Role of Staphylococcus aureus Global Regulators sae and B in Virulence Gene Expression during Device-Related Infection
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感染与免疫杂志 2005年第6期
Institute for Medical Microbiology and Hygiene, University Hospital Tübingen, Tübingen, Germany
Division of Infectious Diseases, University Hospitals Basel, Basel
Department of Medical Microbiology, University of Zurich, Zurich, Switzerland
Inhibitex, Inc., Alpharetta, Georgia
ABSTRACT
The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and B of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. B seemed to be less active throughout the infection than under induced conditions in vitro.
INTRODUCTION
Staphylococcus aureus causes a variety of local and systemic infections in humans and is one of the most important community-acquired and nosocomial pathogens. Staphylococci are the most frequently implicated etiologic agents in device-related infections, in which the bacteria accumulate locally and often persist until the device is removed. Animal models using tissue cages as devices (37) allow the monitoring of various microbiological and immunological events during the course of infection.
The ability of S. aureus to adapt to different environments is probably due to a global regulatory network comprising the loci agr, sar, sigB, rot, arlR/S, svrA, and saeR/S (1, 6-8, 28, 36). Each of these regulators is involved in the control of the expression of virulence factors such as hemolysins (for instance alpha-hemolysin, encoded by hla), protein A, fibronectin-binding proteins (FnBPA and FnBPB, encoded by fnbA and fnbB), or capsular polysaccharide (CP, encoded by the cap operon). Knowledge about the impact of these regulatory circuits on virulence gene expression during infection is still limited. In certain infections the central regulator agr is not involved in the activation of virulence factors (17, 18, 35). For instance in an experimental infective endocarditis model it was shown that fnbA is positively regulated in the absence of agr and sarA (34), suggesting additional regulatory loci in vivo. We could show that the regulator Sae is essential for the transcription of hla during device-related infection in guinea pigs (18). Recently, the importance of sae was shown in two whole-genome screens for the identification of genes required for full virulence (2, 3). The sae locus consists of four ORFs, two of which (saeR and saeS) show strong sequence homology to response regulators and histidine kinases of bacterial two-component regulators (12). Two additional ORFs, ORF3 and ORF4, located upstream of saeR/S are likely to be important for functionality of the sae operon (29, 31). Four overlapping sae-specific transcripts (T1 to T4) originate from three promoters (P1, P2, and P3): the T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR (Fig. 1). T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. The T1, T2, and T3 mRNAs are supposed to terminate at the same stem-loop sequence downstream of saeS (31). The similar molecular architecture of the sae locus was described by Novick and Jiang (29) using other designations for the components (ORF3 [saeQ], ORF4 [saeP], etc.). The transcription pattern of the sae operon is strongly influenced by other regulators (13, 29), as well as by diverse environmental parameters, such as pH or subinhibitory concentrations of antibiotics (23, 29). The sae operon activates the expression of several virulence factors, including serine proteases, nuclease, coagulase (encoded by coa), hla, and fnbA, but it represses the expression of the cap operon (14, 15, 29, 31). These effects on target genes are not likely to be mediated by RNAIII or sarA activity since the same amounts of these two regulators were transcribed in an sae-transposon mutant (13).
The interaction of sae with the alternative sigma factor B of S. aureus is largely unknown. However, all of the known sae target genes were also found to be influenced by B, albeit not always in the same direction. Using a microarray approach it has been demonstrated that coa, fnbA, cap, and also clumping factor A (encoded by clfA) are activated by B, whereas hla, serine proteases, and nuclease are repressed (4). Interestingly, a typical B consensus promoter could be identified only in front of some of the target genes, indicating that B exerts its effect on virulence factor expression in many cases not by direct activation but via other regulators. In contrast, the transcription of alkaline shock protein 23 (asp23) is solely dependent on B (4, 10, 11, 22, 24), making it an ideal target to monitor B activity.
In this report, we present a detailed study of the interaction between the two regulators sae and B and their influence on virulence gene expression both in vitro and in vivo. Transcription analysis was performed with isogenic sae and sigB mutants of S. aureus strain Newman in batch culture and in vivo in a guinea pig model of implant infection.
