A Novel Type VIII Secretion System in Gram-negative Bacteria

项目摘要/摘要 细菌的毒力通常依赖于病原体分泌促进各种过程的效应物的能力。 例如生物膜的形成和免疫逃避。革兰氏阴性细菌已经进化到编码分泌物 通过细胞被膜运输蛋白质的系统，从而促进致病。典范的第VIII类 在大肠杆菌和沙门氏菌中发现的分泌系统(T8SS)使用外膜 (OM)毛孔CsgG在OM中分泌卷曲亚基，其中广泛的卷曲淀粉样纤维网络 形成的，作为生物膜形成的支架。与T8SS密切相关的一个系统是Holdfast锚 是由CsgG同源物HfaB和分泌的两种蛋白组成的复合体 通过HfaB到达细菌表面。该复合体将固定的多糖锚定在细胞上，促进生物被膜 队形。因此，T8SS及其相关系统通过不同的过程在生物膜的形成过程中发挥作用。 我们最近在革兰氏阴性细菌中发现了一个广泛保守的三基因操纵子，它编码一种 CsgG/HfaB同源基因。值得注意的是，在许多不产生卷曲/Holdfast的病原体中发现了这个操纵子， 这意味着CsgG同源物的作用不同。为了研究这个操纵子的功能，我们首先用 以鲍曼不动杆菌为模型。有趣的是，我们发现操纵子的突变导致了 不动杆菌的主要多糖成分表面相关聚N-乙酰氨基葡萄糖(PNAG) 生物膜。因此，突变体在生物膜的形成中被减弱。接下来，我们将我们对操纵子的研究扩展到 另一种革兰氏阴性杆菌，铜绿假单胞菌。同样，突变导致刚果人口减少 红色结合，表明与生物膜相关的多糖(如PEL和PSL)减少。因此， 生物被膜的形成明显减弱。根据这些数据和已知的HfaB的功能，我们 假设这个三基因操纵子编码一种广泛分布的T8SS的新变体 在革兰氏阴性病原菌中，并在生物膜相关多糖附着到 细胞表面。为了测试这一点，提出了以下目标；在目标1中，将使用分子方法来 表征所建议的T8SS变体在A中的本地化和相互作用。 鲍曼氏杆菌和铜绿假单胞菌，并确定其结构。在目标2中，我们将评估T8SS的作用 在鲍曼不动杆菌和铜绿假单胞菌中将多糖锚定在膜上。此外，我们将确定 两种细菌中假定的T8SS变种的效应物。在目标3中，使用鲍曼氏杆菌作为模型，我们将 使用临床相关肺炎和导管确定假定的T8SS在毒力中的作用- 相关尿路感染(CAUTI)小鼠感染模型。总之，这些目的将评估一部小说(S)的功能 两种革兰氏阴性菌重要致病因素与分泌系统的关系 健康问题。此外，由于这种操纵子在革兰氏阴性细菌中广泛保守，因此 研究将影响我们对不同病原体的分泌和毒力的理解。

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.