Genetic identity and differential gene expression between Trichomonas vaginalis and Trichomonas tenax

Background  

Trichomonas vaginalis is a human urogenital pathogen responsible for trichomonosis, the number-one, non-viral sexually transmitted disease (STD) worldwide, while T. tenax is a commensal of the human oral cavity, found particularly in patients with poor oral hygiene and advanced periodontal disease. The extent of genetic identity between T. vaginalis and its oral commensal counterpart is unknown.     

Characterization of the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> surface-associated AP65 and binding domain interacting with trichomonads and host cells

Background  

AP65 is a prominent adhesin of Trichomonas vaginalis that mediates binding of parasites to host vaginal epithelial cells (VECs). AP65 with no secretion signal sequence, membrane targeting peptide, and anchoring motif was recently found to be secreted.     

Heterologous expression in <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> of functional <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> AP65 adhesin

Background  

Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.     

Antisense RNA decreases AP33 gene expression and cytoadherence by <Emphasis Type="Italic">T. vaginalis</Emphasis>

Background  

Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis

Background

Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis.

Principal Findings

This study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1.

Significance

These results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption.     

Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity

Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 μM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole‐resistant T. vaginalis.     

Oleate lipase activity in Gardnerella vaginalis and reconsideration of existing biotype schemes

Background  

Gardnerella vaginalis is a facultative gram positive organism that requires subculture every 1–2 days to maintain viability. It has been linked with bacterial vaginosis (BV), a syndrome that has been associated with increased risk for preterm delivery, pelvic inflammatory disease and HIV acquisition. About 10% of the G. vaginalis isolates have been reported to produce sialidase, but there have not been any studies relating sialidase production and biotype. Sialidase activity is dramatically increased in the vaginal fluid of women with BV and bacterial sialidases have been shown to increase the infectivity of HIV in vitro. There are 8 different biotypes of G. vaginalis. Biotypes 1–4 produce lipase and were reported to be associated with BV and the association of these biotypes with BV is under dispute. Other studies have demonstrated that G. vaginalis biotype 1 can stimulate HIV-1 production. Because of the discrepancies in the literature we compared the methods used to biotype G. vaginalis and investigated the relationship of biotype and sialidase production.     

Effect of the Antimicrobial Peptide Tritrpticin on the In Vitro Viability and Growth of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>

Antimicrobial peptides are widely distributed in nature; they play important roles in several aspects of innate immunity and may provide a basis for the design of novel therapeutic agents. In this study, C-amidated tritrpticin, a 13 amino acid tryptophan-rich antimicrobial peptide derived from a porcine cathelicidin, was tested against Trichomonas vaginalis, a protozoan that causes a serious non-viral sexually transmitted disease associated with preterm birth, low birth weight, and high risk of HIV-1 infection. Tritrpticin was selected due to its reasonably easy synthesis and because analogs with lower toxicity may be designed. Our results show that tritrpticin-NH₂ at either 100 or 200 μg/ml (52.5 or 105 μM) clearly reduces the viability and growth of Trichomonas vaginalis. Together with tritrpticin-NH₂, sodium bicarbonate further limited trichomonad growth. Additionally, a low concentration of metronidazole (5.8 μM), the most commonly used medication for Trichomonas vaginalis, was more effective against the growth of the parasite when it was combined with tritrpticin-NH₂.     

Extensive genetic diversity, unique population structure and evidence of genetic exchange in the sexually transmitted parasite Trichomonas vaginalis

Background

Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite''s genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes.

Methodology/Principal Findings

Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages.

Conclusions/Significance

Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.     

T. vaginalis Infection Is Associated with Increased IL-8 and TNFr1 Levels but with the Absence of CD38 and HLADR Activation in the Cervix of ESN

Introduction

Trichomonas vaginalis infection is associated with an increased risk of HIV infection in exposed-seronegative women (ESN) despite their unique immune quiescent profile. It is important to understand possible mechanisms, such as recruitment of activated T cells, by which T. vaginalis could facilitate HIV infection in this population.

Methods

We conducted a cross-sectional study exploring the relationships between T. vaginalis infection, inflammatory markers and T cell activation in the cervix of ESN. During scheduled study visits, participants completed a behavioral questionnaire and physical exam, including sexually transmitted infection (STI) screening and collection of endocervical sponge and cytobrush specimens. T cell and monocyte phenotypes were measured in cervical cytobrush specimens using multi-parameter flow cytometry. Cervical sponge specimens were used to measure cytokines (IL-6, IL-8,IL-10, IP-10, RANTES) using Luminex immunoassays and the immune activation marker soluble TNF receptor 1 using ELISA.

Results

Specimens of 65 women were tested. Twenty-one of these women were infected with T. vaginalis. T. vaginalis infection was associated with significantly increased concentrations of IL-8 (1275pg/ml vs. 566pg/ml, p=.02) and sTNFr1 (430 pg/ml vs. 264 pg/ml, p=.005). However, T. vaginalis infection was not associated with increased percent expression of CCR5+ T cells nor increased CD38 and HLADR activation compared to uninfected women. It was also not associated with increased expression of CCR5+ monocytes.

