Identification of antigenic proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage λ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, α-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.     

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

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.     

Delayed Human Neutrophil Apoptosis by Trichomonas vaginalis Lysate

Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis.     

The Dimension of Trichomonas vaginalis as Measured by Scanning Electron Microscopy

It is known that physicochemical conditions (e.g., pH, temperature, and ionic strength) affect the size of trichomonads. In this study, the sizes of 4 isolates of Trichomonas vaginalis cultured for more than a year (called "old T") and 3 isolates freshly isolated from vaginitis cases (called "fresh T") were compared by scanning electron microscopy. Although the fresh T had shorter body length, body width, and flagellar length than old T, total length (about 26 µm), including body length, flagella length, and axostyle length was almost the same in the 2 groups. A striking difference was observed between the axostyles of the 2 groups; the axostyle length of the fresh T (8.2 µm) was more than twice as long as that of the old T (4.0 µm). However, in several parasitology textbooks, the length of T. vaginalis is said to vary widely from 7 to 32 µm, and its undulating membrane is said to extend about half way (53.5%) to the posterior end of the body. On the other hand, in our study, the undulating membrane was observed to extend more than 3/4 of the body length (72.1%) in old T, whereas in fresh T it could not be measured. Taken together, we suggest that T. vaginalis averages 26 (21-32) µm in total length, with 9.5 (7.4-11.4) µm of body length and 6.8 (5.3-7.7) µm of width, and its undulating membrane extending 3/4 of its body length. Therefore, these findings may provide useful information for morphological characteristics of T. vaginalis.     

Trichomonas vaginalis and trichomoniasis in the Republic of Korea

Vaginal trichomoniasis, caused by Trichomonas vaginalis, is the most common sexually transmitted disease. More than 170 million people worldwide are annually infected by this protozoan. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vaginalis. However, despite its high prevalence, the pathogenesis of T. vaginalis infection has not been clearly characterized although neutrophil infiltration is considered to be primarily responsible for the cytologic changes associated with this infection. We hypothesized that trichomonads in the vagina sometime after an acute infection secrete proteins like excretory-secretory product that have a chemotactic effect on neutrophils, and that these neutrophils are further stimulated by T. vaginalis to produce chemokines like IL-8 and GRO-alpha, which further promote neutrophil recruitment and chemotaxis. Thus, neutrophil accumulation is believed to maintain or aggravate inflammation. However, enhanced neutrophil apoptosis induced by live T. vaginalis could contribute to resolution of inflammation. Macrophages may constitute an important component of host defense against T. vaginalis infection. For example, mouse macrophages alone and those activated by lymphokines or nitric oxide are known to be involved in the extracellular killing of T. vaginalis. In the host, T. vaginalis uses a capping phenomenon to cleave host immunoglobulins with proteinases and thus escape from host immune responses. Recently, we developed a highly sensitive and specific diagnostic polymerase chain reaction (PCR) technique using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650), and found that the method enables the detection of T. vaginalis at concentrations as low as 1 cell per PCR mixture.     

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital,Argentina

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.     

PCR for diagnosis of male Trichomonas vaginalis infection with chronic prostatitis and urethritis

The aim of this study was to assess the usefulness of PCR for diagnosis of Trichomonas vaginalis infection among male patients with chronic recurrent prostatitis and urethritis. Between June 2001 and December 2003, a total of 33 patients visited the Department of Urology, Hanyang University Guri Hospital and were examined for T. vaginalis infection by PCR and culture in TYM medium. For the PCR, we used primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Voided bladder urine (VB1 and VB3) was sampled from 33 men with symptoms of lower urinary tract infection (urethral charge, residual urine sensation, and frequency). Culture failed to detect any T. vaginalis infection whereas PCR identified 7 cases of trichomoniasis (21.2%). Five of the 7 cases had been diagnosed with prostatitis and 2 with urethritis. PCR for the 5 prostatitis cases yielded a positive 330 bp band from bothVB1 and VB3, whereas positive results were only obtained from VB1 for the 2 urethritis patients. We showed that the PCR method could detect T. vaginalis when there was only 1 T. vaginalis cell per PCR mixture. Our results strongly support the usefulness of PCR on urine samples for detecting T. vaginalis in chronic prostatitis and urethritis patients.     

