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: 10391067
• 负责人: Catherine Jane Hunter
• 金额: $ 5.97万
• 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/10391067
• 关键词: Adherence; Advisory Committees; Affect; Apoptosis; Apoptotic; Bacteria; Binding; Biological Assay; Biological Models; CREB1 gene; Calendar; Caspase; Cell Line; Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Disease Outbreaks; Dose; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Evaluation; Failure; Funding; Genetic; Human; Immunofluorescence Immunologic; In Situ Nick-End Labeling; In Vitro; Infant; Intestinal permeability; Intestines; Location; Measurement; Measures; Mediating; Mentors; Mentorship; Microbiology; Microscopy; Mission; Modeling; Molecular Biology; Mutagenesis; Necrotizing Enterocolitis; Neonatal Intensive Care Units; Operative Surgical Procedures; Oral Administration; PKA inhibitor; Pathogenesis; Pathway interactions; Pharmacologic Substance; Premature Infant; Publishing; Rattus; Research; Research Design; Research Methodology; Research Personnel; Resistance; Role; Scientist; Sepsis; Serum; Signal Pathway; Signal Transduction; Small Interfering RNA; Specimen; Supervision; Surgeon; Testing; Therapeutic; Time; Tissues; Training; Translational Research; United States National Institutes of Health; Virulence; Virulence Factors; Western Blotting; Work; apical membrane; base; career; career development; fluorescein isothiocyanate dextran; in vitro Model; innovation; intestinal barrier; intestinal epithelium; intestinal injury; microbial; mutant; novel; pathogen; programs; pup; response; responsible research conduct; success

PROJECT SUMMARY Objectives: 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.

项目概要 目标：该应用程序定义了一个计划，以促进有前途的初级研究员的研究生涯 在有指导的环境下成功完成将允许调查员开始职业生涯。 美国国立卫生研究院 (NIH) 资助的独立外科医生科学家，开展转化研究，旨在确定关键 涉及坏死性小肠结肠炎（NEC）和肠道败血症其他原因的途径一旦明确。 确定了涉及 NEC 发病机制的途径，可以开发更好的治疗方法。 背景：NEC 影响所有住院早产儿的 5%，最严重的形式可能是致命的。 细菌与疾病的发病机制有关，坂崎克罗诺杆菌 (CS) 已被确定为致病菌 根据初步数据和已发表的工作，我们认为 CS 遵守 肠上皮的顶膜，对于增加细胞内 cAMP、PKA 激活和 上皮细胞凋亡，导致肠屏障衰竭和 NEC。 研究设计和方法：目标 1 将确定 CS 是否刺激 cAMP、PKA 和 CREB ​​激活 在实验 NEC 中，cAMP 水平将在不同剂量和浓度的 CS 后通过 ELISA 进行测定。 将测试NEC的体外肠细胞系模型和大鼠幼仔模型。 取自 NEC 婴儿的手术肠道标本中的水平将与对照结果进行比较。 此外，NEC 期间激活的 PKA 的亚细胞位置将是 目标 2 将确定上皮细胞凋亡和肠道屏障功能丧失是否是由 引起的。 实验性 NEC 中 PKA 介导的途径体外模型的细胞凋亡和屏障反应。 药物 PKA 抑制剂和激活剂，以及使用 siRNA 标记的 PKA 基因抑制。 我们将通过蛋白质印迹分析和免疫荧光来测量细胞凋亡（半胱天冬酶和 TUNEL）。 确定 PKA 激活的时间，并确定其与细胞凋亡屏障功能变化的关系。 通过体外跨上皮电阻测量来测量PKA抑制剂在NEC幼鼠中的作用。 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模型。 将比较各组之间的分数，以及比较幼崽之间的屏障功能。 通过基于血清的测定口服 FITC-葡聚糖进行分组，目的 3 将确定 CS 毒力的作用。 实验性 NEC CS 突变体中缺乏促进宿主细胞结合的毒力因子的因子。 评估了它们诱导上皮细胞凋亡的能力，并且可能存在实验性 NEC 突变体。 该项目是新颖的，因为之前没有研究调查过转座子诱变的作用。 NEC 中的 PKA 项目新颖且创新，提出了 CS 触发 cAMP 释放的机制， 改变 PKA 介导的活性，导致细胞凋亡和 NEC 的作用。 NEC中的cAMP、PKA和CREB以及可能引发NEC的毒力因子尚未明确。 研究环境：候选人建议在已建立的环境中开发该项目 在杰出导师的监督下成功培养初级研究员的职业生涯。 团队，该项目将为候选人的背景增添新的专业知识，包括分子生物学培训， 细胞信号通路、免疫染色、肠道通透性评估和微生物诱变。 该提案中的开发活动包括分子生物学、细胞信号传导方面的教学课程 机制和微生物学、职业咨询委员会的定期评估以及相关培训 候选人有 75%（9 个日历月）的受保护研究时间。

项目成果

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis.

坏死性小肠结肠炎的新型人上皮肠样模型。

• 发表时间: 2019
• 作者: Ares, Guillermo J;Buonpane, Christie;Yuan, Carrie;Wood, Douglas;Hunter, Catherine J
• 通讯作者: Hunter, Catherine J

Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis.

顶端出肠样作为坏死性小肠结肠炎的创新模型。

• DOI: 10.1016/j.jss.2022.11.048
• 发表时间: 2023-03-01
• 期刊: The Journal of surgical research
• 作者: Heather L. Liebe;Camille Schlegel;Xue Cai;A. Golubkova;Christopher Loerke;Tyler Leiva;C. Hunter
• 通讯作者: C. Hunter

Experimental Modeling of Necrotizing Enterocolitis in Human Infant Intestinal Enteroids.

人类婴儿肠类坏死性小肠结肠炎的实验模型。

• 发表时间: 2022-01
• 作者: Buonpane, Christie;Ares, Guillermo;Yuan, Carrie;Schlegel, Camille;Liebe, Heather;Hunter, Catherine J
• 通讯作者: Hunter, Catherine J

Rho kinase inhibition maintains intestinal and vascular barrier function by upregulation of occludin in experimental necrotizing enterocolitis.

在实验性坏死性小肠结肠炎中，Rho 激酶抑制通过上调 Occludin 来维持肠道和血管屏障功能。

• DOI: 10.1152/ajpgi.00357.2017
• 发表时间: 2018-10-01
• 期刊: American journal of physiology. Gastrointestinal and liver physiology
• 作者: Justyna S Grothaus;Guillermo Ares;Carrie Y. Yuan;D. Wood;C. Hunter
• 通讯作者: C. Hunter

ROCK1 inhibitor stabilizes E-cadherin and improves barrier function in experimental necrotizing enterocolitis.

ROCK1 抑​​制剂可稳定 E-钙粘蛋白并改善实验性坏死性小肠结肠炎的屏障功能。

• 发表时间: 2020
• 作者: Buonpane, Christie;Yuan, Carrie;Wood, Douglas;Ares, Guillermo;Klonoski, Samuel C;Hunter, Catherine J
• 通讯作者: Hunter, Catherine J