Role of mgrA and sarA in Methicillin-Resistant Staphylococcus aureus Autolysis and Resistance to Cell Wall-Active Antibiotics

Journal of Infectious Diseases, Jan 2009

Background. We have previously shown the importance of mgrA and sarA in controlling autolysis of Staphylococcus aureus, with MgrA and SarA both being negative regulators of murein hydrolases.Methods. In this study, we analyzed the effects of mgrA and sarA on antibiotic-mediated lysis in vitro and on the responses to cell wall-active antibiotic therapy in an experimental endocarditis model by use of 2 representative MRSA strains: the laboratory strain COL and the community-acquired clinical strain MW2.Results. Wefound that mgrA and sarA independently down-regulated sarV (a marker for autolysis), although the alteration in sarV expression did not correlate directly with the autolysis profiles of single mgrA and sarA mutants. Importantly, the mgrA/sarA double mutants of both strains were more autolytic than the single mutants in vitro. We demonstrated that, despite equivalent intrinsic virulences of the parent strains and their isogenic mgrA/sarA double mutants in the endocarditis model, oxacillin and vancomycin treatment of the mgrA/sarA double mutants yielded significant reductions in vegetation bacterial densities in vivo, compared with treatment of their respective parent strains.Conclusions. These results suggest that down-regulation of mgrA/sarA in combination with use of cell wall-active antibiotics may represent a novel approach to treat MRSA infections.

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/199/2/209.full.pdf

Role of mgrA and sarA in Methicillin-Resistant Staphylococcus aureus Autolysis and Resistance to Cell Wall-Active Antibiotics

MRSA, mgrA, sarA, and Autolysis ● JID Role of mgrA and sarA in Methicillin-Resistant Staphylococcus aureus Autolysis and Resistance to Cell Wall-Active Antibiotics María Pilar Trotonda 2 Yan Q. Xiong () 0 1 Guido Memmi 2 Arnold S. Bayer 0 1 Ambrose L. Cheung 2 0 David Geffen School of Medicine at UCLA , Los Angeles, California 1 Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA , Torrance 2 Department of Microbiology, Dartmouth Medical School , Hanover, New Hampshire Background. We have previously shown the importance of mgrA and sarA in controlling autolysis of Staphylococcus aureus, with MgrA and SarA both being negative regulators of murein hydrolases. Methods. In this study, we analyzed the effects of mgrA and sarA on antibiotic-mediated lysis in vitro and on the responses to cell wall-active antibiotic therapy in an experimental endocarditis model by use of 2 representative MRSA strains: the laboratory strain COL and the community-acquired clinical strain MW2. Results. We found that mgrA and sarA independently down-regulated sarV (a marker for autolysis), although the alteration in sarV expression did not correlate directly with the autolysis profiles of single mgrA and sarA mutants. Importantly, the mgrA/sarA double mutants of both strains were more autolytic than the single mutants in vitro. We demonstrated that, despite equivalent intrinsic virulences of the parent strains and their isogenic mgrA/sarA double mutants in the endocarditis model, oxacillin and vancomycin treatment of the mgrA/sarA double mutants yielded significant reductions in vegetation bacterial densities in vivo, compared with treatment of their respective parent strains. Conclusions. These results suggest that down-regulation of mgrA/sarA in combination with use of cell wall-active antibiotics may represent a novel approach to treat MRSA infections. - Staphylococcus aureus is an opportunistic pathogen that can cause a broad spectrum of human infections [1, 2]. Recent reports of methicillin-resistant S. aureus (MRSA) strains with resistance to newer antibiotics have highlighted the increasing threat to public health posed by MRSA [3–5]. The virulence of S. aureus is generally attributable to a diverse array of pathogenetic factors [6 – 8]. The coordinated expression of these virulence determinants has been shown to be controlled by multiple genetic loci, including 2-component regulatory systems and global transcriptional regulators [6 –13]. Besides their roles in virulence, some of the aforementioned regulators (e.g., SarA, SarV, and MgrA) have also been found to control autolysis in S. aureus [7, 11–15]. The integrity of the cell wall is generally maintained by 2 competing processes: cell wall synthesis and cell wall lytic activity. The enzymes involved in the synthesis of peptidoglycan, the major component in the cell wall of S. aureus, are penicillin-binding