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

Abstract

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.

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

“…Conversely, the silencing of ehadh3b b did not increase the virulence of non-pathogenic amoebae. To assign the expression of the ehadh3b a gene directly to a phenotype, transfectants were used whose expression of ehadh3b b was either enhanced or silenced [ 20 , 21 ].…”

“…Generation of the clones A1 np , B2 p and B8 np derived from cell lines HM-1:IMSS-A and HM-1:IMSS-B has been described elsewhere [ 20 ]. Overexpression and silencing transfectants were generated and cultivated as described previously [ 20 , 21 ]. For overexpression of ehadh3b b ( EHI_160670 ) clone B2 p was transfected with the expression plasmid pNC containing the ehadh3b b gene under control of the E. histolytica lectin promotor (B2 p _pNC- ehadh3b b ).…”

“…The cells were cultivated in TYI-S-33 medium containing 20 µg/mL G-418 for 3 weeks. After cloning of the transfectants by limited dilution, the cells were cultivated for at least 4 months without selection until the plasmid was completely lost (B8 np _Si- ehadh3 b b ) [ 21 ]. B8 np trophozoites were used as controls.…”

“…Interestingly, overexpression of ehadh3b b in the pathogenic clone B2 p reduced the ability of the amoebae to form ALAs significantly [ 20 ]. However, pathogenicity was not affected by silencing the ehadh3b expression [ 21 ].…”

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“…RNA interference (RNAi) is a gene silencing technology (Fire et al, 1998) with broad applications, from reverse genetics and functional genomics (Suzuki et al, 2018), to treating diseases (Matthiesen et al, 2019), combating cancer (Ganesh et al, 2018), generating animal models (Guerreo-Rubio et al, 2019) and biotechnological products (Metwali et al, 2015).…”