Isolates of methicillin-resistant Staphylococcus aureus (MRSA) were once linked uniformly with hospitalassociated infections; however, community-acquired MRSA (CA-MRSA) now represents an emerging threat worldwide. To examine the association of differential virulence gene expression with outcomes of human infection, we measured transcript levels of target staphylococcal genes directly in clinical samples from children with active known or suspected CA-MRSA infections. Virulence genes encoding secreted toxins, including Panton- Valentine leukocidin, were highly expressed during superficial and invasive CA-MRSA infections. In contrast, increased expression of surface-associated protein A was linked only with invasive disease. Comparisons with laboratory-grown corresponding clinical isolates revealed that tissue-specific expression profiles reflect the activity of the staphylococcal accessory gene regulator during human infection. These results represent the first demonstration of staphylococcal gene expression and regulation directly in human tissue. Such analysis will help to unravel the complex interactions between CA-MRSA and its host environmental niches during disease development.
Staphylococcus aureus is a persistent human pathogen that is responsible for a range of diseases that vary widely in clinical presentation and severity. The capacity of S. aureus to cause a spectrum of human diseases reflects an ability to adapt to distinct microenvironments in the human body and suggests that the pathogenesis of S. aureus infection is a complex process involving a diverse array of secreted and surface-associated virulence determinants that are coordinately expressed at different stages of infection . Distinct networks of virulence genes are likely activated in response to host signals, including those found in target tissues and those related to innate defenses activated during the infectious process. This expectation manifests in vitro as a growth phase- dependent pattern of virulence determinant expression that is established by global regulatory elements (reviewed in ). During the exponential-growth phase, the organism synthesizes cell-wall proteins with adhesive functions, including protein A and fibrinogen-binding, fibronectin-binding, and collagen-binding proteins; such expression might augment the initial establishment of colonization in the host. During the transition from the exponential phase to the stationary phase of growth, the expression of cell-wall proteins is repressed, whereas the synthesis of extracellular toxins and enzymes predominates. Through their proteolytic activities and toxic effects on host cells, these exotoxins might facilitate local invasion and dissemination during infection.
Protein expression during the transition from the exponential phase to the stationary phase is coordinately controlled by global regulators and the accessory gene regulator (Agr) quorum-sensing system . During this transition, secretion of a modified peptide pheromone signals cell density-dependent gene expression via RNAIII, the regulatory effector molecule of the Agr system [4–6], resulting in up-regulation of the expression of exoprotein genes (e.g., hla and hlb) and down-regulation of the expression of cell surface adhesins, such as protein A (encoded by spa) . Although this mechanism establishes temporal regulation in vitro, it also likely contributes to spatial regulation by limiting the expression of target genes to compartments where the signal molecule reaches a high concentration. Evidence of an in vivo role for this regulatory pathway is restricted to animal models of S. aureus infection. For example, agr inactivation resulted in reduced virulence in experimental staphylococcal musculoskeletal infection models [8, 9]. Recently, Agr-mediated expression of cytolytic peptides by community-acquired methicillin-resistant S. aureus (CA-MRSA) was shown to be important for human neutrophil lysis and pathogenesis in a murine model of soft-tissue infection . Animal studies have yielded conflicting conclusions regarding the pathogenic importance of Panton-Valentine leukocidin (PVL) [11, 12] but indicate that PVL expression may alter agr expression patterns . Although animal models of staphylococcal infection are valuable tools for relating the contribution of putative virulence factors identified in vitro to pathogenesis, their relevance to clinical disease is inherently limited and may not reflect human-specific adaptive behavior. To address this, we identified patients with active community-acquired S. aureus infections and defined bacterial gene expression profiles for multiple types of infection directly in tissue specimens.
Patients, Materials and Methods
Bacterial strains and culture.S. aureus was grown in tryptic soy broth (TSB [BD Biosciences]). Overnight broth cultures were inoculated from frozen stocks of S. aureus strains first isolated from patient specimens on 5% sheep blood agar plates (BD Biosciences) by the clinical bacteriology laboratory at St. Louis Children's Hospital (SLCH). Fresh broth was inoculated with bacteria from the overnight culture (dilution, 1:100) and incubated at 37°C with shaking (250 rpm). There were no observable differences in the growth rates of the various S. aureus strains in TSB. Therefore, for gene expression analyses, strains were cultured through the transition from the exponential phase to the stationary phase (OD600, 1.0) and were harvested immediately for RNA isolation. For comparison to mid-exponential phase gene expression, a subset of strains was grown to an OD600 of 0.30 in TSB.
Isolation of RNA from infected human tissue. Patients who presented to the SLCH emergency department with cutaneous infection suspected to be due to CA-MRSA and who required abscess drainage were identified by emergency department staff. No patient had a clinical history suggestive of immune deficiency. Written informed consent was obtained from the parent or guardian, and assent was obtained from the child when appropriate; guidelines of the US Department of Health and Human Services and of the Washington University Human Research Protection Office (HRPO) were followed for all human studies. Upon abscess drainage, a routine sample of abscess contents was collected and transported to the SLCH clinical bacteriology laboratory for aerobic culture as part of routine diagnostic analysis (table 1, which appears only in the electronic edition of the Journal). In addition, a second aliquot of purulent material was collected using a sterile syringe or a second sterile swab. Samples were immediately submerged and retained in 2 mL of a bacterial RNA-stabilization reagent (RNeasy Protect Bacteria [Qiagen]) in glass vials that had previously been heated to 240°C for at least 4 h to prevent RNase contamination. Samples were incubated at room temperature for ∼5 min and stored at 4°C for 1–96 h. It was determined experimentally that this range of storage times did not significantly affect RNA stability (data not shown). Samples were centrifuged for 5 min at 16,000 g, and the supernatant was carefully removed. Cells were resuspended in lysis buffer (RLT [Qiagen]), and lysates were further homogenized with a FastPrep FP120 reciprocal shaking device and a commercially available extraction reagent, lysing matrix B (MP Biomedicals). Total RNA was isolated by silica membrane binding (RNeasy [Qiagen]), and contaminating chromosomal DNA was removed by treatment with DNase (Qiagen). Absence of DNA was confirmed by polymerase chain reaction (PCR), and the quality and quantity of RNA were determined by spectrophotometry and agarose gel electrophoresis. Criteria for inclusion in downstream applications were an OD260/280 of >2.0 and the absence of visible degradation. The yield of total RNA varied but was typically 1-3 μg per sample. A similar procedure was used to isolate total RNA from material collected during operative drainage of subperiosteal abscesses. In all, 78 patients were enrolled; 15 were excluded because cultures of abscess specimens did not grow S. aureus, and 20 were excluded because the quantity of RNA yielded was insufficient for analysis.
Interaction ofS. aureuswith human polymorphonuclear leukocytes (PMNs) in vitro. In accordance with a protocol approved by the HRPO, PMNs (or neutrophils) were isolated from the venous blood of healthy volunteers as described previously . Briefly, dextran sedimentation of erythrocytes was followed by Ficoll density gradient centrifugation (Ficoll-Paque Plus [GE Healthcare]) and hypotonic lysis of contaminating erythrocytes. PMN viability was >99% as assessed by trypan blue exclusion, and purity was >99% as determined by visualization of nuclear morphology after staining (Hema3 [Fisher Scientific]). Cells were resuspended in prewarmed RPMI 1640 medium (Gibco) buffered with 10 mmol/L HEPES (RPMI/H; pH 7.2) at a concentration of 107 cells/mL and were used immediately. To measure S. aureus gene expression during in vitro PMN encounters, bacteria were grown to the phase transition between exponential and stationary growth (OD600, 1.0) in TSB, washed once in phosphate buffered saline (PBS), and resuspended in RPMI/H to a concentration of ∼109 cfu/mL. S. aureus (∼108 cfu) was combined with either PMNs (107 cells) or media alone in wells of a 24-well tissue culture plate and centrifuged at 400 g for 5 min at 4°C. Plates were incubated at 37°C in 5% CO2 for 60 min. The cells from each well were collected in RLT lysis buffer (Qiagen). The lysates were homogenized, and total RNA was isolated as described above.
Analysis ofS. aureusgene expression. For analysis of transcript levels in infected human tissue, cDNA was prepared from isolated RNA with random primers and SuperScript II reverse transcriptase (Invitrogen) according to the instructions of the manufacturer. In most cases, 50–100 ng of cDNA was used as a template for TaqMan real-time PCR performed with an ABI 7500 Fast thermocycler (Applied Biosystems). To compare transcript levels in vitro, the S. aureus strain isolated from each patient was cultured as described above, and total RNA was isolated from ∼109 cells in RLT buffer (Qiagen) as described for the in vivo samples. Approximately 100 ng of cDNA from in vitro samples was used as a template for TaqMan real-time PCR. For all samples, transcript levels were normalized to the level of the endogenous control gene rrsA. This gene was selected from several candidates because of the documented stability of rrsA expression and mRNA in a variety of conditions , as well as the comparable efficiency of the rrsA PCR with the reagents and conditions used in these studies. Relative target expression was calculated according to the Δ(ΔCt) method as described elsewhere , in which the fold-change in expression is equal to 2−Δ(ΔCt). Data are presented as the fold-change in transcript levels in infected human tissue relative to those for the broth-cultured S. aureus clinical isolate in the indicated calibrator condition and represent the mean value (±SD) from replicate assays. The in vivo samples were analyzed in triplicate; for in vitro samples, triplicate assays were performed with RNA prepared from each of at least 2 independent cultures. Primers and probes were purchased from a commercial vendor (Integrated DNA Technologies). Primer sequences are listed in table 2, which appears only in the electronic edition of the Journal, and were designed using available genomic sequence and Primer Express software (Applied Biosystems).