proteins 1– 4, which have been traditional targets for antimicrobial therapy (e.g., penicillins and cephalosporins). Autolysis, on the other hand, is mediated by autolytic enzymes (also called “autolysins” or “murein hydrolases”), which cleave the covalent bonds that confer stability to the cross-linked peptidoglycan chain in order to form the rigid cell wall. An imbalance between synthesis and lysis can lead to cell death, as has been the case with penicillin-induced lysis, wherein cell wall synthesis is disrupted while lytic activity remains unchecked. Available data support the notion that murein hydrolases are controlled by global regulators. For instance, sarA and mgrA repress the expression of autolysins, whereas sarV promotes their expression [14, 16]. Additionally, we have shown that both sarA and mgrA downStrain or plasmid pALC3371 pALC5449 pALC1484 pALC2366 Description Mutant strain of 8325–4 that accepts foreign DNA agr laboratory strain related to 8325–4 Methicillin-resistant laboratory strain SCCmec type I; MLST ST250; spa type YHGFMBQBLO; agr group I; TSST-1 ; pvl ; splE ; cna ; seb sec ; seh Community-associated methicillin-resistant laboratory strain SCCmec type IVa; MLST ST1; spa type UJJJJFE; agr group III; TSST-1 ; pvl ; splE ; cna ; seb sec ; seh COL mgrA COL with a sarA::kan mutation mgrA/sarA mutant in COL COL with a sarV::ermC mutation COL with pALC2366 COL with pALC2489 MW2 mgrA MW2 with a sarA::kan mutation mgrA/sarA mutant in MW2 MW2 sarV MW2 with pALC2366 MW2 with pALC2489 Host strain for cloning Reference regulate sarV, which can serve as a marker of murein hydrolase activity [14]. Thus, inhibition of mgrA and sarA could conceivably enhance bacterial lysis and, in conjunction with cell wall– lytic agents, might augment the net bactericidal effect. In this study, we investigated the role of mgrA and sarA in autolysis and resistance to 2 cell wall–active antibiotics, oxacillin and vancomycin, in 2 MRSA strains, COL and MW2. MATERIALS AND METHODS Bacterial strains, plasmids, and genetic manipulations in Escherichia coli and S. aureus. The phenotypic and genotypic properties of 2 representative MRSA strains, the laboratory strain COL and the community-acquired clinical strain MW2, and the plasmids used in this study are listed in table 1. Standard molecular biology and recombinant DNA techniques were used as previously described [17, 26, 27]. The sarA and sarV genes were inactivated in S. aureus COL and MW2 by transducing sarA::kan and sarV::ermC mutations, respectively, using phage 11. To construct mgrA mutants, we used PCR to amplify 2 fragments flanking the gene sequences to be deleted. The PCR primers yielded a complementary region of at least 16 bases, allowing the first PCR product (the left fragment) to anneal to the second PCR product (the right fragment); a second PCR reaction was performed using products of the first PCR reaction, to obtain a single fragment. The fusion product was purified, digested with SmaI, and ligated into the temperature-sensitive shuttle plasmid pMAD [24]. Allelic exchange in the absence of selection markers was performed [28, 29]. Determination of minimum inhibitory concentrations (MICs). MICs of oxacillin and vancomycin were determined using cation-supplemented Mueller-Hinton broth according to Clinical and Laboratory Standards Institute guidelines [30]. Impact of oxacillin on growth of COL and MW2 and their isogenic mutants. To assess the susceptibility of mgrA, sarA, and double mutants of COL and MW2 to oxacillin, bacterial cultures were incubated overnight, and supernatants were diluted into fresh TSB to yield a starting OD600 of 0.05. Oxacillin was added at sub-MIC concentrations. The mixtures were then incubated with shaking at 37°C, with the OD600 measured hourly for 7 h. Impact of Triton X-100 and oxacillin on cellular lysis of COL and its isogenic mutants. To eliminate possible variations due to growth in nutrient media, Triton X-100 and oxacillin were used to lyse the COL strain set [5]. In brief, overnight cultured strains were adjusted to an OD580 of 0.7, washed, exposed to 50 mmol/L of Tris-HCl (pH 7.2) containing 0.05% of Triton X-100 or 1/10 MIC of oxacillin, and incubated at 30°C with agitation. Cellular lysis was measured by determining the changes in OD580. Zymographic analysis. To detect extracellular murein hydrolases, SDS-PAGE– based zymographic analysis was performed with modifications [31]. In brief, overnight cultures of S. aureus strains were diluted and grown at 37°C with shaking (250 rpm) in TSB until reaching an OD650 of 0.7. The supernatants were harvested, concentrated, and analyzed