Trypanothione reductase from the human parasite Entamoeba histolytica: a new drug target.

Although there is a general agreement that the protist Entamoeba histolytica lacks glutathione, it has been a matter of dispute as to whether this human parasite contains the glutathione derivative known as trypanothione. In the present study, we describe a gene for the TR (trypanothione reductase) obtained from E. histolytica by PCR amplification of its DNA. After Northern-blot analysis, the radiolabelled DNA probe from Trypanosoma cruzi hybridizes with the total RNA of Entamoeba, showing that the TR gene is expressed as mRNA. We have demonstrated the presence of the NADPH-dependent TR activity in vitro with partially purified extracts and showed also that the thiol-bimane compound isolated and purified from E. histolytica trophozoites, unequivocally corresponds, by matrix-assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) with m/z 1104.4 and the trypanothione-(bimane) with m/z 914.3. The PCR product consisted of 1476 bp (491 deduced amino acids), has sequences diagnostic for the reducing catalytic site (CVNVGC) as well as domains for binding NADPH, FAD I and FAD II that are present in all members of this group of disulphide-reducing enzymes, as well as those unique to TRs. The putative protein sequence is 86% identical with that of TR from T. cruzi and it is also clearly distinguishable from other related reductases by phylogenetic analysis. We can conclude, from these highly reliable experiments, that E. histolytica contains the TR enzyme and the thiol compound trypanothione that was previously supposed to occur only in trypanosomatids.

Characterization of two long vesicle-associated membrane proteins or longins genes from Entamoeba histolytica.

BACKGROUND: This study characterizes two long vesicle-associated membrane protein (VAMP) genes (SYBL-1 and SYBL-2) obtained from DNA of Entamoeba histolytica by PCR amplification. (Nucleotide sequences of the Entamoeba histolytica SYBL-1 and SYBL-2 genes appear in the GeneBank under accession numbers AY256852 and AY309014.) METHODS: The two cDNA products include one of 663 bp with sequence of 220 deduced amino acids, and a second of 693 bp with 230 deduced amino acid residues. Both products possess corresponding sequences for longin domain at N-terminus, a soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor (SNARE) coiled-coil region, a transmembrane domain (TM), and an intravesicular tail C-terminal, characteristics of all long VAMPs or longins. The current study identified presence of deduced peptide bonds in SYBL-1 and -2, which indicates that these two long VAMPs from E. histolytica could be appropriate substrates for zinc endopeptidases from tetanus and botulinum neurotoxins with specific activity toward neuronal synaptobrevins. RESULTS: Alignment by Basic Local Alignment Search Tool (BLAST) of deduced amino-acid sequence of long VAMPs genes SYBL-1 and -2 showed identity of between 20 and 40% with Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus, Homo sapiens, and Plasmodium falciparum. CONCLUSIONS: It is possible that these two putative transport proteins are involved in endocytosis/exocytosis during a biological membrane fusion process, which may make them suitable candidates as targets for new drugs. According to published data, this is the first time that two such genes have been isolated from E. histolytica.