Previously we identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function -factor, S , involved in stress response and disease causation. Here we present additional characterization of S , demonstrating a role for it in protection against DNA damage, cell wall disruption, and interaction with components of the innate immune system. Promoter mapping reveals the existence of three unique sigS start sites, one of which appears to be subject to autoregulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild types but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates that sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Significantly, expression of sigS is highly induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Phenotypically, S mutants display sensitivity to a broad range of DNA-damaging agents and cell wall-targeting antibiotics. Furthermore, the survivability of S mutants is strongly impacted during challenge by components of the innate immune system. Collectively, our data suggest that S likely serves dual functions within the S. aureus cell, protecting against both cytoplasmic and extracytoplasmic stresses. This further argues for its important, and perhaps novel, role in the S. aureus stress and virulence responses. Staphylococcus aureus is an exceedingly virulent and successful pathogen, capable of causing a wide range of infections, from relatively benign skin lesions to life-threatening septicemia. With an overwhelming ability to adapt to its environment, S. aureus has become the most common cause of both hospital-and community-acquired infections and is believed to be the leading cause of death by a single infectious agent in the United States (20, 34). The threat posed by this organism to human health is further heightened by the rapid and continued emergence of multidrug-resistant isolates (1,20,34,43).Many components govern the adaptive nature of S. aureus, including complex regulatory networks, which allow it to respond to constantly changing environments via rapid shifts in gene expression. There are a number of different elements that mediate this fine-tuning, including DNA-binding proteins, two-component systems, regulatory RNAs, and alternative factors (10,11,18,21,22,32,44,50,51). The last class acts by binding to core RNA polymerase and redirecting promoter recognition to coordinate gene expression, bringing about expedient and wide-reaching alterations within the cell.From a classification perspective, factors are divided into five discrete subfamilies, with the essential housekeeping factors ( A or 70 ), which are responsible for the majority of transcription, constituting group 1. The remaining families (groups 2 to 5) contain alterna...

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of ␣-hemolysin and other virulence factors in Staphylococcus aureus. In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB, saeR, and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I (coa) and class II (hla) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated ␣-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureus.IMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus. Although the regulon of this twocomponent system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase VfrB as a positive regulatory modulator of SaeRS-mediated transcription of virulence factors in S. aureus. In addition to describing a new regulatory aspect of SaeRS, this study establishes a link between fatty acid kinase activity and virulence factor regulation.

To persist within the host and cause disease, Staphylococcus aureus relies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon, yjbIH, resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observed yjbIH mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of crtOPQMN and aur. Previous studies found that YjbH targets the disulfide-and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence of yjbH or yjbI resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the yjbH and yjbI mutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in the yjbH mutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of the yjbIH deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.

No abstract

SummaryPrenylation is the addition of prenyl groups to peptide chains or metabolites via the condensation of geranyl-or isopentenyl-diphosphate moieties by geranyltranstransferases. Although this process is extensively studied in eukaryotes, little is known about the influence of prenylation in prokaryotic species. To explore the role of this modification in bacteria, we generated a mutation in the geranyltranstransferase (IspA) of Staphylococcus aureus. Quite strikingly, the ispA mutant completely lacked pigment and exhibited a previously undescribed small colony variant-like phenotype. Further pleiotropic defects in cellular behavior were noted, including impaired growth, decreased ATP production, increased sensitivity to oxidative stress, increased resistance to aminoglycosides and cationic antimicrobial peptides, and decreased resistance to cell wall-targeting antibiotics. These latter effects appear to result from differences in envelope composition as ispA mutants have highly diffuse cell walls (particularly at the septum), marked alterations in fatty acid composition and increased membrane fluidity. Taken together, these data present an important characterization of prokaryotic prenylation and demonstrate that this process is central to a wealth of pathways involved in mediating cellular homeostasis in S. aureus.

No abstract

A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus aureus USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for Staphylococcus species. LAC-p01 was genetically manipulated into an Escherichia coli-S. aureus shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double-and single-stranded (dso and sso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LACp01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in S. aureus USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Staphylococcus epidermidis. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of S. aureus systemic infection and successfully complemented an hla mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both in vitro and in vivo experiments are powerful new tools for studies of Staphylococcus. IMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during in vivo studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an S. aureus USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations in vitro and in vivo without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of Staphylococcus species.

BackgroundWe previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression.ResultsTo identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS.ConclusionsCollectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0280-9) contains supplementary material, which is available to authorized users.