SarA is a repressor of hla (alpha-hemolysin) transcription in Staphylococcus aureus: its apparent role as an activator of hla in the prototype strain NCTC 8325 depends on reduced expression of sarS.
ABSTRACT: In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.
Project description:Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 strains appeared to be protease negative. The major protease genes were present in all protease-positive (n = 5) and protease-negative (n = 12) strains analyzed. Northern blotting showed that transcription of the protease genes was suppressed due to increased sigma factor B (SigB)-dependent expression of the protease repressor SarA. Other SigB-dependent traits such as pigmentation and expression of asp 23 were also increased in protease-negative compared to protease-positive strains. Inactivation of sarA in three protease-negative strains resulted in increased transcription of all protease genes and increased protease production, while overexpression of sarA in a strain producing protease at high levels repressed protease production. Our results suggest that the protease genes are conserved among clinical S. aureus strains and that the level of SigB-dependent expression of the protease repressor sarA determines the level of protease production in each strain.
Project description:Derivatives of the widely used laboratory strain Staphylococcus aureus NCTC8325, which are natural rsbU mutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor sigma(B). The lack of RsbU prevented the heat-dependent production of sigma(B)-controlled transcripts and resulted in reduced H2O2 and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shock protein Asp23. After 48 h of growth, rsbU mutant strains failed to accumulate staphyloxanthin, the major stationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by sigma(B), making it an excellent target for the study of sigma(B) activity in S. aureus. Reporter gene experiments, using the firefly luciferase gene (luc+) fused to the sigma(B)-dependent promoter(s) of asp23, revealed that sigma(B) is almost inactive in 8325 derivatives. cis complementation of the 8325 derivative BB255 with the wild-type rsbU gene from strain COL produced the rsbU(+) derivative GP268, a strain possessing a sigma(B) activity profile comparable to that of the rsbU(+) wild-type strain Newman. In GP268, the heat inducibility of sigma(B)-dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H2O2 were restored to the levels observed in strain Newman, clearly demonstrating that RsbU is needed for activation of sigma(B) in S. aureus.
Project description:The staphylococcal accessory regulator (encoded by sarA) is an important global regulator of virulence factor biosynthesis in Staphylococcus aureus. To further characterize its role in virulence determinant production, an sarA knockout mutant was created by insertion of a kanamycin antibiotic resistance cassette into the sarA gene. N-terminal sequencing of exoproteins down-regulated by sarA identified several putative proteases, including a V8 serine protease and a novel metalloprotease, as the major extracellular proteins repressed by sarA. In kinetic studies, the sarA mutation delays the onset of alpha-hemolysin (encoded by hla) expression and reduces levels of hla to approximately 40% of the parent strain level. Furthermore, SarA plays a role in signal transduction in response to microaerobic growth since levels of hla were much lower in a microaerobic environment than after aerobic growth in the sarA mutant. An exoprotein exhibiting hemolysin activity on sheep blood, and up-regulated by sarA independently of the accessory gene regulator (encoded by agr), was specifically induced microaerobically. Transcriptional gene fusion and Western analysis revealed that sarA up-regulates both toxic shock syndrome toxin 1 gene (tst) expression and staphylococcal enterotoxin B production, respectively. This study demonstrates the role of sarA as a signal transduction regulatory component in response to aeration stimuli and suggests that sarA functions as a major repressor of protease activity. The possible role of proteases as regulators of virulence determinant stability is discussed.
Project description:Salicylic acid (SAL) may impact Staphylococcus aureus virulence by activating the sigB operon (rsbU-V-W-sigB), thus leading to reductions in alpha-toxin production and decreased fibronectin binding (L. I. Kupferwasser et al., J. Clin. Investig. 112:222-233, 2003). As these prior studies were performed in strain RN6390 (an rsbU mutant) and its rsbU-repaired variant, SH1000, the current investigation was designed to determine if the SAL effect occurs via rsbU- and/or rsbV-dependent pathways in an rsbU-intact S. aureus strain (FDA486). We thus quantified the transcription from two sigB-dependent promoters (asp23 and sarA P3) in FDA486 in response to SAL exposure in vitro, using isogenic single-knockout constructs of rsbU, rsbV, or rsbW and a green fluorescent protein reporter system. SAL induced sarA P3 and asp23 promoter activities in a dose-dependent manner in the parental strain. In contrast, sigB activation by SAL was progressively more mitigated in the rsbU and rsbV mutants. As predicted, SAL caused significant reductions in both alpha-toxin production and fibrinogen and fibronectin binding in the parental strain. The extent of these reductions, compared with the parent, was reduced in the rsb mutants (rsbV > rsbU), especially at low SAL concentrations. Since generation of the free SigB protein usually requires a sequential rsbU-V-W-sigB activation cascade, the present phenotypic and genotypic data suggest key roles for both rsbU and rsbV in SAL-mediated activation of sigB in strains with a fully intact sigB operon.
