RESEARCH PROJECT-RIESTRA/SUSSMAN - Project Summary The public health threat posed by infection with the sexually transmitted parasite Trichomonas vaginalis, its disproportionate affliction of racial and ethnic minorities, and its vastly understudied status have led to classification of trichomoniasis as one of our country’s most neglected parasitic infection. The first part of this research project will investigate the molecular mechanisms by which T. vaginalis kills Lactobacillus crispatus and Lactobacillus iners-cervicovaginal bacteria that generally have a protective role in the female reproductive tract. Specifically, we will observe the parasite-lactobacilli interactions using live/dead staining and assess the dynamics of bacterial killing. Using pharmacological inhibitors and by generation of clathrin light chain knockout parasites, we will determine the contribution of phagocytosis towards T. vaginalis antibacterial activity. A second part of our project will assess if T. vaginalis infection-mediated activation of the inflammatory cell death called pyroptosis leads to indirect antibacterial effects against lactobacilli. Specifically, we will test whether a recombinant form of the gasdermin D N-terminal cleavage fragment has antibacterial effects against lactobacilli that constitute the different cervicovaginal microbial communities present in women. Additionally, using ectocervical gasdermin D knockout cells we will further delineate how induction of pyroptotic cell death during T. vaginalis infection contributes to killing of cervicovaginal lactobacilli, and whether killing of these protective bacteria results in increased inflammation. Lastly, we will investigate the interaction of T. vaginalis with L. crispatus, L. iners, and human vaginal cells using a vagina-on-a-chip model. Indirect immunofluorescence assays of these chips will reveal the spatial organization of these microbes on stratified vaginal epithelium. We will also quantify the differential viability of each microbe upon triad interaction compared to colonization with each single microbe. Lastly, we will profile differences in secretion of inflammasome-mediated cytokines and processed gasdermin D release. Together, our work will elucidate how T. vaginalis exerts antibacterial effects during infection and how it mechanistically promotes inflammation, processes that are associated with increased predisposition of women to adverse health effects and which are currently untreated with standard trichomoniasis treatment. Our research efforts could thus help guide the development of future targeted therapeutics to ameliorate the impact of T. vaginalis infection, particularly those from lower socioeconomic and underrepresented backgrounds disproportionately affected by T. vaginalis infection.