Coordinate Regulation of Bacterial Virulence Factors

描述（由申请人提供）：蛋白质输出或分泌是病原微生物损伤受感染动物和人类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粘附并阻断宿主的防御机制。革兰氏阴性细菌病原体利用至少六种不同的细胞外蛋白质分泌系统通过其多层细胞包膜输出蛋白质，并且在某些情况下输出到宿主细胞中。我们建议研究一个这样的系统，VI型分泌系统（T6 SS），是由许多细菌病原体，包括铜绿假单胞菌表达的功能。铜绿假单胞菌是一种机会致病菌，经常引起囊性纤维化患者的慢性肺部感染。该生物体的T6 SS是肺内的重要毒力因子，因此是疫苗和药物发现的令人兴奋的新靶点。T6 S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T6 SS功能的蛋白质是噬菌体T4的称为“噬菌体尾”的蛋白质复合物的结构类似物。这种复合物是一种特殊的机器，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T6 S装置是否是类似于噬菌体尾复合物的“纳米机器”，并通过类似的膜穿刺装置将输出的蛋白质引入靶细胞。因此，该提案寻求资助一项研究计划，以解决以下关于T6 SS的一般性问题：1）我们能否可视化T6 S装置的组件并确认其与噬菌体尾状结构的结构关系？2)T6 S装置是否在膜中形成可能参与蛋白质递送到靶细胞中的通道？3)是什么控制了铜绿假单胞菌中另外两个高度保守的T6 SS基因簇的表达？4)T6 S装置的组成部分是否出现在细胞表面，并且它们实际上是否旋转？5)是否有可能通过开发针对T6 SS成分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。为了引起疾病，这种细菌需要一个称为VI型分泌系统（T6 SS）的微小机器。T6 SS可能将有毒蛋白质转运到人体细胞中作为其功能的一部分。这项拟议中的研究旨在了解T6 SS“纳米机器”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。由于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.