Mechanistic Studies Of The Mode Of Action Of The Staphylococcus aureus LukAB Cyto

描述(申请人提供)：金黄色葡萄球菌是导致医院和社区感染的最重要的人类病原体之一。由于高度的抗生素耐药性和缺乏有效的疫苗，抗击金黄色葡萄球菌感染的治疗选择有限。因此，有必要开发有效的治疗方法来对抗这种微生物。金黄色葡萄球菌致病成功的关键是 这种细菌通过定向杀灭中性粒细胞来避免宿主清除的能力；控制葡萄球菌感染所必需的先天免疫细胞。因此，这项研究计划的长期目标是了解金黄色葡萄球菌耗尽这些关键吞噬细胞的机制。最近，我们描述了白毒素A/B(LukAB)作为一种毒力因子，通过靶向和消除这些细胞，在保护金黄色葡萄球菌免受中性粒细胞介导的杀伤方面发挥了不可或缺的作用。此外，我们还证明了LukAB在全身播散的小鼠模型中对社区相关的耐甲氧西林金黄色葡萄球菌(CA-MRSA)的致病作用。我们发现：LukAB在金黄色葡萄球菌中是保守的，有助于包括甲氧西林敏感和甲氧西林耐药在内的各种菌株的细胞毒性，不仅负责杀死中性粒细胞，还负责单核细胞、巨噬细胞和树突状细胞，是在金黄色葡萄球菌中发现的双组分成孔毒素家族中最具差异性的成员，并且它具有其他白毒素没有观察到的独特性质。基于我们的发现，我们提出了一个模型，即LukAB选择性地与吞噬细胞结合，导致毒素寡聚和孔洞形成，最终导致膜损伤和靶细胞死亡。这个应用程序的主要目标是测试这个模型。为此，我们提出了三个具体目标。在目标1中，我们计划阐明LukAB趋向性吞噬细胞的候选细胞因子的作用。在目标2中，我们试图定义涉及LukAB介导的毒性的功能区域。最后，在目标3中，我们建议确定LukAB在体内对金黄色葡萄球菌致病作用的机制。为了实现这些目标，我们建议采用多学科的方法，结合分子生物学、遗传学、免疫学和生物化学，以及体外和体内感染模型。了解LukAB如何介导靶向和杀伤吞噬细胞的分子细节对于开发新的治疗方法以抑制这种毒素作为对抗金黄色葡萄球菌感染的新方法至关重要。

DESCRIPTION (provided by applicant): Staphylococcus aureus is one of the most important human pathogens responsible for infections in both hospital and community settings. Therapeutic options to combat S. aureus infections are limited due to the high level of antibiotic resistance and lack of an effective vaccine. Thus, there is a significant need for the development of effective therapeutics against this organism. Critical to the pathogenic success of S. aureus is the ability of this bacterium to avoid clearance by the host via targeted killing of neutrophils; innate immune cells integral to the control of Staphylococcal infections. Thus, the long-term goal of this research program is to understand the mechanism employed by S. aureus to deplete these critical phagocytes. Recently, we have described the leukotoxin A/B (LukAB) as a virulence factor that plays an integral role in protecting S. aureus from neutrophil-mediated killing by targeting and eliminating these cells. In addition, we have demonstrated that LukAB contributes to the pathogenesis of community-associated, methicillin-resistant Staphylococcus aureus (CA-MRSA) in a murine model of systemic dissemination. We have found that: LukAB is conserved in S. aureus, contributes to the cytotoxicity of a variety of strains including methicilln-sensitive and methicillin- resistant S. aureus, is responsible for the killing of not only neutrophls, but also monocytes, macrophages and dendritic cells, is the most divergent member of the bi-component pore-forming family of toxins found in S. aureus, and it exhibits unique properties that have not been observed by the other leukotoxins. Based on our findings, we propose a model whereby LukAB selectively binds to phagocytes resulting in toxin oligomerization and pore-formation, which ultimately leads to membrane damage and killing of the target cell. The primary goal of this application is to test this model. To this end, we propose three Specific Aims. In Aim 1 we plan to elucidate the contribution of candidate cellular factors for LukAB tropism towards phagocytes. In Aim 2 we seek to define functional regions involved in LukAB-mediated toxicity. Lastly, in Aim 3 we propose to determine the mechanism by which LukAB contributes to S. aureus pathogenesis in vivo. To accomplish these Aims, we propose to employ a multidisciplinary approach that combines molecular biology, genetics, immunology, and biochemistry, together with ex vivo and in vivo infection models. Understanding the molecular details of how LukAB mediates targeting and killing of phagocytes is crucial for the development of novel therapeutic modalities to inhibit this toxin as a new approach to combat S. aureus infections.