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.

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.Trichomonas vaginalis, an extracellular protozoan parasite, is the cause of trichomonosis, the most prevalent nonviral sexually transmitted disease (47). In women, vaginitis due to T. vaginalis clinically manifests with symptoms of vaginal itching, odor, and discharge. Adverse health outcomes for women with this sexually transmitted disease include cervical cancer (46) and preterm delivery and low-birth-weight infants (25). There is a relationship between seropositivity to T. vaginalis and prostate cancer (43). This disease is significant due to its association with human immunodeficiency virus (33, 45). More recently, persistent, undetected T. vaginalis infections associated with asymptomatic carriage were found among women (40).T. vaginalis penetration of the mucous layer (28), followed by adherence to vaginal epithelial cells (VECs), is preparatory for colonization (9, 10). VEC adherence by parasites is mediated by numerous distinct trichomonad surface adhesins (5, 10, 18). Brief contact of T. vaginalis with VECs and fibronectin (FN) elicited dramatic changes in parasite morphology, suggesting a host-specific signaling of parasites (8, 9). Importantly, iron and cell contact by parasites each upregulated the expression of adhesins in a coordinated fashion via distinct mechanisms (2,4,6,21,29). Genet...

Trichomonas vaginalis is a protist that causes the most common human sexually transmitted infection. A T. vaginalis cDNA expression library was screened with pooled sera from patients with trichomoniasis. A highly reactive cDNA clone of 1,428 bp encoded a trichomonad protein of 472 amino acids with sequence identity to ␣-enolase (tv-eno1). The sequence alignment confirmed the highly conserved nature of the enzyme with 65% to 84% identity among organisms. The expression of tv-eno1 was up-regulated by contact of parasites with vaginal epithelial cells, and this is the first report demonstrating up-regulation by cytoadherence of a plasminogenbinding ␣-enolase in T. vaginalis. Immunofluorescence with monoclonal antibody of nonpermeabilized trichomonads showed tv-ENO1 on the surface. The recombinant tv-ENO1 was expressed in Escherichia coli as a glutathione S-transferase (GST)::tv-ENO1 fusion protein, which was cleaved using thrombin to obtain affinity-purified recombinant tv-ENO1 protein (tv-rENO1) detectable in immunoblots by sera of patients. Immobilized tv-rENO1 bound human plasminogen in a dose-dependent manner, and plasminogen binding by tv-rENO1 was confirmed in a ligand blot assay. The plasminogen-specific inhibitor -aminocaproic acid blocked the tv-rENO1-plasminogen association, indicating that lysines play a role in binding to tv-rENO1. Further, both parasites and tv-rENO1 activate plasminogen to plasmin that is mediated by tissue plasminogen activator. These data indicate that as with other bacterial pathogens, tv-ENO1 is an anchorless, surfaceassociated glycolytic enzyme of T. vaginalis.Trichomonas vaginalis is responsible for the number one, nonviral sexually transmitted disease worldwide (56). There are 250 million new cases of trichomoniasis worldwide and ϳ9 million cases in the United States (15,55,57). Trichomoniasis is known to increase the portal of entry and exit for human immunodeficiency virus (51). A recent study showed serological evidence linking trichomoniasis with prostate cancer (52). Given the significant human morbidity caused by T. vaginalis, there is a need for identifying virulence factors and elucidating the mechanisms of disease pathogenesis.A prerequisite for T. vaginalis infection is its ability to colonize the vaginal epithelium. Surface-associated adhesin proteins were shown to be involved in parasite adherence to vaginal epithelial cells (VECs) (4,19,20). There is a direct relationship between the amount of surface adhesin that binds to host cells in a ligand-receptor type interaction (8,20) and the level of cytoadherence (8,20). Contact with VECs produces a dramatic change in morphology accompanied by synthesis of adhesins (9, 26). A recent study using antisense RNA reaffirmed the importance of AP65 and AP33 in parasite associations with VECs (37, 38). In addition, heterologous expression of AP65 and AP33 on the Tritrichomonas foetus surface provided evidence that both are bona fide adhesins of T. vaginalis (25,38). Interestingly, these adhesins show sequence identity to metab...

SummaryWe 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.

Host parasitism byTrichomonas vaginalis colonizes the urogenital tract of humans, causing trichomonosis, the number one nonviral sexually transmitted disease worldwide. Despite an estimated 8 million new cases per year in the United States alone (53), this health disparities disease (48) remains poorly studied. T. vaginalis infection is associated with adverse health consequences to both men and women, including infertility (20, 46), atypical pelvic inflammatory disease (35), and increased human immunodeficiency virus transmission (24,49). Trichomonosis is also associated with preterm birth, low-birth-weight infants (16), predisposition to development of cervical neoplasia in women (51), and nongonococcal urethritis (9) and chronic prostatitis (10) in men. The complex interplay of trichomonad responses and that of the host cells during infection have not been investigated so far. Of the few reports concerning host-parasite interactions, one study showed that after short contact of trichomonads with host vaginal epithelial cells (VECs), but not HeLa cells, the parasite morphology is transformed from an ellipsoid to an amoeboid form (8), suggesting host-specific signaling of parasites. In addition, synthesis of all four adhesin proteins in the amoeboid forms bound to VECs was enhanced (8,22). In a separate study using different forms of parasites grown in culture flasks, ␣-actinin was shown to be overexpressed in amoeboid parasites compared to batch-cultured ellipsoid trichomonads (1).The transcriptional regulation of parasite genes in response to interactions with host cells using in vitro models has been studied in Neisseria meningitides (23), Porphyromonas gingivalis (25, 41), and Helicobacter pylori (26). Due to the lack of a good animal model system to study T. vaginalis pathogenesis, we have used an in vitro model of immortalized human VECs (22) in our present study. Because of the unavailability of genome sequence data at the time this study was initiated, we used the subtraction cDNA library approach to identify the transcriptional changes in gene expression during the initial step of T. vaginalis attachment to VECs. Differentially expressed gene profiling using cDNA subtraction has been an ideal tool in identifying novel genes and transcripts of low abundance (13). Our data identify numerous T. vaginalis genes that are upregulated upon contact, which was confirmed by semiquantitative reverse transcription-PCR (RT-PCR) and protein immunoblot analyses. We believe that functional analyses of up-regulated genes of both VECs, as done recently (29), and of the organisms after the adherence event will contribute to our understanding of the host-pathogen interrelationship and the elucidation of the mechanisms of pathogenesis. MATERIALS AND METHODSParasites and host cells. T. vaginalis isolate T016 was grown in trypticase-yeast extract-maltose (TYM) medium supplemented with 10% serum at 37°C (19). For iron-replete parasites, TYM serum was supplemented with 200 M ferrous ammonium sulfate, and iron-depleted parasite...