Inter-Bacterial Competition Systems in Burkholderia pseudomallei

描述(由申请人提供)：假鼻疽伯克霍尔德氏菌是类鼻疽的病原体，也是潜在的生物恐怖主义威胁，它对最常用的抗生素具有内在的抗药性，表明对替代抗菌剂的明确要求，以及对这种新兴微生物所采用的致病策略的更好理解。在大肠杆菌中，发现了一个由两个伙伴分泌的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CDIA外蛋白C末端的核酸酶活性，可通过与系统的同源免疫蛋白CdiI的直接结合而被阻断。该系统的同源物在假鼻疽杆菌和相关的非病原菌泰兰杆菌的基因组中被发现。然而，伯克霍尔德氏菌和大肠杆菌CDI系统之间存在明显的差异，这些系统在伯克霍尔德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机制的完全了解。初步数据表明，假鼻疽杆菌CDIA同源物BtpA的C-末端区域与Colicin E5 tRNase有相似的序列，当在几种细菌中产生时是有毒的，除非系统的同源免疫蛋白BtpI也产生。为了了解这些系统在假鼻疽杆菌生命周期中的作用这一长期目标，这一应用将检验BtpA是一种表面定位的蛋白质的假设，它作为种内和/或种间核酸酶来调节细菌间的抑制。目标1中的研究将确定BtpA蛋白的拓扑结构和加工过程，目标2中的实验将确定BtpA毒性的机制并确定最小抑制单位。最后，目标3中提出的研究将评估BtpA对其他细菌病原体的毒性，并检查其调节物种内和/或物种间细菌抑制的能力。这项拟议的研究将有助于了解一种新出现的病原体，并通过研究一类新的细胞毒性蛋白，满足追求新的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.