for protein concentrations with the Bradford assay (BioRad). Equivalent amounts of extracellular proteins were electrophoretically resolved on an 8% SDS–polyacrylamide gel containing either lyopholized cells of Micrococcus lysodeikticus or S. aureus RN4220, after which they were washed and incubated overnight at 37°C with agitation. Gels were then stained with 0.1% methylene blue (Sigma), with the clear bands indicating regions of murein hydrolase activity. Transcriptional fusion of the sarV promoter. To confirm the effect of mgrA and/or sarA mutations on sarV promoter activity, we used plasmid pALC2489, which contains the sarV promoter that drives the gfpuvr reporter gene (table 1). The plasmid was introduced into COL and MW2 strain sets. For the assay, S. aureus strains were diluted in TSB containing chloramphenicol (10 g/mL) and grown at 37°C. Aliquots were assayed for cell density (at OD650) and fluorescence in a FL600 fluorescence reader (BioTek Instruments). Promoter activation was plotted as mean fluorescence/OD. Purification of SarV and production of anti-SarV antibody. The 351-bp sarV coding region was amplified by PCR, using strain RN6390 chromosomal DNA as the template, and then was cloned into the NdeI and BamHI site of expression vector pET14b (Novagen) and transformed to E. coli BL21(DE3)pLysS. Recombinant protein expression was induced by adding 1 mmol/L of ITPG to a growing culture. After a 4-h period of growth at 37°C, cells were harvested and resuspended in binding buffer, frozen, thawed overnight, and sonicated. Clarified supernatant was applied to a nickel affinity column (Novagen) and eluted with 1 mol/L of imidazole. The authenticity and purity of SarV were confirmed by N-terminal sequencing and Coomassie brilliant blue staining of an SDS–polyacrylamide gel containing the protein. To raise polyclonal sera, 50 g of SarV in complete Freund’s adjuvant was injected subcutaneously into mice. This was followed by biweekly immunization (50 g) in incomplete adjuvant and serial testing of sera with strips of immunoblot containing 5 ng of SarV. When the anti-SarV antibody titer in serum reached 100,000 against preimmune serum, animals were sacrificed and blood was collected. Western blot analysis. Cellular extracts were obtained from bacteria grown overnight in the presence of a hypertonic buffer containing lysostaphin (12.5 g/mL; AMBI), PMSF, iodoacetamide, and DNase. The pellet was lysed, and the supernatant was harvested. Equivalent amounts of supernatant proteins (40 –50 g each) were resolved in 16% SDS–polyacrylamide gels, blotted onto Immobilon P membrane (Millipore) and blocked [27]. The blot was incubated for 2 h with serum containing anti-SarV antibody (dilution, 1:8000), washed, and further incubated for 1 h with antimouse peroxidase-conjugated secondary antibodies diluted to 1:10,000 (Pierce). Blots were developed using the enhanced chemiluminescence system ECL Plus (Amersham Bioscience). Rabbit endocarditis model. To determine whether there is a linkage between in vitro autolysis profiles and improved therapeutic outcomes with cell wall–active agents in vivo, a rabbit endocarditis model was used [32]. In brief, anesthetized rabbits (New Zealand white [Irish Farms]) underwent transcarotidtransaortic valve catheterization. Twenty-four hours after catheterization, rabbits were inoculated intravenously with different S. aureus strains at the inoculum of interest. All rabbits were treated in accordance with institutional and US Public Health Service guidelines for the humane care and treatment of animals. We compared the virulence of COL and MW2 parent strains with their respective mgrA/sarA double mutants, using inocula of 104, 105, or 106 cfu/animal. As established by pilot studies, these inocula encompass the ID95 for inducing endocarditis. Twenty-four hours after infection, rabbits were sacrificed for microbiological evaluation (see below). Before therapeutic experiments were performed, pharmacokinetic profiles of oxacillin (intramuscular doses of 50 or 100 mg/kg) and vancomycin (an intravenous dose of 7.5 mg/kg) were investigated. These doses represent strategies used in previous studies of experimental S. aureus endocarditis [33, 34]. Oxacillin and vancomycin serum concentrations were determined by agar diffusion assays, using S. aureus RN6390 and Bacillus subtilis ATCC 6633, respectively [35]. The limit of detection for these bioassays was 1 g/mL for each antibiotic. Twenty-four hours after infection, rabbits were randomized to receive no therapy, oxacillin (50 or 100 mg/kg intramuscularly 3 times/day), or vancomycin (7.5 mg/kg intravenously 