Characterization of a Type II secretion system in a Gram-positive pathogen

革兰氏阳性病原体 II 型分泌系统的表征

• 批准号: 8877396
• 负责人: STEPHEN MELVILLE
• 金额: $ 18.58万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8877396
• 关键词: Adherence; Bacteria; Binding; Biological Assay; Cell Wall; Cell membrane; Cells; Chimeric Proteins; Clostridium; Clostridium perfringens; Complement; Complex; Computer software; Core Protein; Defect; Development; Disease; Drug Design; Elements; Enzymes; Fimbriae Proteins; Fluorescence; Fluorescence Microscopy; Genes; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Health; Human; Image; Immunoprecipitation; Infection; Kinetics; Lead; Measures; Membrane; Methods; Minor; Modeling; Molecular; Molecular Conformation; Mus; Mutate; Mutation; Pathway interactions; Peptidoglycan; Physiologic pulse; Pilum; Process; Protein Export Pathway; Proteins; Research; Role; System; Testing; Thick; Tissues; Toxin; Type II Secretion System Pathway; Virulence; Virulence Factors; base; cell envelope; macrophage; mutant; novel; pathogen; periplasm; programs; protein folding; secretion process; signal peptidase

DESCRIPTION (provided by applicant): A major factor in diseases caused by Gram-positive (Gr+) pathogens is secretion of toxins and other virulence factors. While the mechanism for transport across the cytoplasmic membrane is known in most Gr+ secretion systems, the process by which secreted proteins traverse the thick Gr+ peptidoglycan (PG) layer is not. Once thought to be produced by only Gram-negative (Gr-) bacteria, our lab discovered that Gr+ bacteria also produce Type IV pili (T4P). Type II secretion (T2S) systems share a high degree of similarity to proteins found in T4P. In Gr- bacteria, the main function of T2S systems is to transport folded proteins from the periplasm through the outer membrane. Clostridium perfringens and many of the Clostridium species examined thus far have two sets of T4P assembly apparatuses. Analysis of one set of T4P-associated genes leads us to hypothesize that they encode the Gr+ equivalent of a T2S system. One protein, CPE0517, which depends on a putative T2S pilin for secretion, binds to host cells, making it a potential virulence factor. The project has two aims. The first aim is to identify and characterize the major components of the T2S system in C. perfringens. In-frame deletions will be introduced into each gene that encodes components of the putative T2S and T4P systems, since they may share some components. The mutants will be tested for loss of secretion of CPE0517, T4P pilus assembly and adherence to host cells. The second aim is to determine the pathway of CPE0517 secretion. Our hypothesis is that CPE0517 undergoes the same overall process for secretion that a T2S substrate would in a Gr- bacteria, but in the context of a Gr+ cell envelope: (1) Sec-dependent translocation across the cytoplasmic membrane (2) folds into its final conformation in the space between the cytoplasmic membrane and PG layer (CM/PG space) and (3) undergoes export across the PG layer via the T2S system. Each of these processes will be tested by (1) Mutating a Sec-dependent signal peptidase that likely processes CPE0517, (2) Determining if CPE0517 forms a complex with other proteins in the CM/PG space and measuring folding of a CPE0517-mCherry protein fusion in the CM/PG space using fluorescence microscopy, (3) using a fluorescence-based kinetic assay to measure the rate of secretion from the CM/PG space. Discovery of a T2S in Gr+ bacteria would be significant, since T2S systems have not been identified in Gr+ bacteria before and would provide a mechanism for export of some proteins through the PG layer. The C. perfringens T2S is also an excellent model to study T2S systems in both Gr+ and Gr- pathogens, since it appears to be very simple, with significantly fewer proteins than those seen in Gr- bacteria.