MATERIALS AND METHODS
Bacterial strains and growth conditions. S. aureus strains Newman (9), AS3 (Newman, sae::Tn917; Emr) (18), and IK184 (Newman rsbUVW-sigB; Emr) (22) were routinely cultured on sheep blood agar or grown in CYPG medium (27). When included, erythromycin was used in a concentration of 10 μg ml–1. For transcript analysis in vitro, the cells were inoculated from an overnight culture to an initial optical density at 600 nm (OD600) of 0.05 in CYPG and grown to the exponential (OD600 = 0.8, 2.5 h) or postexponential (OD600 = 8, 8 h) phase.
Animal model of device-related infection. The guinea pig model of implant infection was used (37). Four perforated Teflon tubes were inserted in the flanks of guinea pigs (30). Two weeks after the implantation of the tissue cages, 105 CFU of the test strain were inoculated in the tissue cages. Before inoculation, the interstitial fluid that had accumulated in the tissue cages was checked for sterility. The exudate was aspirated 2 and 8 days after infection. One aliquot of the exudate was immediately stored in liquid nitrogen for subsequent RNA preparation. A second aliquot was used for quantitative bacteriology. A third aliquot was mixed with the same volume of 8% paraformaldehyde for immunofluorescence analysis.
RNA isolation and Northern analysis. For RNA preparation from exudates, the frozen samples were thawed rapidly and 200-μl aliquots were used. RNA was isolated and purified as described previously (16). S. aureus cells were lysed directly in 1 ml of TRIzol LS reagent (Invitrogen Life Technologies, Karlsruhe, Germany) with 0.5 ml of zirconia/silica beads (0.1 mm in diameter) in a high-speed homogenizer (Savant Instruments, Farmingdale, N.Y.). RNA was isolated as described in the instructions provided by the manufacturer of TRIzol LS. In order to remove PCR inhibitors, the RNA was further purified with the viral nucleic acid kit (Roche Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Contaminating DNA was degraded by digesting RNA samples with DNase as described elsewhere (17).
RNA preparation from culture and Northern analysis were performed as described previously (17). Briefly, ca. 109 S. aureus cells were lysed in 1 ml of TRIzol reagent (Invitrogen Life Technologies) in a high-speed homogenizer (Savant Instruments). RNA was isolated as described in the instructions provided by the manufacturer of TRIzol. Several DIG-labeled probes for the detection of specific transcripts were generated by using a DIG-Labeling PCR kit according to the manufacturer's instructions (Roche Biochemicals). Primers are listed in Table 1.
Construction of RNA standards. Sequence-specific RNA standards for LightCycler reverse transcription-PCR (RT-PCR) were engineered by using the following protocol: PCR was performed with a gene-specific primer with a 5'-extension encompassing the T7 phage promoter sequence, thus generating transcription-competent amplicons. Primers for standard construction are listed in Table 1. T7-driven in vitro transcription was performed by using a standard transcription assay (T7-MEGAShortscript; Ambion, Huntingdon, United Kingdom). The reaction mixture was subjected to DNase I treatment (Roche Biochemicals), and the RNA was recovered with the MEGAclear kit (Ambion). Quantification of the transcripts was done spectrophotometrically and verified by ethidium bromide staining on agarose gels.
Quantification of specific transcripts with LightCycler RT-PCR. LightCycler RT-PCR was carried out by using the LightCycler RNA amplification kit for hybridization probes or with the LightCycler RNA amplification kit SYBR Green I (Roche Biochemicals). Master mixes were prepared according to the manufacturer's instructions with the oligonucleotides shown in Table 1. Specific primers were selected in such a way that they bind to an internal part of the respective RNA standard. Standard curves were generated by using 10-fold serial dilutions (104 to 108 copies/μl) of the specific RNA standards. The number of copies of each sample transcript was then determined with the aid of the LightCycler software. At least two independent RT-PCR runs were performed for each sample. The specificity of the PCR was verified by ethidium bromide staining on 3% agarose gels. To check for DNA contamination, each sample and RNA standard was subjected to PCR by using the LightCycler DNA amplification kit SYBR Green I (Roche Biochemicals). In none of the cases was an amplification product detectable.
ClfA detection by indirect immunofluorescence in vivo. ClfA production in vivo was determined in exudates that were mixed with the same volume of 8% paraformaldehyde immediately after aspiration and then spotted on poly-L-lysine-coated slides. The slides were washed three times with phosphate-buffered saline (PBS)-Tween and incubated with human serum (1:10 in PBS) for 30 min to prevent unspecific binding of immunoglobulin G by cell-wall-associated protein A. The slides were incubated with a ClfA-specific monoclonal antiserum from the mouse (19) (1:200 in PBS-Tween) for 1 h, followed by incubation of CY2-conjugated anti-mouse F(ab)2 fragment (Dianova, Hamburg, Germany) (1:50 in PBS-Tween) for 1 h. Bacteria were also stained with 2 μg DAPI (4',6'-diamidino-2-phenylindole)/ml for 5 min, washed three times with water, and air dried. The slides were then mounted with fluorescent mounting medium (DakoCytomation, Hamburg, Germany), and positively stained bacteria were detected by using fluorescence microscopy.
