Skip to main content
NCBI home page
mBio logoLink to mBio
. 2015 Feb 17;6(1):e02272-14. doi: 10.1128/mBio.02272-14

Differential Expression and Roles of Staphylococcus aureus Virulence Determinants during Colonization and Disease

Amy Jenkins a, Binh An Diep b,c, Thuy T Mai b, Nhung H Vo b, Paul Warrener a, Joann Suzich a, C Kendall Stover a, Bret R Sellman a,✉
Editor: Michael S Gilmored
PMCID: PMC4337569  PMID: 25691592

ABSTRACT

Staphylococcus aureus is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of sdrC, fnbA, fhuD, sstD, and hla was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens.

IMPORTANCE

Many bacteria, such as Staphylococcus aureus, asymptomatically colonize human skin and nasal passages but can also cause invasive diseases, such as bacteremia, pneumonia, sepsis, and osteomyelitis. The goal of this study was to analyze differences in the expression of selected S. aureus genes during a commensal lifestyle and as an invasive pathogen to gain insight into the commensal-to-pathogen transition and how a bacterial pathogen adapts to different environments within a host (e.g., from nasal colonization to invasive pathogen). The gene expression data were also used to select genes for which to construct knockout mutants to assess the role of several proteins in nasal colonization and lethal bacteremia. These results not only provide insight into the factors involved in S. aureus disease pathogenesis but also provide potential therapeutic targets.

INTRODUCTION

Staphylococcus aureus is a Gram-positive bacterium that causes both community- and hospital-acquired infections (1). Approximately 20 to 30% of the population is reported to be permanent carriers in the nose and 60% are intermittent carriers (2). Nasally colonized individuals are at increased risk for developing an infection with their colonizing isolate (2, 3). S. aureus infections range from mild skin and soft tissue infections to more severe infections, including bacteremia, sepsis, and osteomyelitis (1).

Differential gene expression has been described for many bacteria under various growth conditions or during infection (4–8). Due to the difficulty in studying bacterial gene expression in vivo, many studies have been performed in in vitro systems meant to mimic the host environment, including iron and nutrient limitation, conditions of low oxygen, biofilm versus planktonic bacterial growth, and following exposure to blood in vitro (4–8). These studies have provided valuable information about genes involved in the bacterial response to particular host environments when mimicked in vitro; however, the in vivo setting is far more complex.

In addition to in vitro studies, S. aureus gene expression has been characterized in vivo. Expression of a limited number of genes has been investigated in both device-related and wound infections (9–11), during skin infections (12, 13), human nasal colonization (14), kidney infection in mice (13), and in a cotton rat nasal colonization model (15). In these studies, transcript levels in vivo were compared with transcript levels measured under in vitro growth conditions. A comparison of gene expression in S. aureus between disease states has not been reported. The goal of this study was to compare the expression profile of 23 putative S. aureus virulence determinants in two clinical isolates during three stages of infection: nasal colonization, early bacteremia, and infected heart tissue (thromboembolic lesions) in a sepsis model. These models were chosen to represent the stepwise progression of individual S. aureus strains from a commensal (asymptomatic nasal colonization) to a pathogen (from early bacteremia and finally to an invasive infection during sepsis). Our results revealed upregulation of sdrC, fnbA, fhuD, sstD, and hla in S. aureus as an invasive pathogen compared to a commensal. These findings suggest roles for these proteins in the progression of S. aureus infection and support further investigation of these factors as therapeutic targets.

RESULTS

Quantitative real-time PCR (qRT-PCR) was utilized to determine how S. aureus gene expression responds to environmental changes in the host, using different animal models (cotton rat nasal colonization and murine bacteremia and lethal sepsis). Cotton rat nasal colonization was chosen because it is well characterized and closely resembles chronic human nasal colonization. Alternatively, S. aureus is either cleared or requires antibiotics for chronic murine nasal colonization (16–20). Murine bacteremia and sepsis models were used because these models are not established for cotton rats. Four S. aureus gene classes were chosen for analysis: protein and carbohydrate adhesins, metal cation transporters, exotoxins, and immune evasion proteins (see Table S1 in the supplemental material).

One concern when running qRT-PCR on samples purified from an in vivo setting is that contaminants could copurify with the cDNA that inhibit the PCR. To address this concern, cDNA samples from each of the different infection sites (cotton rat nose and mouse blood and heart) were spiked into a control PCR assay specific for Pseudomonas aeruginosa pcrV on a plasmid. All of the samples amplified the pcrV gene with similar efficiency, indicating that none of the in vivo purified cDNA samples regardless of infection site adversely affected PCR efficiency (see Fig. S1 in the supplemental material).

Nasal colonization.

