Description

The protozoan parasite T. vaginalis causes Trichomoniasis, the most common non-viral, sexually transmitted infection (STI) in the world. T. vaginalis infections have been found to induce pyroptosis, a pro-inflammatory cell death in human macrophages. However the molecular mechanisms of infection of female reproductive tract (FRT) cells is poorly understood. We hypothesize that T. vaginalis induces pyroptotic cell death in epithelial cells of the female reproductive tract. Pyroptosis is executed by gasdermin D (GSDMD) cleavage by an inflammatory caspase protease. To begin to investigate if pyroptosis is activated upon infection of female reproductive tract epithelial cells, we co-incubated T. vaginalis with ectocervical (Ect-1) or vaginal (VK2) epithelial cells during a 2, 4 and 6 hrs infection time series. At each time point we collected whole cell lysates (WCL) and performed western blot analysis utilizing an anti-GSDMD antibody. We detected the presence of full-length GSDMD (~53 kDa) in Ect-1 and VK2 cell lines under uninfected and infected conditions. Upon T. vaginalis infection, we also detected the presence of the N-terminal GSDMD cleavage fragment (~31 kDa). Furthermore, as the multiplicity of infection (MOI) ratio of T. vaginalis: host cell was increased, an increased amount of the GSDMD N-terminal cleavage fragment was also detected, with densitometric analysis revealing that the GSDMD cleavage fragment was present at 3-fold higher levels in the 5:1 MOI compared to the 1:1 ratio. To further test the specificity of the detected GSDMD cleavage product, we performed an immunoprecipitation of WCLs collected from VK2 cells exposed to T. vaginalis and an update on these results will be presented. Our results have identified expression of GSDMD in Ect-1 and VK2 cells and novel GSDMD cleavage during infection of Ect-1 cells with T. vaginalis. Detection of the GSDMD N-terminal cleavage fragment during T. vaginalis and Ect-1 co-incubation may implicate pyroptosis as a potential mechanism for cell death in ectocervical epithelium. Together, the results of our work investigating the molecular mechanisms contributing to T. vaginalis pathogenesis will contribute to an increased understanding of trichomoniasis and may aid in development of new pharmacological treatments, a growing necessity as strains acquire antibiotic resistance.