Point-of-Care Diagnostic for Trichomonas vaginalis, the Most Prevalent, Non-Viral Sexually Transmitted Infection.

A point-of-care (POC) diagnostic is needed for both women and men to establish universal screening and surveillance for the number one, non-viral sexually transmitted infection (STI) caused by Trichomonas vaginalis. We developed a POC diagnostic for this STI using the MedMira Rapid Vertical Flow (RVF®) Technology test cartridge with a membrane that includes a Vertical procedural/reagent control line (referred to as CVL) and spotted with 1 µg of a 72.4-kDa truncated version of α-actinin called ACT::SOE3. This protein is a specific diagnostic target for antibody in sera of individuals with trichomoniasis. Serum antibody to ACT::SOE3 is a positive reaction with the test spot. Specificity of ACT::SOE3 was revealed with monoclonal antibodies (MAbs) generated to ACT::SOE3. Addition of negative control serum with MAb 67B reactive to ACT::SOE3 shows detection of both ACT::SOE3 and the CVL. Only positive sera of individuals had antibody reactive with ACT::SOE3 and detected the presence of the spot and the CVL. Negative control sera were unreactive with ACT::SOE3 and only showed the presence of the CVL. Importantly, to show proof-of-principle for POC application, ACT::SOE3 was detected with the positive patient sera spiked with whole blood. Finally, packaged cartridges stored with desiccant packs at 37 °C for one year gave identical results with the positive and negative human sera. Our results show the validity of this new POC serodiagnostic for this STI.

Advancing Prevention of STIs by Developing Specific Serodiagnostic Targets: Trichomonas vginalis as a Model.

Point-of-Care (POC) serum antibody screening of large cohorts of women and men at risk for the sexually transmitted infection (STI) caused by Trichomonas vaginalis requires the availability of targets with high specificity. Such targets should comprise epitopes unique to T. vaginalis immunogenic proteins detected by sera of women and men patients with trichomonosis but not uninfected controls. Three enzymes to which patients make serum IgG antibody were identified as fructose-1,6-bisphosphate aldolase (A), α-enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G). Epitopes within these proteins were identified that had no sequence identity to enzymes of humans and other pathogens. Therefore, I constructed a chimeric recombinant String-Of-Epitopes (SOE) protein consisting of 15-mer peptides, within which are the epitopes of A, E, and G. This novel protein of ~36-kD is comprised of two epitopes of A, ten epitopes of E, and seven epitopes of G (AEG::SOE2). The AEG::SOE2 protein was detected both by immunoblot and by enzyme-linked immunosorbent assay (ELISA) using highly reactive sera of women and men but not negative serum unreactive to T. vaginalis proteins. Finally, AEG::SOE2 was found to be immunogenic, as evidenced by serum IgG from immunized mice. I discuss how this approach is important in relation to infectious disease diagnostic targets for detection of serum IgG antibody in exposed and/or infected individuals and how such novel targets may have potential as subunit vaccine candidates against microbial pathogens.

Trichomonas vaginalis infection and prostate-specific antigen concentration: Insights into prostate involvement and prostate disease risk.

BACKGROUND: The protist Trichomonas vaginalis causes a common, sexually transmitted infection and has been proposed to contribute to the development of chronic prostate conditions, including benign prostatic hyperplasia and prostate cancer. However, few studies have investigated the extent to which it involves the prostate in the current antimicrobial era. We addressed this question by investigating the relation between T. vaginalis antibody serostatus and serum prostate-specific antigen (PSA) concentration, a marker of prostate infection, inflammation, and/or cell damage, in young, male, US military members. METHODS: We measured T. vaginalis serum IgG antibodies and serum total PSA concentration in a random sample of 732 young, male US active duty military members. Associations between T. vaginalis serostatus and PSA were investigated by linear regression. RESULTS: Of the 732 participants, 341 (46.6%) had a low T. vaginalis seropositive score and 198 (27.0%) had a high score, with the remainder seronegative. No significant differences were observed in the distribution of PSA by T. vaginalis serostatus. However, slightly greater, nonsignificant differences were observed when men with high T. vaginalis seropositive scores were compared with seronegative men, and when higher PSA concentrations were examined (≥0.70 ng/mL). Specifically, 42.5% of men with high seropositive scores had a PSA concentration greater than or equal to 0.70 ng/mL compared with 33.2% of seronegative men (adjusted P = .125). CONCLUSIONS: Overall, our findings do not provide strong support for prostate involvement during T. vaginalis infection, although our suggestive positive findings for higher PSA concentrations do not rule out this possibility entirely. These suggestive findings may be relevant for prostate condition development because higher early- to mid-life PSA concentrations have been found to predict greater prostate cancer risk later in life.

