Type VI Protein Secretion in an Emerging Multidrug-Resistant Pathogen

描述(由申请人提供)：由多重耐药细菌引起的医院感染是免疫力低下患者发病率和死亡率增加的主要原因。这种感染的主要病原体是鲍曼不动杆菌。这种微生物会引起侵袭性感染，包括肺炎和败血症，近年来这些感染变得越来越严重，对抗生素产生了抗药性，这引发了人们的担忧，即有一天它们可能无法治愈。人们迫切需要预防和治疗鲍曼不动杆菌感染的新方法，但令人担忧的是，人们对这些细菌如何致病知之甚少。最近的证据表明，这些细菌对培养的上皮细胞产生黏附依赖性的细胞毒性；然而，鲍曼不动杆菌在黏附时使细胞中毒的机制尚不清楚。拟议研究的目标是了解这些微生物如何与人类细胞相互作用，以及这种相互作用如何破坏正常的细胞过程，促进感染。为了实现这一目标，这项建议旨在详细描述一种接触依赖型蛋白递送系统，称为VI型分泌系统(T6SS)，该系统存在于导致最近疫情的鲍曼不动杆菌临床菌株中。这些系统在一系列微生物的宿主感染中发挥作用，但在鲍曼不动杆菌中没有定义。这项拟议的工作将确定鲍曼不动杆菌T6SS针对哪些细胞，识别它向这些细胞发送的效应蛋白，并研究该系统及其效应器在宿主相互作用和发病机制中的功能作用。这些目标将通过分析报告融合到靶细胞的转移，对效应器进行定向全基因组筛选，并在培养和肺炎小鼠模型中检查T6SS和效应器缺失突变体在与宿主细胞相互作用过程中对细菌毒性和宿主先天免疫反应的影响来实现。这些研究将在鲍曼不动杆菌促进感染的机制上开辟新的天地，并将解决我们对T6SS病理生物学理解中的关键空白。最终，这些信息将揭示新的抗感染治疗的目标。 以及针对高度抗药性细菌的疫苗。培训经验将拓宽我在分子生物学方面的专业知识，教会我哺乳动物细胞培养和动物模型方面的新技术，并增进我对传染病发病机制的理解。

DESCRIPTION (provided by applicant): Hospital-acquired infections due to multidrug-resistant bacteria are a major cause of increased morbidity and mortality in patients with weakened immunity. A leading agent of such infections is the bacterium Acinetobacter baumannii. This microbe causes invasive infections, including pneumonia and sepsis, that in recent years have become increasingly severe and antibiotic-resistant, raising the specter that they may one day be untreatable. New ways to prevent and treat infections with A. baumannii are urgently needed, but alarmingly little is known about how these bacteria cause disease. Recent evidence indicates that these bacteria induce adherence-dependent cytotoxicity toward cultured epithelial cells; however, the mechanisms used by A. baumannii to intoxicate cells upon adhesion are unclear. The goal of the proposed studies is to understand how these microbes interact with human cells and how such interactions undermine normal cellular processes, promoting infection. Towards this goal, this proposal aims to characterize in detail a contact-dependent protein delivery system, known as a Type VI Secretion System (T6SS) that is present in clinical strains of A. baumannii responsible for recent epidemics. These systems play a role in host infections by a range of microbes, but the system is undefined in A. baumannii. The proposed work will determine which cells are targeted by the A. baumannii T6SS, identify the effector proteins it sends into these cells, and examine the functional roles of the system and its effectors in host interactions and pathogenesis. These aims will be accomplished by analyzing the transfer of reporter fusions into target cells, conducting directed genome-wide screens for effectors, and examining the effects of T6SS- and effector-null mutants on bacterial toxicity and the host innate immune response during interactions with host cells in culture and in a murine model of pneumonia. These studies will break new ground on the mechanisms used by A. baumannii to promote infection and will address key gaps in our understanding of T6SS pathobiology. Ultimately, this information will uncover targets for novel anti-infective treatments and vaccines against highly drug- resistant bacteria. The training experience will broaden my expertise in molecular biology, teach me new techniques in mammalian cell culture and animal models, and advance my understanding of the pathogenesis of infectious diseases.