Bacterial Survival in the Mammalian Urothelium

哺乳动物尿路上皮中的细菌存活

• 批准号: 7147860
• 负责人: Joseph Palermo
• 金额: $ 12.56万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147860
• 关键词: Escherichia coli; adhesin; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial genetics; cell differentiation; disease /disorder model; electron microscopy; fluorescence microscopy; gene expression; host organism interaction; immunocytochemistry; interstitial cystitis; laboratory mouse; microorganism culture; phenotype; polymerase chain reaction; quorum sensing; urinary bladder epithelium; urinary tract infection

DESCRIPTION (provided by applicant): Urinary tract infections (UTIs) are the second most common infectious disease in the United States and result in medical expenditures exceeding one billion dollars annually. Despite the prevalence of the UTIs current medical regimens are unable to eradicate infections or prevent their occurrence. E. coli cause 85% of UTIs and are able to thrive within host cells, but the mechanisms allowing for internalization and survival are unclear. Recently, we have shown that during acute infections uropathogenic E. coli (UPEC) form intracellular bacterial communities (IBCs), which provide a survival advantage and are a source for ongoing disease. Moreover, the IBCs are present in the cytoplasm of the superficial umbrella cells, not membrane bound vacuoles as seen in cancer cell monolayers. We wish to understand how UPEC enter superficial umbrella cells, localize to the cytoplasm to form IBCs and adapt to survive long term within the bladder. This proposal will use a novel in vitro model of IBCs in conjunction with our well defined murine cystitis model to study bacterial entry in differentiated umbrella cells and identify processes, such as uroplakin recycling, cytoskeletal rearrangement, membrane trafficking, protein synthesis and cell differentiation required for IBC formation. Routine microbiology and invasion assays will be used along with immunohistochemistry and fluorescent confocal microscopic techniques to track bacteria internalization, and their localization will be confirmed by electron microscopy. The FimH adhesin, vital for binding and internalization, will specifically be assessed for its ability to target bacterial delivery to the umbrella cell cytoplasm. We will use co-infection of UPEC and K-12 bacteria to determine how UPEC alter the urothelium to improve bacterial survival. Finally, we will use targeted inactivation of a quorum sensing signaling pathway to determine its role in IBC maturation and dispersal. The specific knowledge gained from these studies will enhance our understanding of bacterial pathogenesis, intracellular trafficking in umbrella cells and communication within bacterial communities. Using the in vivo mouse model, I have already made contributions to the understanding of UTI pathogenesis, and I have developed an in vitro model of IBC formation that will greatly enhance our ability to dissect the early stages of UPEC infection. My ongoing relationship with Dr. Scott Hultgren, a recognized leader in this field, will provide the guidance and support needed for continued success. I am currently an instructor in Pediatric Gastroenterology with greater than75% effort committed to research. Over the next 5 years, I will develop a research course focusing on the host-pathogen interactions of intestinal flora in the urinary and gastrointestinal tracts that will be the basis of an NIH RO1 application and future independent investigations. The services and infrastructure at Washington University are of the highest caliber and together with the leadership of senior faculty, will provide an ideal environment for a young investigator to establish a solid foundation in academic science.

描述（由申请人提供）： 尿路感染 (UTI) 是美国第二大常见传染病，每年造成的医疗支出超过 10 亿美元。尽管尿路感染很普遍，但目前的医疗方案无法根除感染或预防其发生。 85% 的尿路感染由大肠杆菌引起，并且能够在宿主细胞内大量繁殖，但其内化和存活的机制尚不清楚。最近，我们发现，在急性感染期间，尿路致病性大肠杆菌（UPEC）形成细胞内细菌群落（IBC），这提供了生存优势，并且是持续疾病的来源。此外，IBC 存在于表面伞状细胞的细胞质中，而不是像癌细胞单层中所见的膜结合液泡。我们希望了解 UPEC 如何进入表面伞细胞、定位于细胞质形成 IBC 并适应在膀胱内长期生存。该提案将使用一种新型的 IBC 体外模型与我们定义明确的小鼠膀胱炎模型相结合，研究细菌进入分化的伞状细胞，并确定尿斑蛋白回收、细胞骨架重排、膜运输、蛋白质合成和细胞分化所需的过程。 IBC 形成。常规微生物学和侵袭检测将与免疫组织化学和荧光共聚焦显微镜技术一起使用来追踪细菌的内化，并通过电子显微镜确认它们的定位。 FimH 粘附素对于结合和内化至关重要，将专门评估其将细菌递送至伞状细胞细胞质的能力。我们将利用 UPEC 和 K-12 细菌的共同感染来确定 UPEC 如何改变尿路上皮以提高细菌的存活率。最后，我们将利用群体感应信号通路的靶向失活来确定其在 IBC 成熟和分散中的作用。从这些研究中获得的具体知识将增强我们对细菌发病机制、伞状细胞的细胞内运输以及细菌群落内通讯的理解。使用体内小鼠模型，我已经为理解 UTI 发病机制做出了贡献，并且我开发了 IBC 形成的体外模型，这将大大增强我们剖析 UPEC 感染早期阶段的能力。我与该领域公认的领导者 Scott Hultgren 博士的持续关系将为持续成功提供所需的指导和支持。我目前是一名儿科胃肠病学讲师，75% 以上的精力致力于研究。在接下来的 5 年里，我将开发一门研究课程，重点关注泌尿道和胃肠道肠道菌群的宿主与病原体相互作用，这将成为 NIH RO1 申请和未来独立研究的基础。华盛顿大学的服务和基础设施都是最高水平的，再加上高级教师的领导，将为年轻的研究人员提供一个理想的环境，以在学术科学方面打下坚实的基础。