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

BACKGROUND: Trichomonosis 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. RESULTS: An 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. CONCLUSION: This 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

The Interaction of Canine Plasminogen with Streptococcus pyogenes Enolase: They Bind to One Another but What Is the Nature of the Structures Involved?

For years it has been clear that plasminogen from different sources and enolase from different sources interact strongly. What is less clear is the nature of the structures required for them to interact. This work examines the interaction between canine plasminogen (dPgn) and Streptococcus pyogenes enolase (Str enolase) using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), fluorescence polarization, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and simple pull-down reactions. Overall, our data indicate that a non-native structure of the octameric Str enolase (monomers or multimers) is an important determinant of its surface-mediated interaction with host plasminogen. Interestingly, a non-native structure of plasminogen is capable of interacting with native enolase. As far as we can tell, the native structures resist forming stable mixed complexes

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

Host 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. We 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. These 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

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

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

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

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 ɛ-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

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- neo S (S) and pBS- neo AS (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 novel surface protein of is regulated independently by low iron and contact with vaginal epithelial cells-3

<p><b>Copyright information:</b></p><p>Taken from "A novel surface protein of is regulated independently by low iron and contact with vaginal epithelial cells"</p><p>BMC Microbiology 2006;6():6-6.</p><p>Published online 31 Jan 2006</p><p>PMCID:PMC1403785.</p><p>Copyright © 2006 Mundodi et al; licensee BioMed Central Ltd.</p>sites grown in normal medium (lane 1) or in low- (lane 2) versus high-iron (lane 3) medium. The bottom panel shows the RT-PCR products for the α-tubulin (-) gene as a control to show equal amounts of RNA in the PCR reactions. B. Immunoblot detection by mAb 6B8 of a 44-kDa protein after SDS-PAGE and blotting as described in the legend of Figure 1 of total proteins derived from trichomonads grown in normal medium (lane 1) and high- (lane 2) versus low-iron (lane 3) medium, as indicated. Numbers on the right are molecular weight standards in kilodaltons (× 1000)

A novel surface protein of is regulated independently by low iron and contact with vaginal epithelial cells-4

<p><b>Copyright information:</b></p><p>Taken from "A novel surface protein of is regulated independently by low iron and contact with vaginal epithelial cells"</p><p>BMC Microbiology 2006;6():6-6.</p><p>Published online 31 Jan 2006</p><p>PMCID:PMC1403785.</p><p>Copyright © 2006 Mundodi et al; licensee BioMed Central Ltd.</p> evident by uniform fluorescence seen in panel B1. In contrast, parasites isolated after contact with MS-74 VECs and handled identically had little or no detectable fluorescence as seen in panel B2. Brightfield pictures show the integrity of parasites used for the assay. No fluorescence was detectable in the absence of the mAb 6B8 as negative controls (not shown)