The Recombinase FimX: Role in type 1 pili regulation and E. coli-induced cystitis

描述(由申请人提供)： 1型菌毛是大肠埃希氏菌相关性膀胱炎的主要毒力决定因素，它促进与膀胱表面的结合，促进上皮细胞的定植和侵袭。在细胞内，大肠杆菌聚集成具有生物膜性质的公共结构，称为细胞内细菌群落(IBC)，然后从受感染的细胞分散和流动到膀胱腔。在分泌时，1型菌毛可能再次介导与幼稚上皮细胞的黏附，重新启动侵袭和IBC的形成。由于1型菌毛在泌尿发病机制中的核心作用，了解这些纤维的调节对于完整的感染分子描述和最终确定新的治疗靶点是至关重要的。1型菌毛受两种重组酶FIMB和FIME的调控，这两种重组酶共同通过相位变化动态调节纤维的表达，从而使启动子区域反转到开和关位置。初步研究已确定FimX是第三种位点特异性重组酶，尽管没有FIMB和FIME，但它会产生时相变化和1型菌毛的表达，并支持完全毒力。FimX和其他两个重组酶的消融使生物体无毒。我们推测，FimX作为1型菌毛表达的调节因子，是大肠杆菌尿路感染的核心毒力决定因素。在这项建议的目标1中，我们将结合分子流行病学、遗传学和动物模型来描述FimX的流行特征，并研究FimX作为1型菌毛调节因子在大肠杆菌膀胱炎中的体内作用。在目标2中，我们将通过遗传学和生物化学的方法，研究FimX及其靶DNA序列的结构与功能的关系。鉴于Fim重组酶在调节1型菌毛表达中的核心作用，了解它们的功能和限制因素可能会为尿路感染的治疗和预防提供新的切入点。

DESCRIPTION (provided by applicant): Type 1 pili, the preeminent virulence determinant in Escherichia coli-associated cystitis, promote binding to the bladder surface and facilitate the colonization and invasion of the epithelium. In their intracellular residence, E. coli amass into communal formations with biofilm-like properties termed intracellular bacterial communities (IBC) and then disperse and flux from the infected cell into the lumen of the bladder. Upon fluxing, type 1 pili may again mediate adherence to naive epithelial cells, reinitiating invasion and IBC formation. Because of the central role of type 1 pili in uropathogenesis, understanding the regulation of these fibers is essential for a complete molecular description of the infection and ultimately to identify novel targets for therapeutics. Type 1 pili are regulated by two recombinases, FimB and FimE, that together dynamically modulate the expression of the fibers through phase variation whereby the promoter region is inverted into ON and OFF positions. Preliminary studies have identified FimX, a third site-specific recombinase that, despite the absence of FimB and FimE, produces phase variation and the expression of type 1 pili and supports full virulence. Ablation of FimX and the other two recombinases renders the organism avirulent. We hypothesize that FimX, as regulator of type 1 pili expression, is a central virulence determinant in E. coli urinary tract infections. In Aim 1 of this proposal, we will use a combination of molecular epidemiology, genetics, and animal modeling to characterize the prevalence of fimX and study the in vivo role of FimX as a regulator of type 1 pili in E. coli cystitis. In Aim 2, through the use of genetics and biochemistry, we will study the structure-function relationships of FimX and its target DNA sequences. Given the central role of the Fim recombinases in regulating type 1 pili expression, understanding their functions and constraints may yield novel points for treatment and prevention in urinary tract infections.