Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

BACKGROUND: The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells. RESULTS: The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis. CONCLUSIONS: The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination

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

<p>Abstract</p> <p>Background</p> <p><it>Trichomonas vaginalis </it>is a human urogenital pathogen responsible for trichomonosis, the number-one, non-viral sexually transmitted disease (STD) worldwide, while <it>T. tenax </it>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 <it>T. vaginalis </it>and its oral commensal counterpart is unknown.</p> <p>Results</p> <p>Genes that were differentially expressed in <it>T. vaginalis </it>were identified by screening three independent subtraction cDNA libraries enriched for <it>T. vaginalis </it>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 <it>T. vaginalis </it>cDNA expression library was screened with patient sera that was first pre-adsorbed with an extract of <it>T. tenax </it>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 <it>T. vaginalis </it>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 <it>T. vaginalis </it>and that these genes were also present in <it>T. tenax</it>, albeit at very low levels of expression.</p> <p>Conclusion</p> <p>These results suggest that <it>T. vaginalis </it>and <it>T. tenax </it>have remarkable genetic identity and that <it>T. vaginalis </it>has higher levels of gene expression when compared to that of <it>T. tenax</it>. The data may suggest that <it>T. tenax </it>could be a variant of <it>T. vaginalis</it>.</p

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

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

BACKGROUND: Trichomonosis, 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. RESULTS: In this study, we show stable transfection and expression of the T. vaginalis ap65 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. CONCLUSION: The 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

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

Trichomonas vaginalis adherence mediates differential gene expression in human vaginal epithelial cells

Trichomonas vaginalis, an ancient protist, colonizes the vaginal mucosa causing trichomonosis, a vaginitis that sometimes leads to severe health complications. Preparatory to colonization of the vagina is the adhesion to vaginal epithelial cells (VECs) by trichomonads. We hypothesized that VECs alter the gene expression to form a complex signalling cascade in response to trichomonal adherence. In order to identify the genes that are upregulated, we constructed a subtraction cDNA library after contact with parasites that is enriched for differentially expressed genes from the immortalized MS-74 VECs. Sixty cDNA clones were sequenced and to our knowledge for the first time, differentially regulated genes were identified in response to early trichomonal infection. The identified genes were found to encode functional proteins with specific functions associated with cell structure maintenance and extracellular matrix components, proinflammatory molecules and apoptosis. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed expression of selected genes. Further, cyclooxygenase 2 (COX-2) protein expression was analysed using Western blot and immunofluorescence assays. Data suggest that p38 mitogen-activated protein (MAP) kinase and tyrosine kinases play a role in COX-2 induction. Finally, T. vaginalis and Tritrichomonas foetus but not Pentatrichomonas hominis induce expression of COX-2. This is a first attempt at elucidating the basis of interaction of trichomonads with host cells and the corresponding host responses triggered by the parasites

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

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