Experiments were performed with two S. aureus clinical isolates, SF8300 (CC8; USA300 methicillin-resistant S. aureus [MRSA]) and ARC633 (CC15; methicillin-susceptible S. aureus [MSSA]) obtained from a skin abscess and from a nasal carrier, respectively. To establish a baseline condition (commensal), transcript levels were measured during cotton rat nasal colonization. To avoid the pitfalls of comparing expression data to data for expression levels under an arbitrary in vitro growth condition, expression levels were represented as the fold change from the minimum and maximum in vitro expression levels (a description of the in vitro growth conditions that resulted in these expression levels can be found in Table S2 in the supplemental material). 16S rRNA was selected as an internal control for normalization because its expression varied less than 2-fold for both SF8300 and ARC633 under various in vitro growth conditions (e.g., early-log, mid-log, late-log, and stationary phases in trypticase soy broth [TSB], RPMI, and human serum) (see Fig. S2 in the supplemental material). As such, a change in gene expression was defined as a greater-than-2-fold increase or decrease in transcript levels. During nasal colonization, transcript levels of four adhesin genes (clfB, sdrC, sdrD, and tarK) increased over the highest observed in vitro expression levels in both strains. An additional four genes exhibited increased expression over the highest in vitro expression levels in one strain (clfA, icaB, efb, and sasG) (Fig. 1). Increased clfB, sdrC, sdrD, sasG, and tarK transcript levels support previous results implicating their corresponding proteins in nasal colonization (14, 15, 21–23). Metal ions are limiting in vivo; therefore, one would expect metal cation transporters to be upregulated (24, 25). As expected, most metal cation transporter genes were upregulated relative to the minimal in vitro expression level (Fig. 2). The genes encoding SpA and Sbi showed increased expression over their lowest in vitro expression level and were unchanged or decreased from the highest in vitro expression level. Toxin gene expression decreased compared to the lowest in vitro expression condition (Fig. 3). Of note, ARC633 does not encode lukF-PV.

FIG 1 .

FIG 1 

Open in a new tab

Protein and carbohydrate adhesin genes in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression in vitro (A) and the maximum expression in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P  < 0.05 (Student’s t test). clfB expression was not detected in vitro, and so calculations for clfB expression were based on an in vitro threshold cycle CT value equal to the limit of detection (40 cycles).

FIG 2 .

FIG 2 

Open in a new tab

Metal cation acquisition gene expression in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression level in vitro (A) and the maximum expression level in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P  < 0.05 (Student’s t test).

FIG 3 .

FIG 3 

Open in a new tab

Immune evasion and exotoxin gene expression in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression level in vitro (A) and the maximum expression level in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P < 0.05 (Student’s t test).

Nasal colonization versus early bacteremia.

Gene expression was then assessed in the blood of bacteremic mice 1 h postinfection (via intravenous [i.v.] injection). Transcript levels were compared to those observed in nasal colonization to determine which genes the bacteria modulate upon exposure to the bloodstream. Of the adhesins, only fnbA increased expression in both strains compared to nasal colonization, whereas sdrC and icaB showed increased expression in SF8300 and sasA, tagO, efb, and ebpS showed increased expression in ARC633 upon exposure to the bloodstream. Three metal cation acquisition genes had increased expression (mntC, fhuD, and sstD) compared to nasal colonization in ARC633. The hla gene was upregulated in both strains, whereas lukF-PV (SF8300) and sbi and spa (ARC633) were upregulated in one strain (Fig. 4). Taken together, these results indicate that individual S. aureus isolates respond differently to environmental change and may use different virulence determinants during the same type of infection.

FIG 4 .

FIG 4 

Open in a new tab

Differential gene expression between nasal colonization and early bacteremia. Transcripts were assessed relative to 16S rRNA, and results from the bloodstream were compared to the expression results from nasal colonization for protein and carbohydrate adhesins (A), metal cation acquisition genes (B), and immune evasion genes and exotoxins (C). Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 10 animals/experiment. *, P < 0.05 (Student’s t test). clfA, clfB, and sasG expression was not detected in the blood samples, and so calculations for clfA, clfB, and sasG expression were based on the threshold cycle (CT) value equal to the limit of detection (40 cycles).

Transition from early bacteremia to heart lesions.

Transcript levels were measured in bacteria present in saline-perfused hearts 14 h postchallenge (i.v.) and compared to those in blood 1 h postinfection. The hearts were flushed with saline to ensure transcript levels were measured from bacteria in the heart tissue and not in residual blood. It should be noted that, similar to previous reports (26), we did observe histological evidence of thromboembolic lesions in the hearts of infected mice (data not shown). The comparisons performed here were undertaken to highlight genes regulated upon transition to the thromboembolic heart lesions characteristic of S. aureus sepsis (26), relative to the initial stages of bacteremia. Six adhesin genes (clfA, sdrC, sasF, tagO, fnbA, and tarK) displayed increased expression in both strains, and sasA (SF8300) and icaB (ARC633) showed increased expression in one strain in thromboembolic lesions relative to early bacteremic blood. Of these, clfA, tagO, and fnbA have been implicated in establishing organ colonization (26–29). Four cation transport genes (mntC, isdB, fhuD, and isdA) exhibited increased expression in both strains relative to early bacteremic blood, whereas isdH and sstD showed increased expression in ARC633. Upon seeding of the heart, hla, sbi, and lukF-PV all showed increased expression in SF8300, while hla and sbi remained constant in ARC633 (Fig. 5). In general, these results suggest that S. aureus utilizes multiple adhesins in the process of bloodstream escape and organ invasion. Additionally, upregulation of the metal cation transporters points to the iron-limiting nature of the heart, while exotoxins and immune evasion genes are likely utilized to escape the bloodstream and host defenses.

FIG 5 .

FIG 5 

Open in a new tab

Differential gene expression in the transition from the bloodstream to heart tissue. Transcripts were assessed relative to the 16S rRNA, and results from the heart tissue were compared to the expression results from the bloodstream for protein and carbohydrate adhesins (A), metal cation acquisition genes (B), and immune evasion genes and exotoxins (C). Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 10 animals/experiment. *, P < 0.05 (Student’s t test). clfB and sasG expression was not detected in the blood samples or the heart samples; therefore, expression numbers are not reported for these genes.

