Wednesday, April 2, 2025
4/2/2025
Impact of Yersinia pseudotuberculosis infection on M cells in murine infections - Abstract/Summary Microfold (M) cells are specialized cells of the intestinal epithelium that are concentrated in the epithelium lying over the lymph follicles, called the follicle associated epithelium (FAE). M cells are designed to deliver antigens to underlying lymph tissues. Their specialized function is facilitated both by a host of receptors on their apical surface that bind to antigens and particles and their ability to rapidly transcytose these bound antigens from the apical surface to the basolateral surface where they are captured by immune cells within the Peyer’s patches. M cells are also exploited as cellular portals by many enteric bacterial and viral pathogens to gain access to the lymph system, including the bacterial pathogen Yersinia pseudotuberculosis (Yptb). In our previous work, we established a differentiated human ileum organoid model system on Transwells and induced M cell development in order to study molecular mechanistic interactions between Yptb and M cells. We found that Yptb that are not expressing the type III secretion system (T3SS) are internalized by M cells and found in the basolateral chamber in high numbers. By contrast, Yptb that are expressing the T3SS remained bound on the extracellular surface of M cells and induced M cell extrusion from the monolayer. Strikingly, YopE, a T3SS effector protein, caused extrusion of M cell from the monolayer. Thus, we hypothesize that oral infection of mice with Yptb leads to transient M cell depletion due to the YopE RhoA-GAP activity and this M cell depletion limits superinfection by other pathogens and re-infection by Yptb. The objectives of this application are two- fold. (1) We will use our expertise in murine infection models and determine if infection with WT-Yptb and a type III secretion mutant, yscNU, impacts the numbers and functions of M cells in the follicle associated epithelium. (2) We will build murine organoid/enteroid cell lines that express Cas9 only in developing M cells and then exploit CRISPR-Cas9 techniques to selectively delete RhoA and other putative Yop targets in M cells. This will permit us to evaluate the roles of the proteins targeted for inactivation by Yops in M cell homeostasis and functions. By building these tools and establishing that Yptb infection depletes M cells, we will have a firm basis to investigate the molecular mechanistic and immunological implications of Yptb-M cell interactions in a planned R01 proposal.
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