Inter-Bacterial Competition Systems in Burkholderia pseudomallei

描述（由申请人提供）：假性伯克氏菌，类鼻疽病的病原体和潜在的生物恐怖主义威胁，本质上对大多数常用抗生素具有耐药性，表明对替代抗菌剂的明确需求，以及对这种新兴生物所使用的致病策略的更好理解。在大肠杆菌中，鉴定了一种由双伴侣分泌蛋白组成的细菌间接触依赖抑制（CDI）系统，该系统以等位基因特异性的方式抑制目标细菌。毒性是由于位于CdiA外蛋白c末端的核酸酶活性，通过直接结合系统的同源免疫蛋白CdiI而被阻断。该系统的同源物存在于假芽孢杆菌和相关的非病原体泰国芽孢杆菌的基因组中。然而，伯克氏菌和大肠杆菌CDI系统之间存在明显差异，这些系统在伯克氏菌生物学中的作用尚不清楚。此外，虽然已经提出了CDI的模型，但CdiA/BtpA蛋白的定位和拓扑结构尚不清楚，这阻碍了对CDI机制的全面了解。初步数据表明，伪芽孢杆菌CdiA同源物的c末端BtpA与colicin E5 tRNase序列相似，在几种细菌中产生时是有毒的，除非该系统的同源免疫蛋白BtpI也产生。为了了解这些系统在假芽孢杆菌生命周期中的作用，本应用程序将验证BtpA是一种表面定位的蛋白质，其功能是作为种内和/或种间核酸酶介导细菌间抑制的假设。Aim 1的研究将确定BtpA蛋白的拓扑结构和加工过程，Aim 2的实验将确定BtpA毒性的机制并确定最小抑制单元。最后，Aim 3中提出的研究将评估双酚a对其他细菌病原体的毒性，并检查其介导种内和/或种间细菌抑制的能力。拟议的研究将有助于理解新出现的病原体，并通过研究一类新的细胞毒性蛋白，满足追求新型抗菌疗法的基本需求。

DESCRIPTION (provided by applicant): Burkholderia pseudomallei, the causative agent of melioidosis and a potential bioterrorism threat, is intrinsically resistant to most commonly-prescribed antibiotics, demonstrating a clear requirement for alternative antimicrobials, as well as a better understanding of the pathogenic strategies utilized by this emerging organism. In Escherichia coli, an inter-bacterial contact-dependent inhibition (CDI) system comprised of two-partner secreted proteins was identified that inhibits target bacteria in an allele-specific manner. Toxicity, which is due to nuclease activity located at the C-terminal end of the CdiA exoprotein, is blocked by direct binding of the system's cognate immunity protein, CdiI. Homologs of this system are found in the genomes of B. pseudomallei and the related non-pathogen B. thailandensis. However, distinct differences exist between the Burkholderia and E. coli CDI systems and the role of these systems in Burkholderia biology is not known. Moreover, while a model for CDI has been suggested, the localization and topology of the CdiA/BtpA proteins are unknown, preventing a complete understanding of the CDI mechanism. Preliminary data indicate that the C-terminal region of the B. pseudomallei CdiA homolog, BtpA, which shares sequence similarity with colicin E5 tRNase, is toxic when produced in several bacterial species unless the system's cognate immunity protein, BtpI, is also produced. Toward the long-term objective of understanding the role of these systems in the lifecycle of B. pseudomallei, this application will test the hypothesis that BtpA is a surface-localized protein that functions as an intra- and/or inter-species nuclease to mediate inter-bacterial inhibition. Studies in Aim 1 will determine the topology and processing of the BtpA protein and experiments in Aim 2 will identify the mechanism of BtpA toxicity and define the minimal inhibitory unit. Finally, the research proposed in Aim 3 will assess the toxicity of BtpA to other bacterial pathogens and examine its ability to mediate intra- and/or inter- species bacterial inhibition. The proposed research will contribute to the understanding of an emerging pathogen and, by investigating a novel class of cytotoxic proteins, fulfills an essential need in the pursuit of novel antimicrobial therapies.