Isogenic mutant analysis.

To compare gene expression analysis with a gene’s role in pathogenesis, isogenic deletion mutants were constructed in SF8300. The genes (clfA, clfB, sdrC, isdH, isdB, hla, and spa) were chosen to represent a variety of expression profiles in the three models. The adhesins clfA and sdrC were chosen based on their consistent upregulation during infection. clfB, isdH, and isdB were chosen based on their upregulation in the nasal colonization model and in the transition from bloodstream to invasive pathogen (isdB). The remaining genes (hla and spa) showed substantially increased expression in two models and are known to play a role in S. aureus pathogenesis. Cotton rats were infected intranasally with isogenic mutants, and CFU were compared to exposuure to wild type (WT) 4 days postinfection (Fig. 6). Only the ΔsdrC mutant showed a trend toward a reduction (~0.5 log) in nasal colonization, and this trend was not significant. When both sdrC and clfB (an adhesin shown to play a role in nasal colonization [21, 30]) were deleted (ΔsdrC ΔclfB), a significant reduction in CFU in the nares was observed.

FIG 6 .

FIG 6 

Open in a new tab

CFU enumeration of SF8300 wild-type and mutant strains in a cotton rat nasal colonization model. Groups of 12 cotton rats were challenged intranasally with 5 × 105 CFU S. aureus in 10 µl (5 µl per nostril). For ΔsdrC ΔclfB, P =0.0047 (Mann-Whitney test). Data shown are from a single replicate of three independent experiments.

Mutant strains were compared to WT for a change in heart CFU following i.v. infection. Knockout mutants for clfA, sdrC, isdB, and hla all resulted in decreased heart CFU, consistent with increased expression observed in thromboembolic lesions (Fig. 7). Knockout mutants of genes that were downregulated or unchanged in thromboembolic lesions did not impact CFU recovery from the heart, with the exception of ΔspA (Fig. 7). It should be noted that all isogenic mutants were determined to have no growth defects in TSB compared to WT SF8300 (data not shown).

FIG 7 .

FIG 7 

Open in a new tab

CFU enumeration of SF8300 wild-type and mutant strains in a murine thromboembolic lesion model. Groups of 10 female BALB/c mice were challenged intravenously (i.v.) with 200 µl of S. aureus (5 × 107 CFU) by tail vein injection. P values were as follows: Δhla, 0.0030; ΔclfA, <0.0001; ΔsdrC, 0.0050; ΔisdB, <0.0001 (Mann-Whitney test). Data shown are from a single replicate of three independent experiments.

DISCUSSION

Understanding how S. aureus regulates gene expression as it transitions from colonization to infection is critical to understanding how S. aureus causes disease. We used qRT-PCR to examine transcript levels of known or putative virulence determinants in bacteria isolated from relevant animal models. In vivo bacterial gene expression data are typically presented relative to an arbitrary in vitro condition, making interpretation of gene expression data in disease states difficult. To avoid this limitation, gene expression patterns were compared directly from nasal colonization to bacteremia to thromboembolic lesions. This reduced the need to compare expression to an in vitro condition and allowed for the assessment of gene expression changes in the bacterium at different stages of infection (e.g., commensal to pathogenic). Out of necessity, nasal colonization expression was compared to in vitro expression to establish a baseline for future comparisons.

Given the well-documented role of adhesins during nasal colonization (14–17, 21–23, 30, 31), it was not surprising that several adhesin genes were dramatically upregulated (5- to 21,000-fold) in at least one strain relative to their highest expression in vitro. For example, ClfB, SdrC, SdrD, and SasG have been proposed to play roles in nasal colonization, in either in vivo (21) or in vitro (22, 23) studies. A central role for SdrC and ClfB in nasal colonization was confirmed, as the ΔsdrC ΔclfB double mutant showed a significant CFU reduction relative to wild-type SF8300. To our knowledge, this is the first direct evidence of a role for SdrC in nasal colonization in vivo. SdrC has been reported to bind β-neurexin, a protein found primarily in brain tissue, not on nasal epithelium (32), suggesting an alternate ligand for SdrC. In addition to clfB and sdrC, transcript levels of clfA, icaB, sdrD, tarK, sasG, and efb were increased, suggesting that they also play a role in nasal colonization. These results support the hypothesis that nasal colonization is a multifactorial process requiring numerous adhesins (23).

Nearly all metal cation acquisition genes analyzed during nasal colonization showed decreased expression relative to their highest in vitro expression, although expression was increased above their lowest in vitro level. This was not unexpected, as the nares are a low-iron environment (33), but their environment may not be as metal ion restricted as RPMI medium. Although isdH expression exhibited a slight increase (3-fold) during nasal colonization compared to the maximum in vitro expression, there was no change in bacterial CFU in the nares of cotton rats colonized with ΔisdH versus WT. This does not rule out a role for IsdH in iron acquisition, but it could reflect the redundant nature of S. aureus iron acquisition systems (33). Similar to published results, hla expression was low in the nares (14), and the Δhla mutant did not affect bacterial CFU in the nares, suggesting hla does not play a significant role in nasal colonization. However, due to the multifactorial nature of nasal colonization, a role for the toxins cannot be ruled out. Finally, the immune evasion proteins exhibited a large increase in expression in nasal colonization. This was expected, as this is the bacterium’s first encounter with the immune system.

