systems).
Analysis of S. aureus gene expression
To compare transcript levels in vitro, S. aureus strains were cultured to the desired phase of growth and 2.5 x 109 cells were lysed with 700 µl of RLT buffer (Qiagen). Lysates were homogenized with an FP120 FastPrep system (QBiogene). Total RNA was isolated with RNeasy kits (Qiagen) and contaminating chromosomal DNA was removed by DNase treatment (Qiagen). Purified bacterial RNA was used to prepare fragmented and biotin-dUTP-labeled cDNA according to standard Affymetrix prokaryotic target preparation (
www.affymetrix.com/support/downloads/manuals/expression_s3_manual.pdf
). Briefly, cDNA was synthesized from 10 µg of total RNA in 1x first-strand buffer containing 25 ng/µl random primers (Invitrogen Life Technologies), 10 mM DTT, 0.5 mM dNTPs (Amersham), 0.5 U/µl SUPERase In (Ambion), and 25 U/µl SuperScript II (Invitrogen Life Technologies). Remaining RNA was removed by hydrolysis with 1 N NaOH and the reaction was neutralized with 1 N HCL. cDNA (3–6 µg/sample) was fragmented using 0.6 U of DNase I (Amersham Pharmacia Biotech) per µg of cDNA in One-Phor-All Buffer (Amersham). The 3' termini of the fragmented cDNA was labeled using an Enzo BioArray Terminal Labeling kit with Biotin-dUTP (Enzo Diagnostics). The reaction was completed as suggested by Affymetrix using the Midi format. Biotinylated S. aureus cDNA was hybridized to custom Affymetrix GeneChips (RMLChips) containing 3961 probe sets from eight different S. aureus strains (COL, EMRSA16, MSSA476, RF122, TSS.8325, Mu50, and N315) and scanned according to standard GeneChip protocols (Affymetrix). Specific details for Affymetrix hybridization and scanning protocols can be found at the internet address provided above. Each experiment was repeated in triplicate. Data were analyzed with GeneChip Operating Software version 1.1 (Affymetrix) and GeneSpring version 6.0 (Silicon Genetics). Data was normalized using the mean signal from the S. aureus probe sets multiplied by a scale factor to obtain the target signal. Default detection thresholds of p = 0.04 and p = 0.06 were used to determine genes that were present and absent calls were assigned to values of p 
0.06. The Pearson correlation (GeneSpring) was used to evaluate the replicate correlation. Any nongrouping replicates were deleted from further evaluation. Quality filters based upon Present-Absent calls and signal intensities were used to create final gene lists (Microsoft Excel 2003). Microarray data are posted on the Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo/, accession number GSE2728).
To measure S. aureus gene expression during S. aureus–PMN interaction, bacteria were grown to E1, washed once in DPBS, and opsonized in 50% normal human serum at 37°C for 30 min. PMNs (107) were combined with 108 opsonized S. aureus in wells of a 12-well tissue culture plate on ice and centrifuged at 380 x g for 10 min at 4°C to synchronize phagocytosis (17, 18). Plates were transferred to a 37°C incubator with 5% CO2 for the desired times and samples were lysed as described above. For the microarray data derived from S. aureus-PMN interaction, RNA was isolated from the combination of three populations of bacteria: 1) S. aureus bound to PMNs; 2) S. aureus ingested by PMNs; and 3) free (uningested) S. aureus. However, we determined in separate experiments using fluorescence microscopy, and transmission and scanning electron microscopy, that there were few, if any, free bacteria after 30 min of S. aureus-PMN interaction (see Fig. 2, and data not shown). Further, after 60 min of S. aureus-PMN interaction, typically all neutrophil-associated bacteria were internalized (data not shown). At desired times, PMNs and bacteria in each well were lysed with 700 µl of RLT buffer (Qiagen). Isolation of S. aureus RNA and microarray analyses were performed as described above, except that fold-changes for each gene were determined by comparing RMLChips hybridized with cDNA from S. aureus alone to those with cDNA from S. aureus during PMN phagocytosis (time-matched). To be included in the final gene lists (e.g., see Figs. 5–9, and supplemental Table V4), genes must have met the quality filters described above, and changes in gene expression must have been at least 2-fold in one of the strains. For those genes (>2-fold change in one strain), changes of >1.5-fold were also indicated for the other strains. PMN-S. aureus microarray experiments were performed three separate times with PMNs from three blood donors. Although the RMLChip was designed to avoid cross-hybridization with human RNA or DNA, we determined empirically (using neutrophils from three individuals) that PMN RNA failed to hybridize with RMLChips. In our experiments, 3957 of 3961 probe sets had no significant cross-hybridization with PMN RNA. The four probe sets with noted binding to neutrophil RNA were eliminated from the analysis.
