Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V⁺) and uninfected (V⁻) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V⁺ compared with V⁻ isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V⁺ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V⁺ and V⁻ isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.
Glucose-6-Phosphate Isomerase ; Glycogen Phosphorylase ; Heat-Shock Proteins ; Host-Parasite Interactions ; Malate Dehydrogenase ; Metabolism ; Polymerase Chain Reaction ; Proteome ; Reticuloendotheliosis virus ; Ribosomal Proteins ; RNA, Double-Stranded ; RNA, Messenger ; Trichomonas vaginalis* ; Trichomonas* ; Triose-Phosphate Isomerase ; Virulence

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.
Area Under Curve ; Bacteria/*genetics/isolation & purification ; Bacterial Proteins/genetics ; Candida/*genetics/isolation & purification ; Female ; Fungal Proteins/genetics ; Gardnerella vaginalis/genetics/isolation & purification ; High-Throughput Nucleotide Sequencing ; Humans ; *Microbiota ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; ROC Curve ; Sequence Analysis, DNA ; Trichomonas vaginalis/genetics/isolation & purification ; Vagina/*microbiology ; Vaginitis/*diagnosis/microbiology

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.
Animals ; Antibodies/metabolism ; Cell Proliferation/drug effects ; Epithelial Cells/parasitology ; Host-Pathogen Interactions/drug effects/*physiology ; Iron/pharmacology ; Mice ; Pyruvate Synthase/*metabolism ; Rabbits ; Trace Elements/pharmacology ; Trichomonas Infections/*parasitology ; Trichomonas vaginalis/drug effects/genetics/metabolism/*pathogenicity

Trichomonas vaginalis induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-alpha production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-alpha production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-alpha production was significantly decreased compared to the control; however, TNF-alpha reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-alpha production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-alpha production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.
Cell Line ; Cervix Uteri/enzymology/metabolism/*parasitology ; Epithelial Cells/*enzymology/metabolism/parasitology ; Female ; Humans ; *MAP Kinase Signaling System ; Mucous Membrane/*enzymology/metabolism/parasitology ; Phosphatidylinositol 3-Kinases/genetics/*metabolism ; Proto-Oncogene Proteins c-akt/genetics/*metabolism ; Trichomonas Vaginitis/*enzymology/genetics/metabolism/parasitology ; Trichomonas vaginalis/*physiology ; Tumor Necrosis Factor-alpha/genetics/*metabolism

Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47phox in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling.
Cell Degranulation ; Cell Line ; *Exocytosis ; Humans ; Mast Cells/*drug effects/*metabolism ; Mitogen-Activated Protein Kinases/*metabolism ; Reactive Oxygen Species/*metabolism ; Trichomonas vaginalis/*metabolism ; Virulence Factors/*metabolism

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.
Blotting, Western ; Cell Line, Tumor ; Epithelial Cells/metabolism/parasitology ; Humans ; Metalloproteases/genetics/*metabolism ; Proteolysis ; Sequence Analysis, DNA ; TOR Serine-Threonine Kinases/*metabolism ; Trichomonas vaginalis/*enzymology/genetics

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2(.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.
Anions/*metabolism ; Female ; Humans ; Neutrophils/enzymology/*metabolism/parasitology ; Peroxidase/metabolism ; Superoxides/*metabolism ; Trichomonas Infections/enzymology/*metabolism/parasitology ; Trichomonas vaginalis/*isolation & purification/physiology

Trichomonas vaginalis is a flagellated lumen-dwelling extracellular protozoan parasite that causes human trichomoniasis via sexual intercourse. Human neutrophils play a crucial role in acute tissue inflammatory responses in T. vaginalis infection. In this study, we investigated the signaling mechanism of neutrophil responses when stimulated with T. vaginalis-derived secretory products (TvSP), which were collected from 1x10(7) live trichomonads. Incubation of human neutrophils isolated from peripheral blood with TvSP induced up-regulation of IL-8 protein secretion. In addition, stimulation with TvSP induced phosphorylation of NF-kappaB and CREB in neutrophils. Moreover, TvSP-induced IL-8 production was also significantly inhibited by pretreatment of neutrophils with ikappaB inhibitor or CREB inhibitor. These results suggest that transcription factors NF-kappaB and CREB are involved in IL-8 production in human neutrophils induced by stimulation with T. vaginalis infection.
Cyclic AMP Response Element-Binding Protein/*metabolism ; Human Experimentation ; Humans ; Interleukin-8/*metabolism ; Male ; NF-kappa B/*metabolism ; Neutrophils/*immunology ; Phosphorylation ; Trichomonas vaginalis/*immunology

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.
Trichomonas vaginalis/*growth & development ; Reverse Transcriptase Polymerase Chain Reaction ; Pyruvate Synthase/genetics/metabolism ; Organelles/*enzymology/metabolism/*physiology ; Membrane Potentials ; Malate Dehydrogenase/genetics/metabolism ; Iron/*metabolism ; Hydrogenase/genetics/metabolism ; Hydrogen/*metabolism ; Humans ; Gene Expression Regulation, Enzymologic ; *Gene Expression Regulation ; Ferredoxins/genetics/metabolism ; Culture Media ; Animals

OBJECTIVETo investigate the effects of the metabolite produced by Trichomonas vaginalis on human sperm motility in vitro.

METHODSTrichomonas vaginalis having been cultured, the culture solution containing metabolite was obtained by removing the protozoa, then diluted into 3 kinds of concentration. Sperm was obtained from 10 healthy fertile men by masturbation and prepared by swim-up technique to produce a spermatozoon solution of high motility. Every sperm sample was divided into 4 groups (A, B, C, D). Unused culture solution was added to Group A as control, and the other 3 groups (B, C, D) were respectively incubated with the above used culture solution at 3 kinds of concentration (1.2 x 10(9)/L, 6 x 10(8)/L, 1.2 x 10(8)/L). Measurements were carried out at 30 s, 1 h, 2 h, 4 h, 6 h by CASA.

RESULTSSperm motility decreased in both Group B and C markedly, and the effects displayed a concentration- and time-dependent manner.

CONCLUSIONThe metabolite of Trichomonas vaginalis can reduce human sperm motility in vitro, and may be one of the causes of infertility.