Several adhesin genes (fnbA, sdrC, iCab, sasA, tagO, efb, and ebpS), all of the immune evasion genes, and both toxins increased in expression at least 2-fold in the bloodstream of mice relative to nasal colonization, while the metal cation acquisition genes were largely unchanged or decreased relative to expression in the nares. Increased adhesin gene expression suggests that, upon exposure to the bloodstream, S. aureus begins to adhere to host cells or tissue. While a role for icaB during bacteremia has been reported (34), sdrC, sasA, tagO, efb, and ebpS have not been reported to play a role during bacteremia. Several of these adhesins have been studied in both in vitro and in vivo systems, and those results, combined with the results presented here, provide insights into a role for these genes in S. aureus pathogenesis. For example, FnbA and TagO have been reported to play a role in adherence to endothelial cells (29, 35, 36). Although SasA has not been implicated in bloodstream infection, it has been reported that SasA antibodies are detected in convalescent patient sera, providing further evidence that SasA is expressed by S. aureus during systemic infection (37). These data suggest S. aureus, even early in bacteremia, rapidly expresses adhesins to facilitate binding to endothelial surfaces, presumably to escape the bloodstream and colonize host tissues. The three other upregulated adhesin genes, efb, ebpS, and sdrC, have not been implicated in S. aureus bacteremia; however, our data suggest a role for these genes in S. aureus bacteremia and/or endothelial adherence.

The metal cation acquisition proteins (except for isdA and isdB) were still highly expressed in the bloodstream, where they aid in scavenging iron. The difference in isdB and isdA expression relative to nasal colonization could highlight differences in nutrient availability and host iron-scavenging proteins in the nares versus the blood (38). Increased expression of sbi, spA, and lukF-PV suggests that the bacterium is actively engaged in evading the host immune system upon introduction into the bloodstream. Increased hla expression during bacteremia supports previous studies describing a role for alpha-toxin (AT) in bloodstream infections (39).

Transition from bacteremia to heart tissue led to drastically increased expression of most adhesin genes and all metal cation acquisition, toxin, and immune evasion genes, with the exception of spa, relative to bloodstream expression. Increased adhesin gene expression may be expected, as these proteins are generally thought to facilitate adherence to and colonization of host tissues (40, 41). Although some of the upregulated adhesins have been shown to play a role in establishing organ lesions (ClfA [26, 27], FnbA [35], and TagO [29]), many of them do not have a reported role in organ lesion formation. Increased expression of sdrC (13-fold), sasA (4-fold), and sasF (11-fold) are of particular interest in that little is known about their roles in S. aureus virulence. While a ΔsdrC mutant strain led to a relative decrease in CFU recovered from heart lesions compared to WT, further experiments are required to elucidate the role of these putative adhesins in severe S. aureus infections. The upregulation of all metal ion acquisition genes (with the exception of isdH and sstD in strain SF8300) indicates that heart lesions may be more iron limiting than blood. Previous studies have shown iron content varies among organs, with heart muscle containing less iron than other organs, although free blood iron levels were not determined in those studies (42). The expression of spa in the heart is decreased relative to that in the bloodstream; however, Δspa resulted in decreased heart CFU relative to infection with WT. This may be due to high spa expression in the bloodstream; therefore, a decrease in spa between the bloodstream and the heart still results in a significant amount of protein A present on the bacterial surface. Alternatively, perhaps Δspa reduces bacterial survival in the blood, resulting in lower bacterial numbers invading the heart tissue. Although AT has been shown to be important in establishing S. aureus infections, including infective endocarditis (43), there is no evidence that it plays a role in thromboembolic lesions. The data presented here suggest AT plays a role in infecting the heart tissue, both through increased transcript levels and decreased CFU recovered from heart tissue following infection with Δhla relative to infection with WT. One hypothesis for AT’s role in establishing the heart lesions involves its activation of ADAM10-mediated E-cadherin cleavage, resulting in vascular leakage and bacterial invasion of heart tissue (44).

To our knowledge, these results are the first example of a study designed to directly compare virulence factor expression by a bacterium during commensal and pathogenic states. Additionally, this study has shown that this type of analysis is possible, and valuable information can be garnered by comparing expression patterns between colonization and disease states, laying the groundwork for large-scale microarray studies for a broader view of S. aureus as a commensal and a pathogen. Recently, a microarray study examining S. aureus isolated from human skin infections was performed and, as expected, many of the upregulated genes correlated well with our in vivo results (e.g., hla, isdB, isdA, and sitC) (13). The results obtained in this study support previous reports describing the involvement of individual S. aureus genes and proteins in the three disease states, and they provide new information about genes which may play a role in nasal colonization (icaB, clfA, and efb), early bloodstream infection (sdrC, sasA, tagO, fnbA, efb, ebpS, mntC, fhuD, and sstD), and thromboembolic lesions (sdrC, sasA, sasF, isdH, mntC, sstD, fhuD, spA, sbi, and hla). The steadily increasing expression of sdrC, fnbA, fhuD, sstD, and hla (in at least one strain) from nasal colonization to bacteremia to heart lesions suggests a role for these genes in the transition from commensal to pathogen. The expression patterns of the chosen genes often varied between the strains tested, highlighting S. aureus diversity and stressing the need to evaluate multiple clinical isolates when identifying important virulence factors. The information collected provides a basic understanding of S. aureus disease pathogenesis and how a commensal becomes a pathogen, and this understanding may ultimately lead to the development of targeted therapeutics.

