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.

Innate immunity is the first line of defense against invading pathogens. Toll-like receptors (TLRs) act as sentinels of the innate immune system, sensing a variety of ligands from lipopolysaccharide to flagellin to dsRNA through their ligand-binding domain that is composed of leucine-rich repeats (LRRs). Ligand binding initiates a signaling cascade that leads to the up-regulation of inflammation mediators. In this study, we have expressed and crystallized the ectodomain (ECD) of human TLR3, which recognizes dsRNA, a molecular signature of viruses, and have determined the molecular structure to 2.4-Å resolution. The overall horseshoe-shaped structure of the TLR3-ECD is formed by 23 repeating LRRs that are capped at each end by specialized non-LRR domains. The extensive ␤-sheet on the molecule's concave surface forms a platform for several modifications, including insertions in the LRRs and 11 N-linked glycans. The TLR3-ECD structure indicates how LRR loops can establish distinct pathogen recognition receptors.dsRNA ͉ innate immunity ͉ pathogen recognition receptor

SUMMARY Staphylococcus aureus is both a colonizer of humans and a cause of severe invasive infections. Although the genetic basis for phenotype switching from colonizing to invasive has received significant study, knowledge of host factors that antagonize the switch is limited. We show that VLDL and LDL lipoproteins prevent this switch by antagonizing a quorum sensing operon agr that upregulates genes required for invasive infection. The mechanism of antagonism entails binding of the major structural protein of these lipoproteins, apolipoprotein B, to an autoinducing pheromone preventing its attachment to the bacteria and subsequent signaling through its receptor, AgrC. Mice deficient in plasma apolipoprotein B, either genetically or pharmacologically, are significantly more susceptible to invasive agr+ infection but not to infection with an agr deletion mutant. Therefore, apolipoprotein B at homeostatic levels in blood is an essential innate defense effector against invasive S. aureus infection.