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/总结 细菌的毒力通常依赖于病原体分泌促进不同过程的效应子的能力 例如生物膜形成和免疫逃避。革兰氏阴性菌已经进化到编码分泌物 系统转运蛋白质穿过细胞包膜，从而促进发病机制。典型的VIII型 分泌系统（T8 SS），其存在于大肠杆菌和沙门氏菌属中，采用了外膜 (OM)孔CsgG分泌卷曲亚单位跨越OM，在OM处广泛的卷曲淀粉样纤维网络被 形成，作为生物膜形成的支架。与T8 SS密切相关的系统是固定锚 新月柄杆菌，这是一个复合物组成的CsgG同源物，HfaB，和两个蛋白分泌 通过HfaB到达细菌表面。这种复合物将多糖固定在细胞上， 阵因此，T8 SS和相关系统通过不同的过程在生物膜形成中起作用。 我们最近在革兰氏阴性菌中发现了一个广泛保守的三基因操纵子， CsgG/HfaB同系物。值得注意的是，这种操纵子存在于许多不产生卷曲/固着的病原体中， 暗示CsgG同源物的不同作用。为了研究这个操纵子的功能，我们首先使用了 鲍曼不动杆菌作为模型。有趣的是，我们发现操纵子的突变会导致 表面结合的聚-N-乙酰葡糖胺（PNAG），不动杆菌的主要多糖成分， 生物膜因此，突变体在生物膜形成中减弱。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 红结合，指示生物膜相关多糖的减少（例如，Pel和Psl）。因此，委员会认为， 生物膜形成显著减弱。基于这些数据和HfaB的已知功能，我们 我假设这个三基因操纵子编码一种广泛分布的T8 SS的新变体 在革兰氏阴性病原体中，并在生物膜相关多糖附着于 细胞表面为了验证这一点，提出了以下目标：在目标1中，分子方法将用于 表征所提出的T8 SS变体在A中的定位和组分之间的相互作用。 鲍曼不动杆菌和铜绿假单胞菌，以及确定其结构。在目标2中，我们将评估T8 SS的作用 在A.鲍曼不动杆菌和铜绿假单胞菌。此外，我们将确定 两种细菌中假定的T8 SS变体的效应子。在目标3中，使用A.鲍曼不动作为一个模型，我们将 使用临床相关肺炎和导管- 相关UTI（UTI）鼠感染模型。总之，这些目标将评估小说的功能 分泌系统参与两种革兰氏阴性菌致病的关键方面的重要公共 健康问题。此外，由于该操纵子在革兰氏阴性菌中广泛保守，因此， 研究将影响我们对不同病原体的分泌和毒力的理解。