MATERIALS AND METHODS

S. aureus strains, media, and growth.

SF830 is a prototypical USA300-0114 community-associated methicillin-resistant S. aureus strain (45). ARC633 (CC15; MSSA) is a nasal colonization isolate provided by David A. Bruckner, UCLA Medical Center. Bacteria were cultured with shaking (250 rpm) at 37°C in TSB for growth curves and for in vitro gene expression cultures. For RNA purification from in vitro cultures, samples were mixed 1:2 with RNAprotect (Qiagen), incubated at room temperature for 5 min, and pelleted by centrifugation. The pellet was resuspended in 1 ml of TRIzol (Invitrogen). The TRIzol-treated samples were lysed by using a FastPrep 24 homogenizer with lysing matrix B tubes (MP Biomedicals). RNA purification was performed as described below. Animal challenge stocks were prepared by growing bacteria overnight in TSB, then diluting cultures into fresh TSB and growing until the optical density at 600 nm reached ~0.8. The bacteria were collected by centrifugation and resuspended in phosphate-buffered saline (PBS; Invitrogen) plus 10% glycerol and frozen at −80°C in aliquots for use in all animal studies.

Cotton rat nasal colonization model.

Cotton rats (n = 12) were challenged intranasally with 5 × 105 CFU S. aureus in 10 µl (5 µl per nostril). Four days postchallenge, the nares were harvested and placed into 20 ml of RNAprotect (for RT-PCR) and incubated for 5 min, or placed into 2 ml of PBS plus 0.1% Tween 20 (for CFU enumeration). For RT-PCR, nares were placed into 10 ml of TRIzol (Invitrogen) and vigorously vortexed to remove bacteria. The TRIzol-treated samples were lysed using the FastPrep 24 homogenizer with lysing matrix B tubes (MP Biomedicals). RNA purification was performed as described below. All RT-PCR data are reported as means of 3 independent experiments using 12 animals/experiment. For CFU enumeration, nares were homogenized using a Polytron PT-10-35 GT apparatus. The samples were serially diluted and plated onto Staph Chrome agar plates (BD Biosciences).

All animal use protocols were reviewed and approved by MedImmune’s IACUC and complied with the animal welfare standards of the USDA, Guide for the Care and Use of Laboratory Animals, and AAALAC International.

Murine bacteremia.

Female BALB/c mice (n = 10) were challenged by tail vein injection of 1 × 108 CFU of S. aureus (200 µl). One hour postchallenge, blood was collected from and pooled in 20 ml of RNAprotect. The bacteria were pelleted by centrifugation, resuspended in 1 ml of TRIzol, and treated as described above. All RT-PCR data are reported as means of 3 independent experiments using 10 animals/experiment.

Murine sepsis.

Female BALB/c mice (n = 10) were challenged by tail vein injection of 1 × 107 CFU S. aureus (200 µl). Fourteen hours postchallenge, hearts were harvested and placed into 20 ml of RNAprotect (for RT-PCR) and incubated for 5 min, or tissue was placed in 1 ml of PBS plus 0.1% Tween 20 (for CFU enumeration). Bacteria and heart tissue were lysed, and RNA was purified as described above. All RT-PCR data are reported as means of 3 independent experiments using 10 animals/experiment. For CFU enumerations, hearts were homogenized in 1 ml of PBS plus 0.1% Tween 20 in lysing matrix A tubes (MP Biomedicals) in a FastPrep 24 homogenizer. Samples were serially diluted and plated onto trypticase soy agar plates (BD Biosciences).

Construction of in-frame gene deletions.

In-frame deletions of selected genes were constructed by allelic replacement using pKOR1 (46). Primers X1-X2 (see Table S3 in the supplemental material) were used to amplify approximately 1,000 bp that corresponded to the first 84 to 153 nucleotides from the start codon and flanking 5′ region. Primers X3-X4 were used to amplify approximately 1,000 bp that corresponded to the last 33 to 159 nucleotides from the stop codon and flanking 3′ region (see Table S3). X1-X2 and X3-X4 PCR products were spliced together by overlap PCR using primers X5 and X6. Attachment sites (attB), appended to 5′ ends of primers X5 and X6 were recombined with the attP sequences flanking a lambda recombination cassette on pKOR1 in the presence of bacteriophage lambda integrase and Escherichia coli integration host factor (Clonase; Invitrogen) and electroporated into E. coli. The in-frame deletion constructs were electroporated into S. aureus RN4220 and then transduced with Φ11 into SF8300. Allelic replacement was performed as described elsewhere (46). Allelic replacement mutants were identified by PCR and DNA sequencing using primers X1 to X4 and primers X1, X4, S1, and S2, respectively (see Table S3). Multiple gene deletions were carried out sequentially.

RNA preparation.

Chloroform (200 µl) was added to 1 ml of lysed bacteria and centrifuged at 14,000 × g, and the top clear layer was removed, added to 1 ml of isopropanol, and then incubated at −20°C overnight. RNA was pelleted by centrifugation (14,000 × g) for 30 min. The pellet was washed with 70% ethanol, dried, resuspended in 100 µl of RNase-free water, and digested with RNase-free DNase (Promega) for 1 h. Following digestion, 1 ml of TRIzol and 200 µl of chloroform were added. RNA precipitation and DNase digestion procedures were repeated. RNA was then purified using the RNeasy minikit (Qiagen).

RT-PCR.

