Rho-Modifying Cytotoxic Necrotizing Factor of E. coli

细胞毒性坏死性因子1型（CNF1）是针对哺乳动物细胞中小gtp结合蛋白Rho家族的细菌毒素家族的一员。CNF1在RhoA、Cdc42和Rac1中脱酰胺一个谷氨酰胺残基，但在Ras中不脱酰胺。这种脱酰胺导致这些gtpase的组成性激活，根据目标细胞和毒素剂量的不同，可以触发肌动蛋白应激纤维的形成、多核或细胞死亡。CNF1通常由引起尿路感染（uti）的大肠杆菌菌株产生，如膀胱炎、前列腺炎和肾盂肾炎。为了支持这一流行病学联系，我们最近发现，与CNF1阴性等基因突变体相比，CNF1不仅在5637个人尿路上皮细胞中诱导凋亡，而且在尿路感染小鼠模型中，与CNF1阴性等基因突变体相比，CNF1还为尿路致病性大肠杆菌（UPEC）提供生长优势。此外，我们发现CNF1增强了感染小鼠膀胱的炎症程度和由此导致的组织损伤，以及经尿道受到产生CNF1的UPEC攻击的大鼠的前列腺。最后，我们发现在人类多形核白细胞（PMNs）存在下，产生CNF1的upc比CNF1阴性的等基因突变体存活得更好。综上所述，这些发现使我们提出了以下假设。CNF1通过以下途径增强UPEC的致病性：1)促进尿上皮细胞脱落；ii.)引起pmn的大量涌入，同时提供产生毒素的大肠杆菌保护，防止pmn介导的杀伤；（Iii）促使感染菌株深入侵入膀胱或前列腺。验证这一理论的具体目的是：1.)通过分析CNF1或表达CNF1的UPEC与来自这些动物的PMN的相互作用，并通过定义CNF1介导的引起PMN内流的细胞因子反应，进一步确定CNF1在UPEC介导的小鼠膀胱炎和大鼠前列腺炎发病机制中所起的作用；2)研究人类膀胱类器官对CNF1或产生CNF1的UPEC菌株的细胞和细胞因子反应；3)通过序列生化和分子方法鉴定CNF1的功能受体；4.)通过鉴定中和单克隆抗体识别的CNF1表位，以及分析CNF1与相关毒素CNF2、多杀性巴氏杆菌毒素和皮肤坏死杆菌毒素组成的嵌合分子，继续评估CNF1的结构和功能。

Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that target the Rho family of small GTP-binding proteins in mammalian cells. CNF1 deamidates a single glutamine residue in RhoA, Cdc42, and Rac1 but not in Ras. This deamidation results in the constitutive activation of these GTPases which can trigger actin stress fiber formation, multinucleation, or cell death, depending on the target cell and dose of toxin. CNF1 is frequently produced by Escherichia coli strains that cause urinary tract infections (UTIs), such as cystitis, prostatitis, and pyelonephritis. In support of this epidemiological connection, we recently showed that CNF1 not only induces apoptosis in 5637 human uroepithelial cells but also provides a growth advantage to uropathogenic E. coli (UPEC) in a mouse model of ascending UTI when compared to CNF1-negative isogenic mutants. Additionally, we found that CNF1 enhances the degree of inflammation and resulting tissue damage in bladders of infected mice and in prostates of rats challenged intraurethrally with CNF1-producing UPEC. Finally, we discovered that CNF1- producing UPEC survive better than CNF1-negative isogenic mutants in the presence of human polymorphonuclear leukocytes (PMNs). Taken together, these findings have led us to propose the following hypothesis. CNF1 enhances the pathogenicity of UPEC by: i.) promoting uroepithelial cell shedding; ii.) evoking a large influx of PMNs while providing toxin-producing E. coli protection against PMN-mediated killing, and; iii.) facilitating deeper invasion of the bladder or prostate by the infecting strain. The specific aims designed to test this theory are to: 1.) further define the role that CNF1 plays in the pathogenesis of UPEC-mediated cystitis in the mouse and prostatitis in the rat by analyzing the interaction of CNF1 or CNF1-expressing UPEC with PMNs from these animals and by defining the CNF1-mediated cytokine response that evokes PMN influx; 2) investigate the cellular and cytokine responses of a human bladder organoid to CNF1 or a CNF1-producing UPEC strain; 3.) identify the functional receptor for CNF1 by sequential biochemical and molecular approaches, and; 4.) continue to evaluate CNF1 structure and function by characterizing the CNF1 epitopes recognized by neutralizing monoclonal antibodies and by analyzing chimeric molecules comprised of portions of CNF1 and the related toxins CNF2, Pasteurella multocida toxin, and the Bordetella dermonecrotic toxin.