RESULTS AND DISCUSSION
Influence of sae and B on target genes during growth in culture. In a first step the interaction between the global regulators sae and B was assessed in isogenic mutants of strain Newman (wild type [WT]) by Northern analysis. In addition, selected target genes were analyzed that had already been reported to be influenced by both regulators.
sae-specific transcripts T1 to T4 were discerned by using probes specific for saeR to detect T1, T2, and T3 and probes specific for ORF4 to detect T1 and T4. The constitutively expressed T3 transcript is less distinct in strain Newman (31). There was no indication of direct cross talk between the global regulators sae and B (Fig. 2). None of the transcripts of the sae system are altered with respect to expression in the sigB mutant of strain Newman at any time point analyzed. These results were corroborated by LightCycler RT-PCR detecting saeR (exponential phase: 1.41 copies of sae/copy of gyr in the WT, 1.08 copies of sae/copy of gyr in the sigB mutant; postexponential phase: 10.87 copies of sae/copy of gyr in the WT, 12.65 copies of sae/copy of gyr in the sigB mutant).
These findings are consistent with those of Bischoff et al. (4), who found no influence of B on sae expression in the genetically distinct S. aureus strains COL, Newman, and GP268. Interestingly, Novick et al. (29) described an inhibitory effect of RsbU and B on sae expression in strain 8325-4. This discrepancy in the results is most probably strain dependent. We were able to show in an earlier study that 8325-4 exhibits markedly lower overall transcription of sae than strain Newman (31). Possibly, the sigB effect is not evident in the high-level sae producer.
The independency of the regulators was further supported by analysis of sigB expression in the sae mutant. Comparable levels of sigB were observed in both the WT and the sae mutant under both in vitro conditions analyzed (Fig. 2). The described additional transcripts of the sigB operon (21) were not detectable in our system. In agreement with a recent microarray study (4), B-dependent genes, such as asp23 and clfA, were transcribed mainly during the stationary-growth phase. In addition, our data indicate that asp23 and clfA expression was not affected by sae.
The situation is somewhat different for the expression of the cytotoxic hla, which is influenced contrarily by both regulators; whereas hla transcription is completely abolished in the sae mutant, it is clearly elevated in the sigB mutant compared to the WT. Thus, sae seems to be essential for hla activation, whereas the presence of B seems to inhibit hla. Since B did apparently not affect sae expression, other factors have to be postulated that are responsible for the increase in hla expression observed in the sigB mutant. A likely candidate for such a scenario might be RNAIII of the agr locus, which on the one hand exerts a positive effect on hla expression (25) and on the other hand was shown to be influenced negatively by B activity (5, 20).
Interestingly, although appearing to act mainly independent of each other, both regulators seem to be important for coa and fnbA transcription (Fig. 2). The most profound effect on transcription was observed in the sae mutant. A B consensus sequence was predicted only in the promoter region of coa (24, 26). A comparison of the upstream sequences of fnbA and coa revealed no obvious common motifs for sae activation (31). Thus, additional factors are needed to explain the observed regulatory pattern of fnbA and coa expression. This is further emphasized by the observation that both genes are strictly repressed during the late growth cycle, which cannot be ascribed to any of the regulatory loci studied thus far.
Influence of sae and B on target genes during device-related infection. The impact of sae and B on target gene expression was further analyzed during device-related infection by quantitative LightCycler RT-PCR. All strains established infection after inoculation of the tissue cages with 105 CFU/ml, reaching a density of ca. 109 CFU/ml after 8 days. In general, there were no significant differences between strain Newman and its isogenic sae and sigB mutants with respect to the densities found in the exudates (data not shown).