RNA was reverse transcribed into cDNA by using the SuperScript III cDNA synthesis kit (Invitrogen). TaqMan primers (see Table S4 in the supplemental material) containing a 6-carboxyfluorescein reporter and nonfluorescent quencher were designed using the TaqMan design tool (Life Technologies). cDNA samples were assayed in triplicate using 16S rRNA as a control. Samples were assayed using TaqMan universal PCR master mix (Applied Biosystems) on an Applied Biosystems 7900HT apparatus with standard cycling protocols and analyzed using SDS software. Relative expression values were calculated using the ΔΔCT method with 16S RNA as the normalizer (47–49). 16S RNA was determined to be the most stable housekeeping gene in our experiments, based on expression under a variety of in vitro growth conditions (rich medium, minimal medium, or in the presence of serum).

SUPPLEMENTAL MATERIAL

Figure S1 

Impact of cDNA from different RNA source tissues on the efficiency of amplification of a test amplicon. Download

Figure S1, DOCX file, 0.02 MB (16.6KB, docx)
Figure S2 

16S rRNA levels in S. aureus strains SF8300 and ARC633 under various in vitro growth conditions. Download

Figure S2, DOCX file, 0.1 MB (72.6KB, docx)
Table S1 

Genes analyzed in this study

Table S1, DOC file, 0.1 MB. (56KB, doc)
Table S2 

Growth conditions for the highest and lowest in vitro expression levels

Table S2, DOC file, 0.05 MB. (52KB, doc)
Table S3 

Primers used for in-frame gene deletions using pKOR1

Table S3, DOCX file, 0.01 MB. (15KB, docx)
Table S4 

Primers and probes used for TaqMan qPCR

Table S4, DOCX file, 0.01 MB. (16KB, docx)

Footnotes

Citation Jenkins A, Diep BA, Mai TT, Vo NH, Warrener P, Suzich J, Stover CK, Sellman BR. 2015. Differential expression and roles of Staphylococcus aureus virulence determinants during colonization and disease. mBio 6(1):e02272-14. doi:10.1128/mBio.02272-14.