2 times/day). Treatment was given for 3 days. These dose strategies were based on pharmacokinetics data and were intentionally designed to achieve peak serum levels that were approximately at or below the MICs for these agents, to divulge the potential combined impacts of autolysis enhancement plus cell wall–active antibiotics. Control rabbits were sacrificed 24 h after receipt of the infective inoculum. Antibiotic-treated rabbits were sacrificed 24 h after receipt of the last antibiotic dose. At sacrifice, all aortic valve vegetations were removed and quantitatively cultured. Statistical comparisons. For comparison of in vivo microbiologic data, Kruskal-Wallis analysis of variance with post hoc correction for multiple comparisons was used. Impact of oxacillin on growth of the COL and MW2 parent strains and their isogenic mutants. The oxacillin MICs for COL and MW2 were 256 and 32 g/mL, respectively. The MIC of vancomycin was 2 g/mL for both strains. For the COL strain set exposed to 25 and 50 g/mL of oxacillin (representing 1/10 and 1/5 MIC, respectively, for the parent strain), both mgrA and sarA single mutants exhibited decreasing ODs, compared with the parent. Remarkably, the mgrA/sarA double mutant exhibited the greatest decrease in growth, compared with the other strains (figure 1A). The mgrA/sarA double mutant of MW2 also exhibited the most potent growth inhibition in the presence of oxacillin at fractions of the MIC, compared with the parent strain and mgrA single mutant (figure 1B). Impact of Triton X-100 and oxacillin on cellular lysis of the COL parent strain and its isogenic mutants. In the presence of Triton X-100, only 40% of the COL parent cells and 60% of the single mutant cells lysed 7 h after incubation (figure 2A). However, 80% of the mgrA/sarA double mutant cells lysed in Figure 2. Autolysis induced by Triton X-100 (A) and 1/10 MIC of oxacillin (B) over time at 30°C in the parent methicillin-resistant Staphylococcus aureus COL strain ( ) and its mgrA mutants ( ), sarA mutants (‘), and mgrA/ sarA mutants ( ). the presence of Triton X-100 (figure 2A). For oxacillin-induced autolysis, the mgrA/sarA double mutant had the highest lysis rates, compared with the single mutants and parent strain (figure 2B). Effect of mgrA and/or sarA on expression of autolysins in strains COL and MW2. The major known autolysin, Atl, is proteolytically cleaved to generate a 62-kDa amidase and a 51kDa glucosaminidase [36]. M. lysodeikticus (figure 3A and 3C) and S. aureus RN4220 (figure 3B and 3D) were used because they are susceptible to glucosaminidase and amidase, respectively [31, 37]. As shown in figure 3A and 3C, the sarA mutants of COL and MW2 displayed a reduction in the level of Atl ( 115 kDa), accompanied by enhanced glucosaminidase ( 51 kDa) and amidase ( 62 kDa) activity, compared with parent strains. The identity of amidase was confirmed by increased lytic activity with the 62-kDa band in RN4220 (figure 3B and 3D). Of note, the double mgrA/sarA mutants displayed a zymographic profile similar to that of the sarA mutants. Interestingly, the mgrA mutant only showed unprocessed Atl and glucosaminidase activities. Of interest, neither the sarV mutant nor the sarV hyperexpression mutant had increased glucosaminidase or amidase activity, compared with the respective parent strains. Lytic activity of mgrA/sarA double mutants is probably independent of sarV expression. As shown in figure 4A, both mgrA/sarA double mutants had higher sarV promoter activation than the single mutants and respective parent strains. Interestingly, the impact of the mgrA mutation on sarV expression was higher than that observed for the sarA mutant, which exhibited a level of sarV activation similar to that of the parent strain. Given that MgrA and SarA can bind to the sarV promoter [14] and that there is a differential effect on sarV expression by these 2 regulators, we also evaluated autocrine regulation by assaying for sarV promoter activation in sarV mutants; however, we were unsuccessful in detecting any evidence of autocrine regulation with this assay (data not shown). Concordant with results of the promoter fusion assays, the double mgrA/sarA mutants expressed a higher level of SarV than their respective parent strains (figure 4B). However, in contrast to the results of the promoter fusion assay, the mgrA mutant did not express a higher level of SarV than the parent strain (figure 4B). Similarly, the sarA mutants also expressed a lower level of SarV than the respective parent strains. We included the corresponding sarV mutant as a negative control and the parent strain carrying