In agreement with our in vitro findings, no influence of B on sae transcription was discernible. Quantifying either the transcripts T1, T2, or T3 (saeR) of the sae operon, which are initiated from the promoters P1, P2, and P3, respectively, or the transcripts T1 and T4 (ORF4) initiating from P1 yielded comparable copy numbers in the WT and the sigB mutant cells (Fig. 3A and B). Interestingly, in vivo the copy number of ORF4-containing transcripts was significantly lower than that of saeR-containing transcripts. The same analysis in vitro revealed nearly identical levels of saeR and of ORF4 transcripts in the WT, independent of the growth phase (for instance, 10.9 copies of saeR/copy of gyr and 14.1 copies of ORF4/copy of gyr in postexponential phase). It is conceivable that in vivo P2, P3, or other as-yet-unidentified promoters downstream of ORF4 are induced. In fact, additional transcripts are detectable under certain growth conditions in vitro (unpublished data).
In order to analyze B effects in vivo, we first focused on the transcription of the target genes asp23 and clfA with known B consensus promoters. A high copy number of asp23 transcripts was detectable in Newman WT early in infection (day 2), which seemed to decline in the later infection stage (day 8). The asp23 in vivo expression pattern was again not influenced by the sae mutation, whereas almost no asp23-specific transcript was detectable in the sigB mutant in both infection stages analyzed (Fig. 3C). This emphasizes the predominant role of B for the regulation of this gene also during infection. However, the finding that asp23-specific transcripts were still detectable in a sigB mutant was somewhat surprising, since asp23 transcription is believed to rely exclusively on B activity (4, 10, 11, 22, 24). One explanation for this finding might be that S. aureus might produce basal amounts of asp23-containing transcripts in a B-independent manner, which are sufficient to be detected by real-time PCR but are not identified by less sensitive methods such as Northern blotting and microarray analysis. Interestingly, transcription levels of asp23 found in vivo were well below the amounts found in the postexponential phase (data not shown). Thus, B seems to be less active throughout the device-related infection than under induced conditions in vitro.
Unlike asp23, clfA expression seemed to increase with the infection time. Although 1.4 copies of clfA/copy of gyr were detectable in exudates from infected devices 2 days after inoculation with the WT, a fourfold increase was observed in exudates from day 8 (Fig. 3D), which is in agreement with previous observations showing that clfA expression is induced late during infection (33). In accordance with the in vitro findings, no effect of sae on clfA expression was detectable. Interestingly, transcription of clfA was only diminished in the sigB mutant compared to the WT, and the temporal expression pattern was still traceable. Additional regulatory pathways are obviously needed for the full expression of ClfA, leading to induction, especially late during infection. However, thus far none of the described regulators have been shown to influence clfA expression.
In analyzing genes that are mutually influenced by B and sae, it became evident that sae had a dominant effect on these target genes. In the sigB mutant hla transcription was only marginally higher than in the WT in vivo (Fig. 3E), whereas in the sae mutant hla expression was totally abolished. Surprisingly, the expression of coa, a B-activated gene, was not altered in the sigB mutant compared to the WT under in vivo conditions (Fig. 3F), which contradicts the in vitro findings, where coa expression clearly decreased in the sigB mutant (Fig. 2). One possible explanation for this discrepancy might be that coa expression is mainly driven by a B-independent mechanism in vivo, which is not that important under in vitro conditions.
When we compared gene expression 2 and 8 days after inoculation it became evident that the sequential activation of virulence genes in vivo was profoundly different from that seen during growth in vitro. It has already been shown that the maximum expression of hla occurs early and that of clfA late during infection (18, 33). Here, we could also demonstrate that the expression patterns of hla and coa were closely linked to sae expression. Thus, sae may be the predominant regulator determining expression of these genes in vivo. The signal leading to downregulation of sae during infection remains to be clarified. It has been shown that sae reacts to diverse environmental parameters such as pH or subinhibitory concentrations of antibiotics (23, 29, 32).
Correlation between transcript analysis and protein expression. In order to correlate transcript quantification with protein expression, LightCycler data were compared to an immunofluorescence assay for ClfA. The assay was performed with exudates derived from animals infected with strain Newman and its isogenic sae and sigB mutant 2 and 8 days after inoculation. A clear increase of the fluorescence signal was detected during the course of infection in the WT and in the sae mutant (Fig. 4). The sigB mutant showed markedly diminished signals at both time points. The results obtained by quantitative transcript analysis of clfA (Fig. 3D) correlated well with the detection of ClfA by immunofluorescence staining, indicating that bacterial transcript analysis is an appropriate tool for discerning the phenotype of bacteria in the host.
ACKNOWLEDGMENTS
We thank Zarko Rajacic for expert technical assistance in the animal experiments.
