Coordinate Regulation of Bacterial Virulence Factors

描述(申请人提供)：蛋白质输出或分泌是病原微生物伤害受感染动物和人类的宿主细胞的一种方式。例如，细菌输出的蛋白质可以杀死宿主细胞或促进对组织的黏附，并阻止宿主防御机制。革兰氏阴性细菌病原体利用至少六个不同的细胞外蛋白质分泌系统，通过其多层细胞膜输出蛋白质，在某些情况下进入宿主细胞。我们建议研究一个这样的系统，即VI型分泌系统(T6SS)，它由包括铜绿假单胞菌在内的许多细菌病原体表达。铜绿假单胞菌是一种机会性病原体，经常导致囊性纤维化患者的慢性肺部感染。这种生物的T6SS是肺内的一个重要毒力因子，因此是疫苗和药物开发的一个令人兴奋的新靶点。T6S装置被认为由15-20种蛋白质组成，其生化功能尚不清楚。我们的初步数据表明，参与T6SS功能的蛋白质是被称为T4噬菌体尾巴的蛋白质复合体的结构类似物。这种复合体是一种特殊的机器，在噬菌体感染过程中攻击宿主细胞的外表面。我们建议研究铜绿假单胞菌的T6S装置是否是类似于噬菌体尾部复合体的“纳米机器”，并通过类似的膜穿透装置将输出的蛋白质引入靶细胞。因此，本提案寻求资金用于一项研究计划，以解决以下关于T6SS的一般性问题：1)我们能否可视化T6S装置的组件并确认其与噬菌体尾状结构的结构关系？2)T6S装置是否在膜上形成通道，参与蛋白质输送到靶细胞？3)是什么控制了另外两个高度保守的T6SS基因簇在铜绿假单胞菌中的表达？4)T6S装置的组件是否出现在细胞表面，这些组件是否真的旋转？5)是否有可能通过开发针对T6SS组件的药物或疫苗来控制细菌感染？公共卫生相关性：铜绿假单胞菌是烧伤、免疫功能低下和囊性纤维化患者严重感染的原因。为了致病，这种细菌需要一台名为VI型分泌系统(T6SS)的微型机器。作为其功能的一部分，T6SS可能会将有毒蛋白质运输到人类细胞中。这项拟议的研究试图了解T6SS“纳米机器”是如何在宿主细胞中钻孔，然后注入杀死或改变人类细胞功能的蛋白质的。由于目前还没有针对这种微生物的疫苗，这些研究也可能提供一种新的方法来预防这种细菌引起的疾病。

DESCRIPTION (provided by applicant): Protein export or secretion is one means by which pathogenic microbes injure the host cells of infected animals and humans. For example, bacteria export proteins that can kill host cells or promote adherence to tissues and block host defense mechanisms. Gram- negative bacterial pathogens utilize at least six distinct extracellular protein secretion systems to export proteins through their multi-layered cell envelope and in some cases into host cells. We propose to study the function of one such system, the Type VI secretion system (T6SS) that is expressed by numerous bacterial pathogens including Pseudomonas aeruginosa. P. aeruginosa is an opportunistic pathogen that frequently causes chronic lung infections in cystic fibrosis patients. The T6SS of this organism is an important virulence factor within the lung and thus is an exciting new target for vaccine and drug discovery. T6S apparatus is thought to be composed of 15-20 proteins whose biochemical function is not well understood. Our preliminary data suggest that proteins involved in T6SS function are structural analogs of a protein complex called the `phage tail' of the bacteriophage T4. This complex is a special machine that attacks the outer surface of host cells during the phage infection process. We propose to study whether the T6S apparatus of P. aeruginosa is a "nano-machine" similar to the phage tail complex, and introduces exported proteins into target cells via a similar membrane puncturing device. Thus, this proposal seeks funding for a research program to address the following general questions about T6SS: 1) Can we visualize components of the T6S apparatus and confirm its structural relationship to phage tail-like structures? 2) Does the T6S apparatus form channels in membranes that could be involved in protein deliver into target cells? 3) What controls the expression of the two other highly conserved T6SS gene clusters in P. aeruginosa? 4) Do components of the T6S apparatus appear on the surface of cells and do these actually spin? 5) Is it possible to control bacterial infection by developing drugs or vaccines targeting components of the T6SS? PUBLIC HEALTH RELEVANCE: The bacterium Pseudomonas aeruginosa is the cause of severe infections in burn, immunocompromised and cystic fibrosis patients. In order to cause disease, this bacterium requires a tiny machine called the Type VI secretion system (T6SS). The T6SS likely transports toxic proteins into human cells as part of its function. The proposed study seeks to understand how the T6SS `nanomachine' drills holes into host cells and then injects proteins that kill or alter the function of human cells. Because no vaccine exists for this organism, these studies may also provide a new way to prevent disease due to this bacterium.