SummaryThe 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 immunocytochemical 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. Twodimensional 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.

SummaryLevels of adherence of Trichomonas vaginalis to epithelial cells was found to be modulated by iron . Cytoadherence values were greater than or equal to twofold higher for trichomonads grown in a complex cultivation medium supplemented with iron . This increase in adherence levels was specifically mediated by iron ; parasites cultured in a low-iron medium in the presence of salts other than iron were unresponsive to changes in adherence levels. Expression of the higher adherence property, by parasites grown first in low-iron medium followed by supplementation with iron, was a function of time, and the extent of cytoadherence was proportional to the concentration of iron added to the medium . Lactoferrin, an important iron source for trichomonads at the site of infection, elevated adherence of the parasite to epithelial cells, demonstrating the likely in vivo modulation of adherence by iron. The alteration of levels of adherence caused by iron was determined to be a reflection of gene expression of previously characterized trichomonad adhesins. Parasites grown under iron-replete conditions had higher quantities of surface-exposed adhesins, and this was a result of increased synthesis of adhesins. Actinomycin D and a-amanitin prevented expression of adhesin molecules, which resulted in decreased cytoadherence, showing that adhesin synthesis was dependent on gene transcription . Data indicated that genes encoding the four trichomonad adhesins are coordinately regulated by iron .Specificadherenceofmucosal pathogens to host cells leads to colonization, and this interaction is fundamental and prerequisite for infection and pathogenesis. Cytoadherence is an important mechanism by which infecting organisms overcome the flushing effect of mucosal secretions (1) . Trichomonas vaginalis is a flagellated protozoan responsible for one of the most common, world-wide sexually transmitted diseases, and the parasite affects mostly women. The emotional and economic consequences to all world societies caused by this protozoan are significant . This microorganism colonizes the vaginal epithelium by specific, receptor-ligand interactions (2) . Adherence to squamous vaginal epithelial cells by this parasite is mediated by four surface proteins (3) . The ability of T. vaginalis parasites to cytoadhere is the result of a complex cascade of events involving adhesin proteins (3) and proteinase activity (4) .These earlier studies were conducted with organisms grown in a complex, nutrient-rich medium different from that encountered by parasites in the vagina, a nutritional environment which itself is changing during the menstrual cycle . Recent continuous flow culture experiments demonstrated that T. vaginalis organisms were capable of altering several properties in response to varying culture environments (5) . In addition, growth and multiplication (6), and certain virulence traits of T. vaginalis, were found to be modulated by iron, which is an essential nutrient for this parasite (6, 7). This ability of pathogenic human trichomon...

Iron is an essential nutrient for Trichomonas vaginalis and is acquired via highly specific receptor-mediated mechanisms from the host. Responses of T. vaginalis to conditions of iron limitation or iron excess were analysed in order to determine whether iron levels in the growth medium regulate certain properties of the parasite. When compared with organisms grown in excess iron, iron limitation resulted in greater than or equal to 80% lower rates of protein synthesis and greater than or equal to 3-fold decreases in cell densities. These parasites also exhibited generation times of approximately 10 hours, 2.5-fold longer than organisms grown in the usual complex medium. Iron-restricted growth also resulted in increased binding of lactoferrin by trichomonads, which paralleled elevated expression of the lactoferrin-binding receptor protein having a relative molecular mass of 136,000 daltons (136 kDa). A Mr 126 kDa protein was concomitantly repressed in low-iron-grown parasites. The greater amounts of lactoferrin bound by iron-depleted T. vaginalis organisms corresponded with both the expression of additional receptors onto trichomonal surfaces and increased affinity of the receptor for the lactoferrin molecule. Finally, immunoblot analysis of parasites grown under high- and low-iron conditions using sera from patients with trichomoniasis further revealed the synthesis by T. vaginalis of at least 19 iron-regulated immunogens, and patients' sera also detected the lactoferrin receptor. These data not only show the overall importance of iron to the biology of this protozoan, but illustrate the in vivo iron modulation of gene expression of the biofunctional lactoferrin receptor and other immunogens.

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Background/objective: Trichomonas vaginalis, the causal agent of trichomonosis, is a flagellated parasitic protozoan that colonises the epithelial cells of the human urogenital tract. The ability of T vaginalis to colonise this site is in part a function of its ability to circumvent a series of non-specific host defences including the mucous layer covering epithelial cells at the site of infection. Mucin, the framework molecule of mucus, forms a lattice structure that serves as a formidable physical barrier to microbial invasion. The mechanism by which trichomonads traverse the mucous covering is unknown. Proteolytic degradation of mucin, however, may provide for a mechanism to penetrate this layer. The goal, therefore, was to determine how trichomonads cross through a mucous layer. Methods: Secreted trichomonad proteinases were analysed for mucinase activity by mucin substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The importance of trichomonad mucinases for traversing the mucous layer was examined on an artificial mucin layer in invasion chambers. Adherence to mucin and tissue culture cells was measured using a microtitre plate assay. Results: Trichomonad isolate 24402 secreted five proteinases when incubated in PBS. All five proteinases were shown to possess mucinase activity. These mucinases were able to degrade bovine submaxillary mucin and to a lesser extent porcine stomach mucin. These enzymes were active over a pH range of 4.5-7.0 and were inhibited with cysteine proteinase inhibitors. Furthermore, T vaginalis was shown to bind to mucin possibly via a lectin-like adhesin. Adherence to mucin was increased threefold when parasites were grown in iron deficient medium. Adherence to soluble mucin prevented attachment to HeLa cells. Proteinase activity, adherence, and motility were required for trichomonads to traverse a mucin layer in vitro. Conclusions: These results show that trichomonads can traverse the mucous barrier first by binding mucin followed by its proteolytic degradation. The data further underscore the importance of trichomonad proteinases in the pathogenesis of trichomonosis. Finally, this study suggests that interference with trichomonad mucin receptors and proteinases may be a strategy to prevent colonisation by this parasite. (Sex Transm Inf 1999;75:231-238)