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）：此应用程序定义了一个程序，以进一步在一个有前途的初级研究人员的研究生涯辅导设置。成功完成将使研究者开始作为一个独立的NIH资助的外科医生，科学家的职业生涯，进行翻译研究，旨在确定参与坏死性小肠结肠炎（NEC）和肠败血症的其他原因的关键途径。一旦确定了NEC病因中涉及的特定途径，就可能开发出更好的治疗方法。背景：NEC影响所有住院早产儿的5%，并且在其最严重的形式中可能是致命的。细菌参与疾病的发病机制，坂崎克罗伊氏菌（CS）已被确定为引起NEC的爆发。基于初步数据和已发表的工作，我们假设CS粘附于肠上皮的顶膜，对于细胞内cAMP、PKA活化和上皮细胞凋亡的增加是必不可少的， 导致肠屏障衰竭和NEC。研究设计和方法：目的1：确定CS是否刺激实验性NEC中cAMP、PKA和CREB的激活。在CS的不同剂量和浓度后，通过ELISA测定cAMP水平。将测试NEC的体外肠细胞系模型和大鼠幼仔模型。将取自NEC婴儿的手术肠标本中的cAMP、PKA和CREB水平与对照组进行比较。结果将在模型系统之间进行比较。此外，将确定NEC期间激活的PKA的亚细胞位置。目的2：探讨在实验性NEC中，PKA介导的细胞凋亡和肠屏障功能的丧失是否与细胞凋亡有关。体外模型对药物PKA抑制剂和激活剂的凋亡和屏障反应，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法测量细胞凋亡标志物（半胱天冬酶和TUNEL）。我们将确定PKA激活的时间，并确定其与细胞凋亡的关系。屏障功能的变化将通过体外跨上皮电阻测量来测量。将通过组织显微镜检查、免疫荧光和蛋白质印迹分析评估PKA抑制剂在NEC大鼠幼仔模型中的作用。将比较组间的肠损伤评分以及幼仔存活率。将通过基于血清的测定，通过口服给予FITC-葡聚糖，比较各组间的屏障功能。目的3明确CS毒力因子在实验性NEC中的作用。将评估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和实验NEC的能力。另外的突变体可以通过转座子诱变产生。这个项目是新颖的，因为没有以前的研究调查PKA在NEC中的作用。该项目在提出CS触发cAMP释放、改变PKA介导的活性、导致细胞凋亡和NEC的机制方面是新颖的和创新的。以前没有研究检查cAMP，PKA和CREB在NEC中的作用，可能触发NEC的毒力因子也没有定义。研究环境：候选人建议在一个成功培养初级调查员职业生涯的环境中发展这一项目。在优秀导师团队的监督下，该项目将为候选人的背景增加新的专业知识，包括分子生物学，细胞信号传导途径，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分子生物学、细胞信号机制和微生物学的教学课程，职业咨询委员会的定期评估，以及负责任地开展研究的培训。候选人有75%（9个日历月）的受保护的研究时间。