Cell and molecular analysis of intestinal colonization by IBD associated opportunistic microbes
Creators: name: Flores, Juan
Publisher: name: No Publisher Supplied
Year: 2023

description: The gastrointestinal tract is the location that houses the largest and most diverse microbial communities living in a symbiotic relationship with host cells in both human and rodents. This microbiome can be a constant source of extracellular cues that stimulate immunological responses in the host epithelial and immune cells. In addition to the resident gut commensal microbiota, opportunistic microorganisms commonly ingested with the diet also attempt to colonize and exploit the nutrient rich environment of the intestinal lumen. In healthy individuals, many of these opportunistic microorganisms can be managed and cleared from the host with a timely and proper cellular response. However, certain environmental or genetic predispositions can elicit or potentiate unwanted inflammatory responses to such so-called pathobiont microorganisms leading to acute or chronic disease states. The molecular basis underlying the interaction between the host and disease-relevant opportunistic microorganisms remains not fully understood.Inflammatory bowel disease (IBD) is one such clinical manifestation of abnormal and prolonged inflammatory responses that damage the gastrointestinal mucosa. The prevalence of IBD in adults is on the rise in the U.S and worldwide. Traditional therapeutic interventions are typically designed to inhibit several prominent pro-inflammatory pathways such as the TNF-alpha signaling cascade. While 60% of individuals respond initially to such treatments, half of those individuals fail to respond subsequently and relapse, while a significant portion of patients never respond at all. In addition, some patients become susceptible to opportunistic pathogens, such as microsporidian. We studied the interaction of human enterocytes with Encephalitozoon intestinalis, a microsporidian species first isolated from HIV patients but also found in IBD patients taking immunosuppressives. Using a combination of high-throughput transcriptomic approaches and electron microscopy analysis, this work reports the development of a system for the continuous culture of E. intestinalis in Caco2 cells. This system recapitulated the in vivo life cycle of E. intestinalis in Caco2. Additionally, two types of spore development were identified, a typical environmental spore that leaves the host and autoinfective spores that immediately germinate and spread infection to neighboring cells. Furthermore, we found that infection altered the host cell mitochondria, nucleus and brush-boarder structures. Untargeted transcriptomic analysis demonstrated that E. intestinalis infection had an impact on cell signaling networks related to energy, metabolism and membrane trafficking. These results suggest that E. intestinalis exploits intestinal epithelial cells for propagation and evasion of host detection systems. This research may shed light on therapeutic interventions for E. intestinalis, which are currently limited. In addition, using multiomics approach combined with gnotobiotic mice, we investigated the impact of colonization by Ruminococcous gnavus, an obligate gut commensal anaerobic bacterium, on host gut physiology. One of the hallmarks of intestinal dysbiosis is the expansion of pathobionts, including opportunistic bacteria that may drive pathogenicity in patients with high-risk environmental or genetic conditions. Ruminococcous gnavus is significantly associated with gut dysbiosis in multiple chronic autoimmune or metabolism disorders, and consistently expanded in patients of IBD, diabetes, obesity, nonalcoholic fatty liver disease, and coronary artery disease, etc. There is a fundamental lack of mechanistic research on whether and how R. gnavus confers its effects on intestinal epithelial homeostasis and the overall host physiology. Initial transcriptomic analysis comparing R. gnavus and germ-free mice showed an increase in genes related to amino acid transport at the plasma membrane. Furthermore, Nos2 expression was higher in R. gnavus ileal tissue compared to their germ-free counterparts. Fecal metabolome analysis via LC-MS detected increased levels of Urea-NOS cycle metabolites, especially ornithine and citrulline, in R. gnavus colonized mice. Lastly, in vitro studies suggest that ornithine may suppress the host’s ability to secrete nitric oxide in response to bacterial stimulation. This work highlights another molecular mechanism by which R. gnavus modulates host response to microbes. Taken together, to better understand the contribution of opportunistic microorganisms to the development of gut pathology, this thesis took advantage of high throughput analysis coupled with cell biology and animal models to characterize their cellular and molecular effects on intestinal epithelial cells. The results may open future avenues for therapeutic intervention of IBD and other intestinal disorders.