Defining bacterial virulence, cAMP and PKA in necrotizing enterocolitis

坏死性小肠结肠炎中细菌毒力、cAMP 和 PKA 的定义

• 批准号: 10093945
• 负责人: Catherine Jane Hunter
• 金额: $ 7.29万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-30 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093945
• 关键词: Defining; bacterial; virulence; cAMP; PKA

 DESCRIPTION (provided by applicant): This application defines a program to further the research career of a promising junior investigator within a mentored setting. Successful completion would allow the investigator to initiate a career as an independent NIH-funded surgeon-scientist, conducting translational research directed at identifying key pathways involved in necrotizing enterocolitis (NEC) and other causes of intestinal sepsis. Once specific pathways involved in the pathogenies of NEC are identified, better therapeutics may be developed. Background: NEC affects 5% of all hospitalized premature infants, and may be fatal in its most severe forms. Bacteria are implicated in disease pathogenesis, and Cronobacter sakazakii (CS) has been identified as causing outbreaks of NEC. Based on preliminary data and published work, we hypothesize that CS adherence to the apical membrane of the intestinal epithelium, is essential for increased intracellular cAMP, PKA activation and epithelial apoptosis, resulting in intestinal barrier failure and NEC. Research Design and Methods: Aim 1 will determine whether CS stimulates cAMP, PKA and CREB activation in experimental NEC. cAMP levels will be assayed by ELISA following various doses and concentrations of CS. Both in vitro intestinal cell line models and the rat pup model of NEC will be tested. cAMP, PKA and CREB levels in surgical intestinal specimens taken from infants with NEC will be compared to controls. Results will be compared among model systems. Furthermore, the subcellular location of activated PKA during NEC will be identified. Aim 2 will determine whether epithelial apoptosis and loss of intestinal barrier function is induced by PKA-mediated pathways in experimental NEC. The apoptotic and barrier responses of the in vitro models to pharmaceutical PKA inhibitors and activators, as well as genetic inhibition of PKA using siRNA. Markers of apoptosis (caspase and TUNEL) will be measured by western blot analysis and immunofluorescence. We will determine the timing of PKA activation, and define its relationship to apoptosis. Changes in barrier function will be measured by transepithelial resistance measurement in vitro. The effect of PKA inhibitors in the NEC rat pup model will be assessed by tissue microscopy, immunofluorescence and western blot analysis. Intestinal injury scores will be compared between groups, as well as pup survival. Barrier function will be compared between groups by oral administration of FITC-Dextran by serum based assay. Aim 3 will define the role of CS virulence factor(s) in experimental NEC. CS mutants lacking virulence factors that facilitate host cell binding will be assessed for their ability to induce epithelial apoptosis and experimental NEC. Additional mutants may be generated by transposon mutagenesis. This project is novel because no prior study has investigated the role of PKA in NEC. This project is novel and innovative in proposing a mechanism by which CS trigger cAMP release, alter PKA mediated activity, resulting in apoptosis and NEC. No study has previously examined the role of cAMP, PKA and CREB in NEC, and the virulence factors that may trigger NEC are not defined. Research environment: The candidate proposes to develop this project within an environment with established success at nurturing the careers of junior investigators. Under the supervision of an outstanding mentorship team, this project will add new expertise to the candidate's background, including training in molecular biology, cell signaling pathways, immunostaining, intestinal permeability assessment, and microbial mutagenesis. Career development activities within the proposal include didactic coursework in molecular biology, cell signaling mechanisms and microbiology, regular evaluations by a career advisory committee, and training in the responsible conduct of research. The candidate has 75% (9 calendar months) of protected research time.

 描述（由申请人提供）：本申请定义了一个计划，旨在在指导环境中促进有前途的初级研究者的研究生涯。成功完成将使研究者能够开始作为独立的 NIH 资助的外科医生科学家的职业生涯，进行转化研究。旨在确定坏死性小肠结肠炎 (NEC) 和肠道脓毒症其他病因中涉及的关键途径。一旦确定了涉及 NEC 发病机制的特定途径，就可以开发出更好的治疗方法。 5% 的住院早产儿（最严重的形式可能是致命的）细菌与疾病发病机制有关，并且根据初步数据和已发表的工作，我们希望阪崎克罗诺杆菌 (CS) 已被确定为导致 NEC 爆发的原因。 CS 粘附于肠上皮顶膜，对于增加细胞内 cAMP、PKA 激活和上皮细胞凋亡至关重要， 研究设计和方法：目的 1 将确定 CS 是否刺激实验 NEC 中的 cAMP 水平，并在体外肠细胞中通过 ELISA 进行测定。将测试取自 NEC 婴儿的手术肠道标本的 cAMP、PKA 和 CREB ​​水平，并将结果与​​对照进行比较。目标 2 将确定 NEC 期间激活的 PKA 的亚细胞位置，以确定实验性 NEC 中 PKA 介导的途径是否会诱导上皮细胞凋亡和肠屏障功能丧失。激活剂以及使用 siRNA 对 PKA 的遗传抑制将通过蛋白质印迹分析和免疫荧光进行测量，我们将确定 PKA 激活的时间。并通过体外跨上皮电阻测量来测定其与细胞凋亡的关系。将通过组织显微镜检查、免疫荧光和蛋白质印迹分析来评估PKA抑制剂在NEC幼仔模型中的作用。组间比较，以及通过基于血清的测定口服 FITC-葡聚糖来比较幼仔的屏障功能，目的 3 将确定 CS 毒力的作用。将评估实验性 NEC 中缺乏促进宿主细胞结合的毒力因子的 CS 突变体诱导上皮细胞凋亡的能力，并且可以通过转座子诱变产生其他突变体。该项目是新颖的，因为之前没有研究。研究了 PKA 在 NEC 中的作用，该项目提出了一种机制，通过 CS 触发 cAMP 释放，改变 PKA 介导的活性，导致细胞凋亡和 NEC，以前没有研究检查过 cAMP 的作用。 NEC 中的 PKA 和 CREB ​​以及可能引发 NEC 的毒力因素均未定义。 研究环境：在培养初级研究人员的职业生涯方面取得成功的环境中开发该项目的候选人在杰出导师的监督下。团队中，该项目将为候选人的背景增添新的专业知识，包括分子生物学、细胞信号通路、免疫染色、肠道通透性评估和微生物诱变方面的培训，该提案中的职业发展活动包括教学课程。分子生物学、细胞信号机制和微生物学、职业咨询委员会的定期评估以及负责任的研究行为培训候选人有 75%（9 个日历月）的受保护研究时间。