Genes differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity-associated phenotypes by multiple mechanisms

Recently, two genes involved in amoebic liver abscess formation in a mouse model were identified by their differential expression of non-pathogenic (A1(np)) and pathogenic (B2(p)) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 reduces the virulence of the pathogenic clone B2(p), overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1(np), while silencing this gene in the pathogenic B2(p) reduces virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing, and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or both in non-pathogenic A1(np) trophozoites significantly altered the transcript levels of 347, 216, and 58 genes, respectively. This strong change in the expression profiles caused by the silencing of ehmp8-1 and ehmp8-2 implies that these peptidases regulate the expression of numerous genes. Consequently, numerous phenotypic characteristics, including cytopathic, hemolytic, and cysteine peptidase activity, were altered in response to their silencing. Silencing of ehhp127 in pathogenic B2(p) trophozoites did not affect the expression of other genes, whereas its overexpression in non-pathogenic A1(np) trophozoites results in an altered expression of approximately 140 genes. EhHP127 is important for trophozoite motility, as its silencing reduces, while its overexpression enhances movement activity. Interestingly, the specific silencing of ehhp127 also significantly affects cytopathic, cysteine peptidase, and hemolytic activities. All three molecules characterized in this study, namely EhMP8-1, EhMP8-2, and EhHP127, are present in amoeba vesicles. The results show that ehmp8-2 and ehhp127 are not only differentially expressed between pathogenic and non-pathogenic amoebae, but that they also significantly affect amoeba pathogenicity-associated phenotypes by completely different mechanisms. This observation suggests that the regulation of amoeba pathogenicity is achieved by a complex network of molecular mechanisms rather than by single factors.