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Phagocytosis experiments and RNA preparation for TaqMan analysis were performed with procedures and conditions identical to those used for the PMN-S. aureus microarray experiments. TaqMan real-time RT-PCR analysis of two separate phagocytosis experiments (each assayed in triplicate) using PMNs from two human blood donors was performed with 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 (housekeeping or calibration gene) 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'-CGGCATCAGTCATAATGACGAT3'; cap8C forward primer, 5'-CGTGATTATTATGAAGATATGAACGGATT-3', cap8C probe 5'-ATTAGTAATGCGAAGTTAGTTGTTGATG-3', cap8C reverse primer 5'-CTTGTTGTGGCATTCGTTTAGG-3'; mraY forward primer, 5'-TGGATTAGCAACTGGACTGTCAAT-3', mraY probe, 5'-ATCGGATTTACAATGTATGCCATCAT-3', mraY reverse primer, 5'-GCCGTTTCTCCTAACACAAAGC-3'; vraR forward primer 5'-CGATGCAGTTCGTAAAACTTCTAGAG-3', vraR probe, 5'-AGAATCTGTTTTTGAACCGGAAGTT-3', vraR reverse primer, 5'-TTCATACGGTTACGCATTTTCACT-3'; agrA forward primer 5'-CGTAAGCATGACCCAGTTGGT-3', agrA probe, 5'-ATTATTTTCGTTACGAGTCACAGTGAAC-T-3', agrA reverse primer 5'-CCATCGCTGCAACTTTGTAGAC-3'; hlgA forward primer 5'-ACTTATTTGCACAAGACCCAACTG-3', hlgA probe 5'-CAGCAGCAAGAGACTATTTCGTCCCAG-3', hlgA reverse primer, 5'-CCACTTTGAATTAAAGGAGGTAATTGAT-3'; sarA forward primer, 5'-CAATGGTCACTTATGCTGACAAATT-3', sarA probe 5'-AGCTTTGAAGAATTCGCTGTATTGAC-3', sarA reverse primer 5'-TCTTTCTCTTTGTTTTCGCTGATG-3'; katA forward primer 5'-CCGTTTCTCTACTGTAGCAGGAGAAC-3', katA probe, 5'-CTGCTGATGCGGAGCGTGACATTC-3', katA reverse primer, 5'-TCAGTGTAGAACTTTAACGCAAATCC-3'; lukD forward primer, 5'-TCAAATCATCAGTTGTTACATCAAT-3', lukD probe, 5'-CTGCTTTTGCTATCGAATACAGTTGATGCAGC-3', lukD reverse primer, 5'-TCTCGCTTACAGGTGTGATATGTTG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To gain new insight into S. aureus pathogenesis, we tested virulence of strains originally isolated from individuals with CA (MW2, MnCop, and LAC) and hospital-acquired (HA; COL and MRSA252) infections in a mouse model of bacteremia (Table I, and Fig. 1). All mice infected with MW2, MnCop, or LAC were moribund or noticeably sick (10 of 10 animals infected with each strain, see Materials and Methods for details). In contrast, those infected with COL or MRSA252, a strain reported as highly virulent in humans (13), appeared relatively healthy (0 of 10 animals infected with COL and 1 of 10 of those infected with MRSA252 were sick). Correspondingly, mortality was significantly increased in those infected with MW2 or MnCop (p = 0.015, log-rank test) (Fig. 1A). At the time of death, major vital organs were evaluated for signs of infection (Fig. 1, B–E). On average, more bacteria were recovered from the livers of animals infected with MW2, MnCop, or LAC compared with the other strains (Fig. 1B). There were also a greater number of S. aureus recovered from lungs of mice infected with MW2 or MnCop compared with the other strains (Fig. 1C). These observations are consistent with the known capacity of MW2 and similar strains to cause necrotizing pneumonia in healthy individuals (20).
