Regulation of Staphylococcus aureus α-Toxin Gene (hla) Expression by agr, sarA and sae In Vitro and in Experimental Infective Endocarditis

Journal of Infectious Diseases, Nov 2006

BackgroundThe Staphylococcus aureus global regulators—agr, sarA and sae—coordinately control α-toxin gene (hla) expression in vitro. However, their relative in vivo contributions to hla expression, particularly in endovascular infections, have not been defined MethodsA plasmid-based hla-promoter:green fluorescent protein reporter system was constructed in 2 genetically related S. aureus strains: RN6390 (a natural sigma factor B [sigB]–deficient mutant), SH1000 (a sigB-repaired variant of RN6390 lineage), and their respective agr, sarA, agr/sarA and sae mutants. These strain sets were used to quantify hla expression in vitro and in an experimental infective endocarditis (IE) model using flow cytometry ResultsIn vitro, hla expression was positively modulated by all 3 regulons (sae > agr/sarA > agr and sarA) in both RN6390 and SH1000 backgrounds. In the IE model, hla expression in cardiac vegetations was lower in all single mutants than in the respective parental strains (P<.05 for sae mutant) but was maintained at near-parental levels in the agr/sarA double mutant in both backgrounds. A similar finding was also observed in kidneys and spleens ConclusionsThese results indicate that, although several distinct regulatory circuits can affect hla expression in vitro and in vivo, sae appears to play a crucial role in this context

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://jid.oxfordjournals.org/content/194/9/1267.full.pdf

Regulation of Staphylococcus aureus α-Toxin Gene (hla) Expression by agr, sarA and sae In Vitro and in Experimental Infective Endocarditis

JID Regulation of Staphylococcus aureus a-Toxin Gene (hla) Expression by agr, sarA, and sae In Vitro and in Experimental Infective Endocarditis Yan Q. Xiong 1 2 Julie Willard 2 Michael R. Yeaman 1 2 Ambrose L. Cheung () 0 Arnold S. Bayer 1 2 0 Department of Microbiology, Dartmouth Medical School , Hanover, New Hampshire 1 David Geffen School of Medicine at University of California Los Angeles , Los Angeles 2 Department of Medicine, Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-University of California Los Angeles Medical Center , Torrance Background. The Staphylococcus aureus global regulators-agr, sarA, and sae-coordinately control a-toxin gene (hla) expression in vitro. However, their relative in vivo contributions to hla expression, particularly in endovascular infections, have not been defined Methods. A plasmid-based hla-promoter:green fluo escent protein reporter system was constructed in 2 genetically related S. aureus strains: RN6390 (a natural sigma factor B [sigB]-deficien mutant), SH1000 (a sigBrepaired variant of RN6390 lineage), and their respective agr, sarA, agr/sarA, and sae mutants. These strain sets were used to quantify hla expression in vitro and in an experimental infective endocarditis (IE) model using flo cytometry. Results. In vitro, hla expression was positively modulated by all 3 regulons (sae 1 agr/sarA 1 agr and sarA) in both RN6390 and SH1000 backgrounds. In the IE model, hla expression in cardiac vegetations was lower in all single mutants than in the respective parental strains (P ! .05 for sae mutant) but was maintained at nearparental levels in the agr/sarA double mutant in both backgrounds. A similar findin was also observed in kidneys and spleens. Conclusions. These results indicate that, although several distinct regulatory circuits can affect hla expression in vitro and in vivo, sae appears to play a crucial role in this context. - Staphylococcus aureus a-toxin (a 33.2-kDa polypeptide encoded by hla) is a pore-forming cytotoxin that is produced by the majority of S. aureus strains [1] and targets a broad range of host cell types [1, 2]. Like most staphylococcal extracellular proteins, a-toxin is not expressed constitutively but is tightly regulated by an array of extracellular and intracellular signals. At least 3 global regulatory loci—the accessory gene regulator (agr), the staphylococcal accessory gene regulator (sarA), and the staphylococcal accessory protein effector (sae)—appear to coordinately control hla expression in S. aureus in vitro [3–7]. In addition, 2 sarA homologues, rot and sarT, repress hla expression [8–10]. The agr locus exerts a direct positive