Project description:Two genes of the sigB operon, rsbU and rsbV, were deleted in an rsbU(+) strain (FDA486) to evaluate the contribution of these two genes to sigma(B) activity in Staphylococcus aureus. The sigma(B) protein level and the transcription of two sigma(B)-dependent promoters (sigB and sarA P3 transcripts) were analyzed in the constructed mutants. A deletion of the first gene (rsbU) within the sigB operon led only to a partial reduction in sigma(beta) activity. A deletion of the second gene (rsbV) resulted in a more dramatic reduction in the sigma(B) protein level and its activity than did the deletion of rsbU, thus indicating that RsbV can be activated independent of RsbU. In the parental strain, the sigma(B)-dependent transcript initiated upstream of rsbV was 28-fold higher than the sigma(A)-dependent transcript originating from the rsbU promoter. The level of the sigma(B)-dependent transcript decreased up to 50% in the rsbU mutant and up to 90% in the rsbV mutant compared with the transcript in the wild type. The yellow pigment of S. aureus colonies, a sigma(B)-dependent phenotype, was partially reduced in the rsbU and rsbV mutants, whereas alpha-hemolysin was increased. Additionally, the sarA P3 promoter activity of the parental strain was induced to a higher level in response to pH 5.5 than was that of the rsbU or rsbV mutant, indicating that RsbU is the major activator of the sigma(B) response to acid stress. Using a tetracycline-inducible system to modulate the expression of RsbW, we progressively repressed pigment production, presumably by reducing the free sigma(B) level. Collectively, our data indicated that RsbU and RsbV in S. aureus contributed to different levels of sigma(B) protein expression and varying sigma(B) activities. Although RsbV can activate sigma(B) independent of RsbU, RsbU remains the major activator of sigma(B) during acid stress.
Project description:Objectives:Staphylococcus aureus small colony variants (SCVs) cause persistent infections and are resistant to cationic antibiotics. Antimicrobial peptides (AMPs) have been suggested as promising alternatives for treating antibiotic-resistant bacteria. We investigated the capacity of the human cationic AMP LL-37 to kill SCVs in the presence of physiological concentrations of bicarbonate, which are reported to alter bacterial membrane permeability and change resistance of bacteria to AMPs. Methods:MBCs of LL-37 for S. aureus SCVs with mutations in different genes in the presence and absence of bicarbonate were determined. Results:In the absence of bicarbonate, SCVs of S. aureus strains LS-1 and 8325-4 had the same level of resistance to LL-37 as the parental strain (8?mg/L). In the presence of bicarbonate, hemB, menD and aroD SCVs of LS-1 had high-level resistance to LL-37 (?128 mg/L) compared with the parental strain (16 mg/L). However, only the aroD SCV of strain 8324-5 showed high-level resistance. 8325-4 harbours mutations in two genes, tcaR and rsbU, which are involved in antimicrobial sensing and the stress response, respectively. When rsbU was repaired in 8325-4 it displayed high-level resistance to LL-37 in the presence of bicarbonate. This phenotype was lost when tcaR was also repaired, demonstrating that RsbU and TcaR are involved in LL-37 resistance in the presence of bicarbonate. Conclusions:S. aureus SCVs would be resistant to high concentrations of LL-37 in niches where there are physiological concentrations of bicarbonate and therefore this AMP may not be effective in combating SCVs.
Project description:The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains.
Project description:Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.
Project description:Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected ?11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (?63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin ?3 (PSM?3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.
Project description:The GraS/GraR two-component system has been shown to control cationic antimicrobial peptide (CAMP) resistance in the major human pathogen Staphylococcus aureus. We identified a highly conserved ten base pair palindromic sequence (5’ ACAAATTTGT 3’) located upstream from GraR-regulated genes (mprF and the dltA and vraFG operons), which we show to be essential for transcriptional regulation by GraR and induction in response to colistin, a bacterial CAMP, suggesting it is the likely GraR binding site. Genome-based predictions and transcriptome analysis revealed several novel GraR target genes. Global expression changes between the wild type strain HG001 (a rsbU+ variant of strain NCTC 8325) and a DgraSR mutant, grown to mid-exponential phase in TSB with 50 µg/ml colistin, were examined. Hybridizations were performed in triplicate using RNA isolated from independent cultures.