Investigation of Yop activities in M cells during murine infection

Tufts University Boston, Boston MA

Investigators

Abstract

Project Abstract Microfold (M) cells are specialized cells in the gastrointestinal (GI) epithelium. They are important sentinel cells of the gut-associated lymphoid tissue (GALT), because they deliver foreign antigens and particles to the immune cells that underlie the intestinal epithelium. Their specialized function is facilitated both by numerous receptors on their apical surface that bind to antigens and their ability to rapidly transcytose bound antigens to the Peyer’s patches. In their absence, immune responses are muted. However, M cells are also exploited by many enteric bacterial and viral pathogens as cellular portals that permit the pathogens to gain access to the lymph system and deeper tissues. Study M cells has been challenging because they are a minor fraction of the gut epithelium, are terminally differentiated, and until recently, could not be grown in culture. We established a differentiated human ileum organoid model system using Transwells. This allows us to induce M cell development simultaneously with other major and minor epithelial cell types and form a polarized model ileal monolayer. Infection of these model intestinal systems with the enteric pathogen, Yersinia pseudotuberculosis (Yptb) revealed that Yptb bound extensively to the apical surface of M cells, and minimally to other cell types, using its adhesin, Invasin. Yptb carries a type 3 secretion system (T3SS) that is expressed at 37°C, injects effector proteins called Yops into host cells and is essential for Yptb survival in the GI tract and Peyer’s patches. Yptb expressing the T3SS remained bound extracellularly to the apical surface of M cells in these monolayers. By contrast, Yptb was internalized and delivered 20 times more efficiently to the basolateral side in the absence of the T3SS. The combined activities of the translocated effectors, YopE, a GTPase activating protein (GAP) that inactivates Rac1, RhoG and RhoA, and YopH, a tyrosine phosphatase, prevented internalization. Strikingly, YopE caused extrusion of M cell from the monolayer by an unknown mechanism. These results demonstrate a pronounced impact of Yptb infection on M cells and provoke the hypothesis that Yptb infection of M cells induces M cell depletion, and that the loss of this component of the mucosal immune system profoundly impacts responses to subsequent immune challenge and infection. To test this hypothesis and gain insights into M cell functions at a molecular mechanistic level, we are proposing the following two aims: (1) Determine how infection with Yptb impacts M cell abundance and function in the Peyer’s patches and in pyogranuloma areas in the GI tract: and (2) Evaluate the roles of Yop targets in M cell activities, including M cell extrusion, particle internalization and transcytosis. These studies will define how infection with an enteric pathogen alters M cell physiology and functions during and after infection.