REFERENCES

  • 1.Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med 339:520–532. doi: 10.1056/NEJM199808203390806. [DOI] [PubMed] [Google Scholar]
  • 2.Kluytmans J, van Belkum A, Verbrugh H. 1997. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 10:505–520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Von Eiff C, Becker K, Machka K, Stammer H, Peters G. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. N Engl J Med 344:11–16. doi: 10.1056/NEJM200101043440102. [DOI] [PubMed] [Google Scholar]
  • 4.Lory S, Jin S, Boyd JM, Rakeman JL, Bergman P. 1996. Differential gene expression by Pseudomonas aeruginosa during interaction with respiratory mucus. Am J Respir Crit Care Med 154:S183–S186. doi: 10.1164/ajrccm/154.4_Pt_2.S183. [DOI] [PubMed] [Google Scholar]
  • 5.Sitkiewicz I, Babiak I, Hryniewicz W. 2011. Characterization of transcription within sdr region of Staphylococcus aureus. Antonie Van Leeuwenhoek 99:409–416. doi: 10.1007/s10482-010-9476-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Boyce JD, Cullen PA, Adler B. 2004. Genomic-scale analysis of bacterial gene and protein expression in the host. Emerg Infect Dis 10:1357–1362. doi: 10.3201/eid1008.031036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Malachowa N, Whitney AR, Kobayashi SD, Sturdevant DE, Kennedy AD, Braughton KR, Shabb DW, Diep BA, Chambers HF, Otto M, DeLeo FR. 2011. Global changes in Staphylococcus aureus gene expression in human blood. PLoS One 6:e18617. doi: 10.1371/journal.pone.0018617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Shemesh M, Tam A, Steinberg D. 2007. Differential gene expression profiling of Streptococcus mutans cultured under biofilm and planktonic conditions. Microbiology 153:1307–1317. doi: 10.1099/mic.0.2006/002030-0. [DOI] [PubMed] [Google Scholar]
  • 9.Goerke C, Fluckiger U, Steinhuber A, Bisanzio V, Ulrich M, Bischoff M, Patti JM, Wolz C. 2005. Role of Staphylococcus aureus global regulators sae and sigma B in virulence gene expression during device-related infection. Infect Immun 73:3415–3421. doi: 10.1128/IAI.73.6.3415-3421.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Joost I, Blass D, Burian M, Goerke C, Wolz C, von Müller L, Becker K, Preissner K, Herrmann M, Bischoff M. 2009. Transcription analysis of the extracellular adherence protein from Staphylococcus aureus in authentic human infection and in vitro. J Infect Dis 199:1471–1478. doi: 10.1086/598484. [DOI] [PubMed] [Google Scholar]
  • 11.Wolz C, Goerke C, Landmann R, Zimmerli W, Fluckiger U. 2002. Transcription of clumping factor A in attached and unattached Staphylococcus aureus in vitro and during device-related infection. Infect Immun 70:2758–2762. doi: 10.1128/IAI.70.6.2758-2762.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Loughman JA, Fritz SA, Storch GA, Hunstad DA. 2009. Virulence gene expression in human community-acquired Staphylococcus aureus infection. J Infect Dis 199:294–301. doi: 10.1086/595982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Date SV, Modrusan Z, Lawrence M, Morisaki JH, Toy K, Shah IM, Kim J, Park S, Xu M, Basuino L, Chan L, Zeitschel D, Chambers HF, Tan MW, Brown EJ, Diep BA, Hazenbos WL. 2014. Global gene expression of methicillin-resistant Staphylococcus aureus USA300 during human and mouse infection. J Infect Dis 209:1542–1550. doi: 10.1093/infdis/jit668. [DOI] [PubMed] [Google Scholar]
  • 14.Burian M, Wolz C, Goerke C. 2010. Regulatory adaptation of Staphylococcus aureus during nasal colonization of humans. PLoS One 5:e10040. doi: 10.1371/journal.pone.0010040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Burian M, Rautenberg M, Kohler T, Fritz M, Krismer B, Unger C, Hoffmann WH, Peschel A, Wolz C, Goerke C. 2010. Temporal expression of adhesion factors and activity of global regulators during establishment of Staphylococcus aureus nasal colonization. J Infect Dis 201:1414–1421. doi: 10.1086/651619. [DOI] [PubMed] [Google Scholar]
  • 16.Weidenmaier C, Kokai-Kun JF, Kristian SA, Chanturiya T, Kalbacher H, Gross M, Nicholson G, Neumeister B, Mond JJ, Peschel A. 2004. Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat Med 10:243–245. doi: 10.1038/nm991. [DOI] [PubMed] [Google Scholar]
  • 17.Schaffer AC, Solinga RM, Cocchiaro J, Portoles M, Kiser KB, Risley A, Randall SM, Valtulina V, Speziale P, Walsh E, Foster T, Lee JC. 2006. Immunization with Staphylococcus aureus clumping factor B, a major determinant in nasal carriage, reduces nasal colonization in a murine model. Infect Immun 74:2145–2153. doi: 10.1128/IAI.74.4.2145-2153.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Clarke SR, Brummell KJ, Horsburgh MJ, McDowell PW, Mohamad SA, Stapleton MR, Acevedo J, Read RC, Day NP, Peacock SJ, Mond JJ, Kokai-Kun JF, Foster SJ. 2006. Identification of in vivo-expressed antigens of Staphylococcus aureus and their use in vaccinations for protection against nasal carriage. J Infect Dis 193:1098–1108. doi: 10.1086/501471. [DOI] [PubMed] [Google Scholar]
  • 19.Kiser KB, Cantey-Kiser JM, Lee JC. 1999. Development and characterization of a Staphylococcus aureus nasal colonization model in mice. Infect Immun 67:5001–5006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kokai-Kun JF, Walsh SM, Chanturiya T, Mond JJ. 2003. Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model. Antimicrob Agents Chemother 47:1589–1597. doi: 10.1128/AAC.47.5.1589-1597.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wertheim HF, Walsh E, Choudhurry R, Melles DC, Boelens HA, Miajlovic H, Verbrugh HA, Foster T, van Belkum A. 2008. Key role for clumping factor B in Staphylococcus aureus nasal colonization of humans. PLoS Med 5:e17. doi: 10.1371/journal.pmed.0050017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Corrigan RM, Miajlovic H, Foster TJ. 2009. Surface proteins that promote adherence of Staphylococcus aureus to human desquamated nasal epithelial cells. BMC Microbiol 9:22. doi: 10.1186/1471-2180-9-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Weidenmaier C, Goerke C, Wolz C. 2012. Staphylococcus aureus determinants for nasal colonization. Trends Microbiol 20:243–250. doi: 10.1016/j.tim.2012.03.004. [DOI] [PubMed] [Google Scholar]
  • 24.Mazmanian SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P, Jelenska J, Joachmiak A, Missiakas DM, Schneewind O. 2003. Passage of heme-iron across the envelope of Staphylococcus aureus. Science 299:906–909. doi: 10.1126/science.1081147. [DOI] [PubMed] [Google Scholar]
  • 25.Andrews SC, Robinson AK, Rodríguez-Quiñones F. 2003. Bacterial iron homeostasis. FEMS Microbiol Rev 27:215–237. doi: 10.1016/S0168-6445(03)00055-X. [DOI] [PubMed] [Google Scholar]
