Molecular and Cellular Basis of Toxin Mediated Pathogenesis in Staphylococcus aur

描述（由申请人提供）：金黄色葡萄球菌是一种主要的公共卫生问题，每年在美国导致超过30万人住院（1-2）。这种微生物是化脓性皮肤病变、细菌性心内膜炎、菌血症和肺炎的主要原因。金黄色葡萄球菌感染的治疗因大量耐药菌株的出现而复杂化(2)。一旦以前仅限于医院环境，金黄色葡萄球菌引起的社区获得性感染继续出现，通常在其他健康个体中(2)。金黄色葡萄球菌分泌一系列毒力因子，促进易感宿主的生存。在这些因素中，许多分泌的毒素直接与宿主细胞相互作用并杀死/损伤宿主细胞（3-5）。毒素表达的协同调控对金黄色葡萄球菌的发病至关重要。我们的数据表明，通过转录抑制对毒素产生的调节对白质毒素LukED的最佳表达至关重要。当这种调节模式受到干扰时，金黄色葡萄球菌菌株在小鼠感染模型中由于LukED产量增加而变得高毒力。本申请中描述的实验旨在(i)阐明金黄色葡萄球菌中lukED基因表达的调控机制(s)以及（ii）确定lukED中毒在体外、离体和体内的功能影响。细菌遗传学、启动子结合试验、体外细胞毒性和存活试验、体内败血症感染模型和免疫学技术将是实现上述目标的关键。实验将研究lukED基因在体内协调表达的影响，毒素在细菌介导的哺乳动物细胞杀伤中的作用，以及毒素对体内免疫细胞募集/活力的影响。最终，我们将确定的主要贡献

DESCRIPTION (provided by applicant): Staphylococcus aureus is a major public health concern and is responsible for over 300,000 hospitalizations in the United States each year (1-2). The organism is a leading cause of suppurative skin lesions, bacterial endocarditis, bacteremia, and pneumonia. Treatment of S. aureus infection is complicated by the emergence of numerous antibiotic resistant strains (2). Once previously limited to hospital settings, community acquired infections due to S. aureus continue to arise, often in otherwise healthy individuals (2). S. aureus secretes an arsenal of virulence factors that promote the organism's survival in susceptible hosts. Among these factors are a number of secreted toxins that directly interact with and kill/damage host cells (3-5). The coordinated regulation of toxin expression is critical to S. aureus pathogenesis. Our data demonstrate that regulation of toxin production by transcriptional repression is critical to optimal expression of the leukotoxin LukED. When such regulatory patterns are perturbed, S. aureus strains become hypervirulent in mouse models of infection due to increased LukED production. Experiments described in this application are designed to (i) elucidate the regulatory mechanism(s) of lukED gene expression in S. aureus and (ii) determine the functional impact of LukED intoxication in vitro, ex vivo, and in vivo. Bacterial genetics, promoter binding assays, in vitro cell toxicity and survival assays, in vivo models of septicemic infection, and immunological techniques will be critical to the execution of the above aims. Experiments will investigate the impact of coordinated lukED gene expression in vivo, the role of the toxin in bacterial mediated mammalian cell killing, and the toxin's effecton immune cell recruitment/viability in vivo. Ultimately, we will determine the major contributions of LukED to pathogenesis in vivo and will provide insight into the optimal toxin expression patterns required for infection. Importantly, this work will further the mission of the National Institutes f Health by examining the mechanistic underpinnings of disease caused by S. aureus, with an end goal of improving prevention and treatment strategies.