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Phagocytosis and killing of S. aureus by human PMNs
To determine whether differences in virulence are due in part to varied capacity of the strains to interact with the innate immune system, we evaluated phagocytosis and killing of each by human neutrophils. Although there were differences in early uptake of the strains (e.g., ingestion of COL and MW2 at 5 min was 65.1 ± 7.9% and 46.3 ± 12.9%, respectively, p < 0.05, ANOVA with Tukey’s posttest), phagocytosis of each strain occurred rapidly and was essentially complete by 15 min (Fig. 2, A and B). Notably, uptake of each strain by PMNs was virtually identical by 60 min of S. aureus-PMN interaction and there were few or no free bacteria (Fig. 2, and data not shown). We next determined that ingested S. aureus were exposed to microbicidal PMN ROS and granule contents (Fig. 2, C–E). Despite the exposure to neutrophil antibacterial components, there was significant S. aureus survival after phagocytosis (Fig. 2F), findings consistent with studies by Gresham et al. (21). Further, survival was significantly better for strains isolated from patients with CA infections (e.g., survival at 30 min was 58.7 ± 7.6% for MW2 vs 32.0 ± 3.0% for MRSA252, p < 0.001, ANOVA with Tukey’s posttest) (Fig. 2F). These observations provide strong support to the notion that S. aureus infections are caused at least in part by evasion of neutrophil-mediated killing.
Destruction of human PMNs by S. aureus
Between 3 and 6 h after PMN phagocytosis, net growth of each S. aureus strain increased significantly (Fig. 2F). Inasmuch as virtually all pathogenic S. aureus produce leukocidins, such as 
-hemolysins (22), we tested the hypothesis that late pathogen survival and growth after phagocytosis (i.e., after 3 h) is linked to the ability of each strain to lyse human PMNs (Fig. 3). Compared with COL and MRSA252, strains MW2, MnCop, and LAC caused significantly more neutrophil destruction 6 h after phagocytosis (e.g., at 6 h release of lactate dehydrogenase was 54.3 ± 7.6% for MW2 vs 25.0 ± 4.0% for COL, p < 0.01, ANOVA with Tukey’s posttest) (Fig. 3A). These results were verified by scanning and transmission electron microscopy (Fig. 3, C and D). The observation that heat-killed MW2 failed to lyse neutrophils (Fig. 3A) is consistent with the idea that S. aureus actively produces factors to evade killing by PMNs. Importantly, ability of the strains to survive killing by neutrophils and cause subsequent PMN lysis correlated generally with the mouse pathogenesis data.
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As a first step toward understanding the molecular basis of differences in virulence and interaction with neutrophils, we compared global gene expression in the strains grown in vitro with S. aureus microarrays (Table I, Fig. 4, and supplemental Tables II-IV). Collectively, expression of 210 genes differentiated MW2, MnCop, and LAC from MRSA252 and COL (supplemental Table II). Only 17 genes (not expressed in strains COL or MRSA252) were commonly expressed in the three CA strains during growth in vitro (supplemental Table II). Nine of the 17 genes encoded hypothetical proteins, and 3 others were phage-encoded (supplemental Table II). The finding that MW2, MnCop, and LAC collectively express a limited number of unique transcripts (not present in COL or MRSA252) is consistent with the genetic similarity between LAC and COL based on MLST and spa typing, and gene expression (Fig. 4C, and Table I). Although strain-specific gene expression (Fig. 4) might underlie differences in pathogenesis, it is likely that many pathogen processes used to evade PMN killing are triggered by interaction with the innate immune system.
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To further elucidate the molecular mechanisms used by the pathogen to evade innate host defense, we measured global changes in S. aureus gene expression during phagocytic interaction with human PMNs. We discovered that 21.8–39.1% of the genes in S. aureus were differentially regulated at any given time following phagocytic interaction (e.g., 593, 857, and 1029 genes in COL, MRSA252, and MW2, respectively, were up- or down-regulated at 180 min after phagocytosis) (Figs. 5–9, and supplemental Table V). Genes differentially expressed during PMN phagocytosis were divided into categories based on function or annotation. Genes encoding proteins with undefined function and those involved in metabolism comprised the two largest categories of S. aureus genes modulated by PMN phagocytosis (e.g., n = 348 and 372 genes, respectively, for MW2 30 min after initial PMN interaction) (Figs. 5 and 8, and supplemental Table V). Notably, 26.8–38.8% of S. aureus genes were differentially regulated within 30 min after PMN phagocytosis, depending on the strain analyzed (e.g., 26.8% of those in COL and 38.8% of those in MW2) (Fig. 5).
