Antisense inhibition of amoebapore expression inEntamoeba histolyticacauses a decrease in amoebic virulence

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Abstract
Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasiteEntamoeba 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 theNeoresistance 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 theE. histolyticaribosomal protein (RP‐L21) gene copies. Transfectants of virulentE. histolyticastrain HM‐1:IMSS, in which the expression of amoebapore was inhibited by ~ 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 ofE. histolyticaexert their remarkable cytolytic and tissue destructive activity.