impact on hla expression, whereas sarA positively affects hla expression by both agr-dependent and agr-independent pathways [3, 5–7, 11]. The sae locus consists of a 2-component signal-transduction system encoded by saeS (sensor) and saeR (response regulator) that positively regulates the expression of hla at the transcriptional level [4, 12, 13]. In addition, the complex transcriptional profil of sae activation is influence by agr, as well as by certain environmental stimuli, and it may also be influence by sigma factor B (sigB) [14]. Although the regulation of hla has been studied extensively in vitro, its in vivo regulation remains incompletely defined especially that involved in staphylococcal endovascular infections. Recently, we examined the in vitro versus in vivo expression patterns of a number of key regulatory and structural virulence genes in S. aureus in an experimental infective endocarditis (IE) model. These investigations demonstrated that their in vitro expression and regulation profile were often not precisely mirrored in vivo [15–18]. Moreover, we define target tissue-specifi differences in gene expression, as well as unexpected regulatory pathways that were active exclusively in the IE model. Therefore, these data strongly implicated that host environmental cues and/or additional regulatory circuits play a major role in the in vivo activation of S. aureus virulence genes. The present study was designed to compare hla activation profile in vitro versus in vivo and to examine the effects of the global regulators (agr, sarA, and sae) on such activation profile in 2 genetically related S. aureus strains that differ principally in sigB functionality: RN6390 (a natural sigB-deficien mutant) and SH1000 (a sigB-repaired variant of the RN6390 lineage). A well-characterized S. aureus IE model was used in these investigations. The model mimics its human counterpart microbiologically, immunologically, histopathologically, pathogenetically, and anatomically [19]. At an inoculum of ID95, sequential stages of pathogenesis ensue, including the colonization of damaged cardiac endothelium, microbial persistence and proliferation, eventual hematogenous seeding of distant organs, and reseeding of the endocardial lesions. MATERIALS AND METHODS Microorganisms and plasmids. The S. aureus strains and plasmids used in the study are listed in table 1. S. aureus RN6390 is a a-toxin–producing strain that has been used in numerous studies [5, 15, 20]. It contains a natural 11-bp deletion in the rsbU sensing locus of its sigB operon that renders it functionally sigB deficien [21]. SH1000 is a sigB-repaired variant (rsbU+) in the RN6390 lineage strain, 8325-4, that renders it sigB functional. For construction of the hla promoter:reporter plasmid, pALC1740 in the N315 genome, the promoter fragment of hla (nt 1,141,972–1,140,534) was amplifie by polymerase chain reaction (PCR) on basis of the hla sequence of strain N315 [22]. This PCR fragment was cloned into pCR2.1 and then ligated into pALC1484 upstream of the red-shifted green fluo rescent protein (GFP) gene, gfpuvr, in Escherichia coli [16, 17]. Correct insertion of the hla promoter fragment was confi med by restriction digestion and DNA sequencing. The recombinant plasmid pALC1740, which contains the hla promoter, was electroporated firs into RN4220 to obtain chloramphenicol-resistant transformants. The plasmid was then purifie from RN4220derived transformants and electroporated into RN6390 and SH1000 and their isogenic agr, sarA, agr/sarA, or sae mutants. Of note, the number of copies of this plasmid has been shown to be ∼15 copies/cell [23]. For verificatio of plasmid maintenance and for routine storage, S. aureus strains were grown with appropriate antibiotic selection markers: chloramphenicol (10 mg/mL) for the selection of pALC1740 or its derivatives, tetracycline (5 mg/mL) for Dagr::tetM, and erythromycin (5 mg/ mL) for DsarA::ermC and saeR::Tn551 mutants [4, 24, 25]. Importantly, the pALC1740 plasmid was retained in all constructs after in vitro and in vivo animal model passage, as evidenced by restriction analyses and retention of chloram Description Laboratory strain related to 8325-4 harboring an 11-bp deletion in rsbU RN6390 with pALC1484 RN6390 with pALC1740 agr mutant of RN6390 with pALC1740 sarA mutant of RN6390 with pALC1740 agr/sarA double mutant of RN6390 with pALC1740 sae mutant of RN6390 with pALC1740 Repaired sigB stress response operon in 8325-4 (RN6390 lineage) SH1000 with pALC1484 SH1000 with pALC1740 agr mutant of SH1000 with pALC1740 sarA mutant of SH1000 with pALC1740 agr/sarA double mutant of SH1000 with pALC1740 sae mutant of SH1000 with pALC1740 RN6390 with Dspa::Etbr mutation Shuttle vector containing pUC19 cloned into the HindIII site of pC194 pSK236 containing gfpuvr preceded by the hla promoter fragment Derivative of pSK236 containing the promoterless gfpuvr gene Reference phenicol resistance [26]. All S. aureus strains were stored at 70 C until they were thawed for in vitro and in vivo use. Expression of hla in vitro as determined by flo cytometry. Overnight S. aureus cultures were diluted into fresh tryptic soy broth (TSB) to an initial inoculum of 1 107 cfu/mL and then incubated at 37 C on a rotary shaker (at 200 rpm) for 24 h [17, 18]. At selected time points, 0.5-mL aliquots were obtained for the assessment of hla promoter expression by flo cytometry (FACScalibur; Becton-Dickinson), as described elsewhere [17, 18]. For each sample, 10,000 cells were acquired and analyzed for quantitative GFP fluo escence [16–18]. RN6390 and SH1000 that contained a promoterless gfpuvr plasmid were used as GFP-negative controls. Data were expressed as the mean SD percentage of acquired cells expressing GFP above the baseline (set at 10 fluo escent units in the FL-1 channel, as described elsewhere) [17, 18]. Northern-blot analysis of hla transcription. To verify the in vitro patterns of hla transcription revealed by the GFP reporter methods described above, we compared gene expression in the parental strains with that in selected mutants, using Northern analyses. In brief, S. aureus cells in the early stationary phase were processed by use of a Trizol isolation kit (Gibco BRL) in combination with 0.1-mm sirconia-silica beads in a Biospec reciprocating shaker to yield RNA, as described elsewhere [27]. Then, 15 mg of each sample was electrophoresed in a 1.5% agarose–0.66 mol/L formaldehyde gel in morpholine propanesulfonic acid (MOPS) running buffer (20 mmol/L MOPS, 10 mmol/L sodium acetate, and 2 mmol/L EDTA [pH 7.0]). Blotting of RNA onto Hybond N+ membranes (Amersham) was performed using the Turboblotter alkaline transfer system (Schleicher & Schuell). For detection of the hla transcript, gelpurifie DNA probes were radiolabeled with a-32P dCTP using the random-primed DNA labeling method (Roche Diagnostics) and hybridized under aqueous-phase conditions at 65 C. The blots were subsequently washed and autoradiographed. Chemiluminescent Western-blot analysis. To delineate the correlation between hla transcription and a-toxin production, we tested the specifi production of this protein in both RN6390 and SH1000 backgrounds. S. aureus culture supernatants from equal numbers of cells in the early stationary phase of growth were applied to a 12% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membranes (Millipore). After transfer, the membranes were blocked overnight with 1% nonfat dry milk in Tris-buffered saline that contained 0.05% Tween 20 and then incubated for 5 h with a sheep anti–staphylococcal a–toxin IgG:horseradish peroxidase conjugate (1:600 dilution; Toxin Technology) that contained 0.05% Tween 20. The blot was then developed with supersignal West Dura substrate (Pierce Biotechnology). The chemiluminescent emissions from each construct were quantitatively compared using the Fluorchem 8900 Imaging System (Alpha Innotech). Purifie a-toxin (20 mg) was used as a positive control (Toxin Technology). Hemolytic activity assay. We evaluated the relative hemolytic activities of the parental strains and sae mutants using rabbit erythrocytes, as described elsewhere [28]. Briefl , overnight cultures of S. aureus were equalized to the optical density at 600 nm and then centrifuged at 2500 g to pellet bacteria. Aliquots of serial dilutions of culture supernatants were added to a 1% suspension of washed rabbit erythrocytes in 0.01 mol/ L PBS (pH 7.2) that contained 0.1% bovine serum albumin (Sigma) and were incubated for 30 min at 37 C [28]. Triton (10%) was used as a positive control. The hemolytic activity was expressed as mean units of hemolytic activity ( SD) per milliliter. Hemolytic units (HU) were define as the reciprocal of the highest dilution of the culture supernatant that caused 50% hemolysis [28]. Rabbit IE model. A