Executive Summary: Chronic non-communicable diseases (NCDs) are a public health problem worldwide. CNCDs, including obesity, hypertension, metabolic syndrome, cancer, share characteristics in relation to changes in the intestinal microbiota associated with increased permeability of the intestinal epithelium.
Within this context, short-chain fatty acids (SCFA) such as butyrate contribute to the normal development and maintenance of the intestinal epithelium and immune system. Furthermore, it is clearly established that SCFA are the main link between diet, microbiota and the health status of individuals. Therefore, it is necessary to develop new tools to study and positively modify the metabolism and composition of the intestinal microbiota and thus contribute to the search for treatment alternatives for various CNCDs. The objective of this proposal is to establish a system that allows systematic evaluation of the effects exerted by butyrate produced by a probiotic strain of Saccharomyces boulardii, on the structure and function of the intestinal epithelium as well as monocytes/macrophages in an in vitro model of dual cell culture. With this purpose, initially variants of the butyrate biosynthetic pathway in S. boulardii will be assembled and the best producer will be selected based on a reporter system that activates the expression of a fluorescent protein in response to the presence of butyrate. Simultaneously, the produced butyrate will be quantified through high performance liquid chromatography (HPLC). In addition, a dual culture system of intestinal epithelial cells (IEC) and monocytes/macrophages will be established that will be used to determine the effect of butyrate produced by S. boulardii on the morphology and functionality of the dual culture. Specifically, IEC differentiation and IEC monolayer formation will be determined by measuring the transepithelial electrical resistance (TEER) and the expression of proteins present in the gap junctions (occludin, zonula occludens-1 (ZO-1), polymeric immunoglobulin receptor-IgA transporter (pIgR)) and intestinal microvilli formation by electron microscopy, confocal microscopy and immunocytochemistry. Butyrate produced by S. boulardii is expected to contribute positively to ISC differentiation by increasing the expression of occludin, ZO-1, pIgR, and intestinal microvilli, and by decreasing TEER values and ISC monolayer permeability. . The results of this research will contribute to the development of an experimental model, which allows the systematic evaluation of the role of various metabolites and/or components of the intestinal microbiota in the structure and function of the epithelium, as well as intestinal immune cells.
Objectives General: To evaluate the effect of butyrate produced by Saccharomyces boulardii on the structure and function of the intestinal epithelium and macrophages in an in vitro cell culture model.
Specific objectives:
- Build the in silico metabolic network of S. boulardii for mathematical modeling.
- Select candidate genes for the construction of the synthetic pathway for butyrate production.
- To construct the synthetic pathway for butyrate production in S. boulardii.
- To evaluate the production of butyrate in S. boulardi.
- Establish a dual culture system of intestinal epithelial cells and monocytes/macrophages.
- To evaluate the morphology and functionality of the dual culture in the presence or absence of S. boulardi modified for butyrate production.
Participating Institutions:
UTE, UTA, ESPE.
Participants:
Project Director Linda Priscila Guamán Bautista
- Linda Priscila Guaman Bautista
- Angel Oswaldo Cargua Garcia
- Carlos Arturo Barba Ostria
- Erika Belen Muñoz Salazar
- Federico José Zertuche Dulbeco
- Orestes Darío López Hernández
- Rodrigo Marcelo Grijalva Silva
- Lizeth Andrea Salazar Anchundia
Awarded budget: $63600
Project status: Signing of agreements.