a shuttle plasmid expressing sarV (the sarV hyperexpressor) as a positive control (figure 4). In comparing these data (figure 4B) with those of transcriptional fusion assay, in which the sarV promoter expression was higher in the mgrA mutant than the parent strain (figure 4A), it seems clear that other factors must be involved in the translational or posttranslational regulation of SarV in the mgrA mutant. Additionally, the level of SarV production in the sarA mutants also did not correlate with the lytic activity wherein the sarA mutant exhibited increased autolytic activity (figure 3). Moreover, hyperexpressing SarV strains did not demonstrate enhanced autolytic activity upon zymogram analysis (figure 3). Thus, SarV appears to be a marker of autolysis, whereby one can follow the autolytic activity of the double mutant, although the level of SarV itself is not the determining factor for enhanced lytic activity of the double mutants in strains COL and MW2. Pharmacokinetics of oxacillin and vancomycin in the rabbit endocarditis model. After a single 100-mg/kg dose of oxacillin, serum oxacillin levels peaked at 27 g/mL, a concentration well below the MICs for COL and MW2. The peak serum level for vancomycin (7.5 mg/kg) was 3 g/mL, which was slightly higher than the MICs for each strain, but the level rapidly decreased below the MICs. The half-lives of oxacillin and vancomycin were 30 and 120 min, respectively. In vivo role of mgrA and sarA in the efficacy of cell wall– active antibiotics in the endocarditis model. The efficacies of oxacillin and vancomycin against the COL strain and its mgrA/ sarA double mutant (challenge dose, 105 cfu) in the model are presented in table 2. We focused our in vivo studies on comparing the parent strains and mgrA/sarA double mutants, because the outcomes of in vitro comparisons were maximally distinct in these 2 organism groups. At 100 mg/kg of oxacillin and 7.5 mg/kg of vancomycin, a significant reduction in S. aureus vegetation densities was observed in animals infected with the mgrA/ sarA double mutant, compared with the untreated double mutant. In contrast, in infection with the parent COL strain, neither oxacillin nor vancomycin treatment yielded any significant reduction in vegetation densities. For strain MW2, we used a lower oxacillin dose (50 mg/kg) because of the substantially lower MIC, compared with that for the COL strain. This oxacillin regimen yielded a modest reduction in bacterial vegetation densities in the parent MW2 strain (table 3). Importantly, this treatment caused a reduction of 6 log10 cfu per g of vegetation. in animals infected with the mgrA/sarA double mutant, compared with untreated controls. Similar to the COL strain, vancomycin treatment significantly reduced the vegetation densities of the double mutant, compared with findings for untreated double mutant controls and in vancomycin-treated animals infected with the parent MW2 strain. There were no significant differences in the intrinsic virulence between the parent COL and MW2 strains and their respective mgrA/sarA double mutants in the absence of antibiotics at challenge levels of 104, 105, and 106 cfu (data not shown for 104 and 106 cfu). DISCUSSION It has been shown that mgrA and sarA, both of which are global regulators of virulence determinants [7, 12, 16, 38], are also negative regulators of autolysis [16]. In this study, we extended these findings to ascertain the role of MgrA and SarA in the control of MRSA autolysis and its impact on in vivo antibiotic efficacies. Several interesting observations emerged from the present investigations. For example, we found that concomitant presence of mgrA and sarA mutations led to an enhanced oxacillinmediated autolytic phenotype, compared with the presence of either mutation alone, in both MRSA strains in vitro. More important, our studies emphasized a significant enhanced bactericidal effect of cell wall–active antibiotics in mgrA/sarA double mutants of strains COL and MW2 in a well-characterized endocarditis model. The mechanisms of enhanced oxacillin-induced cell autolysis in the double mutants of these 2 MRSA strains are not precisely defined by our studies. However, previous observations and data in this study enable us to piece together a putative network of genes that help explain our findings. From recent investigations [16], it is known that MgrA positively regulates lytRS and arlRS, two 2-component regulatory systems that repress expression of autolysins. LytRS is also a positive regulator of LrgA [39], a putative antiholin molecule that interferes with the function of the putative holin molecule