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: 8949746
• 负责人: Catherine Jane Hunter
• 金额: $ 15.3万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8949746
• 关键词: Adherence; Advisory Committees; Affect; Apical; Apoptosis; Apoptotic; Award; Bacteria; Bacterial Infections; Binding; Biological Assay; Biological Models; Calendar; Caspase; Cell Line; Cells; Child; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Data; Disease; Disease Outbreaks; Dose; Enteral; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Evaluation; Event; Experimental Models; Failure; Figs - dietary; Functional disorder; Funding; Genetic; Goals; Health; Human; Immunofluorescence Immunologic; Immunofluorescence Microscopy; In Situ Nick-End Labeling; In Vitro; Infant; Infection; Inflammatory disease of the intestine; Injury; Intestines; Knowledge; Lead; Link; Location; Long-Term Survivors; Measurement; Measures; Mediating; Mediator of activation protein; Mentors; Mentorship; Microbiology; Mission; Modeling; Molecular Biology; Mutagenesis; Necrotizing Enterocolitis; Neonatal Intensive Care Units; Neurologic; Operative Surgical Procedures; Oral Administration; Pathogenesis; Pathway interactions; Patients; Permeability; Pharmacologic Substance; Phosphorylation; Premature Infant; Production; Protein Kinase; Protein Kinase Inhibitors; Pseudomonas aeruginosa; Publishing; Rattus; Research; Research Design; Research Methodology; Research Personnel; Resistance; Rodent; Rodent Model; Role; Sampling; Scientist; Sepsis; Serum; Short Bowel Syndrome; Signal Pathway; Signal Transduction; Small Interfering RNA; Specimen; Staging; Supervision; Surface; Surgeon; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Training; Translational Research; United States National Institutes of Health; Virulence; Virulence Factors; Western Blotting; Work; Yersinia pestis; apical membrane; base; career; career development; established cell line; fluorescein isothiocyanate dextran; improved; in vitro Model; in vivo; innovation; intestinal epithelium; knock-down; meetings; microbial; mutant; novel; novel strategies; pathogen; prevent; programs; protein activation; protein kinase inhibitor; public health relevance; pup; response; responsible research conduct; skills; success; tool; transcription factor

 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.