An Alcohol Dehydrogenase 3 (ADH3) from Entamoeba histolytica Is Involved in the Detoxification of Toxic Aldehydes.

Recently, a putative alcohol dehydrogenase 3, termed EhADH3B of the Entamoeba histolytica isolate HM-1:IMSS was identified, which is expressed at higher levels in non-pathogenic than in pathogenic amoebae and whose overexpression reduces the virulence of pathogenic amoebae. In an in silico analysis performed in this study, we assigned EhADH3B to a four-member ADH3 family, with ehadh3b present as a duplicate (ehadh3ba/ehadh3bb). In long-term laboratory cultures a mutation was identified at position 496 of ehadh3ba, which codes for a stop codon, which was not the case for amoebae isolated from human stool samples. When using transfectants that overexpress or silence ehadh3bb, we found no or little effect on growth, size, erythrophagocytosis, motility, hemolytic or cysteine peptidase activity. Biochemical characterization of the recombinant EhADH3Bb revealed that this protein forms a dimer containing Ni2+ or Zn2+ as a co-factor and that the enzyme converts acetaldehyde and formaldehyde in the presence of NADPH. A catalytic activity based on alcohols as substrates was not detected. Based on the results, we postulate that EhADH3Bb can reduce free acetaldehyde released by hydrolysis from bifunctional acetaldehyde/alcohol dehydrogenase-bound thiohemiacetal and that it is involved in detoxification of toxic aldehydes produced by the host or the gut microbiota.

Trigger-induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica.

Recently, Entamoeba histolytica clones derived from isolate HM-1:IMSS that differ in their pathogenicity were identified. Whereas some clones induce amoebic liver abscesses (ALAs) in animal models of amoebiasis, others provoke only minimal liver lesions. Based on transcriptome studies of pathogenic and nonpathogenic clones, differentially expressed genes associated with reduced or increased liver pathology can be identified. Here, to analyze the influence of these genes on ALA formation in more detail, an RNA interference-trigger mediated silencing approach was used. Using newly identified trigger sequences, the expression of 15 genes was silenced. The respective transfectants were analyzed for their ability to induce liver destruction in the murine model for the disease. Silencing of EHI_180390 (encoding an AIG1 protein) increased liver pathology induced by a nonpathogenic parent clone, whereas silencing of EHI_127670 (encoding a hypothetical protein) decreased the pathogenicity of an initially pathogenic parent clone. Additional phenotypical in vitro analyses of EHI_127670 silencing as well as overexpression transfectants indicated that this molecule has an influence on size, growth, and cysteine peptidase activity of E. histolytica. This work describes an example of how the sole operational method for effective gene silencing in E. histolytica can be used for comprehensive analyses of putative pathogenicity factors.-Matthiesen, J., Lender, C., Haferkorn, A., Fehling, H., Meyer, M., Matthies, T., Tannich, E., Roeder, T., Lotter, H., Bruchhaus, I. Trigger-induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica.