THE ROLE OF THE BLADDER EPITHELIUM IN RESPONSE TO ENTEROCOCCUS FAECALIS UTI

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 据估计，粪肠球菌每年在美国引起300，000 - 500，000例尿路感染（UTI）。作为一种医院获得性泌尿道病原体，E. faecalis仅次于E.大肠杆菌在医院尿路感染的数量。粘膜上皮是微生物病原体的重要物理屏障，也被认为在感知和响应外来入侵者的存在方面发挥积极作用。宿主对革兰氏阳性泌尿道病原体（包括大肠杆菌）的反应性质知之甚少。粪便为了在宿主中建立感染，病原体必须适应克服这种先天防御系统的策略。我们以前已经表明，致病性衍生的E。粪肠球菌具有抗吞噬囊，其在体外保护生物体免受嗜中性粒细胞介导的调理吞噬作用。约50%的E。粪球菌分离物具有编码荚膜生物合成机制的遗传基因座。此外，胶囊的存在增强了E.肠外部位的粪菌菌株。包膜在尿路粘膜上皮感染中的作用是本研究的重点之一。为了解决这个问题，我们将使用最近建立的小鼠上行性UTI模型，并将野生型包囊菌株与两种导致胶囊结构改变（cpSF）或完全消除胶囊生产（cpSC）的等基因胶囊突变体进行比较。将通过量化感染动物尿液、膀胱和肾脏中的细菌负荷来评估结局指标。还将处理膀胱和肾脏组织，以检查这些感染引起的组织病理学。此外，将使用Luminex系统分析感染动物尿液中的细胞因子和趋化因子谱，并对从膀胱和肾脏尿路上皮细胞分离的RNA进行RT-PCR，作为确定尿路内细胞因子/趋化因子来源的补充方法。除荚膜外，其他可能改变先天防御系统清除率的微生物因素包括两种细胞外蛋白酶（明胶酶和丝氨酸蛋白酶）。还将在UTI模型中比较同基因蛋白酶突变体，以确定每种蛋白酶对感染严重程度的相对贡献。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is estimated that Enteroccus faecalis causes 300,000-500,000 cases of urinary tract infection (UTI) each year in the United States. As a nosocomial uropathogen, E. faecalis ranks second only to E. coli in the number of nosocomial UTI's. The mucosal epithelium is an important physical barrier to microbial pathogens and is also thought to play an active role in sensing and responding to the presence of foreign invaders. Very little is known about the nature of the host response to Gram-positive uropathogens, including E. faecalis. In order to establish infection in the host, pathogens must adapt strategies to overcome this innate defense system. We have previously shown that pathogenic-derived lineages of E. faecalis possess an antiphagocytic capsule that protects the organism in vitro against neutrophil-mediated opsonophagocytosis. Roughly 50% of E. faecalis isolates possess the genetic locus that encodes the capsule biosynthetic machinary. In addition, the presence of the capsule enhances the survival of E. faecalis strains at extraintestinal sites. What role the capsule plays in establishing infection at the mucosal epithelium in the urinary tract is one focus of the present study. To address this question, we will use a recently established murine ascending UTI model and compare a wild-type encapsulated strain with two isogenic capsule mutants that either result in structural alterations to the capsule (cpsF) or eliminate capsule production altogether (cpsC). The outcome measures will be assessed by quantifying the bacterial burden in the urine, bladder, and kidneys of infected animals. Bladder and kidney tissues will also be processed to examine the histopathology resulting from these infections. In addition, cytokine and chemokine profiles will be analyzed from the urine of infected animals using a Luminex system, and RT-PCR will be perfomed on RNA isolated from bladder and kidney urothelial cells as a complimentary approach to determine the cytokine/chemokine source within the urinary tract. In addition to capsule, other microbial factors that could potentially alter clearance by the innate defense system include two extracellular proteases (gelatinase and serine protease). Isogenic protease mutants will also be compared in the UTI model to determine the relative contribution of each protease to the severity of the infection.