Genes encoding proteins that moderate oxidative stress, and those involved in metabolism and capsule synthesis are induced during phagocytosis
Consistent with the finding that S. aureus was exposed to PMN-derived ROS (Fig. 2C), at least 25 stress response genes were up-regulated in the pathogen during phagocytosis (Fig. 6, and supplemental Table V). For example, genes encoding catalase (katA), thioredoxin (trxA), thioredoxin reductase (trxB), superoxide dismutase (sodA, sodM), alkyl hydroperoxide reductase (ahpC, ahpF), and glutathione peroxidase (gpxA), were up-regulated in S. aureus immediately following phagocytosis (at 30 and 60 min), a period of time that corresponds with maximum generation of neutrophil ROS (Figs. 2C and 6). Genes encoding proteins involved in capsular polysaccharide synthesis (capA-G, capI-P, and cap8I) were up-regulated in all but one of the strains during initial S. aureus-PMN interaction (Fig. 6, and supplemental Table V). Although there is evidence that capsule blocks phagocytosis (23), under our conditions each strain was readily ingested by neutrophils (Fig. 2A). Thus, the data suggest S. aureus capsule is important for survival once the pathogen is internalized. Alternatively, induction of S. aureus capsule by PMN uptake might confer a phagocytosis-resistant phenotype to bacteria that escape destruction from initial neutrophil interaction.
There was striking up-regulation of genes involved in metabolism following pathogen uptake (Fig. 8, and supplemental Table V). Twelve genes encoding proteins of the tricarboxylic acid cycle, including aconitate hydratase (citB), isocitrate dehydrogenase (citC), citrate synthase (citZ), and fumarate hydratase (citG), were up-regulated following phagocytosis by PMNs (Fig. 8, and supplemental Table V). These findings concur with recent studies that link tricarboxylic acid cycle metabolism and pathogenesis (24). In contrast, genes encoding proteins involved in cell envelope synthesis, cell division, and replication were generally down-regulated immediately following phagocytosis (Figs. 6 and 9, and supplemental Table V). Thus, S. aureus gene-regulated processes appear directed toward defense against neutrophil killing rather than for growth immediately after phagocytosis (within 30 or 60 min). This idea is supported by the lack of net S. aureus growth for up to 3 h after ingestion (Fig. 2F).
Phagocytosis up-regulates S. aureus genes encoding proteins that mediate virulence
Within 30 min of S. aureus-PMN interaction, genes involved in virulence were up-regulated in each of the strains (Figs. 5 and 6, and supplemental Table V). For example, at least 36 genes encoding virulence factors and/or toxins were induced in MW2 30 min after phagocytic interaction with neutrophils (Figs. 5 and 6, and supplemental Table V). These genes encode toxins, secreted exoproteins, and cell-wall associated adhesions, such as extracellular matrix and plasma binding protein (ssp), epidermin immunity/lantibiotic proteins (epiE, epiF), fibronectin-binding proteins (fnb, fnbB), staphylocoagulase, and clumping factor (clfA) (Fig. 6). Genes encoding two-component -hemolysins (hlgA, hlgB and hlgC), which are known to destroy leukocytes, were induced in all strains during PMN phagocytosis (Fig. 6) (22). In contrast, genes encoding several toxins, such as an exotoxin 2 homologue, set7, set11, set14, lukD, lukE, and enterotoxin type C3 (sec3), as well as those encoding numerous hypothetical or hypothetical exported/surface-associated proteins, were up-regulated only in strains causing CA infections (Figs. 6 and 7, and supplemental Table V). Further studies are needed to determine whether these genes underlie the noted variances in strain pathogenesis (Fig. 1) and enhanced ability of MW2, MnCop, and LAC to circumvent killing by neutrophils (Fig. 3).
S. aureus genes involved in transcriptional regulation are modulated by neutrophil phagocytosis
S. aureus adapts to varied host environments and thus inhabits skin, mucous membranes, blood, and deeper tissues (1, 2, 3, 4, 5, 6). This characteristic is due partially to several gene-regulatory systems that tightly regulate expression of S. aureus virulence genes (25). Transcripts encoding S. aureus gene-regulatory systems VraSR, SaeSR, and SarA were up-regulated significantly by PMN phagocytosis (Fig. 6). The VraSR two-component gene regulatory system is induced by cell-wall synthesis inhibitors and/or cell envelope damage (26), observations most compatible with our results. SarA is known to control expression of S. aureus virulence factors and is thus important for pathogen survival during host-pathogen interaction (21, 27) (Fig. 6). Our observation that sarA is induced, and alternative 
factor B (sigB) and accessory gene regulator (agr) operons are repressed during PMN phagocytosis, corresponded with concomitant regulation of a number of putative and proven virulence factors, such as the gene encoding -hemolysin (hld) and the anti-holin encoding operon lgrAB (Fig. 6, and supplemental Table V) (28, 29, 30). Taken together, these findings suggest that evasion of human innate immunity by S. aureus is regulated at the level of gene transcription.
