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.
Highlights
The protozoan parasite Entamoeba histolytica is responsible for approximately 50 million cases of invasive amoebiasis per year, resulting in an annual death toll of 40,000–100,000 [1]
To analyse whether the E. histolytica cell lines consisted of a mixture of different cell types with different pathogenic phenotypes, the cell lines were cloned by limited dilution method
This resulted in 12 clones derived from cell line HM-1:IMSS-A and 12 clones derived from cell line HM-1:IMSS-B
Summary
The protozoan parasite Entamoeba histolytica is responsible for approximately 50 million cases of invasive amoebiasis per year, resulting in an annual death toll of 40,000–100,000 [1]. Homologues of the majority of these potential pathogenicity factors are present in the non-pathogenic sister species Entamoeba dispar, a commensal protozoan that is genetically closely related to E. histolytica. It remains to be shown whether one of these factors or their combination is responsible for amoeba pathogenicity or whether additional factors are involved. One straight-forward approach of identifying pathogenicity factors is a direct comparison of pathogenic and non-pathogenic E. histolytica isolates that has been performed using comparative microarray and proteome approaches [7,8,9,10] These studies used two isolates with completely different genetic backgrounds (pathogenic isolate HM-1:IMSS and non-pathogenic isolate Rahman). An in-depth phenotypical characterisation of the Rahman isolate revealed a number of genomic defects that presumably interfere with its virulence capacity [10]
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