Trichomonas vaginalis infection and risk of prostate cancer: associations by disease aggressiveness and race/ethnicity in the PLCO Trial.

PURPOSE: Results from previous sero-epidemiologic studies of Trichomonas vaginalis infection and prostate cancer (PCa) support a positive association between this sexually transmitted infection and aggressive PCa. However, findings from previous studies are not entirely consistent, and only one has investigated the possible relation between T. vaginalis seropositivity and PCa in African-American men who are at highest risk of both infection and PCa. Therefore, we examined this possible relation in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, including separate analyses for aggressive PCa and African-American men. METHODS: We included a sample of participants from a previous nested case-control study of PCa, as well as all additional Caucasian, aggressive, and African-American cases diagnosed since the previous study (total n = 438 Gleason 7 Caucasian cases, 487 more advanced Caucasian cases (≥Gleason 8 or stage III/IV), 201 African-American cases, and 1216 controls). We tested baseline sera for T. vaginalis antibodies. RESULTS: No associations were observed for risk of Gleason 7 (odds ratio (OR) = 0.87, 95% confidence interval (CI) 0.55-1.37) or more advanced (OR = 0.90, 95% CI 0.58-1.38) PCa in Caucasian men, or for risk of any PCa (OR = 1.06, 95% CI 0.67-1.68) in African-American men. CONCLUSIONS: Our findings do not support an association between T. vaginalis infection and PCa.

Sexually transmitted infections, benign prostatic hyperplasia and lower urinary tract symptom-related outcomes: results from the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

OBJECTIVE: To examine whether a history of sexually transmitted infections (STIs) or positive STI serology is associated with prevalent and incident benign prostatic hyperplasia (BPH)/lower urinary tract symptoms (LUTS)-related outcomes in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. METHODS: Self-reported history of STIs (gonorrhoea, syphilis) was ascertained at baseline, and serological evidence of STIs (Chlamydia trachomatis, Trichomonas vaginalis, human papillomavirus (HPV)-16, HPV-18, herpes simplex virus type 2, human herpesvirus type 8 and cytomegalovirus) was detected in baseline serum specimens. We used data collected on the baseline questionnaire, as well as results from the baseline prostate-specific antigen (PSA) test and digital rectal examination (DRE), to define prevalent BPH/LUTS-related outcomes as evidence of LUTS (self-reported diagnosis of an enlarged prostate/BPH, BPH surgery or nocturia [waking ≥2 times/night to urinate]) and evidence of prostate enlargement (PSA > 1.4 ng/mL or prostate volume ≥30 mL) in men without prostate cancer. We created a similar definition of incident BPH using data from the follow-up questionnaire completed 5-13 years after enrolment (self-reported diagnosis of an enlarged prostate/BPH or nocturia), data on finasteride use during follow-up, and results from the follow-up PSA tests and DREs. We used Poisson regression with robust variance estimation to calculate prevalence ratios (PRs) in our cross-sectional analysis of self-reported (n = 32 900) and serologically detected STIs (n = 1 143) with prevalent BPH/LUTS, and risk ratios in our prospective analysis of self-reported STIs with incident BPH/LUTS (n = 5 226). RESULTS: Generally null results were observed for associations of a self-reported history of STIs and positive STI serologies with prevalent and incident BPH/LUTS-related outcomes, with the possible exception of T. vaginalis infection. This STI was positively associated with prevalent nocturia (PR 1.36, 95% confidence interval (CI) 1.18-1.65), prevalent large prostate volume (PR 1.21 95% CI 1.02-1.43), and any prevalent BPH/LUTS (PR 1.32 95% CI 1.09-1.61); too few men had information on both STI serologies and incident BPH/LUTS to investigate the associations between T. vaginalis infection and incident BPH/LUTS-related outcomes. CONCLUSIONS: Our findings do not support associations of several known STIs with BPH/LUTS-related outcomes, although T. vaginalis infection may warrant further study.

