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.

牙周炎与龈下微生物区系向以革兰氏阴性为主的微生物的戏剧性转变有关。在革兰氏阴性牙周病原菌中，直肠弯曲杆菌是唯一一种表达150kd表层(S层)蛋白的细菌。这些丰富的蛋白质形成均匀的阵列，覆盖在许多细菌的表面，是细菌致病所必需的。因此，S层被适当地定位到细胞表面是至关重要的。关于S层蛋白分泌机制的研究报道较少。有趣的是，与大多数其他S层蛋白不同的是，直肌S层蛋白在运输到细胞表面时不会去除任何氨基酸。此外，在直肠弯曲菌crsA基因上游的操纵子中还发现了与I型分泌系统三种成分之一的同源基因crsD。这表明crsA是通过I型途径运输的。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。对构建的一个极性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肠弯曲菌将其S紫菜运输到细胞表面的机制。我们的假设是，crsA蛋白将由一个L系统运输。重要的是，我们假设CRSC将在I型分泌途径中具有新的功能，可能是作为伴侣。这项提议的一个目标是利用定向突变和基因替换技术识别将其定向到细胞表面的直肠直肌S层序列。此外，crsA运输所需的基因将通过克隆和测序其余的crsCD操纵子或通过转座子突变来鉴定。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来表征。由此产生的CRSC突变体的特征将首次确定这种新蛋白在S蛋鸡新陈代谢中的功能。最后，运输突变体将被检查非S-层蛋白可能的错误定位，因为有两个报告的类型I运输途径运输一个以上的毒力蛋白。