Prevalence and Comparison of Diagnostic Methods for Trichomonas vaginalis Infection in Pregnant Women in Argentina

The objectives of this study were to conduct a prevalence survey of trichomoniasis in pregnant women and to evaluate the utility of different methods for its diagnosis. A total of 597 vaginal exudates from pregnant women who were examined at the Hospital de Clinicas in Buenos Aires, Argentina from 1 August 2005 to 31 January 2007, were prospectively and consecutively evaluated. The investigation of Trichomonas vaginalis was made by different microscopic examinations, and culture on liquid medium. The sensitivity and specificity of the microscopic examinations were assessed considering culture on liquid medium as the "gold standard". The prevalence of T. vaginalis obtained by culture on liquid medium was 4.0% (24/597). The prevalence of T. vaginalis obtained by direct wet smear, prolonged May-Grunwald Giemsa staining, and sodium acetate-formalin (SAF)/methylene blue staining-fixing technique was 1.8%, 2.3% and 2.5%, respectively. The sensitivity of the direct wet smear was 45.8%, that of the prolonged May-Grunwald Giemsa staining was 58.3%, and that of the SAF/methylene blue method was 62.5%. Considering the 3 microscopic examinations altogether, the sensitivity rose to 66.7% and the specificity was 100% for all of them. This is the first time that the prevalence data of T. vaginalis by culture in pregnant women are published in Argentina. Due to the low sensitivity obtained by microscopy in asymptomatic pregnant women, the use of the liquid medium is recommended during pregnancy, in order to provide an early diagnosis and treatment.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

Double-stranded RNA virus in Korean isolate IH-2 of Trichomonas vaginalis

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slippage heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV IH-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.     

Prevalence of Trichomonas vaginalis by PCR in Men Attending a Primary Care Urology Clinic in South Korea

Trichomonas vaginalis, a causative agent of trichomoniasis, may trigger symptomatic or asymptomatic nongonococcal urethritis and chronic prostatitis in men. Despite the availability of highly sensitive diagnostic tests, such as nucleic acid amplification tests, including PCR, few prospective studies present data on male T. vaginalis infection in South Korea. In the present study, the prevalence of T. vaginalis and associated clinical conditions were evaluated in 201 male patients from a primary care urology clinic in South Korea. The prevalence of T. vaginalis infection in our cohort was 4% (8/201) by PCR. T. vaginalis infection was common in men older than 40 years (median age, 52 years). Among the 8 Trichomonas-positive patients, 87.5% (7/8) had prostatic diseases, such as prostatitis and benign prostatic hyperplasia, and 25.0% (2/8) and 12.5% (1/8) were coinfected with Chlamydia trachomatis and Mycoplasma genitalium, respectively. Our results suggest that T. vaginalis infection is not rare in men attending primary care urology clinics in South Korea, especially in those older than 40 years, in whom it may explain the presence of prostatic disease. The possibility of T. vaginalis infection should be routinely considered in older male patients with prostatic diseases in South Korea.     

Histamine and TNF-alpha release by rat peritoneal mast cells stimulated with Trichomonas vaginalis

Mast cells have been reported to be predominant in the vaginal smears of patients infected with T. vaginalis. In this study, we investigated whether T. vaginalis could induce mast cells to migrate and to produce TNF-alpha and histamine. Rat peritoneal mast cells (RPMC), a primary mast cell, were used for the study. T. vaginalis induced an increase in chemotactic migration of the mast cells toward excretory and secretory product (ESP) of T. vaginalis, and the mast cells activated with T. vaginalis showed an increased release of histamine and TNF-alpha. Therefore, mast cells may be involved in the inflammatory response caused by T. vaginalis.     

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

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.     

Comparative study of epitopes recognized by two monoclonal antibodies that protects mice against Trichomonas vaginalis challenge

Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.     

Trichomonas vaginalis: in vitro survival in swimming pool water samples

In this work it is shown that Trichomonas vaginalis remains viable and infective in swimming pool water samples for several hours. After survival, trichomonad cytotoxicity was tested on primary cultures of epithelial cells. It demonstrates that the some trichomonad strains are able to survive in water pools and survival time is dependent on the degree of strain infectivity and also if it is a long term cultured or fresh isolate.     

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis

The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.     

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 by HMDM. The involvement of nuclear factor (NF)-κB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-κB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-κB activation and TNF-α production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-κB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-α. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, and NO. In particular, we showed that T. vaginalis induced TNF-α production in macrophages through NO-dependent activation of NF-κB, which might be closely involved in inflammation caused by T. vaginalis.