well-characterized rabbit IE model was used to evaluate regulation of hla expression in vivo. In brief, female New Zealand white rabbits (Irish Farms Products and Services) underwent transcarotid-transaortic valve catheterization [15–18]. At 24 h after catheterization, rabbits were challenged intravenously with each S. aureus construct at the previously define ID95 inoculum (1 107 cfu/rabbit) [17, 18]. Rabbits were euthanized by a rapid intravenous injection of sodium pentobarbital (100 mg/kg; Abbott Laboratories) at 48 h after infection. At death, vegetations, kidneys, and spleen were removed for the determination of bacterial densities, and these were also processed in parallel for the detection of hla expression (see below). Only samples from rabbits with proper catheter placement and macroscopic vegetations were analyzed in the present study. Microbiologic evaluation. Homogenized tissue samples were quantitatively cultured onto TSB agar plates and incubated at 37 C for 24 h. S. aureus densities were expressed as mean SD log10 colony-forming units per gram of tissue. hla regulation in vivo. Tissue samples were prepared for flo cytometric-analysis by a series of processing steps to remove tissue debris, as detailed elsewhere [17, 18]. In addition, to enhance the specificit of these analyses, an immunodetection system featuring a labeled anti–staphylococcal protein A antibody was used to distinguish S. aureus cells from hosttissue debris [17, 18]. This dual fluo ophore system features nonoverlapping emission spectra for GFP and the labeled protein A probe, which causes no quenching of the GFP signal [17, 18]. A protein A knockout mutant of S. aureus (ALC1645) was used to confi m the specificit of this immunolabeling, as described elsewhere [17, 18]. GFP quantitation was limited to protein A–positive S. aureus cells. We recognize that the 3 regulators (agr, sarA, and sae) each modulate the production of protein A in S. aureus. For instance, both agr and sarA repress the transcription of spa (the protein A gene), whereas sae up-regulates the transcription of protein A. However, the flow-cytometri analyses were highly sensitive, and we limited the detection system to only a baseline level of protein A expression in vivo among the 10,000 events captured, which circumvented potential selection bias due to different levels of protein A expression. Statistical analysis. To statistically compare GFP fluo escence and target tissue densities among the various S. aureus strains, the Kruskal-Wallis analysis of variance test was used with Tukey post hoc adjustments for multiple comparison where indicated. P ! .05 was considered to be significant hla regulation in vitro. Figure 1 shows the results of in vitro hla expression and regulation in both RN6390 and SH1000 backgrounds over the course of a 24-h growth cycle. As expected, a growth phase–dependent hla activation profil was observed in both RN6390 and SH1000 parental strains that reached its maximum during the late log/early stationary phases of growth. In RN6390, hla expression decreased by ∼10% in the agr single mutant, compared with the parental strain. For the sarA and agr/sarA mutants, hla activation was substantially decreased throughout the growth cycle, compared with that of the parental strain (∼50% reduction of hla expression during 8–16 h incubation). Of interest, during 16–24 h of incubation, hla activation increased substantially in the agr/sarA mutants (i.e., the percentage of hla-expressing S. aureus cells increased from 16% at 16 h to 56% at 24 h). In SH1000, hla expression was lower than that in the RN6390 parental strain (e.g., 56% vs. 72% hla-expressing cells, respectively, at 24 h). Similar to the RN6390 data, hla activation increased substantially in the agr/sarA double mutant, compared with the parental strain in SH1000, after 16 h of incubation. Minimal GFP expression was observed in the sae mutants in both RN6390 and SH1000 backgrounds (figu e 1). In addition, the promoterless gfpuvr construct in both backgrounds demonstrated !2% GFP expression during 24 h of incubation. Of importance, the above percentage of GFP expression in temporal profile paralleled the GFP fluo rescence intensity per cell in study constructs (data not shown). Northern-blot analysis of hla transcription. RNA transcript analysis confi med several important aspects of the hla expression profile as define by the