This study was supported by the Deutsche Forschungsgemeinschaft (Wo 578/3-3). Research in the laboratory of M.B. is supported by Swiss National Science Foundation grant 3100A0-100234.
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Division of Infectious Diseases, University Hospitals Basel, Basel
Department of Medical Microbiology, University of Zurich, Zurich, Switzerland
Inhibitex, Inc., Alpharetta, Georgia
ABSTRACT
The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and B of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. B seemed to be less active throughout the infection than under induced conditions in vitro.
INTRODUCTION
Staphylococcus aureus causes a variety of local and systemic infections in humans and is one of the most important community-acquired and nosocomial pathogens. Staphylococci are the most frequently implicated etiologic agents in device-related infections, in which the bacteria accumulate locally and often persist until the device is removed. Animal models using tissue cages as devices (37) allow the monitoring of various microbiological and immunological events during the course of infection.
The ability of S. aureus to adapt to different environments is probably due to a global regulatory network comprising the loci agr, sar, sigB, rot, arlR/S, svrA, and saeR/S (1, 6-8, 28, 36). Each of these regulators is involved in the control of the expression of virulence factors such as hemolysins (for instance alpha-hemolysin, encoded by hla), protein A, fibronectin-binding proteins (FnBPA and FnBPB, encoded by fnbA and fnbB), or capsular polysaccharide (CP, encoded by the cap operon). Knowledge about the impact of these regulatory circuits on virulence gene expression during infection is still limited. In certain infections the central regulator agr is not involved in the activation of virulence factors (17, 18, 35). For instance in an experimental infective endocarditis model it was shown that fnbA is positively regulated in the absence of agr and sarA (34), suggesting additional regulatory loci in vivo. We could show that the regulator Sae is essential for the transcription of hla during device-related infection in guinea pigs (18). Recently, the importance of sae was shown in two whole-genome screens for the identification of genes required for full virulence (2, 3). The sae locus consists of four ORFs, two of which (saeR and saeS) show strong sequence homology to response regulators and histidine kinases of bacterial two-component regulators (12). Two additional ORFs, ORF3 and ORF4, located upstream of saeR/S are likely to be important for functionality of the sae operon (29, 31). Four overlapping sae-specific transcripts (T1 to T4) originate from three promoters (P1, P2, and P3): the T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR (Fig. 1). T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. The T1, T2, and T3 mRNAs are supposed to terminate at the same stem-loop sequence downstream of saeS (31). The similar molecular architecture of the sae locus was described by Novick and Jiang (29) using other designations for the components (ORF3 [saeQ], ORF4 [saeP], etc.). The transcription pattern of the sae operon is strongly influenced by other regulators (13, 29), as well as by diverse environmental parameters, such as pH or subinhibitory concentrations of antibiotics (23, 29). The sae operon activates the expression of several virulence factors, including serine proteases, nuclease, coagulase (encoded by coa), hla, and fnbA, but it represses the expression of the cap operon (14, 15, 29, 31). These effects on target genes are not likely to be mediated by RNAIII or sarA activity since the same amounts of these two regulators were transcribed in an sae-transposon mutant (13).
The interaction of sae with the alternative sigma factor B of S. aureus is largely unknown. However, all of the known sae target genes were also found to be influenced by B, albeit not always in the same direction. Using a microarray approach it has been demonstrated that coa, fnbA, cap, and also clumping factor A (encoded by clfA) are activated by B, whereas hla, serine proteases, and nuclease are repressed (4). Interestingly, a typical B consensus promoter could be identified only in front of some of the target genes, indicating that B exerts its effect on virulence factor expression in many cases not by direct activation but via other regulators. In contrast, the transcription of alkaline shock protein 23 (asp23) is solely dependent on B (4, 10, 11, 22, 24), making it an ideal target to monitor B activity.
In this report, we present a detailed study of the interaction between the two regulators sae and B and their influence on virulence gene expression both in vitro and in vivo. Transcription analysis was performed with isogenic sae and sigB mutants of S. aureus strain Newman in batch culture and in vivo in a guinea pig model of implant infection.
MATERIALS AND METHODS
Bacterial strains and growth conditions. S. aureus strains Newman (9), AS3 (Newman, sae::Tn917; Emr) (18), and IK184 (Newman rsbUVW-sigB; Emr) (22) were routinely cultured on sheep blood agar or grown in CYPG medium (27). When included, erythromycin was used in a concentration of 10 μg ml–1. For transcript analysis in vitro, the cells were inoculated from an overnight culture to an initial optical density at 600 nm (OD600) of 0.05 in CYPG and grown to the exponential (OD600 = 0.8, 2.5 h) or postexponential (OD600 = 8, 8 h) phase.
