John F. Alderete

Molecular basis of host epithelial cell recognition by Trichomonas vaginalis

Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65000 daltons (65kDa; AP65), 51 kDa (AP51), 33kDa (AP33), and 23 kDa (AP23) mediate the interaction of T. vaginalis with epithelial cells. Fresh isolates, when compared with long‐term‐grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti‐adhesin antibodies reacted by immunobiol only with the respective protein and detected, by indirect immunofluore‐scence, each adhesin on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact‐dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesins also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.

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 α-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, Pinteraction = 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. (Cancer Epidemiol Biomarkers Prev 2006;15(5):939–45)

Signalling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence

The cytoadherence of Trichomonas vaginalis, the sexually transmitted flagellated protozoan, to vaginal epithelial cells (VECs) is the key to infection. Electron microscopy revealed that in vitro‐grown parasites having typical globular shape transformed rapidly after contact with VECs into thin, flat, amoeboid cells, maximizing the area of adhesion to the surface of VECs. Amoebic trichomonads formed filopodia and pseudopodia, which interdigitated at distinct sites on the plasma membrane of target cells. In contrast, the amoeboid transformation did not occur for T. vaginalis interacting with He La cells, the previously used in vitro host model cell. Initial parasitism of VECs by a single organism was followed by establishment of a monolayer of trichomonads on the host cell. Finally, parasites adhering to either VECs or HeLa cells were induced to synthesize greater amounts of the four previously described adhesins. Therefore, distinct signals after contact with either epithelial cell type leads to the morphological transformation and/or induction of adhesin synthesis by T. vaginalis.

Immunogenic and Plasminogen-Binding Surface-Associated α-Enolase of Trichomonas vaginalis

ABSTRACT 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 α-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 plasminogen-binding α-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 ε-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, surface-associated glycolytic enzyme of T. vaginalis.

Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis

The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high‐iron conditions. Fluorescence and immuno‐cytochemical experiments show that high‐iron‐grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low‐iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti‐AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two‐dimensional electrophoresis and immunoblot of adhesins from surface‐labelled parasites provided evidence that all members of the multigene family were co‐ordinately expressed and placed on the trichomonal surface. Similar two‐dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface‐expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug‐resistant isolate absent in hydrogenosome proteins and adhesins, were non‐adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.

Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers.

Exposure of monolayer cultures of human urogenital and vaginal (HeLa), human epithelial (HEp-2), normal baboon testicular (NBT), and monkey kidney (Vero) cells to live pathogenic Trichomonas vaginalis resulted in extensive disruption of monolayers. Trypan blue was taken up by all host cells released from cell monolayers, which indicated irreversible damage of these cell types by trichomonads. Time and dose related data on cytotoxicity kinetics were obtained using increasing ratios of parasites to cells. All cell types were most sensitive to trichomonads at a multiplicity of infection of one. Release of tritiated thymidine (3H-thymidine) of the deoxyribonucleic acid (DNA) of prelabelled host cells after incubation with T vaginalis corroborated that extensive cytotoxicity was caused by pathogenic trichomonads in man. Only living parasites were cytotoxic, and no trichomonal toxic products were implicated in disruption of the cell monolayer cultures. A pathogenic bovine trichomonad, Tritrichomonas foetus KV-1, produced half as much cell damage as did T vaginalis. Trichomonas tenax, a non-pathogenic member of the normal flora of the oral cavity in man, produced no measurable cytotoxicity to HeLa cells when compared with the pathogenic human trichomonads.

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated, Fibronectin-Binding Protein of Trichomonas vaginalis

ABSTRACT 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.

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

ABSTRACT 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.

Characterization of Trichomonas vaginalis haemolysis.

The haemolytic activity of live Trichomonas vaginalis organisms was investigated. Optimal haemolysis of human erythrocytes was observed at a parasite to erythrocyte ratio of 1:5 during a 2 h incubation period. No haemolytic activity was detected in concentrated culture supernatants after overnight growth of trichomonads or when parasites were separated from erythrocytes by a 3 microns filter, suggesting a contact-dependent mechanism for haemolysis. The haemolytic activity was temperature-dependent and maximal haemolysis occurred at 37 degrees C. Treatment of trichomonads with metronidazole reduced levels of haemolysis by greater than 50%. Maximal haemolysis occurred at the pH range of the vagina during trichomoniasis. N-alpha-tosyl-L-lysyl-chloromethyl ketone and iodoacetamide, inhibitors of trichomonad cysteine proteinases, reduced the haemolytic activity of live parasites.

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 (≥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.