Persistence of Trichomonas vaginalis serostatus in men over time.

PURPOSE: Previous epidemiologic studies have observed positive associations between Trichomonas vaginalis (Tv) serostatus and both prostate cancer (PCa) risk and mortality. However, only a few small older studies have examined Tv antibody persistence over time, all of which were composed mainly of female patients. Therefore, we examined Tv antibody persistence over time, as well as intra-individual variability, among middle- to older-aged men in the Southern Community Cohort Study (SCCS). METHODS: We tested baseline and repeat plasma specimens (collected 1-3 years later) from 248 male participants for Tv antibodies. We used the same enzyme-linked immunosorbent assay as in previous studies of Tv serostatus and PCa. RESULTS: At baseline, 46 (18.5 %) participants were seropositive for Tv infection. Seventy-six percent of these men were still seropositive 1-3 years later. A similar proportion of men "seroconverted" (4.0 %) as "seroreverted" (4.4 %), all of whom had absorbance values near the cutoff point for seropositivity. Overall, substantial agreement was observed between baseline and repeat serostatus (κ = 0.72, 95 % confidence interval 0.60-0.83). CONCLUSION: Tv seropositivity was largely persistent between plasma specimens collected 1-3 years apart from middle- to older-aged men. These high levels of persistence are similar to those observed for other sexually transmitted infections frequently investigated in relation to PCa.

Prospective study of effect modification by Toll-like receptor 4 variation on the association between Trichomonas vaginalis serostatus and prostate cancer.

PURPOSE: In previous studies, we observed a positive association between Trichomonas vaginalis serostatus and risk of prostate cancer, particularly aggressive cancer, which we hypothesized might be due to T. vaginalis-mediated intraprostatic inflammation and cell damage. To explore this hypothesis further, we investigated effect modification by Toll-like receptor 4 (TLR4) variation on this association. We hypothesized that TLR4 variation might serve a marker of the anti-trichomonad immune response because T. vaginalis has been shown to elicit inflammation through this receptor. METHODS: We previously genotyped the non-synonymous TLR4 single nucleotide polymorphism (SNP), rs4986790, and determined T. vaginalis serostatus for 690 incident prostate cancer cases and 692 controls in a nested case-control study within the Health Professionals Follow-up Study. RESULTS: A non-significant suggestion of effect modification was observed by rs4986790 carrier status on the association between T. vaginalis serostatus and prostate cancer risk (p interaction = 0.07). While no association was observed among men homozygous wildtype for this SNP (odds ratio (OR) = 1.23, 95 % confidence interval (CI): 0.86-1.77), a positive association was observed among variant carriers (OR = 4.16, 95 % CI: 1.32-13.1). CONCLUSIONS: Although not statistically significant, TLR4 variation appeared to influence the association between T. vaginalis serostatus and prostate cancer risk consistent with the hypothesis that inflammation plays a role in this association. Larger studies will be necessary to explore this possible effect modification further.

Comparative transcriptomic and proteomic analyses of Trichomonas vaginalis following adherence to fibronectin.