Animal model of device-related infection. The guinea pig model of implant infection was used (37). Four perforated Teflon tubes were inserted in the flanks of guinea pigs (30). Two weeks after the implantation of the tissue cages, 105 CFU of the test strain were inoculated in the tissue cages. Before inoculation, the interstitial fluid that had accumulated in the tissue cages was checked for sterility. The exudate was aspirated 2 and 8 days after infection. One aliquot of the exudate was immediately stored in liquid nitrogen for subsequent RNA preparation. A second aliquot was used for quantitative bacteriology. A third aliquot was mixed with the same volume of 8% paraformaldehyde for immunofluorescence analysis.
RNA isolation and Northern analysis. For RNA preparation from exudates, the frozen samples were thawed rapidly and 200-μl aliquots were used. RNA was isolated and purified as described previously (16). S. aureus cells were lysed directly in 1 ml of TRIzol LS reagent (Invitrogen Life Technologies, Karlsruhe, Germany) with 0.5 ml of zirconia/silica beads (0.1 mm in diameter) in a high-speed homogenizer (Savant Instruments, Farmingdale, N.Y.). RNA was isolated as described in the instructions provided by the manufacturer of TRIzol LS. In order to remove PCR inhibitors, the RNA was further purified with the viral nucleic acid kit (Roche Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Contaminating DNA was degraded by digesting RNA samples with DNase as described elsewhere (17).
RNA preparation from culture and Northern analysis were performed as described previously (17). Briefly, ca. 109 S. aureus cells were lysed in 1 ml of TRIzol reagent (Invitrogen Life Technologies) in a high-speed homogenizer (Savant Instruments). RNA was isolated as described in the instructions provided by the manufacturer of TRIzol. Several DIG-labeled probes for the detection of specific transcripts were generated by using a DIG-Labeling PCR kit according to the manufacturer's instructions (Roche Biochemicals). Primers are listed in Table 1.
Construction of RNA standards. Sequence-specific RNA standards for LightCycler reverse transcription-PCR (RT-PCR) were engineered by using the following protocol: PCR was performed with a gene-specific primer with a 5'-extension encompassing the T7 phage promoter sequence, thus generating transcription-competent amplicons. Primers for standard construction are listed in Table 1. T7-driven in vitro transcription was performed by using a standard transcription assay (T7-MEGAShortscript; Ambion, Huntingdon, United Kingdom). The reaction mixture was subjected to DNase I treatment (Roche Biochemicals), and the RNA was recovered with the MEGAclear kit (Ambion). Quantification of the transcripts was done spectrophotometrically and verified by ethidium bromide staining on agarose gels.
Quantification of specific transcripts with LightCycler RT-PCR. LightCycler RT-PCR was carried out by using the LightCycler RNA amplification kit for hybridization probes or with the LightCycler RNA amplification kit SYBR Green I (Roche Biochemicals). Master mixes were prepared according to the manufacturer's instructions with the oligonucleotides shown in Table 1. Specific primers were selected in such a way that they bind to an internal part of the respective RNA standard. Standard curves were generated by using 10-fold serial dilutions (104 to 108 copies/μl) of the specific RNA standards. The number of copies of each sample transcript was then determined with the aid of the LightCycler software. At least two independent RT-PCR runs were performed for each sample. The specificity of the PCR was verified by ethidium bromide staining on 3% agarose gels. To check for DNA contamination, each sample and RNA standard was subjected to PCR by using the LightCycler DNA amplification kit SYBR Green I (Roche Biochemicals). In none of the cases was an amplification product detectable.
ClfA detection by indirect immunofluorescence in vivo. ClfA production in vivo was determined in exudates that were mixed with the same volume of 8% paraformaldehyde immediately after aspiration and then spotted on poly-L-lysine-coated slides. The slides were washed three times with phosphate-buffered saline (PBS)-Tween and incubated with human serum (1:10 in PBS) for 30 min to prevent unspecific binding of immunoglobulin G by cell-wall-associated protein A. The slides were incubated with a ClfA-specific monoclonal antiserum from the mouse (19) (1:200 in PBS-Tween) for 1 h, followed by incubation of CY2-conjugated anti-mouse F(ab)2 fragment (Dianova, Hamburg, Germany) (1:50 in PBS-Tween) for 1 h. Bacteria were also stained with 2 μg DAPI (4',6'-diamidino-2-phenylindole)/ml for 5 min, washed three times with water, and air dried. The slides were then mounted with fluorescent mounting medium (DakoCytomation, Hamburg, Germany), and positively stained bacteria were detected by using fluorescence microscopy.
