Defining bacterial virulence, cAMP and PKA in necrotizing enterocolitis

  • Hunter, Catherine Jane (PD/PI)

Project: Research project

Project Details

Description

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 and PKA activation. cAMP levels will be assayed by ELISA following various doses and concentrations of CS. Both in vitro cell line models and the rat pup model of NEC will be tested. In Aim 2 it will be determined whether epithelial apoptosis is induced by PKA-mediated pathways in experimental NEC. In vitro intestinal cell lines (IEC-6, CaCO2) will be treated with PKA inhibitors and activators and siRNA. Markers of apoptosis will be measured by western blot analysis and immunohistochemistry. The effect of PKA inhibitors in the NEC rat pup model will be assessed by tissue microscopy, immunohistochemistry and western blot analysis. 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 and PKA 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 cell signaling pathways, immunostaining, and microbial mutagenesis. Career development activities within the proposal include didactic coursework in molecular biology and cell signaling mechanisms, 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.
StatusFinished
Effective start/end date8/1/156/1/19

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases (5K08DK106450-04)

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