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: 6516542
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 16.31万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516542
• 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.

牙周炎与层属菌群的急剧转移有关，主要是革兰氏阴性的生物。弯曲杆菌直肠在革兰氏阴性牙周病原体中是独特的，其表达150 kD表面层（S层）蛋白。这些丰富的蛋白质形成覆盖许多细菌表面的同质阵列，是细菌发病机理所必需的。 因此，至关重要的是，S层适当地定位于细胞表面。关于S层蛋白的分泌机制只有少数报道。有趣的是，与大多数其他S层蛋白不同，由于将其转运到细胞表面时，没有从直肠C. c. tectus s层蛋白（CRSA）中除去氨基酸。此外，在直肌CRSA基因上游的操纵子中，发现了I型分泌系统三个组成部分之一的同源物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，其蛋白质没有已知功能。我们构建的极性CRSC突变体的表型的分析表明，CRSC参与了CRSA分泌，合成或降解。现在，我们建议利用我们开发的分子遗传工具，以确定直肠梭状芽胞杆菌用来将其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型分泌途径中可能具有新的功能，也许是作为伴侣的。该提案的目标之一是确定使用靶向诱变和基因置换技术将其引导到细胞表面的直肠C. c. c. c. s层序列。另外，将通过克隆和测序CRSCD操纵子或转座子诱变来鉴定CRSA转运所需的基因。重要的是，每个推定的转运基因的功能将通过制造非极性突变体来表征。 CRSC突变体的结果表征将首次定义这种新型蛋白质在S层代谢中的功能。最后，将检查转运突变体是否可能会误差非S层蛋白质，因为有两种I型运输途径的报告有两种运输多种毒力蛋白的报告。