Proteins differentially expressed between pathogenic and non-pathogenicEntamoeba histolyticaclones influence pathogenicity by different mechanisms

Abstract

AbstractRecently, two genes involved in pathogenicity in a mouse model of amoebic liver abscess were identified based on their differential expression between non-pathogenic (A1np) and pathogenic (B2p) clones of theEntamoeba histolyticaisolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 decreases the virulence of the pathogenic clone B2p, overexpression of the geneehi_127670(ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in B2pdecreases virulence. To understand the role of both molecules in determining the pathogenicity ofE. histolytica, silencing and overexpression transfectants were characterized in detail. Silencing ofehmp8-2, of the homologous geneehmp8-1, or of both together in A1nptrophozoites significantly altered the transcript levels of 60-350 genes. This strong change in the expression profile caused by the silencing ofehmp8-1and/orehmp8-2implies that these peptidases regulate expression of numerous genes. Consequently, numerous phenotypic characteristics including cytopathic, hemolytic and cysteine peptidase activity were changed in response to their silencing. Silencing ofehhp127in B2ptrophozoites did not affect other genes, whereas overexpression in A1nptrophozoites results in an altered expression of approximately 140 genes. EhHP127 appears to be important for trophozoite movement, as silencing negatively affects and overexpression positively affects trophozoite motility. Interestingly, the specific silencing ofehhp127also impairs cytopathic activity, cysteine peptidase and hemolytic activity. All three molecules of interest, namely EhMP8-1, EhMP8-2, and EhHP127 can be detected in amoeba vesicles. Our results clearly show that the proteins studied here influence the pathogenicity of amoebae in different ways and use entirely different mechanisms to do so.Author summaryThe human pathogenEntamoeba histolyticacan live asymptomatically in the intestine or become invasive and cause fatal liver abscesses. Approximately 15,000 people die each year as a result of an amoebic infection. Recently, two clones with different pathogenicity (A1np: non-pathogenic; B2p: pathogenic) derived from theE. histolyticaisolate HM:1-IMSS were compared at the transcriptome level. Two highly differentially expressed genes (ehhp127encoding a hypothetical protein andehmp8-2encoding a metallopeptidase) were identified. Analysis ofE. histolyticatransfectants showed that silencing ofehhp127and overexpression ofehmp8-2in B2ptrophozoites reduced amoebic liver abscess formation in the mouse model. In this study, we characterizedE. histolyticasilencing and overexpression transfectants ofehmp8-2, as well as of the homologous geneehmp8-1and ofehhp127. It was shown that the altered expression of the metallopeptidase genes has a strong influence on the expression of a large number of genes and that the phenotype is strongly altered as a result. Silencing ofehhp127does not affect the overall expression profile. However, specific silencing has a negative effect on motility, cysteine peptidase, hemolytic and cytopathic activity. All three molecules were shown to be localized in trophozoite vesicles.