The morphological transformation of Trichomonas vaginalis from an ellipsoid form in batch culture to an adherent amoeboid form results from the contact of parasites with vaginal epithelial cells and with immobilized fibronectin (FN), a basement membrane component. This suggests host signaling of the parasite. We applied integrated transcriptomic and proteomic approaches to investigate the molecular responses of T. vaginalis upon binding to FN. A transcriptome analysis was performed by using large-scale expressed-sequence-tag (EST) sequencing. A total of 20,704 ESTs generated from batch culture (trophozoite-EST) versus FN-amoeboid trichomonad (FN-EST) cDNA libraries were analyzed. The FN-EST library revealed decreased amounts of transcripts that were of lower abundance in the trophozoite-EST library. There was a shift by FN-bound organisms to the expression of transcripts encoding essential proteins, possibly indicating the expression of genes for adaptation to the morphological changes needed for the FN-adhesive processes. In addition, we identified 43 differentially expressed proteins in the proteomes of FN-bound and unbound trichomonads. Among these proteins, cysteine peptidase, glyceraldehyde-3-phosphate dehydrogenase (an FN-binding protein), and stress-related proteins were upregulated in the FN-adherent cells. Stress-related genes and proteins were highly expressed in both the transcriptome and proteome of FN-bound organisms, implying that these genes and proteins may play critical roles in the response to adherence. This is the first report of a comparative proteomic and transcriptomic analysis after the binding of T. vaginalis to FN. This approach may lead to the discovery of novel virulence genes and affirm the role of genes involved in disease pathogenesis. This knowledge will permit a greater understanding of the complex host-parasite interplay.

Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis.

SUMMARY: Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (approximately 82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

Prospective study of Trichomonas vaginalis infection and prostate cancer incidence and mortality: Physicians' Health Study.

BACKGROUND: A recent nested case-control study found that the presence of antibodies against Trichomonas vaginalis, a common nonviral sexually transmitted infection, was positively associated with subsequent incidence of prostate cancer. We confirmed these findings in an independent population and related serostatus for antibodies against T vaginalis to prostate cancer incidence and mortality. METHODS: We conducted a case-control study nested within the Physicians' Health Study that included 673 case subjects with prostate cancer and 673 individually matched control subjects who had available plasma samples. Plasma from blood samples collected at baseline was assayed for antibodies against T vaginalis with an enzyme-linked immunosorbent assay. We used conditional logistic regression to estimate the odds ratios (ORs) of incident prostate cancer, extraprostatic prostate cancer, and cancer that would ultimately progress to bony metastases or prostate cancer-specific death. RESULTS: Although not statistically significant, the magnitude of the association between T vaginalis-seropositive status and overall prostate cancer risk (OR = 1.23, 95% confidence interval [CI] = 0.94 to 1.61) was similar to that reported previously. Furthermore, a seropositive status was associated with statistically significantly increased risks of extraprostatic prostate cancer (OR = 2.17, 95% CI = 1.08 to 4.37) and of cancer that would ultimately progress to bony metastases or prostate cancer-specific death (OR = 2.69, 95% CI = 1.37 to 5.28). CONCLUSIONS: This large prospective case-control study obtained further support for an association between a seropositive status for antibodies against T vaginalis and the risk of prostate cancer, with statistically significant associations identified for the risk of extraprostatic prostate cancer and for clinically relevant, potentially lethal prostate cancer.

Correlates of sexually transmitted infection histories in a cohort of American male health professionals.

