# Defining bacterial virulence, cAMP and PKA in necrotizing enterocolitis

> **NIH NIH K08** · UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR · 2021 · $59,698

## Abstract

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 relea...

## Key facts

- **NIH application ID:** 10391067
- **Project number:** 3K08DK106450-06S1
- **Recipient organization:** UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
- **Principal Investigator:** Catherine Jane Hunter
- **Activity code:** K08 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $59,698
- **Award type:** 3
- **Project period:** 2020-03-30 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10391067

## Citation

> US National Institutes of Health, RePORTER application 10391067, Defining bacterial virulence, cAMP and PKA in necrotizing enterocolitis (3K08DK106450-06S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10391067. Licensed CC0.

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