The accessory sigma factor σ^Bcontrols a general stress response that is thought to be important forStaphylococcus aureussurvival and may contribute to virulence. The strain of choice for genetic studies, 8325-4, carries a small deletion inrsbU, which encodes a positive regulator of σ^Bactivity. Consequently, to enable the role of σ^Bin virulence to be addressed, we constructed anrsbU⁺derivative, SH1000, using a method that does not leave behind an antibiotic resistance marker. The phenotypic properties of SH1000 (8325-4rsbU⁺) were characterized and compared to those of 8325-4, thersbUmutant, parent strain. A recognition site for σ^Bwas located in the promoter region ofkatA, the gene encoding the sole catalase ofS. aureus, by primer extension analysis. However, catalase expression and activity were similar in SH1000 (8325-4rsbU⁺), suggesting that this promoter may have a minor role in catalase expression under normal conditions. Restoration of σ^Bactivity in SH1000 (8325-4rsbU⁺) resulted in a marked decrease in the levels of the exoproteins SspA and Hla, and this is likely to be mediated by reduced expression ofagrin this strain. By using Western blotting and asarA-lacZreporter assay, the levels of SarA were found to be similar in strains 8325-4 and SH1000 (8325-4rsbU⁺) andsigBmutant derivatives of these strains. This finding contrasts with previous reports that suggested that SarA expression levels are altered when they are measured transcriptionally. Inactivation ofsarAin each of these strains resulted in an expected decrease inagrexpression; however, the relative level ofagrin SH1000 (8325-4rsbU⁺) remained less than the relative levels in 8325-4 and thesigBmutant derivatives. We suggest that SarA is not likely to be the effector in the overall σ^B-mediated effect onagrexpression.

The Staphylococcus aureus genome encodes three ferric uptake regulator (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of PerR, we inactivated the gene by allelic replacement using a kanamycin cassette, creating strain MJH001 ( perR ). PerR was found to control transcription of the genes encoding the oxidative stress resistance proteins catalase (KatA), alkyl hydroperoxide reductase (AhpCF), bacterioferritin comigratory protein (Bcp), and thioredoxin reductase (TrxB). Furthermore, PerR regulates transcription of the genes encoding the iron storage proteins ferritin (Ftn) and the ferritin-like Dps homologue, MrgA. Transcription of perR was autoregulated, and PerR repressed transcription of the iron homeostasis regulator Fur, which is a positive regulator of catalase expression. PerR functions as a manganese-dependent, transcriptional repressor of the identified regulon. Elevated iron concentrations produced induction of the PerR regulon. PerR may act as a peroxide sensor, since addition of external hydrogen peroxide to 8325-4 (wild type) resulted in increased transcription of most of the PerR regulon, except for fur and perR itself. The PerR-regulated katA gene encodes the sole catalase of S. aureus, which is an important starvation survival determinant but is surprisingly not required for pathogenicity in a murine skin abscess model of infection. In contrast, PerR is not necessary for starvation survival but is required for full virulence ( P < 0.005) in this model of infection. PerR of S. aureus may act as a redox sentinel protein during infection, analogous to the in vitro activities of OxyR and PerR of Escherichia coli and Bacillus subtilis , respectively. However, it differs in its response to the metal balance within the cell and has the added capability of regulating iron uptake and storage.

The Staphylococcus aureus genome encodes three ferric uptake repressor (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of Fur in S. aureus , we inactivated the fur gene by allelic replacement using a tetracycline resistance cassette, creating strain MJH010 ( fur ). The mutant had a growth defect in rich medium, and this defect was exacerbated in metal-depleted CL medium. This growth defect was partially suppressed by manganous ion, a metal ion with known antioxidant properties. This suggests that the fur mutation leads to an oxidative stress condition. Indeed, MJH010 ( fur ) has reduced levels of catalase activity resulting from decreased katA transcription. Using a katA-lacZ fusion we have determined that Fur functions, either directly or indirectly, as an iron-dependent positive regulator of katA expression. Transcription of katA is coregulated by Fur and PerR, since in MJH010 ( fur ) transcription was still repressed by manganese while transcription in MJH201 ( fur perR ) was unresponsive to the presence of iron or manganese. Siderophore biosynthesis was repressed by iron in 8325-4 (wild-type) but in MJH010 ( fur ) was constitutive. A number of putative Fur-regulated genes were identified in the incomplete genome databases using known S. aureus Fur box sequences. Of those tested, the sstABCD and sirABC operons and the fhuD2 and orf4 genes were found to have Fur-regulated expression. MJH010 ( fur ) was attenuated ( P < 0.04) in a murine skin abscess model of infection, as was double-mutant MJH201 ( fur perR ) ( P < 0.03). This demonstrates the importance in vivo of iron homeostasis and oxidative stress resistance regulation in S. aureus.

Neutrophil responses to commercial LPS, a dual Toll-like receptor (TLR)2 and TLR4 activator, are regulated by TLR expression, but are amplified by contaminating monocytes in routine cell preparations. Therefore, we investigated the individual roles of TLR2 and TLR4 in highly purified, monocyte-depleted neutrophil preparations, using selective ligands (TLR2, Pam₃CysSerLys₄ and Staphylococcus aureus peptidoglycan; TLR4, purified LPS). Activation of either TLR2 or TLR4 caused changes in adhesion molecule expression, respiratory burst (alone, and synergistically with fMLP), and IL-8 generation, which was, in part, dependent upon p38 mitogen-activated protein kinase signaling. Neutrophils also responded to Pam₃CysSerLys₄ and purified LPS with down-regulation of the chemokine receptor CXCR2 and, to a lesser extent, down-regulation of CXCR1. TLR4 was the principal regulator of neutrophil survival, and TLR2 signals showed relatively less efficacy in preventing constitutive apoptosis over short time courses. TLR4-mediated neutrophil survival depended upon signaling via NF-κB and mitogen-activated protein kinase cascades. Prolonged neutrophil survival required both TLR4 activation and the presence of monocytes. TLR4 activation of monocytes was associated with the release of neutrophil survival factors, which was not evident with TLR2 activation, and TLR2 activation in monocyte/neutrophil cocultures did not prevent late neutrophil apoptosis. Thus, TLRs are important regulators of neutrophil activation and survival, with distinct and separate roles for TLR2 and TLR4 in neutrophil responses. TLR4 signaling presents itself as a pharmacological target that may allow therapeutic modulation of neutrophil survival by direct and indirect mechanisms at sites of inflammation.

Summary: The Staphylococcus aureus DtxR‐like protein, MntR, controls expression of the mntABC and mntH genes, which encode putative manganese transporters. Mutation of mntABC produced a growth defect in metal‐depleted medium and increased sensitivity to intracellularly generated superoxide radicals. These phenotypes resulted from diminished uptake of manganese and were rescued by the addition of excess Mn(II). Resistance to superoxide was incompletely rescued by Mn(II) for STE035 (mntA mntH), and the strain had reduced virulence in a murine abscess model of infection. Expression of mntABC was repressed by Mn(II) in an MntR‐dependent manner, which contrasts with the expression of mntH that was not repressed in elevated Mn(II) and was decreased in an mntR mutant. This demonstrates that MntR acts as a negative and positive regulator of these loci re‐spectively. PerR, the peroxide resistance regulon repressor, acts with MntR to control the expression of mntABC and manganese uptake. The expression of the PerR‐regulated genes, katA (catalase), ftn (ferritin) and fur (ferric uptake regulator), was diminished in STE031 (mntR) when grown in excess Mn(II). Therefore, the control of Mn(II)‐regulated members of the PerR regulon and the Fur protein is modulated by MntR through its control of Mn(II) uptake. The co‐ordinated regulation of metal ion homeostasis and oxidative stress resistance via the regulators MntR, PerR and Fur of S. aureus is discussed.

Microbes can defend their host against virulent infections, but direct evidence for the adaptive origin of microbe-mediated protection is lacking. Using experimental evolution of a novel, tripartite interaction, we demonstrate that mildly pathogenic bacteria (Enterococcus faecalis) living in worms (Caenorhabditis elegans) rapidly evolved to defend their animal hosts against infection by a more virulent pathogen (Staphylococcus aureus), crossing the parasitism–mutualism continuum. Host protection evolved in all six, independently selected populations in response to within-host bacterial interactions and without direct selection for host health. Microbe-mediated protection was also effective against a broad spectrum of pathogenic S. aureus isolates. Genomic analysis implied that the mechanistic basis for E. faecalis-mediated protection was through increased production of antimicrobial superoxide, which was confirmed by biochemical assays. Our results indicate that microbes living within a host may make the evolutionary transition to mutualism in response to pathogen attack, and that microbiome evolution warrants consideration as a driver of infection outcome.