Conference Agenda

Trichomonas vaginalis is a protozoan parasite responsible for the most common non-viral sexually transmitted infection, trichomoniasis. T. vaginalis has striking motility driven by its 5 flagella. We hypothesize that T. vaginalis motility plays a key role in mediating infection. To investigate this, we performed the first characterization of kinesin-2 proteins in T. vaginalis. Kinesin-2 proteins are motor proteins that mediate anterograde intraflagellar transport that is necessary for flagellar assembly and maintenance. To identify putative flagellar-localized kinesin-2 proteins, we performed bioinformatic analysis and identified five putative kinesin-2 genes with similarity to kinesins found in the flagella of other single cell protozoan parasites and the flagellar model organism Chlamydomonas reinhardtii. Utilizing NEB HiFi DNA assembly, fusion proteins of the candidate genes were generated with two C-terminal HA tags and mNeonGreen, a green fluorescent protein. Using confocal microscopy, we found that two putative kinesin-2 fusion proteins indeed localize to the flagella. We are in the process of optimizing CRISPR/Cas9 methodology to knockout these genes, and an update on these research efforts will be presented. Our work will generate novel knowledge about the cellular biology of T. vaginalis flagella and the contribution of T. vaginalis motility towards pathogenesis.

The sexually transmitted parasite Trichomonas vaginalis (Tv) secretes extracellular vesicles (TvEVs) which are internalized by host cell and mediate immune responses. To identify the underlying mechanisms driving TvEV induced responses, we analyzed the effect of TvEVs on host gene expression. RNA-seq analyses revealed that TvEVs have a suppressive effect on host cell innate immune gene expression. Interestingly, interferon epsilon (IFNε), a cytokine which is constitutively expressed by vaginal epithelial cells, except in peri and post-menopausal women where it is undetectable, is strongly downregulated. Epidemiological studies showing that Tv positivity is unusually high in older women suggest that physiological and immunological changes make the female reproductive tract of older women more conducive to infection. Thus, we tested the effect of pretreatment of host cells with IFNε on Tv infection. We found that pretreatment is protective against Tv killing of host cells, which may, at least in part, explain why older women are more susceptible to Tv infection. We also showed that TvEVs block IFNε-mediated nuclear translocation of transcription factors pSTAT1 and pSTAT2 and increase host cell killing and parasite burden in vitro. Further studies will elucidate how IFNε protects against Tv infection and how TvEVs downregulate IFNε to promote infection.

The parasite plays critical roles in the training and development of major components of the host’s innate and adaptive immune system. Stimulation with Trichomonas vaginalis-derived secretory products (TvSP), which contains LTB₄, induces NOX2 activation and ROS generation in human mast cells through BLT1-mediated signaling. NOX2 activity is known to be regulated by the proteasomal degradation of NOX2. Here, we investigated whether proteasome-ubiquitin pathway is associated with maturation of NOX2 and its trafficking to the cell surface in TvSP- or LTB₄-stimulated human mast cells. Stimulation with TvSP or LTB₄ for 30 min increased matured form of NOX2 protein and its surface trafficking, which was prevented by pretreatment of cells with BLT1 antagonist. When HMC-1 cells were stimulated with TvSP or LTB₄ for up to 30 min, ubiquitinated proteins and proteasome activity were significantly reduced compared with results for cells incubated with medium alone. Furthermore, treatment of quiescent cells with proteasome inhibitor alone augmented expression of NOX2 and its migration to the cell surface. In IP assay, stimulation with TvSP or LTB₄ diminished amount of ubiquitinated NOX2 compared with control. These results suggest that signaling talk between human mast cells and T. vaginalis provides example of BLT1-mediated maturation mechanism of NOX2.

The host immune response to Trichomonas vaginalis involves the chemokine IL-8 production at the site of infection. Previously, we reported that LTB₄ contained in T. vaginalis-derived secretory products (TvSP) play an essential role in IL-8 production in human mast cell via LTB₄ receptor BLT1 or BLT2. Dynamin, a GTPase, is involved in endocytosis of surface receptors for signaling of production of cytokine or chemokines. In this study, we investigated the role of dynamin in the BLT-dependent IL-8 production induced by T. vaginalis-secreted LTB_4. Pretreatment of HMC-1 cells with a dynamin inhibitor reduced BLT-dependent IL-8 production and NF-kB phosphorylation induced by TvSP or LTB₄. In addition, the exposure of HMC-1 cells with TvSP or LTB₄ for up to 60 min, BLT1 translocated from intracellular compartments to the plasma membrane at 30 minutes. At 60 min after stimulation with TvSP, BLT1 migrated from the cell surface to the intracellular areas. In dynamin-2 siRNA-knockdown cells, TvSP-induced BLT1 internalization was nicely prevented. In co-IP experiment, we also found that dynamin-2 was physically interacted with BLT1 at 60 min after stimulation with TvSP or LTB₄. These results suggest that T. vaginalis-secreted LTB₄ induces IL-8 production via dynamin-mediated endocytosis of BLT1 in HMC-1 cells.