Molecular Pathogenesis of Enterotoxigenic Escherichia coli Infections

About This Grant

Project Summary/Abstract Significance to VA During deployment to Operation Desert Storm as an Army Medical Officer the Pl of this project cared for many soldiers with severe diarrhea, later shown to be caused primarily by enterotoxigenic E. coli (ETEC)1. These infections remain a major cause of acute diarrheal illness worldwide, have been responsible for major outbreaks in the United States, and fuel the global spread of antibiotic resistance determinants. In addition, these pathogens are closely associated with long-term sequelae including malnutrition and wasting in young children, tropical sprue in adults, and Irritable Bowel Syndrome (IBS) in returning Veteran service members. Gaps addressed by these studies Our understanding of the molecular pathogenesis of gastrointestinal pathogens as well as the composition of the commensal microflora is exceedingly biased by models that fail to faithfully recapitulate key features of the human intestine, particularly the major immunoglobulin superfamily glycoproteins known as CEACAMs. The proposed studies seek to overcome these deficiencies using transgenic mice and human stem cell-derived gastrointestinal organoids that accurately reflect the molecular organization of human small intestine. Innovation and Impact: These studies center on the interaction of ETEC and other intestinal bacteria (including commensals) with Carcinoembryonic Antigen-related Cell Adhesion Molecules (CEACAMs), which we have recently shown in novel human intestinal organoid models and a transgenic mouse model to play essential roles in pathogen­ host interactions, as well as innate response to ETEC. While thousands of intestinal microbiome studies have been performed in conventional mice, they lack CEACAM genes normally expressed in the human gastrointestinal tract. We therefore anticipate that the proposed studies will significantly impact our understanding of intestinal bacteriology and approach to care. Specific Aims: 1. Define molecular interactions between ETEC, EV and CEACAMs. 2. Examine the interplay of CEACAMs, intestinal microbiota and ETEC 3. Examine the impact of ETEC pathogen-host interactions on gene expression. Methodology: Aim 1 we will use recombinant CEACAMs to investigate interactions between the bacteria and these receptors. In addition, we will map glycosylation sites on CEACAM6 before and after treatment with ETEC heat labile toxin by mass spectrometry, mutate the respective sites, and examine their impact as recombinant proteins and in transformed cells. Aim 2: Here we will examine the impact of CEACAM expression and the introduction of ETEC on defined microbiota in a gnotobiotic core facility by next generation metagenomic sequencing. Aim 3: To examine the impact of EV- interactions with ETEC we plan to perform RNA sequencing (RNAseq) of bacterial RNA following contact with CEACAM-laden extracellular vesicles (EV). In addition, we will explore the impact of heat-labile toxin on activation of gene expression pathways that lead to CEACAM upregulation by this toxin. Path to Translation/Implementation: Although basic in nature, we anticipate that these studies will provide a more detailed understanding of both host-pathogen interactions and the intestinal microbiota that can readily be applied to improving preclinical testing of orally delivered drugs, vaccines, and other therapeutics.