CidA [40]. The holin protein has been found to form pores in the cell membrane that Strain, treatment S. aureus density, log10 cfu/g of vegetation NOTE. The challenge inoculum of each strain was 105 cfu. a Denotes comparison with untreated controls. Table 3. Efficacies of oxacillin and vancomycin against Staphylococcus aureus strain MW2 and its mgrA/sarA double mutant in an experimental rabbit endocarditis model. Strain, treatment Rabbits, no. S. aureus density, log10 cfu/g of vegetation NOTE. The challenge inoculum of each strain was 105 cfu. a Denotes comparison with untreated controls. b P .05, compared with the oxacillin-treated MW2 parent group. c P .05, compared with the vancomycin-treated MW2 parent group. enable murein hydrolase export from the cytosol to the cell wall. Accordingly, we speculate that an mgrA mutation would lead to down-regulation of LrgA and ArlRS, which would, in theory, increase the expression of murein hydrolases. However, zymogram analysis of the mgrA mutant failed to reveal a significant overall effect on the expression of major autolysins, compared with the parent strains (figure 3). Of importance, however, the mgrA mutants were more likely than the parent strains to be autolytic in the presence of oxacillin (figure 1). In contrast, the sarA mutants (which displayed increased autolysin expression on zymograms) also exhibited augmented autolysis in the presence of oxacillin concentrations below the MIC, compared with the mgrA mutants and the parent strains (figure 1). Remarkably, the double mutants were clearly more autolytic than either single mutant at oxacillin concentrations below the MIC, and they also expressed a greater level of autolysins than their respective parent strains (figures 1 and 3). On the basis of these data, we speculate that the synergistic autolytic activity of the double mgrA/sarA mutants may be due to a combination of factors individually contributed by mgrA and sarA, including the complex regulation of genes in the net autolytic cascade and the production of autolysins. Additionally, we analyzed the impact of sarV, a positive regulator of autolysis that is repressed by sarA and mgrA [14]. Although the double mutants clearly demonstrated an increased level of sarV expression (figure 4), enhanced expression of sarV by itself is not enough to initiate significant autolysis. Thus, an mgrA mutant that displayed elevated sarV expression (figure 4) neither significantly increased oxacillin-induced autolysis (figure 1) nor produced increased levels of autolysins (figure 3). This lack of correlation between sarV gene expression and autolysin expression in mgrA mutants may, in part, be due to subsequent translation of sarV; thus, despite expression of greater levels of sarV promoter (figure 4A), the mgrA mutants did not produce increased levels of SarV, compared with the parent strains (figure 4B). Nonetheless, we could not rule out the possibility that the stability of SarV may have been altered in the mgrA mutants. Given that protease expression is generally down-regulated in an mgrA mutant as a result of lower agr expression [10, 41], a reduction in the SarV level in mgrA mutants cannot be attributable to augmented proteolytic activity. Oxacillin-induced lysis is dependent on murein hydrolase activity [31]. This class of antibiotics (i.e., cell wall–active antibiotics) can exert a potent bactericidal effect without lysis in mutants lacking murein hydrolases [15]. Conversely, the lytic effect of oxacillin might be enhanced in mutants with an elevated level of murein hydrolases (e.g., mgrA/sarA strains). Such an event in vitro could be translated into an augmented therapeutic efficacy of oxacillin in treating MRSA infections. Indeed, we found this to be true in both mgrA/sarA double mutants, compared with their respective parent strains, by use of the endocarditis model. In this model, clearance of the organism from infected heart valves is principally dependent on the bactericidal effects of the antibiotics, without substantial contributions from the adaptive immune system [42, 43]. Our studies emphasized the enhanced effect between autolysis regulatory mutants and oxacillin. Thus, in endocarditis caused by COL and MW2, which are normally resistant to oxacillin, a mgrA/sarA double mutation significantly enhanced the bactericidal effect of oxacillin in vivo, presumably because of enhanced lysis in the double mutant, even at sub-MICs of an antibiotic to which it is resistant. This enhanced in vivo bactericidal effect between the mgrA/sarA double mutants of strains COL and MW2 and cell wall–active antibiotics was also