CDI System-mediated Intra- and Inter-species Interactions in Burkholderia

描述(申请人提供)：革兰氏阴性菌假鼻疽伯克霍尔德氏菌是一种土壤腐生菌，也是一种新出现的传染病病原体。它是类鼻疽的病原体，可导致急性肺部疾病、败血症，如果不治疗，可能是致命的。虽然它天然存在于东南亚和澳大利亚北部，但由于其被武器化的潜力而引起全球关注。细菌通常生活在多菌群落中，因此进化出了与邻居交流、合作和竞争的机制。一种这样的机制是接触依赖生长抑制(CDI)系统。这个系统由两个伙伴的分泌蛋白组成，被细菌用来杀死邻近的细胞，并潜在地控制周围的种群。这个系统的主要成分是一个大的表面外蛋白BCPA，它含有一个有毒的C-末端(CT)。CT被传递到目标细菌细胞以诱导死亡，除非目标细胞含有免疫蛋白来阻止毒性活动。CDI系统的一个标志性功能是 CTs及其同源免疫蛋白之间的多态性，以等位基因特异性的方式保护。本实验室的研究表明，非病原菌泰兰杆菌产生的CDI系统用于细菌间的竞争和生物膜的形成。在本提案中，我们将解决有关Burkholderia类型CDI系统的几个问题。首先，我们将评估假鼻疽杆菌利用其CDI系统进行细菌间杀灭的能力。然后，当细菌与密切相关的生物体接触时，CDI系统将在物种间杀戮中发挥作用。为了实现这一点，我们将评估假鼻疽杆菌和泰兰杆菌之间的物种间竞争。此外，通过使用BCPA嵌合蛋白的研究，我们将分析BCPA的区域，这些区域可能对系统的活性和特异性起作用。最后，我们将进行筛选分析，以确定目标细胞上被毒素识别的蛋白质。这一步骤对于促进细胞间的接触是必不可少的，它定义了系统的特异性。我们将在这项研究中调查的不同方面将有助于理解CDI系统在微生物群落中的作用。这一知识可以应用于土壤中存在的生物膜群落，这是假鼻疽杆菌向人类传播的来源。

DESCRIPTION (provided by applicant): The Gram-negative bacterium Burkholderia pseudomallei is a soil saprotroph and an emerging infectious disease agent. It is the causative agent of melioidosis, which can result in acute pulmonary disease, septicemia, and can be fatal if left untreated. Although naturally found in Southeast Asia and Northern Australia, it is of globl concern because of its potential to be weaponized. Bacteria typically live in polymicrobial communities and hence have evolved mechanisms to communicate, cooperate and compete with their neighbors. One such mechanism is the contact-dependent growth inhibition (CDI) system. This system is composed of two-partner secretion proteins, and is utilized by the bacterium to kill neighboring cells and potentially control the surrounding population. The main component of this system is a large surface exoprotein, BcpA, which contains a toxic C-terminus (CT). The CT is delivered to a target bacterial cell to induce death, unless the target cell contains an immunity protein to block the toxic activity. A hallmark feature of CDI systems is polymorphism among CTs and their cognate immunity proteins that protect in an allele specific manner. Studies in our laboratory have shown that the CDI system produced by the non-pathogen B. thailandensis is used for interbacterial competition and biofilm formation. In this proposal we will address several questions regarding Burkholderia-type CDI systems. First we will assess the ability of B. pseudomallei to utilize its CDI systems for interbacterial killing. W will then establish a role for CDI systems in interspecies killing when the bacterium comes into contact with closely related organisms. To achieve this we will evaluate interspecies competition between B. pseudomallei and B. thailandensis. Additionally, through studies using BcpA chimeric proteins, we will analyze regions of BcpA that potentially play a role in activity and specificity of the system. Lastly, we will perform screening analyses to identify the proteins on the target cell that are recognized by the toxin. This step is essential to promote cell-to-cell contact and it defines the specificity of the system. The different aspects that we will investigat in this study will contribute to the understanding of the role of CDI systems within a microbial community. This knowledge can be applied towards biofilm communities that are present in the soil, which is the source of transmission of B. pseudomallei to humans.