Our long-term goal is to understand how pathogen-mediated genotoxicity, which is increasingly associated with pathogenic bacteria, fungi, and viruses, impacts microbe-host interactions and disease pathogenesis. We are studying the cytolethal distending toxins (CDTs), a conserved, but broadly distributed family of intracellular- acting genotoxins, which are secreted by pathogenic bacteria that infect and damage mucocutaneous tissues at different sites within the body, including the gastrointestinal tract. Despite increasing evidence of the importance of CDTs as determinants of pathogenesis, a major deficit in our understanding of CDT biology is the exact role of these genotoxins during infection. In this application, we propose studies to address the impact of genotoxicity at the intestine mediated by CDT produced by the human intestinal pathogen Campylobacter jejuni (Cj-CDT), which has been demonstrated to be important for C. jejuni (Cj) invasion of the epithelial barrier, and subsequent dissemination to the blood, spleen and liver in mice. Although the mechanism of invasion is poorly understood, Cj-CDT-mediated cytotoxicity, which has been examined primarily in vitro using immortalized cell lines, is widely believed to be a major driver of damage to the epithelial barrier during infection. However, preliminary studies in our lab revealed that Cj-CDT alters epithelial barrier integrity and function, in the absence of toxin-mediated cell death, within a murine-derived intestinal organoid model that recapitulates the dynamic regeneration of the crypt/villus architecture of the intestinal epithelium. These findings challenge the importance of Cj-CDT cytotoxicity as a mechanism of toxin-dependent changes at epithelial barriers, by suggesting possible associations between Cj-CDT-mediated DNA damage and/or host DNA damage repair and the mechanisms that regulate cell proliferation and differentiation during normal regeneration of the intestinal epithelium every 3-5 days. Based on preliminary data from our laboratory, our overall model of this potential association is that Cj- CDT-dependent DNA damage and subsequent DNA damage response (DDR) promote intestinal barrier dysfunction by modulating cell proliferation and cellular lineage allocation, both of which are critical for regeneration. In this exploratory R21 application, we address several existing gaps in knowledge.
In Aim 1, we propose studies to test predictions of the hypothesis that Cj-CDT-dependent genotoxicity is causal for intestinal barrier dysfunction. We will investigate the causal relationship between Cj-CDT-dependent DNA damage/repair and alterations in cellular proliferation and differentiation, and assess the essentiality of Cj-CDT for inducing epithelial barrier function during Cj infection of intestinal organoids.
In Aim 2, we will investigate the relationship between Cj-CDT-dependent DNA damage/repair, and the reduction in cellular levels of SNAI1, an important transcriptional factor involved in regulating intestinal lineage allocation. Because subversion of epithelial barrier integrity is associated with human disease and pathology, we are ultimately interested in extending these studies to investigate the potential negative impact of Cj-CDT-mediated genotoxicity on human intestinal health.
A normal, functioning intestine is critical for human health. In this application, we propose studies to evaluate the mechanism by which some pathogenic bacteria damage intestinal health by producing genotoxins that cause damage to DNA inside of intestinal cells. Understanding the full spectrum of consequences stemming from microbial-mediated DNA damage will provide new insights into the negative impact of genotoxin-producing microbes on human health, and will better inform clinical decisions about how to most effectively treat infected individuals.