Assays for molecular typing ofS. aureusclinical isolates. The results of molecular typing assays are summarized in table 3; detailed results for each isolate are reported in table 4, which appears only in the electronic edition of the Journal. Total DNA was extracted from staphylococci grown on agar plates by silica membrane binding (QIAamp DNA [Qiagen]) and used as an amplification template for PCR-based assays performed with primers described in table 2. The agr allele group was determined as previously described  with primers designed to amplify specific agr alleles. The presence of bsaB, lukS-PV, lukF-PV, and arcA of the arginine catabolic mobile element(ACME)originally identified in a USA300 isolate of S. aureus  was detected with allele-specific primers. In addition to Taq DNA polymerase and reaction buffer (Invitrogen), the reaction contained deoxyribonucleotides (final concentration, 200μ;mol/L), MgCl2 (final concentration, 2 mmol/L), and oligonucleotide primers (final concentration, 500 nmol/L). Amplification was performed under the following conditions: an initial 5-min denaturation step at 95°C; 25 cycles involving a 30-s denaturation step at 95°C, a 30-s annealment step at 55°C, and a 1-min extension step at 72°C; and a final 10-min extension step at 72°C. For MRSA isolates, the SCCmec allotype was determined by PCR as described elsewhere .
Statistical analysis. Statistical analysis was performed using GraphPad Prism, version 4.03 for Windows. The Wilcoxon signed rank test was used to determine statistical significance for expression differences of target genes in the experimental condition relative to the calibrator condition. The Mann-Whitney U test was used for comparison of target gene expression in different experimental conditions. For comparison of in vitro and in vivo gene expression profiles, the log-transformed values for fold change in expression of individual genes in each data set were plotted in the x (in vitro) and y (in vivo) dimensions, and the best-fit straight line was determined by the least-squares method as described previously . The Pearson correlation coefficient, R, is reported for selected experiments.
Results and Discussion
The prevalence of community-acquired S. aureus infections, coupled with the accessibility of infected tissue in typical cutaneous disease, provided a unique opportunity to develop methods for direct analysis of gene expression during human infection. Characteristics of the patients and corresponding cutaneous S. aureus isolates used in this study are summarized in table 3 (see table 1 and table 4 for additional details). Although the patient population was diverse, the staphylococcal isolates were comparatively homogeneous and representative of circulating community-acquired strains. Phenotypic and genotypic analysis of these strains showed that all 40 cutaneous isolates encoded PVL. Furthermore, 33 strains (83%) were resistant to methicillin and contained SCCmec type IV, and 32 (80%) were similar to USA300, the clone that causes the majority of CA-MRSA infections in the United States [17, 20].
To profile the transcriptional pattern of S. aureus virulence gene expression during human infection, we used real-time reverse-transcriptase PCR (RT-PCR) to quantify selected virulence gene transcripts directly in material evacuated from cutaneous abscesses of enrolled patients. Target genes chosen for analysis included several characterized S. aureus virulence factors as well as putative CA-MRSA virulence factors, namely lukS-PV (which encodes a component of PVL), arcA (which encodes a component of a putative arginine deiminase pathway horizontally acquired from commensal staphylococci ), bsaB (which encodes an enzyme in a bacteriocin biosynthesis pathway ), hlgB (which encodes γ-toxin), hla (which encodes α-toxin), lukE (which encodes leukocidin), spa, and RNAIII.Wefirst compared the levels of target gene transcripts in each cutaneous abscess sample to those in the corresponding S. aureus strain in stationary-phase broth culture, a condition in which many virulence genes are maximally expressed in vitro . The cutaneous abscess profile was characterized by relative up-regulation of genes encoding components of several cytolytic toxins (i.e., lukS-PV, lukE, hlgB, and hla), including PVL, and a relative decrease in the expression of RNAIII, bacteriocin (by bsaB), and protein A (by spa) (figure 1). When analyzed separately, there was no significant difference between MRSA and methicillin-susceptible S. aureus expression patterns (figure 1). The individual cutaneous abscess samples displayed similar trends in differential gene expression, although there was variation in the magnitude of the changes among samples. In addition to possible strain-specific differences, inherent variability in human samples (e.g., abscess size, time after infection, and host factors) likely contributed to the range of transcript levels measured in different samples.
