Rossana Arroyo

Entamoeba histolytica : a novel cysteine protease and an adhesin form the 112 kDa surface protein

Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non‐coding region. The ORF at the 5′ end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.

CP30, a cysteine proteinase involved in Trichomonas vaginalis cytoadherence.

ABSTRACT We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno‐crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full‐length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701‐base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 63087 daltons, respectively. Comparison of the full‐length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction‐enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65−1 and ap65−2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of ∼1.8 kb for both ap65−1 and ap65‐2 genes in trichomonads grown only in high‐iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.

Localization and identification of an Entamoeba histolytica adhesin

The adherence of Entamoeba histolytica trophozoites to target cells was studied by using monoclonal antibodies (MAbs) and adhesion-deficient mutants of the parasite. MAbs Adh-1 and Adh-2 reacted with a surface protein of approximately 112 kDa of the total proteins of trophozoites from the wild type strain, clone A, strain HM1:IMSS. Both MAbs reacted weakly with the adhesion-deficient mutant clones, C-98, C-919 and C-923, all derived from HM1:IMSS. MAbs Adh-1 and Adh-2 incubated with trophozoites from clone A inhibited adherence to red blood cells, erythrophagocytosis and cytopathic effect on cell culture monolayers. Antibodies against a approximately 112 kDa polypeptide were found in the sera from patients with hepatic abscess. These results demonstrate that the adherence of trophozoites to target cells is a necessary event in order for cytopathogenicity to occur.

The effects of environmental factors on the virulence of Trichomonas vaginalis.

This review focused on potential regulatory mechanisms of Trichomonas vaginalis virulence properties, cytoadherence, cytotoxicity, phagocytosis, hemolysis, induction of apoptosis, and immune evasion in response to environmental factors of the human urogenital tract, iron, zinc, and polyamines. Understanding the multifactorial nature of trichomonal pathogenesis and its regulation may help to unravel the survival strategies of trichomonads and to implement prevention policies, opportune diagnosis, and alternative treatments for control of trichomoniasis.

A Trichomonas vaginalis 120 kDa protein with identity to hydrogenosome pyruvate:ferredoxin oxidoreductase is a surface adhesin induced by iron.

Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 µM iron medium. Antibodies to AP120 and the electro‐eluted AP120 inhibited parasite adherence in a concentration‐dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy‐terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT‐PCR) assays showed that a pfoa‐like gene was better transcribed in trichomonads grown in iron‐rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.

Extensive Genetic Diversity, Unique Population Structure and Evidence of Genetic Exchange in the Sexually Transmitted Parasite Trichomonas vaginalis

Background Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasites genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes. Methodology/Principal Findings Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages. Conclusions/Significance Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.

A 39‐kDa Cysteine Proteinase CP39 from Trichomonas vaginalis, Which Is Negatively Affected by Iron May Be Involved in Trichomonal Cytotoxicity

Trichomonas vaginalis is a flagellate protozoan, which infects the urogenital tract of humans. It is responsible for trichomonosis, one of the most prevalent sexually transmitted diseases (STDs) [26]. Patients with this chronic infection are at a higher risk for HIV seroconversion [18,24], exhibit adverse outcomes during pregnancy [lo], and may be more susceptible for cervical cancer [28]. This parasite has many proteinases; most of them are cysteine proteinases (CPs) [7,9,23]. At least 23 different CPs were identified by two-dimensional (2-D) substrate gel electrophoresis [23]. Some of them are involved in cytotoxicity [3,5], hemolysis [11,13]. immune response evasion [27] or cytoadherence [4,5,22]. Previously, we identified a 30- and a 65-kDa 7: vaginalis proteinases that bound to HeLa and vaginal epithelial cells surfaces [5]. These proteinases are involved in cytoadherence and cytotoxicity, respectively [3,22]. Recently, we have also identified a new 39-kDa cysteine proteinase that binds to HeLa cell surfaces, and degrades different biological substrates. The 39-kDa proteinase is secreted by the parasite in vivo and in vitro, is active in the broad pH range (3.6- 9.0) found in human vagina and prostate, and is located in the surface of the parasite. Also, patients with trichomonosis have circulating antibodies to this CP, named CP39 (Hernandez-Gutierrez, R., Avila- Gonzilez, L., Ortega-L6pez. J., Cruz-Talonia, F., G6mez-Gutierrez, G.

The trichomonad cysteine proteinase TVCP4 transcript contains an iron-responsive element

The differential expression of the Trichomonas vaginalis cysteine proteinase TVCP4 by iron at the protein synthesis level and the prediction of an iron‐responsive element (IRE)‐like stem‐loop structure at the 5′‐region of the T. vaginalis cysteine proteinase 4 gene (tvcp4) mRNA suggest a post‐transcriptional mechanism of iron regulation in trichomonads mediated by an IRE/IRP‐like system. Gel‐shifting, UV cross‐linking and competition experiments demonstrated that this IRE‐like structure specifically bound to human iron regulatory protein‐1. IRP‐like cytoplasmic proteins that bound human ferritin IRE sequence transcripts at low‐iron conditions were also found in trichomonads. Thus, a post‐transcriptional regulatory mechanism by iron for tvcp4 mediated by IRE/IRP‐like interactions was found.

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.