SECRETION MECHANISM OF A NOVEL C. RECTUS S-LAYER PROTEIN

一种新型直肌 S 层蛋白的分泌机制

• 批准号: 6045448
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 15.75万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6045448
• 关键词: Helicobacter; bacterial genetics; bacterial proteins; cell membrane; gene expression; gene mutation; immunoelectron microscopy; immunoprecipitation; laboratory mouse; membrane proteins; molecular chaperones; mutant; operon; oral bacteria; polymerase chain reaction; protein sequence; protein signal sequence; protein structure function; protein transport; secretion; virulence; western blottings; yeast two hybrid system

Periodontitis is associated with a dramatic shift in the subgingival microflora towards predominantly gram negative organisms. Campylobacter rectus is unique among the gram negative periodontal pathogens in its expression of a 150 kd surface-layer (S-layer) protein. These abundant proteins form homogeneous arrays covering the surfaces of numerous bacteria and are required for bacterial pathogenesis. Thus , it is critical that S-layers are properly localized to the cell surface. There are only a few reports on the secretion mechanisms of S-layer proteins. Interestingly, unlike most other S-layer proteins, no amino acids are removed from the C. rectus S-layer protein (crsA) as it is transported to the cell surface. In addition, a homologue, crsD, to one of the three components of type I secretion systems is found in an operon upstream of the C. rectus crsA gene. This suggests that crsA is transported by a type I pathway. Most excitingly, the crsD operon contains another gene, crsC, whose protein has no known function. Analysis of the phenotype of a polar crsC mutant we constructed suggests that crsC is involved in crsA secretion, synthesis or degradation. We now propose to exploit the molecular genetic tools we have developed in order to determine the mechanisms used by C. rectus to transport its S-laver to the cell surface. It is our hypothesis that the crsA protein will be transported by a type l system. Importantly, we hypothesize that crsC will have a novel function in the type I secretion pathway perhaps as a chaperone. One goal of this proposal is to identify the C. rectus S-layer sequences that direct it to the cell surface using targeted mutagenesis and gene replacement technology. In addition, the genes required for crsA transport will be identified by cloning and sequencing the rest of the crsCD operon or by transposon mutagenesis. Importantly, the function of each putative transport gene will be characterized by making non-polar mutants. The resulting characterization of the crsC mutants will define, for the first time, the function of this novel protein in S-layer metabolism. Finally, the transport mutants will be examined for the possible mis-localization of non-S-layer proteins since there are two reports of type I transport pathways transporting more than one virulence protein.

牙周炎与牙龈下菌群向革兰氏阴性菌为主的急剧转变有关。在革兰氏阴性牙周病原体中，直弯曲杆菌是独特的，它表达一种150 kd的表层（s层）蛋白。这些丰富的蛋白质形成均匀阵列，覆盖在许多细菌的表面，是细菌发病所必需的。因此，s层适当地定位到细胞表面是至关重要的。目前关于s层蛋白分泌机制的报道很少。有趣的是，与大多数其他s层蛋白不同，直梭菌s层蛋白（crsA）在运输到细胞表面时没有氨基酸被移除。此外，在直梭菌crsA基因上游的一个操纵子中发现了与I型分泌系统的三个组成部分之一的同源物crsD。这表明crsA是通过I型途径转运的。最令人兴奋的是，crsD操纵子包含另一个基因，crsC，其蛋白质没有已知的功能。我们构建的一个极性crsC突变体的表型分析表明，crsC参与了crsA的分泌、合成或降解。我们现在建议利用我们开发的分子遗传学工具来确定直梭菌将其S-laver运输到细胞表面的机制。我们的假设是，crsA蛋白将通过l型系统运输。重要的是，我们假设crsC在I型分泌途径中可能作为伴侣具有新的功能。本研究的目标之一是利用靶向诱变和基因替代技术鉴定直梭菌s层序列，将其引导到细胞表面。此外，通过克隆和测序crsCD操纵子的其余部分或通过转座子诱变来鉴定crsA转运所需的基因。重要的是，每个假定的运输基因的功能将通过制造非极性突变体来表征。crsC突变体的最终特征将首次定义这种新蛋白在s层代谢中的功能。最后，将检查运输突变体是否存在非s层蛋白的错误定位，因为有两份关于I型运输途径转运不止一种毒力蛋白的报告。