  • 26.McAdow M, Kim HK, Dedent AC, Hendrickx AP, Schneewind O, Missiakas DM. 2011. Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood. PLoS Pathog 7:e1002307. doi: 10.1371/journal.ppat.1002307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Cheng AG, Kim HK, Burts ML, Krausz T, Schneewind O, Missiakas DM. 2009. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB J 23:3393–3404. doi: 10.1096/fj.09-135467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Menzies BE. 2003. The role of fibronectin binding proteins in the pathogenesis of Staphylococcus aureus infections. Curr Opin Infect Dis 16:225–229. doi: 10.1097/01.qco.0000073771.11390.75. [DOI] [PubMed] [Google Scholar]
  • 29.Weidenmaier C, Peschel A, Xiong YQ, Kristian SA, Dietz K, Yeaman MR, Bayer AS. 2005. Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis. J Infect Dis 191:1771–1777. doi: 10.1086/429692. [DOI] [PubMed] [Google Scholar]
  • 30.O’Brien LM, Walsh EJ, Massey RC, Peacock SJ, Foster TJ. 2002. Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization. Cell Microbiol 4:759–770. doi: 10.1046/j.1462-5822.2002.00231.x. [DOI] [PubMed] [Google Scholar]
  • 31.Edwards AM, Massey RC, Clarke SR. 2012. Molecular mechanisms of Staphylococcus aureus nasopharyngeal colonization. Mol Oral Microbiol 27:1–10. doi: 10.1111/j.2041-1014.2011.00628.x. [DOI] [PubMed] [Google Scholar]
  • 32.Barbu EM, Ganesh VK, Gurusiddappa S, Mackenzie RC, Foster TJ, Sudhof TC, Höök M. 2010. Beta-neurexin is a ligand for the Staphylococcus aureus MSCRAMM SdrC. PLoS Pathog 6:e1000726. doi: 10.1371/journal.ppat.1000726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Haley KP, Skaar EP. 2012. A battle for iron: host sequestration and Staphylococcus aureus acquisition. Microbes Infect 14:217–227. doi: 10.1016/j.micinf.2011.11.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cerca N, Jefferson KK, Maira-Litrán T, Pier DB, Kelly-Quintos C, Goldmann DA, Azeredo J, Pier GB. 2007. Molecular basis for preferential protective efficacy of antibodies directed to the poorly acetylated form of staphylococcal poly-N-acetyl-beta-(1-6)-glucosamine. Infect Immun 75:3406–3413. doi: 10.1128/IAI.00078-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Edwards AM, Potts JR, Josefsson E, Massey RC. 2010. Staphylococcus aureus host cell invasion and virulence in sepsis is facilitated by the multiple repeats within FnBPA. PLoS Pathog 6:e1000964. doi: 10.1371/journal.ppat.1000964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sinha B, François PP, Nüsse O, Foti M, Hartford OM, Vaudaux P, Foster TJ, Lew DP, Herrmann M, Krause KH. 1999. Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin α5β1. Cell Microbiol 1:101–117. doi: 10.1046/j.1462-5822.1999.00011.x. [DOI] [PubMed] [Google Scholar]
  • 37.Roche FM, Massey R, Peacock SJ, Day NP, Visai L, Speziale P, Lam A, Pallen M, Foster TJ. 2003. Characterization of novel LPXTG-containing proteins of Staphylococcus aureus identified from genome sequences. Microbiology 149:643–654. doi: 10.1099/mic.0.25996-0. [DOI] [PubMed] [Google Scholar]
  • 38.Hammer ND, Skaar EP. 2012. The impact of metal sequestration on Staphylococcus aureus metabolism. Curr Opin Microbiol 15:10–14. doi: 10.1016/j.mib.2011.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Berube BJ, Bubeck Wardenburg J. 2013. Staphylococcus aureus alpha-toxin: nearly a century of intrigue. Toxins (Basel) 5:1140–1166. doi: 10.3390/toxins5061140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Patti JM, Höök M. 1994. Microbial adhesins recognizing extracellular matrix macromolecules. Curr Opin Cell Biol 6:752–758. doi: 10.1016/0955-0674(94)90104-X. [DOI] [PubMed] [Google Scholar]
  • 41.Patti JM, Allen BL, McGavin MJ, Höök M. 1994. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 48:585–617. doi: 10.1146/annurev.mi.48.100194.003101. [DOI] [PubMed] [Google Scholar]
  • 42.Bogniard RP, Whipple GH. 1932. The iron content of blood free tissues and viscera. J Exp Med 55:653–655. doi: 10.1084/jem.55.4.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Bayer AS, Ramos MD, Menzies BE, Yeaman MR, Shen AJ, Cheung AL. 1997. Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins. Infect Immun 65:4652–4660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Powers ME, Kim HK, Wang Y, Bubeck Wardenburg J. 2012. ADAM10 mediates vascular injury induced by Staphylococcus aureus alpha-hemolysin. J Infect Dis 206:352–356. doi: 10.1093/infdis/jis192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Diep BA, Stone GG, Basuino L, Graber CJ, Miller A, des Etages SA, Jones A, Palazzolo-Ballance AM, Perdreau-Remington F, Sensabaugh GF, DeLeo FR, Chambers HF. 2008. The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus. J Infect Dis 197:1523–1530. doi: 10.1086/587907. [DOI] [PubMed] [Google Scholar]
  • 46.Bae T, Schneewind O. 2006. Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55:58–63. doi: 10.1016/j.plasmid.2005.05.005. [DOI] [PubMed] [Google Scholar]
  • 47.Sellman BR, Timofeyeva Y, Nanra J, Scott A, Fulginiti JP, Matsuka YV, Baker SM. 2008. Expression of Staphylococcus epidermidis SdrG increases following exposure to an in vivo environment. Infect Immun 76:2950–2957. doi: 10.1128/IAI.00055-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Marco ML, Kleerebezem M. 2008. Assessment of real-time RT-PCR for quantification of Lactobacillus plantarum gene expression during stationary phase and nutrient starvation. J Appl Microbiol 104:587–594. doi 10.1111/j.1365-2672.2007.03578.x. [DOI] [PubMed] [Google Scholar]
  • 49.Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔCT) method. Methods 25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 

Impact of cDNA from different RNA source tissues on the efficiency of amplification of a test amplicon. Download

Figure S1, DOCX file, 0.02 MB (16.6KB, docx)
Figure S2 

16S rRNA levels in S. aureus strains SF8300 and ARC633 under various in vitro growth conditions. Download

Figure S2, DOCX file, 0.1 MB (72.6KB, docx)
Table S1 

Genes analyzed in this study

Table S1, DOC file, 0.1 MB. (56KB, doc)
Table S2 

Growth conditions for the highest and lowest in vitro expression levels

Table S2, DOC file, 0.05 MB. (52KB, doc)
Table S3 

Primers used for in-frame gene deletions using pKOR1

Table S3, DOCX file, 0.01 MB. (15KB, docx)
Table S4 

Primers and probes used for TaqMan qPCR

Table S4, DOCX file, 0.01 MB. (16KB, docx)

Articles from mBio are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES