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.

牙周炎与龈下菌群向主要革兰氏阴性菌群的显著转变有关。直弯曲杆菌是唯一的革兰氏阴性牙周病原体中，其表达的150 kd的表面层（S-层）蛋白。这些丰富的蛋白质形成覆盖许多细菌表面的均质阵列，并且是细菌致病所需的。 因此，S层适当地定位于细胞表面是至关重要的。关于S层蛋白的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，C层蛋白中没有氨基酸被去除。腹直肌S层蛋白（crsA），因为它被运送到细胞表面。此外，在C.直肌crsA基因这表明crsA通过I型途径转运。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。我们构建的极性crsC突变体的表型分析表明，crsC参与crsA分泌，合成或降解。我们现在建议利用我们已经开发的分子遗传工具，以确定C。腹直肌将其S-紫菜运输到细胞表面。 我们假设crsA蛋白将由I型系统转运。 重要的是，我们假设crsC在I型分泌途径中可能作为分子伴侣具有新的功能。该提案的一个目标是确定C。腹直肌S层序列，使用靶向突变和基因替换技术将其引导至细胞表面。此外，crsA转运所需的基因将通过对crsCD操纵子的其余部分进行克隆和测序或通过转座子诱变来鉴定。重要的是，每个推定的转运基因的功能将通过产生非极性突变体来表征。 由此产生的crsC突变体的特性将定义，第一次，这种新的蛋白质在S-层代谢的功能。最后，运输突变体将检查非S层蛋白的可能的错误定位，因为有两个报告的I型运输途径运输一个以上的毒力蛋白。