GFP reporter described above in both the RN6390 and SH1000 backgrounds (figu e 2). In particular, the extent of hla activation was lower in the SH1000 than in the RN6390 parental strains. Additionally, the expression of hla in the agr/sarA double mutants was maintained but was lower than that of the parent strains in both backgrounds. Furthermore, hla transcription was undetectable in both sae mutants. Chemiluminescent Western-blot analysis. Paralleling the hla transcription data above, the RN6390 parental strain produced higher amounts of a-toxin than the SH1000 parental strain (figu e 3). In addition, the hierarchy of a-toxin production paralleled the transcriptional data by GFP reporter and Northern-blot analyses (i.e., parent 1 agr/sarA 1 sae mutants) (figu e 3). Hemolytic activity assay. As expected, the RN6390 parental strain had greater hemolytic activity (221 27 HU/mL) than that of SH1000 parental strain (17 1 HU/mL), which paralleled the higher a-toxin secretion by RN6390 than that by SH1000 that was determined by Western-blot analysis. The sae mutants exhibited no detectable hemolytic activity (!10 HU/mL). Microbiologic evaluation in the rabbit IE model. S. aureus RN6390 densities achieved in vegetations were significantl higher than those in kidneys and spleen for each individual study construct (P ! .05) (table 2). However, only the sarA mutant exhibited a significan reduction in bacterial counts in kidneys, compared with the parental strain (P ! .05) (table 2). Similar to RN6390, S. aureus counts in vegetations in all SH1000 strains were significantl higher than those in the respective kidneys and spleen (P ! .05) (table 3). The sae mutant Table 2. Staphylococcus aureus RN6390 densities in 48-h target tissues from rabbits with endocarditis. RN6390 strain (no. of rabbits) Density, mean SD, log10 cfu/g tissue Kidneys Spleen 7.05 6.51 5.92 NOTE. P ! .05, vegetation counts vs. those in kidneys or spleen of each individual strain. a P ! .05 vs. control. Table 3. Staphylococcus aureus SH1000 densities in 48-h target tissues from rabbits with endocarditis. SH1000 strain (no. of rabbits) Density, mean SD, log10 cfu/g tissue Kidneys Spleen 7.96 7.57 NOTE: P ! .05, vegetation counts vs. those in kidneys or spleen of each individual strain. a P ! .05 vs. the parental strain. of SH1000 achieved significantl lower S. aureus densities in all 3 target tissues, compared with the parental strain (P ! .05). When RN6390 was compared with SH1000, RN6390 strains had somewhat lower S. aureus counts than SH1000 strains in vegetations. However, these differences did not reach statistical significance hla regulation in vivo in the rabbit IE model. Figure 4 depicts the in vivo hla expression profile by the isogenic strain sets within vegetations, kidneys, and spleen. Data (mean, 6–10 target tissue assays) are presented that compare the various mutants to the respective parental strains (set at 100%). In the RN6390 strain set, the percentage of hla-expressing cells in cardiac vegetations was modestly (but not significantly lower in the agr and sarA single mutants than in the parental controls. Importantly, hla expression in all target tissues was significantl lower in the sae mutant (P ! .05 vs. the parental strain), whereas the agr/sarA double mutant retained hla expression at or above the parental level. Similarly, in SH1000 strains, hla expression was maintained at or above the parental level in the agr/sarA double mutant in all 3 target tissues. In addition, hla expression was substantially lower in the sae mutant, compared with the other constructs in all 3 target tissues, although this did not reach statistical significanc (P p .08). Moreover, the extent of GFP expression per cell for the various constructs paralleled the percentage of positive GFP expression in both RN6390 and SH1000 backgrounds in all target tissues. For example, for the sae mutants, the mean GFP fluo escence was substantially lower in all target tissues (42%, 41%, and 59% mean GFP fluo escence vs. the parental strain [set at 100%] in vegetations, kidneys, and spleen in the RN6390 strain set, respectively; and 48%, 53%, and 42% mean GFP fluo escence vs. the parental strain [set at 100%] in vegetations, kidneys, and spleen in the SH1000 strain set, respectively). Importantly, 2 promoterless gfp strains (in RN6390 and SH1000) had minimal GFP expression in any target tissue (i.e., !2% GFP