Type VI secretion effectors and interbacterial competition in vivo

描述（由申请人提供）：越来越明显的是，多种微生物聚集体经常是慢性感染的基础，包括囊性纤维化（CF）患者肺部和糖尿病患者伤口中发生的感染。这种感染的复杂性干扰了病因学的建立并使治疗复杂化。临床和体外数据表明，革兰氏阴性变形杆菌铜绿假单胞菌是特别善于建立自己和持久的多微生物感染，然而，这一观察缺乏分子解释。VI型分泌系统（T6 SS）是一种复杂的细胞间效应蛋白递送途径。虽然该系统最初被认为是靶向宿主细胞，但在之前的研究期间，我们的团队发现该途径的主要功能是以细胞接触依赖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T6 SS，称为溶血素共调节蛋白分泌岛I-III（H1-H3-T6 SS）。我们的研究表明，铜绿假单胞菌的H1-T6 SS赋予其Tse（VI型分泌输出）效应底物介导的有效的种内和种间抗菌活性。在本提案中提供的初步数据中，我们证明铜绿假单胞菌使用比以前认识到的更复杂的细菌间竞争策略。事实上，我们已经发现铜绿假单胞菌在细菌间竞争期间使用其三个T6 SS中的至少两个。此外，除了Tse效应物之外，我们还发现它具有最近鉴定的T6 S递送的抗菌磷脂酶超家族的多个成员，称为Tle（VI型分泌脂肪酶效应物）蛋白。我们在此提出测试的假设，广泛和多样化的T6 S依赖性的抗菌谱的铜绿假单胞菌有助于适应的细菌在多微生物感染的情况下。在本提案的目标1中，我们将使用脂质组学分析来定义最近鉴定的Tle效应物的分子靶标，并且我们将确定这些蛋白质靶向临床相关细菌物种的能力。在目的2中，我们使用多微生物糖尿病小鼠伤口模型评估T6依赖性活性在体内的作用。这些实验的结构使得可以确定T6 S效应物的抗微生物活性对慢性感染期间铜绿假单胞菌的疾病结果和体内适应性的具体贡献。最后，在目标3中，我们提出了一种分泌独立的，新的定量质谱法为基础的方法来定义新的T6 S效应。所鉴定的效应子将进行表型分析，并纳入我们的管道中，以评估细菌间T6 S对体内竞争适应性的贡献。本提案中概述的研究对我们对T6 SS的基本理解以及该途径及其效应物在多种微生物感染中的作用都有重大贡献。

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.