Vasanthakrishna Mundodi

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.

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.

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.

Adherence to Human Vaginal Epithelial Cells Signals for Increased Expression of Trichomonas vaginalis Genes

ABSTRACT Host parasitism by Trichomonas vaginalis is complex, and the adhesion to vaginal epithelial cells (VECs) by trichomonads is preparatory to colonization of the vagina. Since we showed increased synthesis of adhesins after contact with VECs (A. F. Garcia, et al., Mol. Microbiol. 47:1207-1224, 2003) and more recently demonstrated up-regulated gene expression in VECs after parasite attachment (A. S. Kucknoor, et al., Cell. Microbiol. 7:887-897, 2005), we hypothesized that enhanced expression of adhesin and other genes would result from signaling of trichomonads following adherence. In order to identify the genes that are up-regulated, we constructed a subtraction cDNA library enriched for differentially expressed genes from the parasites that were in contact with the host cells. Thirty randomly selected cDNA clones representing the differentially regulated genes upon initial contact of parasites with host cells were sequenced. Several genes encoded functional proteins with specific functions known to be associated with colonization, such as adherence, change in morphology, and gene transcription and translation. Interestingly, genes unique to trichomonads with unknown functions were also up-regulated. Semiquantitative reverse transcription-PCR (RT-PCR) confirmed expression of select genes. An increased amount of protein was demonstrated by immunoblotting with monoclonal antibody. Finally, we showed the transcriptional regulation of some genes by iron by using RT-PCR. To our knowledge, this is the first report addressing the differential regulation of T. vaginalis genes immediately upon contact with VECs.

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

BackgroundTrichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.ResultsAn IgA mAb called 6B8 was isolated from a library of mAbs reactive to surface proteins of T. vaginalis. The 6B8 mAb recognized a 44-kDa protein (TV44) by immunoblot analysis, and a full-length cDNA clone encoded a protein of 438 amino acids. Southern analysis revealed the gene (tv44) of T. vaginalis to be single copy. The tv44 gene was down-regulated at both the transcriptional and translational levels in iron-depleted trichomonads as well as in parasites after contact with immortalized MS-74 vaginal epithelial cells (VECs). Immunofluorescence on non-permeabilized organisms confirmed surface localization of TV44, and the intensity of fluorescence was reduced after parasite adherence to VECs. Lastly, an identical protein and gene were present in Tritrichomonas foetus and Trichomonas tenax.ConclusionThis is the first report of a T. vaginalis gene (tv44) encoding a surface protein (TV44) reactive with an IgA mAb, and both gene and protein were conserved in human and bovine trichomonads. Further, TV44 is independently down-regulated in expression and surface placement by iron and contact with VECs. TV44 is another member of T. vaginalis genes that are regulated by at least two independent signaling mechanisms involving iron and contact with VECs.

Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis

Host parasitism by Trichomonas vaginalis is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in T. vaginalis adherence, we silenced expression of ap65 using antisense RNA. The gene for ap65 was inserted into the vector pBS‐neo in sense and antisense orientations to generate plasmids pBS‐neoS (S) and pBS‐neoAS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the neo gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of ap65 transcript in AS transfected parasites. Growth kinetics of the antisense‐transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS‐transfectants compared to control organisms. Antisense silencing of ap65 was not alleviated by growth of trichomonads in high iron, which up‐regulates transcription of ap65. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in T. vaginalis to study the contribution of specific genes in pathogenesis.

A 5′ UTR‐mediated translational efficiency mechanism inhibits the Candida albicans morphological transition

While virulence properties of Candida albicans, the most commonly isolated human fungal pathogen, are controlled by transcriptional and post‐translational mechanisms, considerably little is known about the role of post‐transcriptional, and particularly translational, mechanisms. We demonstrate that UME6, a key filament‐specific transcriptional regulator whose expression level is sufficient to determine C. albicans morphology and promote virulence, has one of the longest 5′ untranslated regions (UTRs) identified in fungi to date, which is predicted to form a complex and extremely stable secondary structure. The 5′ UTR inhibits the ability of UME6, when expressed at constitutive high levels, to drive complete hyphal growth, but does not cause a reduction in UME6 transcript. Deletion of the 5′ UTR increases C. albicans filamentation under a variety of conditions but does not affect UME6 transcript level or induction kinetics. We show that the 5′ UTR functions to inhibit Ume6 protein expression under several filament‐inducing conditions and specifically reduces association of the UME6 transcript with polysomes. Overall, our findings suggest that translational efficiency mechanisms, known to regulate diverse biological processes in bacterial and viral pathogens as well as higher eukaryotes, have evolved to inhibit and fine‐tune morphogenesis, a key virulence trait of many human fungal pathogens.

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

BackgroundTrichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.ResultsIn this study, we show stable transfection and expression of the T.vaginalisap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs.ConclusionThe T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Genetic identity and differential gene expression between Trichomonas vaginalis and Trichomonas tenax.

BackgroundTrichomonas vaginalis is a human urogenital pathogen responsible for trichomonosis, the number-one, non-viral sexually transmitted disease (STD) worldwide, while T. tenax is a commensal of the human oral cavity, found particularly in patients with poor oral hygiene and advanced periodontal disease. The extent of genetic identity between T. vaginalis and its oral commensal counterpart is unknown.ResultsGenes that were differentially expressed in T. vaginalis were identified by screening three independent subtraction cDNA libraries enriched for T. vaginalis genes. The same thirty randomly selected cDNA clones encoding for proteins with specific functions associated with colonization were identified from each of the subtraction cDNA libraries. In addition, a T. vaginalis cDNA expression library was screened with patient sera that was first pre-adsorbed with an extract of T. tenax antigens, and seven specific cDNA clones were identified from this cDNA library. Interestingly, some of the clones identified by the subtraction cDNA screening were also obtained from the cDNA expression library with the pre-adsorbed sera. Moreover and noteworthy, clones identified by both the procedures were found to be up-regulated in expression in T. vaginalis upon contact with vaginal epithelial cells, suggesting a role for these gene products in host colonization. Semi-quantitative RT-PCR analysis of select clones showed that the genes were not unique to T. vaginalis and that these genes were also present in T. tenax, albeit at very low levels of expression.ConclusionThese results suggest that T. vaginalis and T. tenax have remarkable genetic identity and that T. vaginalis has higher levels of gene expression when compared to that of T. tenax. The data may suggest that T. tenax could be a variant of T. vaginalis.

Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

BackgroundHost parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.ResultsWe successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum.ConclusionThese results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis.