Staphylococcus aureus is the most common cause of skin and soft tissue infections, and rapidly emerging antibiotic-resistant strains are creating a serious public health concern. If immune-based therapies are to be an alternative to antibiotics, greater understanding is needed of the protective immune response against S. aureus infection in the skin. Although neutrophil recruitment is required for immunity against S. aureus, a role for T cells has been suggested. Here, we used a mouse model of S. aureus cutaneous infection to investigate the contribution of T cells to host defense. We found that mice deficient in γδ but not αβ T cells had substantially larger skin lesions with higher bacterial counts and impaired neutrophil recruitment compared with WT mice. This neutrophil recruitment was dependent upon epidermal Vγ5 + γδ T cell production of IL-17, but not IL-21 and IL-22. Furthermore, IL-17 induction required IL-1, TLR2, and IL-23 and was critical for host defense, since IL-17R-deficient mice had a phenotype similar to that of γδ T cell-deficient mice. Importantly, γδ T cell-deficient mice inoculated with S. aureus and treated with a single dose of recombinant IL-17 had lesion sizes and bacterial counts resembling those of WT mice, demonstrating that IL-17 could restore the impaired immunity in these mice. Our study defines what we believe to be a novel role for IL-17-producing epidermal γδ T cells in innate immunity against S. aureus cutaneous infection.

Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic infections and antibiotic treatment failure1–3. In Escherichia coli, toxin/antitoxin (TA) modules have been linked to persister formation4–6. The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance into stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers is associated with a 100–1000 fold increase in the likelihood of survival to antibiotic challenge. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

MyD88 is an important signaling adaptor for both TLR and IL-1R family members. Here, we evaluated the role of TLR2/MyD88 and IL-1R/MyD88 signaling in host defense against S. aureus by using a cutaneous infection model in conjunction with bioluminescent bacteria. We found that lesions of S. aureus-infected MyD88- and IL-1R-deficient mice were substantially larger with higher bacterial counts compared with wild-type mice. In contrast, TLR2-deficient mice had lesions that were only moderately larger with minimally higher bacterial counts. In addition, MyD88- and IL-1R- but not TLR2-deficient mice had severely decreased recruitment of neutrophils to the site of infection. This neutrophil recruitment was not dependent upon IL-1R/MyD88 signaling by recruited bone marrow-derived cells, suggesting that resident skin cells utilize IL-1R/MyD88 signaling to promote neutrophil recruitment.

Staphylococcus aureus is an opportunistic pathogen. In response to changing host environments, this bacterium has the capability to switch on selective sets of genes to enhance its chances for survival. This switching process is precisely controlled by global regulatory elements. There are two major groups of global regulatory elements in S. aureus, including two-component regulatory systems (TCRSs) and the SarA protein family. Presumably, the sensor proteins of the 16 TCRSs in S. aureus provide external sensing, while the response regulators, in conjunction with alternative transcription factors and the SarA protein family, function as effectors within the intricate regulatory network to respond to environmental stimuli. Sequence alignment and structural data indicate that the SarA protein family could be subdivided into three subfamilies: (1) single-domain proteins; (2) double-domain proteins; and (3) proteins homologous to the MarR protein family. Recent data using reporter gene fusions in animal models, have confirmed distinct expression profiles of selected regulatory and target genes in vitro vs. in vivo.

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar−) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.

A single insertion of transposon Tn917LTV1 into the chromosome ofa Staphylococcus aureus Cdinical isolate, strain DB, resulted in a pleiotropic effect on the expression of a number of extraceilular and cell-wall-as ted proteins. Detailed comparison ofphenotype associated with the mutant, 11D2, and the parent, DB, indicted that the chromosomal locus inactivated as a result of transposon mutagenesis differs from the S. aureus accessory gene regulator locus (agr). In particular, the expression of a-hemolysin, which is not detectable in Agr mutants, was enhanced in mutant 11D2, while it remained at a low level in strain DB. Likewise, protease activity was significantly enhanced in 11D2 compared with DB. In addition, most of the cell-bound proteins were expressed at lower levels in the mutant than the parent strain. This pattern is contrary to that found in switching from Agr+ to Agrphenotypes. Southern blot hybridization with an agr probe indicated that the inactivated chromosomal locus is distinct from agr. Transduction experiments demonstrated that the phenotypes associated with mutant 11D2 could be transferred to the parental strain DB as well as to RN450, an S. aureus strain with a genetic background similar to strain 8325-4. This locus on the S. aureus chromosome, possibly regulatory in nature, has been designated sar for staphylococcal accessory regulator.

Staphylococcus aureus is a major human pathogen that is associated with diverse types of local and systemic infection characterized by inflammation dominated by polymorphonuclear leukocytes. Staphylococci frequently cause pneumonia, and these clinical isolates often have increased expression of protein A, suggesting that this protein may have a role in virulence. Here we show that TNFR1, a receptor for tumor-necrosis factor-alpha (TNF-alpha) that is widely distributed on the airway epithelium, is a receptor for protein A. We also show that the protein A-TNFR1 signaling pathway has a central role in the pathogenesis of staphylococcal pneumonia.

Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.