描述（由申请人提供）：革兰氏阳性（GR+）病原体引起的疾病的主要因素是毒素和其他毒力因子的分泌。虽然在大多数GR+分泌系统中都知道跨细胞质膜的运输机制，但分泌的蛋白质穿越厚的GR+肽聚糖（PG）层的过程。一旦被认为仅由革兰氏阴性（GR-）细菌产生，我们的实验室发现GR+细菌还会产生IV型Pili（T4P）。 II型分泌（T2S）系统与T4P中发现的蛋白质具有很高的相似性。在gr-细菌中，T2S系统的主要功能是通过外膜从周期转运折叠的蛋白质。迄今为止，透顶芽孢杆菌和许多检查的梭状芽孢杆菌具有两组T4P装配装置。对一组T4P相关基因的分析使我们假设它们编码了T2S系统的GR+等效物。一种蛋白质CPE0517取决于推定的T2S PILIN进行分泌，与宿主细胞结合，使其成为潜在的毒力因子。 该项目有两个目标。第一个目的是识别和表征C. perfringens中T2S系统的主要组成部分。框内缺失将引入每个基因，该基因编码推定的T2S和T4P系统的组件，因为它们可能共享一些组件。将测试突变体的CPE0517分泌，T4P pilus组装和遵守宿主细胞的分泌损失。第二个目的是确定CPE0517分泌的途径。我们的假设是，CPE0517经历了T2S底物在GR细菌中的分泌总体过程，但在GR+细胞包膜的背景下：（1）跨细胞膜膜（2）折叠的Sec依赖性转移，以​​圆柱质量（2）的空间构造（3），PG层（PG）（PG）（PG）（PG）（PG）（PG）（PG）（PG）（MM）（MM）（MM）（PG）的最终构象（M.通过T2S系统在PG层上导出。这些过程中的每一个都将通过（1）突变依赖于SEC的信号肽酶，该信号肽酶可能处理CPE0517，（2）确定CPE0517是否与CM/PG空间中的其他蛋白质形成复合物，并测量使用CPE0517-MCHERRY蛋白质在使用CM/PGESAIN中使用CM/PGS的折叠（使用CM/PG）折叠的折叠（3）测量CM/PG空间的分泌速率。 GR+细菌中T2S的发现将是显着的，因为以前尚未在GR+细菌中鉴定出T2S系统，并且会提供通过PG层出口某些蛋白质的机制。 C. perprringens T2S也是研究GR+和GR-病原体中T2S系统的绝佳模型，因为它似乎非常简单，蛋白质明显少于GR-细菌中的蛋白质。

项目成果

Expansion of the Clostridium perfringens toxin-based typing scheme.

• DOI: 10.1016/j.anaerobe.2018.04.011
• 发表时间: 2018-10
• 期刊: Anaerobe
• 影响因子: 2.3
• 作者: Rood JI;Adams V;Lacey J;Lyras D;McClane BA;Melville SB;Moore RJ;Popoff MR;Sarker MR;Songer JG;Uzal FA;Van Immerseel F
• 通讯作者: Van Immerseel F

Mining transcriptome data: Utilization of environmentally regulated promoters for protein expression and purification in Clostridium perfringens.

挖掘转录组数据：利用环境调节启动子在产气荚膜梭菌中进行蛋白质表达和纯化。

• DOI: 10.1016/j.mimet.2022.106519
• 发表时间: 2022
• 期刊: Journal of microbiological methods
• 影响因子: 2.2
• 作者: Soncini,SamanthaR;Camper,GaryJ;Melville,StephenB
• 通讯作者: Melville,StephenB

Hypermotility in Clostridium perfringens strain SM101 is due to spontaneous mutations in genes linked to cell division.

产气荚膜梭菌 SM101 菌株的过度运动是由于与细胞分裂相关的基因的自发突变所致。

• DOI: 10.1128/jb.01614-14
• 发表时间: 2014
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Liu,Hualan;McCord,KristinD;Howarth,Jonathon;Popham,DavidL;Jensen,RoderickV;Melville,StephenB
• 通讯作者: Melville,StephenB

Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium perfringens.

产气荚膜梭菌对大梭菌毒素 TpeL 的穴蛋白依赖性分泌。

• DOI: 10.1128/jb.00580-20
• 发表时间: 2021
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Saadat,Angela;Melville,StephenB
• 通讯作者: Melville,StephenB

NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

NanR，一种转录调节因子，可与厌氧病原体产气荚膜梭状芽胞杆菌唾液酸代谢相关基因的启动子结合。

• DOI: 10.1371/journal.pone.0133217
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Therit,Blair;Cheung,JackieK;Rood,JulianI;Melville,StephenB
• 通讯作者: Melville,StephenB