A Novel Type VIII Secretion System in Gram-negative Bacteria

革兰氏阴性细菌中的新型 VIII 型分泌系统

• 批准号: 10642097
• 负责人: Mario Feldman
• 金额: $ 23.4万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642097
• 关键词: Acinetobacter; Acinetobacter baumannii; Amyloid fibers; Attenuated; Bacteria; Binding; Bioinformatics; Biology; Carrier Proteins; Caulobacter crescentus; Cell Fractionation; Cell surface; Cells; Co-Immunoprecipitations; Complex; Congo Red; Cryoelectron Microscopy; Data; Escherichia coli; Genes; Gram-Negative Bacteria; Homologous Gene; Hypothetical Protein; Immune Evasion; Infection; Mass Spectrum Analysis; Mediating; Membrane; Microbial Biofilms; Modeling; Molecular; Mus; Mutation; Nutrient; Operon; Pathogenesis; Phenotype; Plasmids; Pneumonia; Polysaccharides; Process; Production; Protein Secretion; Proteins; Proteome; Pseudomonas aeruginosa; Public Health; Repression; Research; Role; Salmonella; Structure; Surface; System; Testing; Variant; Virulence; attenuation; catheter associated UTI; cell envelope; clinically relevant; mutant; novel; pathogen; scaffold

PROJECT ABSTRACT/SUMMARY Bacterial virulence often relies on the ability of pathogens to secrete effectors that facilitate diverse processes such as biofilm formation and immune evasion. Gram-negative bacteria have evolved to encode secretion systems to transport proteins across the cell envelope, thereby facilitating pathogenesis. The canonical type VIII secretion system (T8SS), which is found in Escherichia coli and Salmonella spp., employs the outer membrane (OM) pore CsgG to secrete curli subunits across the OM where extensive networks of curli amyloid fibers are formed, serving as a scaffold for biofilm formation. A system closely related to the T8SS is the holdfast anchor of Caulobacter crescentus, which is a complex consisting of the CsgG homolog, HfaB, and two proteins secreted via HfaB to the bacterial surface. The complex anchors holdfast polysaccharide to the cell, facilitating biofilm formation. Therefore, the T8SS and a related system function in biofilm formation through distinct processes. We recently identified a broadly conserved three-gene operon in Gram-negative bacteria that encodes a CsgG/HfaB homolog. Notably, this operon is found in numerous pathogens that do not produce curli/holdfast, implying a divergent role for CsgG homologs. To investigate the function of this operon, we first employed Acinetobacter baumannii as a model. Intriguingly, we found that mutation of the operon results in the absence of surface-associated poly-N-acetylglucosamine (PNAG), the major polysaccharide component of Acinetobacter biofilms. Accordingly, mutants are attenuated in biofilm formation. We next extended our study of the operon to another Gram-negative pathogen, Pseudomonas aeruginosa. Similarly, mutation resulted in decreased Congo red binding, indicative of a reduction in biofilm-associated polysaccharides (e.g., Pel and Psl). Accordingly, biofilm formation was significantly attenuated. Based on this data and the known function of HfaB, we hypothesize that this three-gene operon encodes a novel variant of the T8SS that is widely distributed in Gram-negative pathogens and plays a role in attachment of biofilm-associated polysaccharides to the cell surface. To test this, the following aims are proposed; In Aim 1, molecular approaches will be used to characterize the localization of and interactions between components of the proposed T8SS variant in A. baumannii and P. aeruginosa, as well as determine its structure. In Aim 2, we will assess the role of the T8SS in anchoring polysaccharides to the membrane in A. baumannii and P. aeruginosa. Additionally, we will identify effectors of the putative T8SS variant in both bacteria. In Aim 3, using A. baumannii as a model, we will determine the role of the putative T8SS in virulence using the clinically-relevant pneumonia and catheter- associated UTI (CAUTI) murine infection models. In all, these Aims will assess the function(s) of a novel secretion system involved in key aspects of pathogenesis in two Gram-negative bacteria of significant public health concern. Moreover, as this operon is broadly conserved in Gram-negative bacteria, results from this research will impact our understanding of secretion and virulence of diverse pathogens.

项目摘要/摘要 细菌毒力通常依赖于病原体分泌促进多种过程的效应子的能力 例如生物膜形成和免疫逃避。革兰氏阴性细菌已经演变为编码分泌 在细胞包膜上运输蛋白质的系统，从而促进发病机理。规范型VIII 分泌系统（T8SS）在大肠杆菌和沙门氏菌属中发现，采用外膜 （OM）毛孔CSGG分泌在OM上分泌curli亚基，其中广泛的Curli淀粉样蛋白纤维网络是 形成，用作生物膜形成的支架。与T8SS密切相关的系统是Holdfast锚 Caulobacter Crescentus的复合物，由CSGG同源物，HFAB和两个分泌的蛋白质组成 通过HFAB到达细菌表面。复合物锚定的固定多糖到细胞，促进生物膜 形成。因此，通过不同的过程，T8S和相关系统在生物膜形成中的功能。 我们最近确定了革兰氏阴性细菌中广泛保守的三基因操纵子，该操纵子编码A CSGG/HFAB同源物。值得注意的是，该操纵子是在许多不产生卷发/holdfast的病原体中发现的 暗示CSGG同源物的作用不同。为了调查该操纵子的功能，我们首先使用 鲍曼尼杆菌作为模型。有趣的是，我们发现操纵子的突变导致不存在 与表面相关的聚-N-乙酰葡萄糖（PNAG）的主要多糖分量的主要多糖成分（PNAG） 生物膜。因此，突变体在生物膜形成中被减弱。接下来，我们将操纵子的研究扩展到 另一种革兰氏阴性病原体铜绿假单胞菌。同样，突变导致刚果减少 红色结合，表明生物膜相关多糖的降低（例如PEL和PSL）。因此， 生物膜形成显着减弱。基于此数据和HFAB的已知功能，我们 假设这个三基因操纵子编码了T8SS的新型变体，该变体被广泛分布 在革兰氏阴性病原体中，并在生物膜相关多糖的附着中起作用 细胞表面。为了测试这一点，提出了以下目标；在AIM 1中，分子方法将用于 表征A中提出的T8SS变体组件之间的定位和相互作用。 Baumannii和铜绿假单胞菌，并确定其结构。在AIM 2中，我们将评估T8SS的作用 在将多糖锚定在鲍曼尼和铜绿假单胞菌的膜上。此外，我们将确定 两种细菌中假定T8SS变体的效应子。在AIM 3中，使用A. Baumannii作为模型，我们将 使用临床上的肺炎和导管 - 确定推定的T8S在毒力中的作用 相关的UTI（CAUTI）鼠感染模型。总之，这些目标将评估小说的功能 在两个革兰氏阴性细菌中，涉及发病机理的关键方面的分泌系统 健康问题。此外，由于该操纵子在革兰氏阴性细菌中广泛保守，因此 研究将影响我们对各种病原体的分泌和毒力的理解。