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 65,000 daltons (65 kDa; AP65), 51 kDa (AP51), 33 kDa (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 immunoblot only with the respective protein and detected, by indirect immunofluorescence, each adhesion 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 adhesions also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.     

Iron regulates growth of Trichomonas vaginalis and the expression of immunogenic trichomonad proteins

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.     

Host and pathogen interaction during vaginal infection by Trichomonas vaginalis and Mycoplasma hominis or Ureaplasma urealyticum

Vaginal infections by Trichomonas vaginalis and Mycoplasma hominis have been shown to be associated. Since M. hominis and Ureaplasma urealyticum are similar pathogens, both belonging to the class of the mycoplasmata, we describe here a molecular study into the interdependence of U. urealyticum and T. vaginalis during infection. Susceptibility towards infection by U. urealyticum depends on genetic polymorphism in the interleukin-1 receptor antagonist (IL-1RA) gene. Now, we defined the relation between IL-1RA genotypes and infection by M. hominis and T. vaginalis. Finally, we also developed a restriction fragment length polymorphism (RFLP) tool for mapping variation in the T. vaginalis AP33 adhesin in order to define putative associations between parasite subtype and mycoplasmata or host. Studies using crudepellets from T. vaginalis culture broth clearly confirm the association between T. vaginalis and M. hominis infection. The association between IL-1RA genotype 2,2 and lack of U. urealyticum infection is corroborated as well. U. urealyticum infection and infection by T. vaginalis are independent. Furthermore, T. vaginalis and M. hominis infection are not depending on IL-1RA genotypes. Interestingly, one of the three AP33 RFLP types identified appeared to be associated with the absence of U. urealyticum infection. In conclusion, the complex interaction between bacterial and parasitic pathogens and the infected host is determined by genetic characteristics of host and microorganisms involved.     

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.     

Effect of iron on the virulence of Trichomonas vaginalis

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T. vaginalis is reduced.     

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.     

Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence

Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens. The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23). To characterize AP33 further, six cDNA clones were examined. Restriction mapping indicated that the six clones represented three similar genes. Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T. vaginalis isolates. Analysis of full-length sequences determined that each contained a 930 bp open reading frame encoding a protein of approximately 33 000 Da. Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels. N-terminal protein sequencing established the presence of leader peptides. Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T. vaginalis AP33 adhesin. A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase α-subunit of several different organisms and virtually 100% identity to the reported T. vaginalis subunit. Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T. vaginalis AP33. The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host–parasite relationship.     

Cysteine proteinase 30 in clinical isolates of T. vaginalis from symptomatic and asymptomatic infected women

A cysteine proteinase of 30 kDa (CP30) of Trichomonas vaginalis, is known to play a role in cytoadherence of the parasite to host cells. However, the CP30 activity in clinical isolates from symptomatic and asymptomatic patients has not been analyzed. In the present study, CP30 was detected in 20 fresh and long-term culture maintained T. vaginalis isolates each from symptomatic and asymptomatic women by substrate gel electrophoresis and immunoblotting. Though CP30 was detected in all the fresh isolates from 20 symptomatic and 20 asymptomatic women, the intensity of CP30 band was significantly higher in isolates from symptomatic as compared to asymptomatic women indicating higher expression in former. CP30 was found in all the 20 long-term cultured isolates from symptomatic whereas only in 70% of asymptomatic women indicating that CP30 expression is a more stable characteristic of symptomatic isolates. The isolates from symptomatic women, demonstrated significantly higher cytoadherence to VECs as compared to asymptomatic women. In both the types of isolates, this cytoadherence was inhibited significantly by CP30 specific hyperimmune serum. These results confirm that CP30 is an important virulence factor of T. vaginalis and has an important role in cytoadherence to VECs and thus has a role in pathogenesis of trichomoniasis.     

Comparative study of epitopes recognized by two monoclonal antibodies that protects mice against Trichomonas vaginalis challenge

Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.     

AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule

The adherence mechanisms of enteropathogenic Escherichia coli (EPEC) to epithelial cells are still not understood. To study the molecular basis of the diffuse adherence (DA) phenotype exhibited by diarrhoeagenic E. coli expressing classical EPEC serotypes we investigated strain 2787 (O126:H27) isolated from a case of infantile diarrhoea. A 6.0 kb plasmid-derived DNA fragment mediates the DA phenotype and encodes the 100 kDa adhesin protein AIDA-I (adhesin involved in diffuse adherence). Sequencing of the entire fragment revealed two open reading frames which encoded proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as an AIDA-I precursor protein. After cleavage of the signal sequence further processing at the C-terminus of the 132 kDa precursor leads to the mature approximately 100 kDa AIDA-I. While the exact function of the cytoplasmic 45 kDa protein is not known, preliminary evidence indicates that it is necessary for the correct maturation of AIDA-I. The AIDA-I precursor exhibits significant homology with the virG(icsA) protein of Shigella flexneri which seems to be involved in the intercellular spread of invasive Shigella organisms.     

Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells

A secreted cysteine protease (CP) fraction from Trichomonas vaginalis is shown here to induce apoptosis in human vaginal epithelial cells (HVEC) and is analyzed by mass spectrometry. The trichomonad parasite T. vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, are shown to induce HVEC apoptosis, as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis is correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. SDS-PAGE analysis of the CP fraction reveals triplet bands around 30 kDa, and matrix-assisted laser desorption ionization time-of-flight MS indicates two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs results in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. The present results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention.     

Iron dictates the virulence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in urinary tract infections

Iron-limiting conditions have been reported to be prevalent in the milieu of urinary tract. In the present investigation, effect of iron on virulence of uropathogenic Pseudomonas aeruginosa in planktonic and biofilm cell mode was studied. Significant enhancement in elaboration of all the virulence traits along with increased adherence to uroepithelial cells and decreased phagocytosis of P. aeruginosa was observed following growth in iron-deplete medium. On the contrary, decrease in all these parameters except phagocytosis was observed when P. aeruginosa was grown in iron-rich medium. In vivo, P. aeruginosa grown in iron-deplete medium showed increased renal bacterial load and tissue pathology in a mouse model of ascending urinary tract infection compared with organisms grown in iron-replete medium. The results of the present study may help in understanding host–parasite interaction and in developing alternative preventive approach against P. aeruginosa induced urinary tract infections.     

Galectin-1 on cervical epithelial cells is a receptor for the sexually transmitted human parasite Trichomonas vaginalis

The extracellular protozoan parasite Trichomonas vaginalis causes the most prevalent non-viral sexually transmitted human infection, yet the pathogenesis of infection is poorly understood, and host cell receptors have not been described. The surface of T. vaginalis is covered with a glycoconjugate called lipophosphoglycan (LPG), which plays a role in the adherence and cytotoxicity of parasites to human cells. T. vaginalis LPG contains high amounts of galactose, making this polysaccharide a candidate for recognition by the galactose-binding galectin family of lectins. Here we show that galectin-1 (gal-1) is expressed by human cervical epithelial cells and binds T. vaginalis LPG. Gal-1 binds to parasites in a carbohydrate-dependent manner that is inhibited in the presence of T. vaginalis LPG. Addition of purified gal-1 to cervical epithelial cells also enhances parasite binding, while a decrease in gal-1 expression by small interfering RNA (siRNA) transfection decreases parasite binding. In contrast, the related galectin-7 (gal-7) does not bind T. vaginalis in a carbohydrate-dependent manner, and is unable to mediate attachment of parasites to host cells. Our data are consistent with the presence of multiple host cell receptors for T. vaginalis of which gal-1 is the first to be identified and highlight the importance of glycoconjugates in host-pathogen interactions.     

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.     

Trichomonas vaginalis lipophosphoglycan mutants have reduced adherence and cytotoxicity to human ectocervical cells

The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.     

The complex fibronectin--Trichomonas vaginalis interactions and Trichomonosis

Trichomonosis is the vaginitis caused by Trichomonas vaginalis. This sexually transmitted agent achieves successful host parasitism through various means including: (1). acquisition of nutrients through specific receptors; (2). recognition and binding to mucin followed by cytoadherence mediated by adhesins that resemble metabolic enzymes; (3). evasion of immune responses through (i). masking of organisms by host proteins, (ii). shedding of trichomonad proteins into the secretions and (iii). secretions of cysteine proteinases that degrade all immunoglobulin subclasses and complement; (4). alternating surface expression of at least two antigen repertoires; and (5) alternate and coordinate expression of virulence genes in response to host environmental factors. The fact that the parasite survives long term in the varying and adverse environment of the vagina attests to the highly evolved nature of this protist. An understanding of the non-self-limiting nature of this infection may come from recent findings illustrating the complexity of Trichomonas vaginalis-fibronectin (FN) interactions. The parasite readily attaches to surfaces with immobilized FN and binds to FN in a highly specific receptor-mediated fashion. The amount and affinity of bound FN by live organisms is influenced by concentrations in medium of both iron and calcium. De novo protein synthesis is required for optimal FN acquisition in the presence of calcium. Furthermore, the parasites bind with differing affinities to the N-terminal domain (NTD), the cell-binding domain (CBD) and the gelatin-binding domain (GBD) of FN. Iron modulates binding of NTD similar to that of FN. This minireview summarizes recent findings on the T. vaginalis-FN associations.