Repression of hla by rot Is Dependent on sae in Staphylococcus aureus

The regulatory locus sae is a two-component system in Staphylococcus aureus that regulates many important virulence factors, including alpha-toxin (encoded by hla) at the transcriptional level. The SarA homologs Rot and SarT were previously shown to be repressors of hla in selected S. aureus backgrounds. To delineate the interaction of rot and sae and the contribution of sarT to hla expression, an assortment of rot and sae isogenic single mutants, a rot sae double mutant, and a rot sae sarT markerless triple mutant were constructed from wild-type strain COL. Using Northern blot analysis and transcriptional reporter gene green fluorescent protein, fusion, and phenotypic assays, we found that the repression of hla by rot is dependent on sae. A rot sae sarT triple mutant was not able to rescue the hla defect of the rot sae double mutant. Among the three sae promoters, the distal sae P3 promoter is the strongest in vitro. Interestingly, the sae P3 promoter activities correlate with hla expression in rot, rot sae, and rot sae sarT mutants of COL. Transcriptional study has also shown that rot repressed sae, especially at the sae P3 promoter. Collectively, our data implicated the importance of sae in the rot-mediated repression of hla in S. aureus

Characterization of sarR, a Modulator of sar Expression in Staphylococcus aureus

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sar and agr). The sar locus is composed of three overlapping transcripts (sar P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp sarA gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of sar promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific colum

sarU, a sarA Homolog, is Repressed by SarT and Regulates Virulence Genes in Staphylococcus aureus

In searching the Staphylococcus aureus genome, we previously identified sarT, a homolog of sarA, which encodes a repressor for alpha-hemolysin synthesis. Adjacent but transcribed divergently to sarT is sarU, which encodes a 247-residue polypeptide, almost twice the length of SarA. Sequence alignment disclosed that SarU, like SarS, which is another SarA homolog, could be envisioned as a molecule with two halves, with each half being homologous to SarA. SarU, as a member of the SarA family proteins, disclosed conservation of basic residues within the helix-turn-helix motif and within the beta hairpin loop, two putative DNA binding domains within this protein family. The transcription of sarU is increased in a sarT mutant. Gel shift and transcriptional fusion studies revealed that SarT can bind to the sarU promoter region, probably acting as a repressor for sarU transcription. The expression of RNAII and RNAIII of agr is decreased in a sarU mutant. As RNAIII expression is up-regulated in a sarT mutant, we hypothesize that sarT may down regulate agr RNAIII expression by repressing sarU, a positive activator of agr expression. We propose that, in addition to the quorum sensing effect of the autoinducing peptide of agr, the sarT-sarU pathway may represent a secondary amplification loop whereby the expression of agr (e.g., those found in vivo) might repress sarT, leading to increased expression of sarU. Elevated sarU expression would result in additional amplification of the original agr signal

Role of Adaptor TrfA and ClpPC in Controlling Levels of SsrA-Tagged Proteins and Antitoxins in Staphylococcus aureus

Staphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus

Contribution of teg49 Small RNA in the 5′ Upstream Transcriptional Region of sarA to Virulence in Staphylococcus aureus

High-throughput RNA sequencing technology has found the 5\u27 untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA

SarS, a SarA Homolog Repressible by agr, Is an Activator of Protein A Synthesis in Staphylococcus aureus

The expression of protein A (spa) is repressed by global regulatory loci sarA and agr. Although SarA may directly bind to the spa promoter to downregulate spa expression, the mechanism by which agr represses spa expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the spa gene. The expression of sarS was almost undetectable in parental strain RN6390 but was highly expressed in agr and sarA mutants, strains normally expressing high level of protein A. Interestingly, protein A expression was decreased in a sarS mutant as detected in an immunoblot but returned to near-parental levels in a complemented sarS mutant. Transcriptional fusion studies with a 158- and a 491-bp spa promoter fragment linked to the xylE reporter gene disclosed that the transcription of the spa promoter was also downregulated in the sarS mutant compared with the parental strain. Interestingly, the enhancement in spa expression in an agr mutant returned to a near-parental level in the agr sarS double mutant but not in the sarA sarS double mutant. Correlating with this divergent finding is the observation that enhanced sarS expression in an agr mutant was repressed by the sarA locus supplied in trans but not in a sarA mutant expressing RNAIII from a plasmid. Gel shift studies also revealed the specific binding of SarS to the 158-bp spa promoter. Taken together, these data indicated that the agr locus probably mediates spa repression by suppressing the transcription of sarS, an activator of spa expression. However, the pathway by which the sarA locus downregulates spa expression is sarS independent

Contribution of teg49 Small RNA in the 5′ Upstream Transcriptional Region of sarA to Virulence in Staphylococcus aureus

High-throughput RNA sequencing technology has found the 5\u27 untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA

Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

The toxin MazFsa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo but spares many essential mRNAs (e.g., secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e., sarA mRNA). We hypothesize that some mRNAs may be protected by RNA-binding protein(s) from degradation by MazFsa. Using heparin-Sepharose-enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities, as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant compared with the parent when grown at 25°C but not at 37°C. Activation of MazFsa in the cshA mutant resulted in lower CFU per milliliter accompanied by a precipitous drop in viability (∼40%) compared to those of the parent and complemented strains. NanoString analysis reveals diminished expression of a small number of mRNAs and 22 small RNAs (sRNAs) in the cshA mutant versus the parent upon MazFsa induction, thus implying protection of these RNAs by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant versus the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNAs in vivo and as a result enhances S. aureus survival upon MazFsa induction during stress

Clumping Factor A Mediates Binding of Staphylococcus aureus to Human Platelets

The direct binding of bacteria to platelets may be an important virulence mechanism in the pathogenesis of infective endocarditis. We have previously described Staphylococcus aureus strain PS12, a Tn551-derived mutant of strain ISP479, with reduced ability to bind human platelets in vitro. When tested in an animal model of endocarditis, the PS12 strain was less virulent than its parental strain, as measured by bacterial densities in endocardial vegetations and incidence of systemic embolization. We have now characterized the gene disrupted in PS12 and its function in platelet binding. DNA sequencing, Southern blotting, and PCR analysis indicate that PS12 contained two Tn551 insertions within the clumping factor A (ClfA) locus (clfA). The first copy was upstream from the clfA start codon and appeared to have no effect on ClfA production. The second insertion was within the region encoding the serine aspartate repeat of ClfA and resulted in the production of a truncated ClfA protein that was secreted from the cell. A purified, recombinant form of the ClfA A region, encompassing amino acids 40 through 559, significantly reduced the binding of ISP479C to human platelets by 44% (P = 0.0001). Immunoprecipitation of recombinant ClfA that had been incubated with solubilized platelet membranes coprecipitated a 118-kDa platelet membrane protein. This protein does not appear to be glycoprotein IIb. These results indicate that platelet binding by S. aureus is mediated in part by the direct binding of ClfA to a novel 118-kDa platelet membrane receptor

Coordinated Regulation by AgrA, SarA, and SarR To Control agr Expression in Staphylococcus aureus

The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that AgrA alone promoted transcription from the agr P2 promoter, with SarA enhancing it and SarR inhibiting agr P2 transcription in the presence of AgrA or with SarA and AgrA. Electrophoretic mobility shift assay (EMSA) analysis disclosed that SarR binds more avidly to the agr promoter than SarA and displaces SarA from the agr promoter. Additionally, SarA and AgrA bend the agr P2 promoter, whereas SarR does not. Collectively, these data indicated that AgrA activates agr P2 and P3 promoters while SarA activates the P2 promoter, presumably via bending of promoter DNA to bring together AgrA dimers to facilitate engagement of RNA polymerase (RNAP) to initiate transcription