expression in vegetations in both backgrounds). DISCUSSION The staphylococcal a-toxin gene, hla, is positively controlled by agr, sarA, and sae [3–5]. It appears that agr activates hla at both the transcriptional and translational levels [3], whereas sarA exerts a complex positive impact on hla expression by both agr-dependent and agr-independent pathways [5, 8, 11]. In addition, sae appears to positively activate hla via an agrdependent pathway in vitro [4, 14]. Moreover, the stress response regulon, sigB, has been demonstrated to be involved indirectly in hla expression by its composite effects on sarA, agr, and sae [1, 14, 25, 29]. These latter interactions can be complex, because sigB can enhance sarA expression but inhibit agr expression during standard in vitro growth conditions [26, 29–31]. Of note, Cheung et al. [32] previously demonstrated enhanced hla expression and a-toxin production in complete sigB operon mutants in RN6390. By contrast, the influenc of sigB on sae is still a matter of debate. For instance, Novick and Jiang [14] demonstrated that sigB activation represses sae expression, whereas other investigators could not confi m these data in different S. aureus genetic background strains [30, 33]. In addition to the in vitro regulatory pathways, there has been recent evidence that the expression of hla and/or production of a-toxin are modifie after interactions with host cells. For example, neutrophils may induce stress responses in the organism that result in the differential activation of key virulence regulons. Alternatively, neutrophils can release elastases and serine proteases that may cleave a-toxin [34]. Moreover, Rothfork et al. [35] documented the capacity of neutrophils to attenuate agr expression (and, presumably, hla activation) by the generation of reactive oxygen and nitrogen intermediates (i.e., HOCL and ONOO , respectively). Such reactive species appear to oxidize the C-terminal methionine and, hence, to inactivate the agr autoinducing peptide of S. aureus cells within neutrophils, thus preventing the full activation of the agr operon [35]. Despite the above in vitro and ex vivo observations, little has been published about the in vivo expression and regulation of hla, particularly in endovascular infections. Thus, the present investigation was designed to defin the regulatory paradigms controlling hla expression in an S. aureus IE model. Several interesting observations emerged from the study. Consistent with the results of prior in vitro studies, hla expression was positively affected by agr, sarA, and especially sae [1, 4, 5, 36]. Interestingly, the expression of hla was increased in agr/sarA double mutants during late periods of stationary growth, albeit to a level lower than that in their respective parental strains. By contrast, minimal hla expression was observed in sae mutants over the course of the same growth periods. These in vitro data suggest that additional regulatory factor(s) other than agr and sarA are responsible for maintaining hla expression, and sae appears to play a key role in this regard. This in vitro paradigm was seen, in part, in vivo. Thus, hla expression in cardiac vegetations was moderately reduced in the agr and sarA single mutants; however, hla activation was markedly reduced in the sae mutants. These finding suggest that in vivo hla activation is more dependent on sae than on agr and/or sarA. This apparent agr/sarA-independent but sae-dependent in vivo regulation of hla expression has recently been confi med in a different animal model: Goerke et al. [5] used transcript analyses in a soft-tissue guinea pig infection model to show retained hla expression in vivo in 2 agr-deficien clinical isolates and in 2 laboratory agr mutants of strains Newman and RN6390 and to show markedly attenuated hla expression in a sae knockout for all study strains discussed above. Collectively, the present data in the IE model and those in the model of Goerke et al., in an entirely different model of pathogenesis, corroborate the role of sae as an important regulatory system controlling hla expression both in vitro and in vivo. Finally, it should be noted that, despite nearly absent hla expression in vitro in each sae mutant in our study, measurable but reduced hla expression was observed in vivo. Such in vitro–in vivo disparities suggest complex influence of host factors (e.g., blood proteins and cells