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Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence
R Bracha1, Y Nuchamowitz, M Leippe
1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel 76100, USA.
Abstract:
Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 5' and 3' untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by approximately 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.
Insights
Amoebapores, key to Entamoeba histolytica virulence, were targeted to reduce parasite pathogenicity. Inhibiting amoebapore gene expression significantly decreased E. histolytica
Area of Science:
- Pathogen biology
- Molecular parasitology
- Cellular cytotoxicity
Background:
- Amoebapores are proposed major virulence factors in Entamoeba histolytica, mediating target cell killing.
- These peptides share structural and functional similarities with cytotoxic lymphocyte molecules like NK-lysin and granulysin.
Purpose of the Study:
- To investigate the role of amoebapores in Entamoeba histolytica pathogenicity.
- To determine if inhibiting amoebapore gene expression reduces parasite virulence and cytolytic activity.
Main Methods:
- Gene silencing of amoebapore A using a reverse orientation plasmid construct (pAP-R2) in virulent E. histolytica strain HM-1:IMSS.
- Assessing parasite pathogenicity, cytopathic/cytolytic activities against mammalian cells and red blood cells.
- Evaluating pore-forming activity, antibacterial effects, and liver abscess formation in a hamster model.
Main Results:
- Transfectants with inhibited amoebapore expression (~60%) showed significantly reduced pathogenicity.
- Cytotoxicity against mammalian cells and red blood cell lysis were markedly inhibited.
- Impaired liver abscess formation in hamsters and reduced pore-forming and antibacterial activities were observed.
Conclusions:
- Amoebapore is confirmed as a critical pathogenicity factor for Entamoeba histolytica.
- Inhibition of amoebapore expression substantially attenuates parasite virulence and tissue-destructive capabilities.
- This study provides direct evidence for amoebapore's role in E. histolytica-mediated disease.