Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

Abstract

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

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

“…Recently, clones of a nonpathogenic cell line (HM-1:IMSS-A) and a pathogenic cell line (HM-1:IMSS-B) were generated and analyzed for their ability to induce ALA in 2 murine models for the disease (8). All clones derived from HM-1: IMSS-A show no or very small abscess formation 7 d after infection.…”

“…This is more divergent for clones derived from HM-1:IMSS-B: for 2/3 of the B-clones, clear abscess formation was visible 7 d after infection, whereas for the other 1/3 abscess formation was significantly lower or absent. The pathogenic phenotype of the respective B-clones remains stable over years without further animal passages (8).…”

“…To identify the factors putatively involved in abscess formation, 3 clones (A1 np , B2 p , and B8 np ) were selected for in-depth transcriptome analyses (8). In total, 76 genes are differentially expressed between clones A1 np and B2 p , and 19 genes are differentially expressed between B2 p and B8 np .…”

“…In total, 76 genes are differentially expressed between clones A1 np and B2 p , and 19 genes are differentially expressed between B2 p and B8 np . Only 6 genes are similarly regulated between the nonpathogenic clones A1 np and B8 np and the pathogenic clone B2 p (8). To investigate the influence of the proteins encoded by the differentially expressed genes, overexpression transfectants of the most promising candidates were generated.…”

“…These genes encode for a metallopeptidase, C2 domain proteins, alcohol dehydrogenases, and hypothetical proteins. Only 1 gene (EHI_127670), encoding a hypothetical protein, increases pathogenicity upon its overexpression (8).…”