Confirmation of microarray data by TaqMan real-time RT-PCR (TaqMan analysis)
We used TaqMan real-time RT-PCR to confirm changes in gene expression identified by microarray analysis (Fig. 10). Eight S. aureus genes identified by microarrays as differentially transcribed during PMN phagocytosis were evaluated by TaqMan analysis. Genes were selected from several functional categories and transcript levels were measured at 60 and 180 min after PMN phagocytosis in strains MRSA252 and MW2. There was a strong correlation between TaqMan and microarray results (83.3%), consistent with previous comparisons (17, 19, 31, 32, 33, 34, 35).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Neutrophils are a critical component of innate immunity and are essential for controlling bacterial infections (44, 45, 46, 47, 48, 49, 50). Defects that alter normal PMN function, such as chronic granulomatous disease and leukocyte adhesion deficiency, predispose individuals to serious S. aureus infections (reviewed in Ref.44). Further, it has long been known that individuals with neutropenia are more susceptible to bacterial infections, including those caused by S. aureus (45, 46, 47, 48). Recent studies in animal models of S. aureus disease support these observations unambiguously (49, 50). Inasmuch as evasion of innate immunity plays a critical role in bacterial pathogenesis (17, 19, 51), a significant part of our study investigated the interaction of human PMNs with S. aureus. Of the S. aureus strains used in our study, those originally isolated from individuals with CA infections (no established risk factors) evaded killing by human neutrophils better than those isolated from hospital infections (Table I and Fig. 2F). Differences in pathogen survival were not due to altered neutrophil function, because phagocytosis, ROS production and degranulation were ultimately comparable for each of the strains tested (Fig. 2). Rather, the data indicate that strains MW2, MnCop, and LAC are more resistant to destruction per se and/or produce factors that cause more rapid host cell lysis by comparison (Fig. 3). Notably, there was no net growth by any of the strains during phagocytic interaction with human neutrophils until sometime after 3 h of culture (Fig. 2F). The increased survival/growth by MW2, MnCop, and LAC at 6 h was reflected generally by the degree of PMN lysis caused by the strains (compare Fig. 2F with Fig. 3, A and B). Thus, our data indicate that some S. aureus survived intraphagosomally without multiplying, but produced factors that cause PMN lysis.
Results from the neutrophil-S. aureus experiments were paralleled by the mouse pathogenesis data, which indicate MW2, MnCop, and LAC (those from community infections) are more virulent than COL or MRSA252 (Fig. 1), consistent with recent studies indicating strain-to-strain variability in S. aureus virulence (52). Strains LAC (pulsed-field type USA300) and MW2 (pulsed-field type USA400) are MRSA strains closely associated with community outbreaks of S. aureus disease in the United States (5, 7, 9, 10, 12, 36, 38, 41, 53). For example, recent reports indicate USA300 is a causative agent of staphylococcal necrotizing fasciitis (9) and severe community-onset pneumonia in healthy adults (12). In contrast, MRSA252 is a leading cause of hospital infections in the United States and United Kingdom (16), but to our knowledge is not a typical source of disease in individuals without associated risk factors. We note also that methicillin resistance has little to do with virulence per se, because MnCop, an MSSA strain closely related to MW2, was similar to MW2 in the mouse model of infection and in each of our host-pathogen assay systems (Figs. 1–3, 6, and 7). Taken together, our findings provide strong support to the idea that enhanced strain virulence is linked to (or results from) evasion of killing by neutrophils, which likely underlies the ability of MW2, MnCop, and LAC to cause disease in individuals without known risk factors.