RESULTS AND DISCUSSION
Influence of sae and B on target genes during growth in culture. In a first step the interaction between the global regulators sae and B was assessed in isogenic mutants of strain Newman (wild type [WT]) by Northern analysis. In addition, selected target genes were analyzed that had already been reported to be influenced by both regulators.
sae-specific transcripts T1 to T4 were discerned by using probes specific for saeR to detect T1, T2, and T3 and probes specific for ORF4 to detect T1 and T4. The constitutively expressed T3 transcript is less distinct in strain Newman (31). There was no indication of direct cross talk between the global regulators sae and B (Fig. 2). None of the transcripts of the sae system are altered with respect to expression in the sigB mutant of strain Newman at any time point analyzed. These results were corroborated by LightCycler RT-PCR detecting saeR (exponential phase: 1.41 copies of sae/copy of gyr in the WT, 1.08 copies of sae/copy of gyr in the sigB mutant; postexponential phase: 10.87 copies of sae/copy of gyr in the WT, 12.65 copies of sae/copy of gyr in the sigB mutant).
These findings are consistent with those of Bischoff et al. (4), who found no influence of B on sae expression in the genetically distinct S. aureus strains COL, Newman, and GP268. Interestingly, Novick et al. (29) described an inhibitory effect of RsbU and B on sae expression in strain 8325-4. This discrepancy in the results is most probably strain dependent. We were able to show in an earlier study that 8325-4 exhibits markedly lower overall transcription of sae than strain Newman (31). Possibly, the sigB effect is not evident in the high-level sae producer.
The independency of the regulators was further supported by analysis of sigB expression in the sae mutant. Comparable levels of sigB were observed in both the WT and the sae mutant under both in vitro conditions analyzed (Fig. 2). The described additional transcripts of the sigB operon (21) were not detectable in our system. In agreement with a recent microarray study (4), B-dependent genes, such as asp23 and clfA, were transcribed mainly during the stationary-growth phase. In addition, our data indicate that asp23 and clfA expression was not affected by sae.
The situation is somewhat different for the expression of the cytotoxic hla, which is influenced contrarily by both regulators; whereas hla transcription is completely abolished in the sae mutant, it is clearly elevated in the sigB mutant compared to the WT. Thus, sae seems to be essential for hla activation, whereas the presence of B seems to inhibit hla. Since B did apparently not affect sae expression, other factors have to be postulated that are responsible for the increase in hla expression observed in the sigB mutant. A likely candidate for such a scenario might be RNAIII of the agr locus, which on the one hand exerts a positive effect on hla expression (25) and on the other hand was shown to be influenced negatively by B activity (5, 20).
Interestingly, although appearing to act mainly independent of each other, both regulators seem to be important for coa and fnbA transcription (Fig. 2). The most profound effect on transcription was observed in the sae mutant. A B consensus sequence was predicted only in the promoter region of coa (24, 26). A comparison of the upstream sequences of fnbA and coa revealed no obvious common motifs for sae activation (31). Thus, additional factors are needed to explain the observed regulatory pattern of fnbA and coa expression. This is further emphasized by the observation that both genes are strictly repressed during the late growth cycle, which cannot be ascribed to any of the regulatory loci studied thus far.
Influence of sae and B on target genes during device-related infection. The impact of sae and B on target gene expression was further analyzed during device-related infection by quantitative LightCycler RT-PCR. All strains established infection after inoculation of the tissue cages with 105 CFU/ml, reaching a density of ca. 109 CFU/ml after 8 days. In general, there were no significant differences between strain Newman and its isogenic sae and sigB mutants with respect to the densities found in the exudates (data not shown).
In agreement with our in vitro findings, no influence of B on sae transcription was discernible. Quantifying either the transcripts T1, T2, or T3 (saeR) of the sae operon, which are initiated from the promoters P1, P2, and P3, respectively, or the transcripts T1 and T4 (ORF4) initiating from P1 yielded comparable copy numbers in the WT and the sigB mutant cells (Fig. 3A and B). Interestingly, in vivo the copy number of ORF4-containing transcripts was significantly lower than that of saeR-containing transcripts. The same analysis in vitro revealed nearly identical levels of saeR and of ORF4 transcripts in the WT, independent of the growth phase (for instance, 10.9 copies of saeR/copy of gyr and 14.1 copies of ORF4/copy of gyr in postexponential phase). It is conceivable that in vivo P2, P3, or other as-yet-unidentified promoters downstream of ORF4 are induced. In fact, additional transcripts are detectable under certain growth conditions in vitro (unpublished data).
