DESCRIPTION (provided by applicant): Infection with Staphylococcus aureus (SA) remains an important clinical challenge despite potent antibiotics. Novel therapeutic advances await elucidation of the molecular bases for persistence, chronicity, and metastatic spread - i.e. the hallmarks of SA infection. The remarkable proclivity of SA to produce severe and chronic human disease reflects, in part, the array of virulence factors it expresses, including a phosphoinositol-specific phospholipase C (PI-PLC) that is secreted during its late logarithmic growth phase. PI-PLC is secreted during infection and is immunogenic, making it a candidate as a virulence factor. Human polymorphonuclear leukocytes (PMN) are at the front-line of cellular innate immune-mediated host defense against SA infection, and PMN exert ~ all of their antimicrobial effort against SA internalized within phagosomes. During PMN phagocytosis and coincident with activation of the phagocyte NADPH oxidase, granules fuse with nascent phagosomes and release their contents into the phagosome, thereby exposing the ingested microbe to an array of toxic agents and a flux of oxidants that collaborate to kill microbes in phagosomes. However, despite the presence of this potent PMN antimicrobial system, 10-20% of ingested SA remain viable, but not replicating, in PMN. In response to PMN toxins in phagosomes, persisting viable SA stimulate PMN to initiate transcriptional programs that dictate the fate of PMN. SA-laden PMN have two potential fates: they may progress from atypical apoptosis to necrosis, with release of viable SA, or can be ingested by macrophages (Mf), thereby triggering phenotypic responses that promote further inflammation. Whereas Mf uptake of typical apoptotic PMN occurs without triggering proinflammatory cascades (i.e. a non phlogistic response), we have recently demonstrated that Mf ingestion of SA-PMN promoted aberrant cytokine responses. Persistence of SA within phagocytes has profound clinical consequences, as signature features of SA infection are relapse, metastases, and failure of antibiotics to which SA are susceptible. In murine infection models, PMN-associated SA recovered from an abscess can transmit infection to na¿ve mice, demonstrating that PMN can serve as a "Trojan horse", covertly entering macrophages (Mf) and then disseminating to cause metastatic infection. Given our preliminary data, we propose studies to test the overall hypothesis that SA PI-PLC serves as a virulence factor for SA by virtue of its ability to undermine phagocyte innate host defense. We hypothesize that PI-PLC may contribute to SA infection at two distinct stages: (1) early in infection, before effective cellular host defenses have been engaged and organisms ingested, extracellular PI-PLC will attack vulnerable substrates, including those on recruited phagocytes, and undermine their capacity to respond normally (Aim 1); and (2) later, after ingestion by phagocytes PI-PLC-expressing SA within phagosomes will alter PMN-Mf interactions, interfere with normal resolution of inflammation, and promote infection and dissemination (Aim 2). Our Specific Aims include: Aim 1. To determine the impact of extracellular SA PI-PLC on phagocyte function and innate host defense - focusing on priming of PMN NADPH oxidase and direct agonist effects phagocytes, and Aim 2. To determine the contribution of PI-PLC expressed within PMN on initiation and promotion of SA infection - focusing on PMN and Mf fate in vitro and consequences in infection in vivo. We have generated the analytical tools and developed experimental models necessary to study the effects of PI-PLC on phagocyte responses, and our ongoing studies of interactions between PMN containing viable SA and Mf provide a framework to model in vitro the fate of SA-laden PMN as might occur in infection.

Staphylococal infections occur commonly in Veterans, both in outpatients as well as hospitalized patients. The VA healthcare system served > 5 million people in 2007 with ~580,000 discharges from 153 medical centers and is thus one of the largest healthcare systems in the US. According to a VA-based study, the prevalence of staphylococcal infections increased from 26.93 per 1,000 discharges in 2000 to 30.59 in 2007, prompting institution of an aggressive and expensive infection control. Furthermore, antibiotic resistance among staphylococci limits therapeutic options. Taken together, the resultant rise in morbidity and mortality due to infection with staphylococci presents significant challenges to our health care system. Understanding the mechanisms by which innate immune elements mediate successful killing of SA will provide fundamental insights that can be translated into the development of novel targets for therapy.