Several observations support the idea that the global pathogen response described herein is used by S. aureus to circumvent innate immunity and thereby promote disease in humans. Notably, global changes in S. aureus gene expression were triggered by PMN phagocytosis, and neutrophils are the predominant host cells used in defense against bacterial infections. Immediate (within 30 min) up-regulation of genes involved in virulence, metabolism, capsule synthesis, and gene regulation after ingestion by neutrophils correlated with S. aureus survival, which was 30% for all strains within the first 90 min after phagocytosis (44% for MW2, MnCop, and LAC) (Figs. 2F and 6). Given the noted increases in expression of ahpC, ahpF, katA, gpxA, sodA, sodM, trxA, and trxB after phagocytosis, the strains used in this study are probably not susceptible to destruction by primary neutrophil-derived reactive oxygen species, i.e., superoxide and hydrogen peroxide. Preliminary studies by our laboratory have shown that there is 60% survival for each of the S. aureus strains after culture with up to 50 mM hydrogen peroxide (K. Braughton and F. DeLeo, unpublished observations).
One of the highlights of our study is that several S. aureus gene regulatory systems were induced after interaction with PMNs. We previously identified a pathogen survival response in group A Streptococcus (GAS, Streptococcus pyogenes) triggered by neutrophils through ihk-irr, a two component gene regulatory system required for virulence (19, 51). The ihk-irr survival response is activated by exposure to neutrophil 
-granule components and reactive oxygen species (51). Because there is significant S. aureus survival after interaction with neutrophils, a similar as yet undefined mechanism must exist in this pathogen. As such, many of the genes reported herein likely comprise a S. aureus pathogen survival response controlled directly or indirectly by one or more of the gene regulatory systems induced by phagocytosis. Consistent with this notion, Gresham et al. (21) reported that survival of S. aureus within PMNs was linked to a global gene regulator called sar, and sarA was up-regulated in all S. aureus strains following PMN phagocytosis in our present work (Fig. 6). Taken together, these findings are consistent with the idea that sarA is important for evasion of killing by neutrophils. A systematic investigation of each of the gene regulators identified in our study by targeted gene mutagenesis and/or knockout studies will be necessary to elucidate the role of each in pathogenesis.
Significant emphasis has been placed on determining principal virulence factors responsible for the spread of CA S. aureus infections. The Panton-Valentine leukocidin (encoded by lukF-PV and lukS-PV, or simply pvl) has been linked to CA infections resulting from methicillin-resistant S. aureus (20, 36, 38, 39, 54). Although our studies failed to address pvl expression in S. aureus during PMN phagocytosis, a recent study by Said-Salim et al. (55) demonstrated that there is no correlation between neutrophil lysis and presence of pvl. Importantly, our work identified many other genes potentially responsible for the differences in PMN lysis and strain virulence, and thus, a possible explanation for the increased virulence of CA strains such as MW2 (Figs. 1, 6, and 7, and supplemental Table V). For example, several putative membrane or exported proteins of uncharacterized or unknown function were up-regulated only in strains that cause CA infections (Figs. 6 and 7, and supplemental Table V). These genes clearly merit further investigation.
An enhanced understanding of the molecular mechanisms used by S. aureus to circumvent the human innate immune system will facilitate development of new treatments for bacterial infections. Our previous work with the prominent human pathogen GAS indicated that 16% of its genes are differentially regulated during phagocytic interaction with neutrophils (19). By comparison, we determined that up to 39% of the genes in S. aureus changed following phagocytosis (Fig. 5, see MW2 at 180 min). Differences in the sensitivity of the methods used (spotted vs oligonucleotide microarrays) may account for some of the differences. However, the two pathogens are quite distinct with regard to genome size, their interaction with human neutrophils, and in the types of disease caused by each. For example, GAS produces numerous molecules that inhibit PMN phagocytosis (reviewed in Ref.56), whereas S. aureus is readily ingested. Nonetheless, each pathogen uses strategies to prolong intraphagosomal survival and cause eventual host cell lysis (Figs. 2 and 3) (19, 51, 56). Thus, there likely exist some common molecular mechanisms for immune evasion between the two pathogens.
We used microarrays to generate a global view of the genes and gene regulatory networks involved in bacterial pathogenesis and therefore generated a vast repository of information from which many new studies of S. aureus pathogenesis will emerge. The global pathogen response revealed by our studies identified dozens of potential vaccine Ags and targets for therapeutics designed to control S. aureus infections.
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1 This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. 
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
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; ROS, reactive oxygen species; CA, community acquired; HA, hospital acquired; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; DPBS, Dulbecco’s PBS; LDH, lactate dehydrogenase; TEM, transmission electron microscopy; GAS group A Streptococcus.
4 The online version of this article contains supplemental material.
Received for publication February 25, 2005. Accepted for publication July 5, 2005.
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