S. aureus virulence factor expression during kidney abscess formation

项目总结 耐药细菌感染正变得越来越普遍，而葡萄球菌 尤其是金黄色葡萄球菌感染对抗生素的耐药率很高，促使人们研究替代方案 治疗策略。美国金黄色葡萄球菌疫苗已经被开发出来，但到目前为止，所有疫苗都可能在临床试验中失败 由于金黄色葡萄球菌能够钝化免疫系统并阻止保护性免疫反应。金黄色葡萄球菌 产生许多促进疾病的毒力因子，包括大量的毒素，这些毒素直接针对和 杀死宿主免疫细胞。几种金黄色葡萄球菌毒素与人体细胞特异结合，并不活跃于 这使得很难剖析毒素在疾病过程中的具体作用。几乎没有模型可以做到 研究金黄色葡萄球菌群落、它们产生的毒素和它们的人体细胞靶标之间的相互作用。 金黄色葡萄球菌在人体宿主中引起多种疾病表现，包括皮肤和软组织。 感染菌血症和全身扩散至深层组织。当金黄色葡萄球菌进入血液时， 细菌被困在肝脏中，然后扩散到肾脏，形成称为脓肿的大病变。 脓肿包含紧密聚集的细菌的中心核心，有同心的坏死层和活体层。 中性粒细胞和巨噬细胞的外层。因为抗生素很难穿透脓肿， 它们可以在药物治疗后持续存在，并可能是复发的原因之一。 感染。更好地了解这些结构中细菌之间的相互作用，以及 与周围宿主细胞的相互作用，将是未来开发更有效的治疗方法的关键 消除这些结构。 这项提议将利用荧光转录记者来确定时空 金黄色葡萄球菌毒素在肾脓肿中的表达模式，并将在体外形成 研究脓肿内宿主-病原体相互作用的系统。我们假设与之直接互动 中性粒细胞和巨噬细胞中可扩散的抗菌素促进毒力因子的表达 尤其是在脓肿的边缘。我们将利用荧光报告菌株和 免疫荧光显微镜确定毒素表达模式是否在疾病过程中发生变化 在我们的小鼠模型中肾脓肿的形成，并确定这是否由中性粒细胞的存在所决定 和巨噬细胞。我们还将开发一个使用3D琼脂糖滴的体外脓肿形成模型 包裹细菌并将宿主细胞附着在液滴表面。在这个模型中，我们将可视化动态 在小鼠和人的原代吞噬细胞存在和不存在的情况下毒力因子的表达 实时成像荧光显微镜。建立这些稳健的系统来研究金黄色葡萄球菌的空间格局 宿主和病原体的相互作用将使我们能够发现金黄色葡萄球菌的关键漏洞，这可能是 开发出针对这种重要的人类病原体的更有效的治疗方法。

PROJECT SUMMARY Antibiotic-resistant bacterial infections are becoming increasingly more prevalent, and Staphylococcus aureus infections in particular have high rates of antibiotic resistance, prompting research into alternative treatment strategies. S. aureus vaccines have been developed, but all have failed in clinical trials to date, likely due to the ability of S. aureus to blunt the immune system and block protective immune responses. S. aureus produces many virulence factors that promote disease, including an arsenal of toxins, which directly target and kill host immune cells. Several S. aureus toxins specifically bind to human cells, and are not active in the mouse, rendering it difficult to dissect the specific role of toxins in the disease process. Few models exist to study interactions between S. aureus communities, the toxins they produce, and their human cell targets. S. aureus causes a wide range of disease manifestations in the human host, from skin and soft tissue infections to bacteremia and systemic spread to deep tissues. When S. aureus enters the bloodstream, bacteria are trapped in the liver, and then spread to the kidney to form large lesions called abscesses. Abscesses contain a central core of tightly clustered bacteria, with concentric layers of necrotic and live neutrophils, and an outer layer of macrophages. Because it is difficult to penetrate abscesses with antibiotics, they can persist follow drug treatment, and may represent one of the reservoirs responsible for recurrent infections. Better understanding of the interactions between bacteria within these structures, and the interactions with surrounding host cells, will be critical in developing future therapeutics to more efficiently eliminate these structures. This proposal will utilize fluorescent transcriptional reporters to determine the spatiotemporal expression patterns of S. aureus toxin expression within kidney abscesses, and will also develop an in vitro system to study host-pathogen interactions within abscesses. We hypothesize that direct interactions with neutrophils, and diffusible antimicrobials from macrophages, promotes expression of virulence factors specifically at the periphery of abscesses. We will utilize fluorescent reporter strains and immunofluorescence microscopy to determine whether toxin expression patterns change over the course of kidney abscess formation in our mouse model, and determine if this is dictated by the presence of neutrophils and macrophages. We will also develop an in vitro model of abscess formation using 3D agarose droplets to encapsulate bacteria and adhere host cells to the droplet surface. In this model, we will visualize the dynamics of virulence factor expression in the presence and absence of mouse and human primary phagocytes using live imaging fluorescence microscopy. Establishing these robust systems to study S. aureus spatial patterning and host-pathogen interactions will enable us to uncover key S. aureus vulnerabilities, which could be exploited to generate more efficacious treatments against this important human pathogen.