Project Summary/Abstract During the past several years, infections with methicillin-resistant Staphylococcus aureus strains (MRSA) have become increasingly prevalent in the US and worldwide. Among a number of mechanisms this bacteria has evolved to evade host defense are the toxins, Panton-Valentine Leukocidin (PVL) and ?-hemolysin (?HL). While virtually all staphylococcal strains carry ?HL, the PVL is increasingly associated with severe necrotizing pneumonia, with alarming susceptibility among non-immunocompromised individuals. These toxins lyse polymorphonuclear neutrophils and macrophages in a highly species-specific manner, with human cells being among the most sensitive. Mouse cells are resistant to the lytic effects of PVL and ?HL, and this difference has made development of an informative animal model difficult. The receptors for human C5a anaphylatoxin, C5aR and C5L2, were recently shown to act as binding sites for both PVL and ?HL/HlgCB, and we hypothesize that small molecule antagonists of the C5a receptor will be useful in treating staphylococcal infections as an adjunct to antibiotic therapy. To facilitate thee studies we have generated a line of humanized mice in which we `knocked in' the human C5aR (hC5aR-KI) on the background of the C5aR knockout animals. In preliminary studies we have shown that these hC5aR-KI mice are responsive to the leukocytic actions of the PVL where wild type animals are not. Following lung infection with PVL+ Staph (USA300), hC5aR-KI mice fail to clear the bacteria as efficiently as wild type animals, and their bronchoalveolar lavage fluid contains significantly elevated inflammatory cells. In a combined approach using the hC5aR-KI mice in addition to cellular and molecular biological studies, we propose 3 specific aims to understand the mechanistic basis for the toxicity of PVL- positive S. aureus. In Aim 1 we will establish a model of S. aureus infection using the humanized mouse line and determine the efficacy of blocking toxin binding to the C5aR with small molecule antagonists. In Aim 2 we will identify the structural specificity of the PVL S component LukS-PV and the related ?HL S component HlgC for the human C5aR and C5L2. Preliminary mutagenesis studies show that LukS-PV recognizes critical sequences in the second extracellular loop (ECL2) of the human C5aR. Binding alone, however, is not sufficient to promote association of LukF-PV and cellular lysis. These functions require more extensive C5aR structures, and their identity may offer additional therapeutic targets against PVL-mediated injury. In Aim 3 we will examine the target cell specificity of the Staph toxins, focusing particular attention on their ability to lyse airway epithelial cells and alveolar macrophages that also express the C5a receptors. Taken together, these investigations will provide preclinical evidence for the therapeutic use of small molecule C5aR antagonists in man to combat staphylococcal infections.