In order to analyze B effects in vivo, we first focused on the transcription of the target genes asp23 and clfA with known B consensus promoters. A high copy number of asp23 transcripts was detectable in Newman WT early in infection (day 2), which seemed to decline in the later infection stage (day 8). The asp23 in vivo expression pattern was again not influenced by the sae mutation, whereas almost no asp23-specific transcript was detectable in the sigB mutant in both infection stages analyzed (Fig. 3C). This emphasizes the predominant role of B for the regulation of this gene also during infection. However, the finding that asp23-specific transcripts were still detectable in a sigB mutant was somewhat surprising, since asp23 transcription is believed to rely exclusively on B activity (4, 10, 11, 22, 24). One explanation for this finding might be that S. aureus might produce basal amounts of asp23-containing transcripts in a B-independent manner, which are sufficient to be detected by real-time PCR but are not identified by less sensitive methods such as Northern blotting and microarray analysis. Interestingly, transcription levels of asp23 found in vivo were well below the amounts found in the postexponential phase (data not shown). Thus, B seems to be less active throughout the device-related infection than under induced conditions in vitro.
Unlike asp23, clfA expression seemed to increase with the infection time. Although 1.4 copies of clfA/copy of gyr were detectable in exudates from infected devices 2 days after inoculation with the WT, a fourfold increase was observed in exudates from day 8 (Fig. 3D), which is in agreement with previous observations showing that clfA expression is induced late during infection (33). In accordance with the in vitro findings, no effect of sae on clfA expression was detectable. Interestingly, transcription of clfA was only diminished in the sigB mutant compared to the WT, and the temporal expression pattern was still traceable. Additional regulatory pathways are obviously needed for the full expression of ClfA, leading to induction, especially late during infection. However, thus far none of the described regulators have been shown to influence clfA expression.
In analyzing genes that are mutually influenced by B and sae, it became evident that sae had a dominant effect on these target genes. In the sigB mutant hla transcription was only marginally higher than in the WT in vivo (Fig. 3E), whereas in the sae mutant hla expression was totally abolished. Surprisingly, the expression of coa, a B-activated gene, was not altered in the sigB mutant compared to the WT under in vivo conditions (Fig. 3F), which contradicts the in vitro findings, where coa expression clearly decreased in the sigB mutant (Fig. 2). One possible explanation for this discrepancy might be that coa expression is mainly driven by a B-independent mechanism in vivo, which is not that important under in vitro conditions.
When we compared gene expression 2 and 8 days after inoculation it became evident that the sequential activation of virulence genes in vivo was profoundly different from that seen during growth in vitro. It has already been shown that the maximum expression of hla occurs early and that of clfA late during infection (18, 33). Here, we could also demonstrate that the expression patterns of hla and coa were closely linked to sae expression. Thus, sae may be the predominant regulator determining expression of these genes in vivo. The signal leading to downregulation of sae during infection remains to be clarified. It has been shown that sae reacts to diverse environmental parameters such as pH or subinhibitory concentrations of antibiotics (23, 29, 32).
Correlation between transcript analysis and protein expression. In order to correlate transcript quantification with protein expression, LightCycler data were compared to an immunofluorescence assay for ClfA. The assay was performed with exudates derived from animals infected with strain Newman and its isogenic sae and sigB mutant 2 and 8 days after inoculation. A clear increase of the fluorescence signal was detected during the course of infection in the WT and in the sae mutant (Fig. 4). The sigB mutant showed markedly diminished signals at both time points. The results obtained by quantitative transcript analysis of clfA (Fig. 3D) correlated well with the detection of ClfA by immunofluorescence staining, indicating that bacterial transcript analysis is an appropriate tool for discerning the phenotype of bacteria in the host.
ACKNOWLEDGMENTS
We thank Zarko Rajacic for expert technical assistance in the animal experiments.
This study was supported by the Deutsche Forschungsgemeinschaft (Wo 578/3-3). Research in the laboratory of M.B. is supported by Swiss National Science Foundation grant 3100A0-100234.
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