or vascular tissue) and/or in vivo microenvironmental cues (e.g., pH, oxygen tension, and nutrient concentrations) on staphylococcal gene expression. In addition, regulatory elements other than agr, sarA, and sae are likely involved. The overall impact of sae on the virulence of S. aureus remains to be elucidated. Because hla expression has been previously documented to be a virulence factor in several animal models, including IE [28], it is reasonable to determine that sae is also a key virulence factor in this latter model. This assumption was supported by bacterial counts within target tissues in the IE model being substantially lower in the sae mutant than in the respective parental strains. Another important findin is that the expression of hla is substantially and negatively influence by sigB both in vitro and in vivo. For instance, higher hla activation in RN6390 than in SH1000 was observed both in vitro (figu e 1) and in all target tissues in the IE model (∼20% higher in RN6390 vs. SH1000; data not shown). These in vitro finding are consistent with those of Horsburgh et al. [21], who found that restoration of jB activity in SH1000 resulted in a marked decrease in the levels of a-toxin. To our knowledge, the confi mation of this in vitro paradigm in an experimental model is reported here for the firs time. These results could be explained by (1) the lack of the repression of sae transcription by sigB in RN6390 [14] and/or (2) a large reduction in expression of agrRNAIII in SH1000, compared with 8325-4 lineage strains (e.g., RN6390) [21]. In summary, the present results suggest that sae plays a critical role in regulating hla expression in vitro and in the experimental IE model. It appears that although agr and sarA are both involved in hla expression, in their absence, sae can directly support hla expression. Acknowledgment We thank Yin Li for her excellent technical assistance. 1. Bhakdi S , Tranum-Jensen J. Alpha-toxin of Staphylococcus aureus . Microbiol Rev 1991 ; 55 : 733 - 51 . 2. Walev I , Martin E , Jonas D , et al. Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions . Infect Immun 1993 ; 61 : 4972 - 9 . 3. Arvidson S , Janzon L , Lofdahl S. The role of the d-lysin gene (hld) in the agr-dependent regulation of exoprotein synthesis in Staphylococcus aureus . In Novick RP, ed. Molecular biology of the staphylococci . New York : VCH, 1990 : 419 - 31 . 4. Giraudo AT , Cheung AL , Nagel R. The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level . Arch Microbiol 1997 ; 168 : 53 - 8 . 5. Goerke C , Flucklger U , Steinhuber A , Zimmerli W , Wolz C. Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of a-toxin during device-related infection resolved by direct quantitative transcript analysis . Mol Microbiol 2001 ; 40 : 1439 - 47 . 6. Morfeldt E , Taylor D , von Gabain A , Arvidson S. Activation of a-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA , RNAIII. EMBO J 1995 ; 14 : 4569 - 77 . 7. Vandenesch F , Kornblum J , Novick RP. A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus . J Bacteriol 1991 ; 173 : 6313 - 20 . 8. Cheung AL , Bayer AS , Zhang G , Gresham H , Xiong YQ. Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus . FEMS Immunol Med Microbiol 2004 ; 40 : 1 - 9 . 9. McNamara PJ , Milligan-Monroe KC , Khalili S , Proctor RA . Identifi cation, cloning, and initial characterization of rot, a locus encoding a regulator or virulence factor expression in Staphylococcus aureus . J Bacteriol 2000 ; 182 : 3197 - 203 . 10. Schmidt KA , Manna AC , Gill S , Cheung AL. SarT, a repressor of alphahemolysin in Staphylococcus aureus . Infect Immun 2001 ; 69 : 4749 - 58 . 11. Cheung AL , Projan SJ . Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr . J Bacteriol 1994 ; 176 : 4168 - 72 . 12. Giraudo AT , Calzolari A , Cataldi AA , Bogni C , Nagel R. The sae locus of Staphylococcus aureus encodes a two-component regulatory system . FEMS Microbiol Lett 1999 ; 177 : 15 - 22 . 13. Novick RP . Pathogenicity factors and their regulation . In: Fischetti VA, Novick RP , Ferretti JJ , Portnoy DA , Rood JI, eds. Gram-positive pathogens. Washington, DC: ASM Press, 2000 : 392 - 407 . 14. Novick RP , Jiang D. The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing . Microbiology 2003 ; 149 : 2709 - 17 . 15. Cheung AL , Nast CC , Bayer AS . Selective activation of sar promoters with the use of green fluo escent protein transcriptional fusions as the detection system in the rabbit endocarditis model . Infect Immun 1998 ; 66 : 5988 - 93 . 