confirmed using vancomycin therapy in the same model. Of note, such significant enhanced impacts of oxacillin or vancomycin in vivo were not observed in single mutants (data not shown). Importantly, comparative virulence assessments showed that both parent strains and their respective double mgrA/sarA mutants retained equivalent intrinsic virulence. In summary, these investigations may open up a new avenue to MRSA infection therapeutics. Given that MgrA and SarA are both dimeric winged helix proteins [44, 45] and that an inhibitory small molecule (e.g., virstatin) has been found to disrupt the dimerization and function of ToxT in Vibrio cholerae [46], it is conceivable that targeting a common functional domain between these 2 proteins with a small synthetic molecule(s) may represent a novel strategy to enhance the effectiveness of oxacillin and other cell wall–lytic agents against MRSA. References 1. Lowy F. Staphylococcus aureus infections . N Engl J Med 1998 ; 339 : 520 - 32 . 2. Crossley KB , Archer GL . The staphylococci in human disease . New York, NY : Churchill Livingston, 1997 . 3. Hiramatsu K , Hanaki H , Ino T , Yabuta K , Oguri T , Tenover FC . Methicillin resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility . J Antimicrob Chemother 1997 ; 40 : 135 - 6 . 4. Bogdanovich T , Ednie LM , Shapiro S , Appelbaum PC. Antistaphylococcal activity of ceftobiprole, a new broad-spectrum cephalosporin . Antimicrob Agents Chemother 2005 ; 49 : 4210 - 9 . 5. Jones T , Yeaman MR , Sakoulas G , et al. Failures in clinical treatment of Staphylococcus aureus infection with daptomycin are associated with alterations in surface charge, membrane phospholipid asymmetry, and drug binding . Antimicrob Agents Chemother 2008 ; 52 : 269 - 78 . 6. Novick RP . Autoinduction and signal transduction in the regulation of staphylococcal virulence . Mol Microbiol 2003 ; 48 : 1429 - 49 . 7. 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 . 8. Arvidson S , Tegmark K. Regulation of virulence determinants in Staphylococcus aureus . Int J Med Microbiol 2001 ; 291 : 159 - 70 . 9. Pragman AA , Yarwood JM , Tripp TJ , Schlievert PM . Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus . J Bacteriol 2004 ; 186 : 2430 - 8 . 10. Ingavale S , van Wamel W , Luong TT , Lee CY , Cheung AL. Rat/MgrA, a regulator of autolysis, is a regulator of virulence genes in Staphylococcus aureus . Infect Immun 2005 ; 73 : 1423 - 31 . 11. Fournier B , Hooper DC . A new two-component regulatory system involved in adhesion, autolysis and extracellular proteolytic activity of Staphylococcus aureus . J Bacteriol 2000 ; 182 : 3955 - 64 . 12. Truong-Bolduc QC , Dunman PM , Strahilevitz J , Hooper DC. MgrA is a multiple regulator of two new efflux pumps in Staphylococcus aureus . J Bacteriol 2005 ; 187 : 2395 - 405 . 13. Truong-Bolduc QC , Hooper DC . The transcriptional regulators NorG and MgrA modulate resistance to both quinolones and beta-lactams in Staphylococcus aureus . J Bacteriol 2007 ; 189 : 2996 - 3005 . 14. Manna AC , Ingavale SS , Maloney M , Van Wamel W , Cheung AL . Identification of sarV (SA2062), a new transcriptional regulator, is repressed by SarA and MgrA (SA0641) and involved in the regulation of autolysis in Staphylococcus aureus . J Bacteriol 2004 ; 186 : 5267 - 80 . 15. Moreillon P , Markiewicz Z , Nachman S , Tomasz A. Two bactericidal targets for penicillin in pneumococci: autolysis-dependent and autolysisindependent killing mechanisms . Antimicrob Agents Chemother 1990 ; 34 : 33 - 9 . 16. Ingavale S , Van Wamel W , Cheung AL . Characterization of RAT, an autolysis regulator in Staphylococcus aureus . Mol Microbiol 2003 ; 48 : 1451 - 66 . 17. Schenk S , Laddaga RA . Improved method for electroporation of Staphylococcus aureus . FEMS Microbiol Lett 1992 ; 73 : 133 - 8 . 18. Chambers HF , Hartman BJ , Tomasz A. Increased amounts of a novel penicillin-binding protein in a strain of methicillin-resistant Staphylococcus aureus exposed to nafcillin . J Clin Invest 1985 ; 76 : 325 - 31 . 19. Fey PD , Said-Salim B , Rupp ME , et al. Comparative molecular analysis of community- or hospital-acquired methicillin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 2003 ; 47 : 196 - 203 . 20. Robinson DA , Monk AB , Cooper JE , et al. Evolutionary genetics of the accessory gene regulator (agr) locus in Staphylococcus aureus . J Bacteriol 2005 ; 187 : 8312 - 21 . 21. Cassat JE , Dunman PM , McAleese F , et al. Comparative genomics of Staphylococcus aureus musculoskeletal isolates . J Bacteriol 2005 ; 187 : 576 - 92 . 22. Yarwood JM , McCormick JK , Pausttian ML , et al. Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3 . J Biol Chem 2002 ; 277 : 13138 - 47 . 23. Baba T , Takeuchi F , Kuroda M , et al. Genome and virulence determinants of high virulence community-acquired MRSA . Lancet 2002 ; 359 : 1819 - 27 . 24. Arnaud M , Chastanet A , Debarbouille M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, grampositive bacteria . Appl Environ Microbiol 2004 ; 70 : 6887 - 91 . 25. Oscarsson J , Tegmark-Wisell K , Arvidson S. Coordinated and differential control of aureolysin (aur) and serine protease (sspA) transcription in Staphylococcus aureus by sarA, rot and agr (RNAIII) . Int J Med Microbiol 2006 ; 296 : 365 - 80 . 26. Novick RP . Genetic systems in staphylococci . Methods Enzymol 1991 ; 204 : 587 - 636 . 27. Maniatis T , Fritsch EF , Sambrook J. Molecular cloning, a laboratory manual . 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory , 1989 . 28. Valle J , Toledo-Arana A , Berasain C , et al. SarA and not B is essential for biofilm development by Staphylococcus aureus . Mol Microbiol 2003 ; 48 : 1075 - 87 . 29. Cheung AL , Eberhardt K , Fischetti VA . A method to isolate RNA from gram-positive bacteria and mycobacteria . Anal Biochem 1994 ; 222 : 511 - 4 . 30. Skov R , Frimodt-Moller N , Espersen F. Correlation of MIC methods and tentative interpretive criteria for disk diffusion susceptibility testing using NCCLS methodology for fusidic acid . Diagn Microbiol Infect Dis 2001 ; 40 : 111 - 6 . 31. Groicher KH , Firek BA , Fujimoto DF , Bayles KW . The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance . J Bacteriol 2000 ; 182 : 1794 - 801 . 32. Xiong YQ , Bayer AS , Yeaman MR , van Wamel W , Manna AC , Cheung AL. Impacts of sarA and agr in Staphylococcus aureus strain Newman on fibronectin-binding protein A gene expression and fibronectin adherence capacity in vitro and in experimental infective endocarditis . Infect Immun 2004 ; 72 : 1832 - 6 . 33. Hirano L , Bayer AS . -lactam- -lactamase inhibitor combinations are active in experimental endocarditis caused by -lactamase producing, oxacillin-resistant staphylococci . Antimicrob Agents Chemother 1991 ; 35 : 685 - 90 . 34. Bayer AS , Lam K. Efficacy of vancomycin plus rifampin in experimental aortic-valve endocarditis due to methicillin-resistant Staphylococcus aureus: in vitro-in vivo correlations . J Infect Dis 1985 ; 151 : 157 - 65 . 35. Xiong YQ , Willard J , Kadurugamuwa JL , Yu J , Francis KP , Bayer AS . Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model . Antimicrob Agents Chemother 2005 ; 49 : 380 - 7 . 36. Sugai M , Yamada S , Nakashima S , et al. Localized perforation of the cell wall by a major autolysin: atl gene products and the onset of penicillininduced lysis of Staphylococcus aureus . J Bacteriol 1997 ; 179 : 2958 - 62 . 37. Fujimoto DF , Bayles KW . Opposing roles of the Staphylococcus aureus virulence regulators, Agr and Sar, in Triton X-100 and penicillin induced autolysis . J Bacteriol 1998 ; 180 : 3724 - 6 . 38. Luong TT , Newell SW , Lee CY . Mgr, a novel global regulator in Staphylococcus aureus . J Bacteriol 2003 ; 185 : 3703 - 10 . 39. Brunskill EW , Bayles KW . Identification of LytRS-regulated genes from Staphylococcus aureus . J Bacteriol 1996 ; 178 : 5810 - 2 . 40. Rice KC , Mann EE , Endres JL , et al. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus . Proc Natl Acad Sci U S A 2007 ; 104 : 8113 - 8 . 41. Luong TT , Dunman PM , Murphy E , Projan SJ , Lee CY . Transcription profiling of the mgrA regulon in Staphylococcus aureus . J Bacteriol 2006 ; 188 : 1899 - 910 . 42. Bayer AS . Staphylococcal bacteremia and endocarditis . Arch Intern Med 1982 ; 142 : 1169 . 43. Bayer AS , Norman DC. Valve site-specific pathogenetic differences between right-sided and left-sided bacterial endocarditis . Chest 1990 ; 98 : 200 - 5 . 44. Chen PR , Bae T , Williams WA , et al. An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus . Nat Chem Biol 2006 ; 2 : 591 - 5 . 45. Liu Y , Manna AC , Pan CH , et al. Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus . Proc Natl Acad Sci U S A 2006 ; 103 : 2392 - 7 . 46. Shakhnovich EA , Hung DT , Pierson E , Lee K , Mekalanos JJ . Virstatin inhibits dimerization of the transcriptional activator ToxT . Proc Natl Acad Sci U S A 2007 ; 104 : 2372 - 7 .


This is a preview of a remote PDF: http://jid.oxfordjournals.org/content/199/2/209.full.pdf

María Pilar Trotonda, Yan Q Xiong, Guido Memmi, Arnold S Bayer, Ambrose L Cheung. Role of mgrA and sarA in Methicillin-Resistant Staphylococcus aureus Autolysis and Resistance to Cell Wall-Active Antibiotics, Journal of Infectious Diseases, 2009, 209-218, DOI: 10.1086/595740