Conclusions

Among ESN T. vaginalis infection is associated with increased levels of genital pro-inflammatory/immune activation markers IL-8 and TNFr1, but was not associated with an increased percentage of activated endocervical T cells along the CD38 and HLADR pathways. Thus, while T.vaginalis infection may result in some reversal of the immune quiescent profile of ESN, enhanced recruitment of activated CD38 and HLADR expressing CD4+ cells into the endocervix may not be part of the mechanism by which Trichomonas infection alters HIV susceptibility in this unique subset of women.     

Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between <Emphasis Type="Italic">Atopobium vaginae</Emphasis>, <Emphasis Type="Italic">Gardnerella vaginalis</Emphasis> and bacterial vaginosis

Background  

The pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora.     

A Freeze-Fracture Electron Microscope Study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller)1

Two strains of Trichomonas vaginalis, JH162A, with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces (PFs) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad. In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of ～9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae, as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta-type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.     

Trichomonas vaginalis Resistance to Mengo Virus Infection*

We have investigated the susceptibility of Trichomonas vaginalis to Mengo virus infection by comparing the outcome of Mengo virus or purified Mengo virus RNA infection in T. vaginalis and in CCL-1 mouse fibroblasts. While the adsorption and entry of Mengo virus into T. vaginalis occurred in the same manner as in fibroblasts, the uncoating was much slower. In addition, Mengo virus infection of T. vaginalis displayed no eclipse nor any subsequent production of infectious virus. Purified RNA failed to initiate productive infection in T. vaginalis, whereas it provoked viral replication in the fibroblast controls. It was shown by assessment of protein synthesis in T. vaginalis and mouse fibroblasts cell-free systems that the protozoan ribosomes were able to translate endogenous mRNA and poly-U, but not viral RNA.     

Quantitative determination by real-time PCR of four vaginalLactobacillus species,Gardnerella vaginalisandAtopobium vaginaeindicates an inverse relationship betweenL. gasseriandL. iners

Background  

Most studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats forLactobacillus crispatus,L. gasseriandGardnerella vaginalisand developed new formats forAtopobium vaginae,L. inersandL. jenseniito obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years.     

Trichomonas vaginalis and early evolving DNA and protein sequences of the CDC2/28 protein kinase family

The human sexually transmittted parasite Trichomonas vaginalis is a representative of one of the three earliest evolving eukaryotic lineages. We investigated whether T. vaginalis has DNA sequences and peptides related to cell division control molecules universal among yeasts and higher eukaryotes. A T. vaginalis ceil division control (CDC2/28) homologue was amplified by the polymerase chain reaction and sequenced. The absolute similarity with other CDC2/28 genes was 47%, with conservative replacement similarity of 67%. Western blots demonstrated a single T. vaginalis peptide reactive with antiserum to the PSTAIRE peptide, an expressed component of CDC2/28 genes in higher eukaryotes. Although eukaryotic, T. vaginalis has properties similar to those of bacteria and is the earlist evolving eukaryote reported to possess CDC2/28 DNA and peptide homologues. These observations suggest that the molecular origins of cell division control in eukaroytes preceded mitochondria, 28S ribosomes and regulated glycolysis.     

Evaluating HIV Prevention Programs: Herpes Simplex Virus Type 2 Antibodies as Biomarker for Sexual Risk Behavior in Young Adults in Resource-Poor Countries

Background

Measuring effectiveness of HIV prevention interventions is challenged by bias when using self-reported knowledge, attitude or behavior change. HIV incidence is an objective marker to measure effectiveness of HIV prevention interventions, however, because new infection rates are relatively low, prevention studies require large sample sizes. Herpes simplex virus type 2 (HSV-2) is similarly transmitted and more prevalent and could thus serve as a proxy marker for sexual risk behavior and therefore HIV infection.

Methods

HSV-2 antibodies were assessed in a sub-study of 70,000 students participating in an education intervention in Western Province, Kenya. Feasibility of testing for HSV-2 antibodies was assessed comparing two methods using Fisher’s exact test. Three hundred and ninety four students (aged 18 to 22 years) were randomly chosen from the cohort and tested for HIV, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. Out of these, 139 students were tested for HSV-2 with ELISA and surveyed for sexual risk behavior and 89 students were additionally tested for HSV-2 with a point-of-contact (POC) test.

Results

Prevalence rates were 0.5%, 1.8%, 0.3% and 2.3% for HIV, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis, respectively. Prevalence of HSV-2 antibodies was 3.4 % as measured by POC test (n=89) and 14.4 % by ELISA (n=139). Specificity of the POC test compared with ELISA was 100%, and the sensitivity only 23.1%. Associations between self-reported sexual behavior and HSV-2 serostatus could not be shown.

Conclusions

Associations between self-reported sexual risk behavior and HSV-2 serostatus could not be shown, probably due to social bias in interviews since its transmission is clearly linked. HSV-2 antibody testing is feasible in resource-poor settings and shows higher prevalence rates than other sexually transmitted diseases thus representing a potential biomarker for evaluation of HIV prevention interventions.