OBJECTIVE: Several epidemiologic studies have investigated sexually transmitted infections (STIs) and later risk of genitourinary conditions with suggestive positive results. While these results may reflect causal associations, other possible explanations include confounding by factors possibly related to both STI acquisition and genitourinary condition risk such as recognized STI-risk factors/correlates, and other factors not typically considered in relation to STIs (e.g., general health-related behaviors or markers of such behaviors). Very few of these factors have been investigated in older populations in which STIs and genitourinary conditions are typically studied. Therefore, we investigated STI history correlates in one such population, the Health Professionals Follow-up Study. METHODS: We ascertained histories of potential correlates, gonorrhea, syphilis by questionnaire (n = 36,032), and performed serologic testing for Chlamydia trachomatis, Trichomonas vaginalis, human papillomavirus, and human herpesvirus type 8 infection in a subset (n = 651). RESULTS: Positive correlations were observed for African-American race, foreign birth, southern residence, smoking, alcohol consumption, ejaculation frequency, vasectomy, and high cholesterol. Inverse correlations were observed for social integration and routine health-related examinations. CONCLUSIONS: These findings provide useful information on potential confounders for epidemiologic investigations of STIs and chronic diseases, and interesting new hypotheses for STI prevention (e.g., STI counseling before vasectomy).

Examination for double-stranded RNA viruses in Trichomonas gallinae and identification of a novel sequence of a Trichomonas vaginalis virus.

To determine if double-stranded RNA (dsRNA) viruses exist and are potential virulence factors in Trichomonas gallinae, virus purification via ultracentrifugation was attempted for 12 T. gallinae isolates recovered from wild birds. Following purification, virus-like particles were not observed by transmission electron microscopy, nor were dsRNA segments visualized in agarose gels after electrophoresis of extracted RNA from any of the 12 T. gallinae isolates. However, virus particles and dsRNA segments were detected from a previously determined virus-infected T. vaginalis isolate as a control using identical purification procedures. Subsequent reverse transcription-polymerase chain reaction analysis of the dsRNA of the virus in this isolate revealed a novel sequence of the RNA-dependent RNA polymerase gene of T. vaginalis viruses.

Comparative analyses among the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S ribosomal RNA genes.

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization.

Trichomonosis and subsequent risk of prostate cancer in the Prostate Cancer Prevention Trial.

We previously observed a positive association between a history of trichomonosis, a sexually transmitted infection caused by the protozoan, Trichomonas vaginalis, and prostate cancer risk in the Health Professionals Follow-up Study. To determine the reproducibility of this finding, we conducted a second, prospective investigation of trichomonosis and prostate cancer in the Prostate Cancer Prevention Trial. Participants were men (>or=55 years of age) with no evidence of prostate cancer at enrollment (n = 18,882). Men were screened annually for prostate cancer, and if not diagnosed during the trial, were offered an end-of-study prostate biopsy. Cases were a sample of men diagnosed with prostate cancer on any biopsy after visit 2 or on their end-of-study biopsy (n = 616). Controls were men not diagnosed with prostate cancer during the trial or on their end-of-study biopsy (n = 616). Controls were frequency-matched to cases by age, treatment arm, and family history of prostate cancer. Serum from visit 2 was tested for anti-T. vaginalis IgG antibodies. No association was observed between T. vaginalis serostatus and prostate cancer. 21.5% of cases and 24.8% of controls had low seropositivity, and 15.2% and 15.0% had high seropositivity. Compared to seronegative men, the odds ratio of prostate cancer for men with low seropositivity was 0.83 [95% confidence interval (CI): 0.63-1.09), and that for men with high seropositivity was 0.97 (95% CI: 0.70-1.34). Given the original strong biologic rationale and potential for prevention, additional studies are warranted to help resolve discrepancies between study findings and to further investigate this hypothesis from a variety of different approaches.

Polyamine depletion down-regulates expression of the Trichomonas vaginalis cytotoxic CP65, a 65-kDa cysteine proteinase involved in cellular damage.

Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage.

First molecular characterisation of hydrogenosomes in the protozoan parasite Histomonas meleagridis.

