The interplay between enterobacteria-mediated inhibition of intestinal epithelial cell extrusion and activation of multiple inflammasomes.

About This Grant

PROJECT SUMMARY In 2019, Escherichia coli (E. Coli) alone was responsible for nearly 1 million deaths worldwide. Difficulties in vaccination and therapeutic development have exposed the need for research on innate immune mechanisms in the intestinal epithelium considering E. coli is usually contracted via an oral route. Among other important defenses, enteropathogenic bacteria like E. coli activate an innate immune sensor termed the NAIP-NLRC4 inflammasome. This leads to inflammatory cell death termed pyroptosis and extrusion of the infected epithelial cell into the lumen of the intestines, which prevents further dissemination of bacteria and maintains the integrity of the epithelium. Several other inflammasomes have been implicated in enteropathogenic infection, however functional redundancies and steady state roles have not been clarified. I propose to study which inflammasomes from this family of sensors are activated and protective during E. coli infection beyond NAIP-NLRC4. Our lab has found that infection with the E. coli murine model, Citrobacter rodentium (C. rodentium) in the absence of the NAIP-NLRC4 inflammasome and the non-canonical inflammasome Caspase 11 still induces pyroptosis. This pyroptosis is accompanied by “specks” of the inflammasome adaptor ASC, indicating that another inflammasome may be active in intestinal epithelial cells (IECs). This project will assess the contribution of another inflammasome, NLRP1, to the host response. NLRP1 is a prime candidate as it is expressed and protective at other barrier tissues, can be activated by bacterial effectors, and has been shown to form ASC specks. This also implies strong evolutionary pressure on the pathogen to circumvent these redundant sensing pathways. I will inquire if E. coli can inhibit multi-inflammasome activation by specifically targeting downstream outcomes of inflammasome activation, such as extrusion. I found that the C. rodentium actin nucleating effector, Tir, reduces the host cell's ability to extrude, a highly actin dependent process. I hypothesize that the NLRP1 inflammasome activation in the intestinal epithelium is protective during C. rodentium infection, however C. rodentium effector Tir is able to circumvent the inflammasome by inhibiting IEC extrusion. To investigate this, my first aim will evaluate the role for NLRP1 in inducing pyroptosis during live imaging of primary colonic monolayer infection by C. rodentium, and the temporal characteristics of the extrusion. Furthermore, using NLRP1 knockout mice I will investigate the contribution of NLRP1 to maintaining protection from C. rodentium in vivo. In my second aim, I plan to evaluate the characteristics of extrusion including timing and actin structure, following infection with a C. rodentium mutant Tir strain. This work will use cutting edge stem cell derived organoid and imaging tools to reveal inflammasome pathway redundancy during enteropathogenic infection and the countermeasures C. rodentium takes to quell this host function. With guidance from my sponsor and cosponsor Dr. Rauch and Dr. Merritt, and the resources at Oregon Health & Science University, I will be able to accomplish these aims and work toward my career goals.