An official website of the United States government.

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

DETERMINING THE MECHANISMS OF PATHOGEN MODULATION BY ADENOSINE METABOLISM IN THE GUT MUCOSA.

Objective

The long-term goal for this project is to elucidate the mechanisms by which adenosine generated by the intestinal epithelium modulates inflammatory diseases. Epithelial cells create a critical boundary between antigens in the intestinal lumen and the underlying submucosa. Pathogens such as Salmonella enterica initiate infection in intestinal epithelial cells (IECs) to cause inflammatory disease. Thus, IECs represent a key point of host defense against pathogens. ATP is released into the intestinal lumen by pathogens, IECs, and infiltrating immune cells and acts as a pro-inflammatory signal during infection. Once near the intestinal epithelial cell surface, extracellular ATP is quickly converted to AMP by ecto-apyrase. AMP must then be converted to the anti-inflammatory molecule adenosine by the ecto-5'-nucleotidase (CD73) for the resolution of inflammation. However, there is a critical knowledge gap in understanding the impact of CD73-generated adenosine on mechanisms of host-microbe interactions in the intestinal epithelium.While inflammation is typically necessary for resolving microbial infections, enteric pathogens have evolved mechanisms to exploit inflammatory responses to intensify virulence. Work from our lab has shown, Salmonella enterica serovar Typhimurium (S. Typhimurium) is unable to carryout normal pathogenesis in the absence of intestinal epithelial CD73 (50). Therefore, we hypothesize that CD73 loss in the intestinal epithelium alters adenosine generation and signaling pathways, limiting the ability for S. Typhimurium to cause disease. Three specific aims are proposed to test this hypothesis:Specific Aim 1: Define the mechanism of S. Typhimurium virulence attenuation by IECs lacking CD73. Work in our lab has shown, that in the absence of CD73, S. Typhimurium can invade cultured IECs, but becomes trapped at the apical surface, suggesting these cells are unable to support bacterial trafficking and replication. Here we will employ a number of approaches to elucidate the underlying mechanisms: 1) we will use confocal fluorescence microscopy to examine the expression and spatial location of host proteins recruited and modulated by well-characterized S. Typhimurium virulence-associated effectors during early stages of S. Typhimurium infection; 2) will employ RT-qPCR and protein analysis to measure changes in gene expression of S. Typhimurium effectors to determine which virulence-associated genes are being expressed; 3) we will utilize this data to guide construction of S. Typhimurium mutants lacking virulence-associated effectors to infect wild type cells to confirm virulence attenuation mechanisms.Specific Aim 2: Identify the primary cause of S. Typhimurium virulence attenuation in CD73f/f VillinCre mice. We have shown that dissemination of S. Typhimurium to distal body sites is attenuated in CD73f/f VillinCre mice, a novel mouse model lacking CD73 only in IECs. These results demonstrate the phenotype that we observe in cultured human IECs can be recapitulated in an in vivo model. However, the microbiota and local tissues can metabolize adenine nucleotides, suggesting the mechanism underlying virulence attenuation could differ in an in vivo model lacking CD73 only in IECs. Here, we will compare disease dynamics between CD73-/- whole body knockout, CD73f/f VillinCre, and control mice infected with S. Typhimurium in conventional and germ free mouse lines to look at cell type-specific phenotypic contributions and the contributions of an intestinal microbiome on our model.Specific Aim 3: Examine the role of CD73 in the modulation of Salmonella infection in bovine IECs. Work in our lab has shown that epithelial CD73 acts as a mediator of S. Typhimurium infection in human cell models and in murine models in vivo. As an extension of these studies, we will examine the dynamics of CD73 modulation of S. Typhimurium infection in an ex vivo bovine intestinal organoid model. Here, we will employ methodologies as outlined in Aim 1 above. Additionally, we will employ the use of CRISPR technology to impact CD73 expression to confirm the importance of CD73 in the bovine cell model.

Investigators
Kominsky, Do, .
Institution
Montana State University
Start date
2018
End date
2021
Project number
MONB00452
Accession number
1015883