Mario A. Rodríguez

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.

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.

Isolation and characterization of Entamoeba histolytica mutants resistant to emetine

Isolation of emetine-resistant mutants of Entamoeba histolytica is described. Spontaneous rate of mutation, obtained from a Luria-Delbrück fluctuation test was 2.5 X 10(-7). The mutagenesis frequency obtained from the number of colonies grown in the presence of emetine, divided by the number of viable trophozoites inoculated in semisolid agar was less than or equal to 10(-7) and it was increased from 10 to 100 fold with ethyl methanesulfonate. Two types of clones were isolated: one was cross-resistant to colchicine and the other was not, indicating that mutation took place at different loci. Protein synthesis in the presence of emetine remained unaltered for colchicine-sensitive mutants, suggesting that the molecular lesion lay in the protein synthesis machinery. Colchicine-resistant mutants showed a lower level of [35S]methionine incorporation, probably due to alterations in the molecular transport of some substances across the membrane. This mutant clone was also deficient in red blood cell adhesion, suggesting membrane alterations.

EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence

EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro‐peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro‐peptide and the mature enzyme expressed a non‐active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti‐tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence‐deficient mutant, and in the wild‐type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.

An Entamoeba histolytica Rab-like encoding gene and protein: function and cellular location

We identified here a 576 bp rab-like gene (EhrabB) in Entamoeba histolytica. EhrabB is located 332 bp upstream from the start codon of the Ehcp112 encoding gene, but is transcribed from the complementary strand. The EhrabB open reading frame predicts a 192 amino acid polypeptide (EhRabB) with 40-42% identity to Rab proteins, involved in vesicle docking regulation in endo and exocytic pathways of eukaryotic cells. Transcripts of 0.6 and 0.97 kb were detected by the EhrabB probe in northern blot assays. Using specific antibodies, EhRabB was located in small cytoplasmic vesicles by confocal microscopy. During phagocytosis, EhRabB was initially translocated to the plasma membrane and to the phagocytic mouths. The protein diminished after 10 min phagocytosis, suggesting that EhRabB could be participating in the regulation of the endocytosis process.

The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites

The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess.

Entamoeba histolytica: molecules involved in the target cell-parasite relationship

To detect molecules of Entamoeba histolytica involved in the trophozoite-target cell interaction, three different antisera were generated: (a) two rabbit antisera, one against total amebic proteins and another directed specifically to the 112-kDa adhesin; and (b) a mouse antiserum against amebic molecules adhering to the red blood cell (RBC) surface after incubation of RBCs with total soluble protein from trophozoites (anti-adhesion serum). All three antisera recognized the 112-kDa adhesion. Adhesion of this molecule to the RBC surface was temperature-dependent. More of the 112-kDa adhesion was found on the surface of RBCs incubated with trophozoites at 37 degrees C than on RBCs incubated at room temperature or at 0 degree C. Experiments using both anti-adhesin and anti-total ambebic protein sera revealed the presence of 210, 160, 112, 90, 70, 50, and 24-kDa proteins on RBC incubated with trophozoites. Surface proteins obtained from iodinated MDCK cells recognized amebic proteins of 112, 90, and 48-50 kDa. Virulence-deficient mutants presented a similar amount of the 112-kDa adhesin to the wild-type strain. However, in mutants, the adhesion was not functional, since they did not adhere to RBCs. 90- and 24-kDa proteins were also found to be altered in mutants.

Cloning and characterization of the Entamoeba histolytica pyruvate: ferredoxin oxidoreductase gene.

In E~ztumoeba histolytica, the microaerophilic protozoan responsible for human amebiasis. glucose is converted to pyruvate by the classical Embden-Meyerhof pathway [l]. In most anaerobic microorganisms, including E. histolvticu, pyruvate is transformed to acetyl-CoA by the pyruvate: ferredoxin oxidoreductase (PFO); a dimeric or tetrameric protein of 240 kDa [2]. In aerobic organisms the same reaction is catalyzed by the pyruvate dehydrogenase complex, which is a multienzymatic system of approximately 10” kDa [3]. The catalytic mechanisms of PFO and the pyruvate dehydrogenase complex are different [4]. The only property in common to both is the presence of thiamine pyrophosphate (TPP) as the

Regulation of Gene Expression in Protozoa Parasites

Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

Cloning and expression of an Entamoeba histolytica NADP+-dependent alcohol dehydrogenase gene

In this paper we cloned, sequenced and expressed a novel Entamoeba histolytica alcohol dehydrogenase gene (Ehadh3). Ehadh3 has a predicted 383 amino acids open reading frame, encoding for a 42.3 kDa protein. The deduced amino acid sequence showed 24 to 26% identity to other type III alcohol dehydrogenases found in prokaryotic and lower eukaryotic organisms, but not in mammalia. There are at least two Ehadh3 gene copies in the genome, but only a 1.2 kb transcript was detected. The EhADH3 fusion protein showed a NADP+(-)dependent ADH activity. Ehadh3 may be a good target for the developing of anti-E. histolytica drugs, without producing damage to the human.