Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40–70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis.

Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30–150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

Levels of most amino acids were higher in GR culture. Arginine was not detectable in either HG or GR cultures; however, its metabolic end-product proline was slightly increased under GR, suggesting that the arginine dihydrolase pathway was more activated by GR. Additionally, methionine catabolism was less stimulated under GR because of greater methionine accumulation. Furthermore, branched chain amino acids (BCAA), including leucine, isoleucine and valine, as well as phenylalanine and alanine, markedly accumulated under GR, indicating that glutamate-related metabolic pathways were remarkably enhanced in this setting. Our metabolomic analysis combined with previous RNA-seq data confirm the existence of several amino acid metabolic pathways in the hydrogenosome and highlight their potentially important roles in T. vaginalis under glucose deprivation.

Background: Iron plays essential roles in the pathogenesis and proliferation of Trichomonas vaginalis, the causative agent of the most prevalent non-viral human sexually transmitted infection. We previously demonstrated that under iron deficiency, the endogenous nitric oxide (NO) is accumulated and capable of regulating the survival of T. vaginalis. Herein, we aim to explore the influence of NO on the activity of the pyruvate-reducing enzyme lactate dehydrogenase in T. vaginalis (TvLDH).Methods: Levels of lactate and pyruvate were detected for determining glycolysis activity in T. vaginalis under iron deficiency. Quantitative PCR was performed to determine the expression of TvLDH. S-nitrosylated (SNO) proteomics was conducted to identify the NO-modified proteins. The activities of glyceraldehyde-3-phosphate dehydrogenase (TvGAPDH) and TvLDH were measured after sodium nitrate treatment. The effects of protein nitrosylation on the production of cellular reducing power were examined by measuring the amount of nicotinamide adenine dinucleotide (NAD) and the ratio of the NAD redox pair (NAD+/NADH).Results: We found that although the glycolytic pathway was activated in cells under iron depletion, the level of pyruvate was decreased due to the increased level of TvLDH. By analyzing the SNO proteome of T. vaginalis upon iron deficiency, we found that TvLDH is one of the glycolytic enzymes modified by SNO. The production of pyruvate was significantly reduced after nitrate treatment, indicating that protein nitrosylation accelerated the consumption of pyruvate by increasing TvLDH activity. Nitrate treatment also induced NAD oxidation, suggesting that protein nitrosylation was the key posttranslational modification controlling cellular redox status.Conclusions: We demonstrated that NO-mediated protein nitrosylation plays pivotal roles in the regulation of glycolysis, pyruvate metabolism, and the activity of TvLDH. The recycling of oxidized NAD catalyzed by TvLDH provided the reducing power that allowed T. vaginalis to adapt to the iron-deficient environment.

Background Iron plays essential roles in the pathogenesis and proliferation of Trichomonas vaginalis, the causative agent of the most prevalent non-viral human sexually transmitted infection. We previously demonstrated that under iron deficiency, the endogenous nitric oxide (NO) is accumulated and capable of regulating the survival of T. vaginalis. Herein, we aim to explore the influence of NO on the activity of the pyruvate-reducing enzyme lactate dehydrogenase in T. vaginalis (TvLDH). Methods Levels of lactate and pyruvate were detected for determining glycolysis activity in T. vaginalis under iron deficiency. Quantitative PCR was performed to determine the expression of TvLDH. S-nitrosylated (SNO) proteomics was conducted to identify the NO-modified proteins. The activities of glyceraldehyde-3-phosphate dehydrogenase (TvGAPDH) and TvLDH were measured after sodium nitrate treatment. The effects of protein nitrosylation on the production of cellular reducing power were examined by measuring the amount of nicotinamide adenine dinucleotide (NAD) and the ratio of the NAD redox pair (NAD+/NADH). Results We found that although the glycolytic pathway was activated in cells under iron depletion, the level of pyruvate was decreased due to the increased level of TvLDH. By analyzing the SNO proteome of T. vaginalis upon iron deficiency, we found that TvLDH is one of the glycolytic enzymes modified by SNO. The production of pyruvate was significantly reduced after nitrate treatment, indicating that protein nitrosylation accelerated the consumption of pyruvate by increasing TvLDH activity. Nitrate treatment also induced NAD oxidation, suggesting that protein nitrosylation was the key posttranslational modification controlling cellular redox status. Conclusions We demonstrated that NO-mediated protein nitrosylation plays pivotal roles in the regulation of glycolysis, pyruvate metabolism, and the activity of TvLDH. The recycling of oxidized NAD catalyzed by TvLDH provided the reducing power that allowed T. vaginalis to adapt to the iron-deficient environment.