Histomonas meleagridis is a trichomonad species that undergoes a flagellate-to-amoeba transformation during tissue invasion and causes a serious disease in gallinaceous birds (blackhead disease or histomoniasis). Living in the avian cecum, the flagellated form can be grown in vitro in the presence of an ill-defined bacterial flora. Its cytoplasm harbours numerous spherical bodies which structurally resemble hydrogenosomes. To test whether these organelles may be involved in anaerobic metabolism, we undertook the identification of H. meleagridis genes encoding some potentially conserved hydrogenosomal enzymes. The strategy was based on several PCR amplification steps using primers designed from available sequences of the phylogenetically-related human parasite Trichomonas vaginalis. We first obtained a C-terminal sequence of an iron-hydrogenase homologue (Hm_HYD) with typical active site signatures (H-cluster domain). Immunoelectron microscopy with anti-Hm_HYD polyclonal antibodies showed specific gold labelling of electron-dense organelles, thus confirming their hydrogenosomal nature. The whole genes encoding a malic enzyme (Hm_ME) and the alpha-subunit of a succinyl coenzyme A synthetase (Hm_alpha-SCS) were then identified. Short N-terminal presequences for hydrogenosomal targeting were predicted in both proteins. Anti-Hm_ME and anti-Hm_alpha-SCS antisera provided immunofluorescence staining patterns of H. meleagridis cytoplasmic granules similar to those observed with anti-Hm_HYD antiserum or mAb F5.2 known to react with T. vaginalis hydrogenosomes. Hm_ME, Hm_alpha-SCS and Hm_HYD were also detected as reactive bands on immunoblots of proteins from purified hydrogenosomes. Interestingly, anti-Hm_alpha-SCS staining of the cell surface in non-permeabilised parasites suggests a supplementary role for SCS in cytoadherence, as previously demonstrated in T. vaginalis.

The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65.

We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65.

Plasma antibodies against Trichomonas vaginalis and subsequent risk of prostate cancer.

BACKGROUND: Although several previous case-control studies have investigated associations between sexually transmitted infections (STI) and prostate cancer, most have focused on gonorrhea and syphilis, two well-recognized, symptomatic STIs. Another STI of interest for prostate carcinogenesis is trichomonosis, a less well recognized and frequently asymptomatic STI with known prostate involvement. We investigated this infection in relation to incident prostate cancer in a nested case-control study within the Health Professionals Follow-up Study. METHODS: Prostate cancer cases were men diagnosed with prostate cancer between the date of blood draw (1993-1995) and 2000 (n = 691). Controls were men who had had at least one prostate-specific antigen test and who were free of prostate cancer and alive at the time of case diagnosis. One control was individually matched to each case by age (n = 691). Serologic evidence of a history of trichomonosis was assessed by a recombinant Trichomonas vaginalis alpha-actinin IgG ELISA. RESULTS: Thirteen percent of cases and 9% of controls were seropositive for trichomonosis (adjusted odds ratio, 1.43; 95% confidence interval, 1.00-2.03). This association persisted after additional adjustment for such factors as a history of other STIs, and was strongest among men who used aspirin infrequently over the course of their lives (odds ratio, 2.05; 95% confidence interval, 1.05-4.02, P(interaction) = 0.11). CONCLUSIONS: Serologic evidence of a history of trichomonosis was positively associated with incident prostate cancer in this large, nested case-control study of male health professionals. As this study is the first, to our knowledge, to investigate associations between T. vaginalis serology and prostate cancer, additional studies are necessary before conclusions can be made.

Trichomonas vaginalis perturbs the junctional complex in epithelial cells.

Trichomonas vaginalis, a protist parasite of the urogenital tract in humans, is the causative agent of trichomonosis, which in recent years have been associated with the cervical cancer development. In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T. vaginalis and the influence of the iron concentration present in the parasite's culture medium on the interaction effects. Our results show that T. vaginalis adheres to the epithelial cell causing alterations in the junctional complex, such as: (a) a decrease in transepithelial electrical resistance; (b) alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin, occludin and ZO-1; and (c) enlargement of the spaces between epithelial cells. These effects were dependent on (a) the degree of the parasite virulence isolate, (b) the iron concentration in the culture medium, and (c) the expression of adhesin proteins on the parasite surface.