Crystallization of outer membrane proteins for export of polysaccharide capsule

多糖胶囊出口用外膜蛋白结晶

• 批准号: 8339443
• 负责人: MARK A SAPER
• 金额: $ 18.61万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8339443
• 关键词: Address; Adhesions; Affect; Antibiotics; Bacteria; Biophysics; Cell surface; Cells; Complex; Crystallization; Crystallography; Detergents; Environment; Escherichia coli; Escherichia coli EHEC; Essential Genes; Foundations; Genes; Goals; Gram-Negative Bacteria; Homologous Gene; Human; Immune system; Infection; Label; Lipopolysaccharides; Lipoproteins; Location; Measures; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular; Molecular Weight; O Antigens; Oligosaccharides; Operon; Orthologous Gene; Pathogenicity; Pharmaceutical Preparations; Polysaccharides; Production; Protein Export Pathway; Proteins; Role; Route; Sequence Analysis; Solubility; Solvents; Structure; Surface; System; United States National Institutes of Health; Universities; Virulence; antimicrobial; capsule; enteropathogenic Escherichia coli; glycosyltransferase; in vivo; interest; mutant; novel strategies; paralogous gene; pathogen; periplasm; porin; research study; scale up; solid state nuclear magnetic resonance

DESCRIPTION (provided by applicant): Bacteria often export high molecular weight polysaccharides that can form a capsule associated with the bacterial surface, or be secreted into the surrounding environment as exopolysaccharide. Capsules can help bacteria evade host immune systems, promote adhesion to host cells, and form biofilms. Like lipopolysaccharide (LPS), group 1 and 4 capsules are assembled from short oligosaccharide repeat units by a Wzy-like glycosyltransferase, but are typically much larger (>500 kDa). Gram-negative bacteria require additional molecular machinery to transport the capsule polysaccharide through the outer membrane where it associates with the LPS by an unknown mechanism. The current model is that Wza, a conserved outer membrane auxiliary protein necessary for capsule production, forms an octamer with a helical pore in the outer membrane from which the polysaccharide chain exits. But are there other proteins that could function in this role? Enteropathogenic and enterohaemorrhagic Escherichia coli require the seven genes of the gfc operon to produce a group 4 capsule comprised of O-antigen repeating units. Unlike the group 1 system, there are four essential genes (gfcABCD) upstream of gfcE (Wza homolog) that encode periplasmic or outer membrane proteins of unknown function. Interestingly, E. coli and many other Gram-negative species encode a similar yjbEFGH operon implicated in biofilm formation and group 1 capsule expression. GfcD and YjbH are predicted to be large outer membrane 2-barrel proteins that also may be acylated. The overall hypothesis is that this protein is part of a more complex secretion system that provides an exit route for the polysaccharide itself or for an accessory molecule through the outer membrane. To clarify the structure and role of GfcD in polysaccharide export we propose two aims for this R21. In Specific Aim 1, experiments will confirm the membrane location of GfcD, characterize the degree to which a gfcD- mutant affects capsule expression in vivo, and identify what other proteins interact with GfcD. In Specific Aim 2, we will prepare expression constructs of GfcD and three homologs and evaluate which will be best suited to for large scale purification. Proteins will be screened with a variety of detergents for optimal solubility and monodispersity. Pure protein will be used to screen for crystallization conditions. Labeled protein will also be prepared for solid-state NMR experiments to examine protein orientation, secondary structure and for the presence of a solvent accessible channel. Results from this short-term project will provide the foundation for a longer term project to determine the GfcD structure, elucidate the function of the other Gfc proteins in polysaccharide export, and evaluate if the export system could be a target for broad spectrum drugs aimed to suppress capsule expression.

描述（由申请人提供）：细菌通常输出高分子量多糖，其可以形成与细菌表面相关的胶囊，或作为胞外多糖分泌到周围环境中。胶囊可以帮助细菌逃避宿主免疫系统，促进与宿主细胞的粘附，并形成生物膜。与脂多糖（LPS）一样，第1组和第4组胶囊通过Wzy样糖基转移酶由短寡糖重复单元组装而成，但通常要大得多（>500 kDa）。革兰氏阴性细菌需要额外的分子机制来将荚膜多糖转运通过外膜，在外膜处它通过未知的机制与LPS结合。目前的模型是，Wza，一个保守的外膜辅助蛋白所必需的胶囊生产，形成一个八聚体与螺旋孔的外膜中的多糖链退出。但是否有其他蛋白质可以发挥这种作用？肠致病性和肠出血性大肠杆菌需要gfc操纵子的7个基因来产生由O-抗原重复单元组成的组4胶囊。与组1系统不同，在gfcE（Wza同源物）上游有四个必需基因（gfcABCD），其编码功能未知的周质或外膜蛋白。有趣的是，E。大肠杆菌和许多其它革兰氏阴性物种编码与生物膜形成和组1荚膜表达有关的类似yjbEFGH操纵子。GfcD和YjbH被预测为也可以被酰化的大的外膜2桶蛋白。总的假设是，这种蛋白质是一个更复杂的分泌系统的一部分，该系统为多糖本身或辅助分子提供了通过外膜的出口途径。为了阐明GfcD在多糖输出中的结构和作用，我们提出了R21的两个目标。在具体目标1中，实验将确认GfcD的膜位置，表征gfcD突变体影响体内胶囊表达的程度，并鉴定哪些其他蛋白质与GfcD相互作用。在特定目标2中，我们将制备GfcD和三种同源物的表达构建体，并评估哪种最适合大规模纯化。将用各种去污剂筛选蛋白质，以获得最佳溶解度和单分散性。纯蛋白将用于筛选结晶条件。标记的蛋白质也将准备用于固态NMR实验，以检查蛋白质取向、二级结构和溶剂可进入通道的存在。这个短期项目的结果将为确定GfcD结构的长期项目提供基础，阐明其他Gfc蛋白在多糖输出中的功能，并评估输出系统是否可以成为旨在抑制胶囊表达的广谱药物的靶点。

项目成果

Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli.

• DOI: 10.1371/journal.pone.0037984
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Nadler C;Koby S;Peleg A;Johnson AC;Suddala KC;Sathiyamoorthy K;Smith BE;Saper MA;Rosenshine I
• 通讯作者: Rosenshine I

Escherichia coli O127 group 4 capsule proteins assemble at the outer membrane.

• DOI: 10.1371/journal.pone.0259900
• 发表时间: 2021
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Larson MR;Biddle K;Gorman A;Boutom S;Rosenshine I;Saper MA
• 通讯作者: Saper MA