Conference Agenda

Trichomonas vaginalis is a sexually transmitted parasite that colonizes the human urogenital tract. As an extracellular pathogen, adherence to host cells is an important step to colonize the human host. Host: parasite interaction is regulated by changes in gene expression, but it is still largely unknown how these changes in transcriptional profiles are controlled. Our recent works highlighted the importance of epigenetics in the regulation of transcription. We identified transcriptionally active or repressive intervals flanked by 6mA-enriched regions, which are in close spatial proximity, suggesting a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression. We now aim to analyze the role of 6mA and chromatin during the host-parasite interaction. We compare MedIP-seq data with ATAC-seq and RNA-seq data in free vs. host cell-attached parasites. We identified 2000 active/repressive intervals flanked by 6mA in the presence and absence of host cells, suggesting a role for 6mA in modulating 3D genome architecture through the formation of chromatin loops. Interestingly, our data showed differentially accessible chromatin regions and differentially expressed genes in pathogenesis-related genes when T. vaginalis is exposed to host cells. These findings underscore the importance of chromatin structure and its role in gene regulation during parasite infection.

To establish infection, T. vaginalis expresses diverse proteins/virulence factors targeted to the trichomonad’s surface and secreted into the vaginal environment. These factors interact with human cells and bacteria, namely lactobacilli, whose population decreases during acute trichomoniasis. We found that T. vaginalis significantly increased the secretion of 19 proteins in response to Lactobacillus jensenii, six of which belong to the TBSR family. This family comprises over 200 members, sorted into two groups based on the presence of a large domain of unknown function (lDUF) and several subgroups based on the number of cadherin-like beta-sheet repeats, presence of a transmembrane domain with a cytosolic tail, and a small domain of unknown function (sDUF). AlphaFold was used to predict potential interacting partners of TBSR1. Beta-catenins appeared among the best candidates to interact with the cytosolic tail, while a surface chaperone-like protein of L. jensenii was predicted to interact with the sDUF. Using Foldseek, the lDUF was identified as part of glycan hydrolases. AutoDock Vina and Alphafold predicted branched N-acetylglucosamines in mucins to represent the best lDUF binding candidates. Altogether, our predictions highlight TBSRs as prominent membrane-associated and secreted proteins potentially involved in recognizing and engulfing lactobacilli and other interactions within the vaginal environment.

Tetraspanins (TSPANs) are a family of proteins highly conserved in all eukaryotes, and known to be involved in adhesion, migration, and intracellular signaling. TSPANS are also often regarded as a marker for extracellular vesicles. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

Entamoeba histolytica is an intestinal protozoan parasite and causes amebiasis, which affects 50 million people annually. Phosphatidyl inositol (PI) metabolism is essential for signaling and trafficking in general and also essential for survival, proliferation, stage conversion, and pathogenesis of this organism. Among PI and phosphorylated PIs, phosphatidylinositol 3-phosphate (PI3P) is in general involved in endocytic and autophagic pathways. PI3P is generated from PI by class III PI-3kinase (C3PI3K). As the E. histolytica genome encodes a single C3PI3K, it is conceivable this gene is expected to play a pivotal role in biology and pathogenesis. However, it remains elusive how C3PI3K regulates endocytosis and autophagy and what accessory molecules are involved in its regulation in E. histolytica. In this study, we identified two major components of C3PI3K, vps34 (Ehvps34) and p150 (Ehp150), from E. histolytica. We also identified a unique E. histolytica-specific C3PI3K component, unlike mammalian counterparts (forming a heterotetramer), by coimmunoprecipitation followed by mass spectrometric analysis. We also demonstrated that EhC3PI3K is necessary for growth. Furthermore, we showed that EhC3PI3K localizes on the cell periphery by confocal laser microscopy. We presume that EhC3PI3K is involved in the endosome maturation, and are further investigating the mechanism how EhC3PI3K regulates endocytosis and autophagy.