16. van Wamel W , Xiong YQ , Bayer AS , Yeaman MR , Nast CC , Cheung AL . Regulation of Staphylococcus aureus type 5 capsular polysaccharides by agr and sarA in vitro and in an experimental endocarditis model . Microb Pathog 2002 ; 33 : 73 - 9 . 17. Xiong Y-Q , Van Wamel W , Nast CC , Yeaman MR , Cheung AL , Bayer AS . Activation and transcriptional interaction between agr RNAII and RNAIII in Staphylococcus aureus in vitro and in an experimental endocarditis model . J Infect Dis 2002 ; 186 : 668 - 77 . 18. Xiong YQ , Bayer AS , Yeaman MR , van Wamel W , Manna AC , Cheung AS . Impacts of sarA and agr in Staphylococcus aureus strain Newman on fib onectin-binding protein A gene expression and fib onectin adherence capacity in vitro and in experimental infective endocarditis . Infect Immun 2004 ; 72 : 1832 - 6 . 19. Moreillon P , Que YA , Bayer AS . Pathogenesis of streptococcal and staphylococcal endocarditis . Infect Dis Clin North Am 2002 ; 16 : 297 - 318 . 20. Blevins JS , Beenken KE , Elasri MO , Hurlburt BK , Smeltzer MS . Straindependent differences in the regulatory roles of sarA and agr in Staphylococcus aureus . Infect Immun 2002 ; 70 : 470 - 80 . 21. Horsburgh MJ , Aish JL , White IL , Shaw L , Lithgow JK , Foster SJ. jB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4 . J Bacteriol 2002 ; 184 : 5457 - 67 . 22. Kuroda M , Ohta T , Uchiyama I , et al. Whole genome sequencing of methicillin-resistant Staphylococcus aureus . Lancet 2001 ; 357 : 1218 - 9 . 23. Novick RP . The Staphylococcus as a molecular genetic system . In: Novick RP, ed. Molecular biology of the staphylococci . New York : VCH, 1990 ; 1 - 37 . 24. Giraudo AT , Mansilla C , Chan A , Raspanti C , Nagel R. Studies on the expression of regulatory locus sae in Staphylococcus aureus . Curr Microbiol 2003 ; 46 : 246 - 50 . 25. Kupferwasser LI , Yeaman MR , Nast CC , et al. Salicylic acid attenuates virulence in endovascular infections by targeting global regulatory pathways in Staphylococcus aureus . J Clin Invest 2003 ; 112 : 222 - 33 . 26. Ziebandt AK , Weber H , Rudolph J , et al. Extracellular proteins of Staphylococcus aureus and the role of SarA and jB . Proteomics 2001 ; 1 : 480 - 93 . 27. Cheung AL , Eberhardt K , Fischetti VA . A method to isolate RNA from gram-positive bacteria and mycobacteria . Anal Biochem 1994 ; 222 : 511 - 4 . 28. Bayer AS , Ramos MD , Menzies BE , Yeaman MR , Shen AJ , Cheung AL. 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 1997 ; 65 : 4652 - 60 . 29. Bischoff M , Entenza JM , Giachino P. Influenc of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus . J Bacteriol 2001 ; 183 : 5171 - 79 . 30. Bischoff M , Dunman P , Kormanec J , et al. Microarray-based analysis of the Staphylococcus aureus jB regulon . J Bacteriol 2004 ; 186 : 4085 - 99 . 31. Gertz S , Engelmann S , Schmid R , et al. Characterization of the jB regulon in Staphylococcus aureus . J Bacteriol 2000 ; 182 : 6983 - 91 . 32. Cheung AL , Chien YT , Bayer AS . Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus . Infect Immun 1999 ; 67 : 1331 - 7 . 33. Goerke C , Fluckiger U , Steinhuber A , et al. Role of Staphylococcus aureus global regulators sae and sigmaB in virulence gene expression during device-related infection . Infect Immun 2005 ; 73 : 3415 - 21 . 34. Worlitzsch D , Kaygin H , Steinhuber A , Dalhoff A , Botzenhart K , Doring G . Effects of amoxicillin, gentamicin, and moxifloxaci on the hemolytic activity of Staphylococcus aureus in vitro and in vivo. Antimicrob Agents Chemother 2001 ; 45 : 196 - 202 . 35. Rothfork JM , Timmins GS , Harris MN , et al. Inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: new role for the NADPH oxidase in host defense . Proc Natl Acad Sci USA 2004 ; 101 : 13867 - 72 . 36. Cheung AL , Koomey JM , Butler CA , Projan SJ , Fischetti VA . Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr . Proc Natl Acad Sci USA 1992 ; 89 : 6462 - 6 .


This is a preview of a remote PDF: http://jid.oxfordjournals.org/content/194/9/1267.full.pdf

Yan Q. Xiong, Julie Willard, Michael R. Yeaman, Ambrose L. Cheung, Arnold S. Bayer. Regulation of Staphylococcus aureus α-Toxin Gene (hla) Expression by agr, sarA and sae In Vitro and in Experimental Infective Endocarditis, Journal of Infectious Diseases, 2006, 1267-1275, DOI: 10.1086/508210