E COLI TRANSLOCATION ACROSS INTESTINAL EPITHELIUM

大肠杆菌跨肠上皮的易位

• 批准号: 6168738
• 负责人: MICHELLE M PIETZAK
• 金额: $ 12.37万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6168738
• 关键词: Escherichia coli; Escherichia coli infections; bacteria infection mechanism; bacterial genetics; biological transport; cell line; gastrointestinal epithelium; genetic strain; host organism interaction; laboratory rat; transposon /insertion element; virulence

This application for a Mentored Clinician Scientist Development Award (K08) seeks support for Michelle Pietzak, M.D., who has recently completed her fellowship in Pediatric Gastroenterology and Nutrition and joined the faculty as an Instructor of Pediatrics at Childrens Hospital Los Angeles (Assistant Professor pending). Under the mentorship of Kwang Sik Kim M.D., Dr. Pietzak will continue to pursue her basic investigations into the mechanisms by which Escherichia coli (E. coli) is able to translocate across intestinal epithelium and cause sepsis. E. coli is a leading cause of severe bacterial infections in premature infants, neonates, immunocompromised hosts, and children with central lines and primary intestinal diseases. Dr. Kim is a prominent researcher in the field of the pathogenesis of E. coli meningitis, having (a) established the virulence roles for the K1 capsular polysaccharide, outer membrane protein A, and S fimbriae using in vitro and in vivo models of the blood brain barrier developed in his lab and (b) identified several novel genes thought to be responsible for invasion of E. coli strain RS218 across the blood brain barrier, using these same models. E. coli strain RS218 is a clinical isolate from the cerebrospinal fluid of a human neonate with E. coli meningitis. Dr. Pietzak's project is focused on investigating the mechanisms by which E. coli strain RS218 is able to penetrate the intestinal epithelial barrier, both in vitro and in vivo. In earlier studies, Dr. Pietzak has used strain E44, a spontaneous rifampin resistant mutant of E. coli strain RS218, to demonstrate that this bacterium is able to invade two intestinal epithelial cell lines, Caco-2 and C2BBe-1. The specific aims of this proposal are to further characterize the invasive phenotype of E-44 in vitro, using both gentamicin invasion assays and a trans-well system. An in vivo model, using neonatal rats, will also be used to test the invasive phenotype of E-44. Environmental factors, which mimic the intraluminal gastrointestinal milieu, will be employed to examine their effects on E. coli invasion and translocation in vitro. The virulence of TnphoA transposon mutants, created in our lab and already shown to be noninvasive to the blood brain barrier in vitro and in vivo, will be determined for the intestinal epithelial barrier both in vitro and in vivo. The precise contribution of these genes to the intestinal translocation of E. coli strain RS218 will then be examined using additional molecular techniques. Under Dr. Kim's mentorship, and with Institutional support, Dr. Pietzak will be able to perfect her techniques in cell tissue culture and animal models of bacterial infection, as well as acquire new knowledge and skills in the sciences of bacterial genetics and molecular pathogenesis. Nurturing these ambitions in a collaborative environment will help Dr. Pietzak achieve her goal of becoming an independent investigator, who hopefully will be able to bring the results of her investigations from the benches of the lab to the bedsides of chronically ill children with gastrointestinal and other diseases.

本申请指导临床医生科学家发展奖 (K08) 寻求对医学博士 Michelle Pietzak 的支持，她最近完成了儿科胃肠病学和营养方面的研究金，并加入洛杉矶儿童医院担任儿科讲师（助理教授待定） 。 在 Kwang Sik Kim 医学博士的指导下，Pietzak 博士将继续对大肠杆菌 (E. coli) 跨肠上皮易位并引起败血症的机制进行基础研究。 大肠杆菌是早产儿、新生儿、免疫功能低下的宿主以及患有中心线和原发性肠道疾病的儿童严重细菌感染的主要原因。 Kim 博士是大肠杆菌脑膜炎发病机制领域的一位杰出研究员，他 (a) 使用大肠杆菌脑膜炎的体外和体内模型确定了 K1 荚膜多糖、外膜蛋白 A 和 S 菌毛的毒力作用。他的实验室开发了血脑屏障，并且 (b) 使用这些相同的模型识别了一些被认为导致大肠杆菌菌株 RS218 穿过血脑屏障入侵的新基因。大肠杆菌菌株 RS218 是从患有大肠杆菌脑膜炎的人类新生儿的脑脊液中临床分离的菌株。 Pietzak 博士的项目重点是研究大肠杆菌菌株 RS218 在体外和体内穿透肠上皮屏障的机制。 在早期的研究中，Pietzak 博士使用菌株 E44（大肠杆菌菌株 RS218 的自发利福平抗性突变体）来证明这种细菌能够侵入两种肠上皮细胞系：Caco-2 和 C2BBe-1。 该提案的具体目的是使用庆大霉素侵袭测定和跨孔系统进一步表征 E-44 的体外侵袭表型。使用新生大鼠的体内模型也将用于测试 E-44 的侵入表型。 将利用模拟肠腔内胃肠环境的环境因素来检查它们对体外大肠杆菌入侵和易位的影响。 我们实验室创建的 TnphoA 转座子突变体的毒力已被证明在体外和体内对血脑屏障无侵入性，将在体外和体内测定肠上皮屏障的毒力。 然后将使用其他分子技术检查这些基因对大肠杆菌菌株 RS218 肠道易位的精确贡献。 在 Kim 博士的指导和机构的支持下，Pietzak 博士将能够完善她在细胞组织培养和细菌感染动物模型方面的技术，并获得细菌遗传学和分子发病机制科学方面的新知识和技能。在协作环境中培养这些雄心壮志将有助于皮扎克博士实现成为一名独立研究者的目标，她希望能够将她的研究结果从实验室的工作台带到患有胃肠道和其他疾病的慢性病儿童的床边。疾病。