To elucidate the underlying regulatory mechanisms that established transcript patterns in our human samples, we grew representative S. aureus clinical isolates under a variety of in vitro conditions and identified the conditions that promoted gene expression most similar to that in infected tissue. Each of the clinical isolates produced a stationary-phase expression profile consistent with the archetypal growth phase-dependent pattern  described above (P < .002 for all genes) (figure 2A). Furthermore, there was a significant correlation between relative expression levels observed in cutaneous abscess samples and those in stationary-phase culture (R = 0.89; P < .003), including high expression of RNAIII, compared with expression in the exponential phase (figure 2B). Taken together, these data indicate that Agr/RNAIII-mediated virulence gene expression during typical cutaneous infection is recapitulated during the stationary phase but that additional host-specific cues apparently augment this transcriptional program in vivo.
Acommon feature of cutaneous infections caused by S. aureus is the presence of neutrophil-rich purulent fluid .We hypothesized that exposure to PMNs (or neutrophils) might contribute to the expression profile of S. aureus at this site of infection. To evaluate the influence of PMN-specific signals, we compared the levels of selected transcripts during in vitro encounters with purified human PMNs versus that in human cutaneous abscess specimens. Previous analysis of the global expression profile of S. aureus during in vitro phagocytic interaction with human PMN revealed a pathogen survival transcriptional pattern characterized by the up-regulation of genes involved in capsule synthesis, oxidative stress, and virulence . Consistent with these findings [11, 22], expression of toxins known to target leukocytes, including PVL, was increased in vitro in the presence of PMNs relative to that in time-matched controls in media alone, and the relative level of the RNAIII transcript was decreased (figure 2C). Compared with expression in human cutaneous infection, PMN-derived expression was similar in direction for 5 of 8 genes analyzed (figure 2C), and there was a strong correlation of the 2 profiles for only one of the strains (R = 0.83, 0.65, 0.37, 0.27, 0.11, and −0.24). These data suggest that this in vitro model of host-pathogen interaction is an insufficient representation of the influence of host-specific cues for S. aureus virulence gene expression in human cutaneous infection.
Community-acquired S. aureus is associated with a broad range of observed disease severities in otherwise healthy patients, yet the host and bacterial factors promoting superficial versus invasive staphylococcal disease are not understood. It has been hypothesized that changes in bacterial virulence gene expression in response to tissue-specific or temporal cues in vivo play an important role in determining the outcome of infection. The in vivo production of virulence factors, inferred by specific humoral and cell-mediated immune responses to human infection , and the localization and kinetics of pathogen gene expression have been investigated in animal models of bacterial infection , but direct measurement of expression in human infection has not been previously reported. We therefore determined the levels of target transcripts in material collected from patients with community-acquired invasive (subperiosteal) abscesses caused by S. aureus for comparison with levels in cutaneous abscess samples. As in cutaneous abscesses, expression of exotoxin genes in strains from invasive abscess samples was significantly higher than that in strains in the stationary phase (P < .05) (figure 3A). RNAIII was present in the invasive abscess samples at a level comparable to that for the corresponding strain grown to the stationary phase (figure 3A) but was not as highly expressed as in the cutaneous samples (figure 2A). Another difference in the invasive profile was the sharp elevation of spa expression relative that observed for the stationary phase (figure 3A). An increase in spa expression in conditions with reduced RNAIII levels is consistent with in vitro transcriptional regulation , and the high level of spa transcription in these samples suggests that protein A might play an important role in dissemination or survival during invasive infections in general, as has been suggested for staphylococcal pneumonia . The invasive-disease expression profiles were strongly correlated with those of S. aureus in PMN encounters (R = 0.86, 0.79, and 0.90; P < .01), suggesting that a decreased RNAIII level and increased spa expression could reflect the influence of PMN interaction (figure 3B).
Our data are therefore consistent with the model of CAMRSA infection described by Cheung et al.  in which the organism interprets host site-specific signals, including cues related to the influx of PMN, to modulate Agr-regulated expression of virulence determinants in vivo (figure 4). Extension of this approach will optimize the establishment of in vitro correlates for in vivo conditions, previously advanced in animal models of gram-positive bacterial infection . The methods described here will also represent a worthwhile approach for analysis of newly identifiedCA-MRSA virulence determinants in human infection. Our findings highlight the importance of understanding gene expression and regulation in the endeavor to determine the mechanisms of CA-MRSA virulence and in the development of antiinfective strategies to combat this emerging pathogen.
We thank the staff of the clinical bacteriology laboratory at Saint Louis Children's Hospital for providing the S. aureus clinical isolates and for assistance with molecular typing of the strains; E. Epplin for assistance with patient enrollment and molecular typing of the strains; and the students of the Washington University Pediatric Emergency Medicine Research Associate Program for assistance with patient enrollment.