Type VI secretion effectors and interbacterial competition in vivo

描述(由申请人提供)：越来越明显的是，多菌组合经常是慢性感染的基础，包括发生在囊性纤维化(CF)患者的肺部和糖尿病患者的伤口。这种感染的复杂性干扰了病原学的确定，并使治疗复杂化。临床和体外数据表明，革兰氏阴性杆菌铜绿假单胞菌特别擅长在多菌感染中自我确立和持续存在；然而，对这种观察到的现象缺乏分子解释。VI型分泌系统(T6SS)是一种复杂的细胞间效应蛋白传递途径。虽然该系统最初被认为是针对宿主细胞的，但在之前的获奖期间，我们的团队发现，该途径的主要功能是以细胞接触的方式在细菌之间传递毒素。有趣的是，铜绿假单胞菌具有三个非冗余的T6SS，称为溶血素共调节蛋白分泌岛I-III(H1-H3-T6SS)。我们的研究表明，铜绿假单胞菌的H1-T6SS具有强大的种内和种间抗菌活性，这是由其TSE(第六型分泌物输出)效应底物介导的。在这项提案中提供的初步数据中，我们证明了铜绿假单胞菌使用的细菌间竞争策略比以前认识到的要复杂得多。事实上，我们已经发现，在细菌间的竞争中，铜绿假单胞菌至少使用了它三个T6SS中的两个。此外，除了TSE效应器外，我们还发现它拥有最近发现的T6S递送抗菌磷脂酶超家族的多个成员，称为TLE(类型VI分泌脂肪酶效应器)蛋白。在此，我们提出了一种假设，即广泛和多样化的依赖T6S的铜绿假单胞菌抗菌谱有助于该细菌在多菌感染的背景下的适应性。在这项提案的目标1中，我们将使用脂组学图谱来定义最近发现的TLE效应器的分子靶点，并确定这些蛋白质针对临床相关细菌物种的能力。在目标2中，我们使用多菌素糖尿病小鼠创伤模型来评估体内T6依赖活性的作用。这些实验的结构是，可以确定T6S效应器的抗菌活性对慢性感染期间铜绿假单胞菌的疾病结局和体内适合性的具体贡献。最后，在目标3中，我们提出了一种不依赖分泌的、基于定量质谱学的新方法来定义新的T6S效应器。确定的效应器将受到表型分析的影响，并纳入我们的管道，以评估细菌间T6S对体内竞争适应性的贡献。这项提案中概述的研究有助于我们对T6SS的基本理解，并有助于该途径及其效应器在多菌感染中所起的作用。

DESCRIPTION (provided by applicant): It is increasingly evident that polymicrobial assemblages frequently underlie chronic infections, including those that occur in the lungs of cystic fibrosis (CF) patients and the wounds of diabetics. The complexity of such infections interferes with establishing etiology and complicates treatment. Clinical and in vitro data indicat that the Gram-negative Proteobacterium Pseudomonas aeruginosa is particularly adept at establishing itself and persisting within polymicrobial infections; however, a molecular explanation for this observation is lacking. The type VI secretion system (T6SS) is a complex intercellular effector protein delivery pathway. Though the system was initially thought to target host cells, during the prior award period our group discovered the primary function of the pathway is to deliver toxins in a cell contact-dependent manner between bacteria. Interestingly, P. aeruginosa possesses three non-redundant T6SSs, termed haemolysin co-regulated protein secretion islands I-III (H1-H3-T6SSs). Our studies revealed that the H1-T6SS of P. aeruginosa confers potent intra- and inter-species antibacterial activity mediated by its Tse (type VI secretion exported) effector substrates. In preliminary data provided in this proposal, we demonstrate that P. aeruginosa uses a far more elaborate interbacterial competition strategy than previously recognized. Indeed, we have discovered that P. aeruginosa employs at least two of its three T6SSs during interbacterial competition. Moreover, in addition to the Tse effectors, we have found that it possesses multiple members of a recently identified superfamily of T6S-delivered antibacterial phospholipases, termed the Tle (type VI secretion lipase effector) proteins. We propose herein to the test the hypothesis that the extensive and diverse T6S-dependent antibacterial repertoire of P. aeruginosa contributes to the fitness of the bacterium in the context of polymicrobial infections. In Aim 1 of this proposal, we will use lipidomic profilingto define the molecular targets of the recently identified Tle effectors and we will determine the capacity of these proteins to target clinically relevant bacterial species. In Aim 2, we evaluate the role of T6-dependent activity in vivo using a polymicrobial diabetic murine wound model. These experiments are structured such that the specific contribution of the antimicrobial activity of T6S effectors to disease outcome and in vivo fitness of P. aeruginosa during chronic infection can be ascertained. Finally, in Aim 3, we propose a secretion-independent, novel quantitative mass spectrometry-based approach for defining new T6S effectors. Effectors identified will be subject to phenotypic analysis and incorporated into our pipeline for evaluating the contribution of interbacterial T6S to competitive fitness in vivo. The studies outlined in this proposal stand t contribute significantly both to our basic understanding of the T6SS, and to the role that the pathway and its effectors play in polymicrobial infections.