Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence

Amoebapores have been proposed to be a major pathogenicity factor of the pr otozoan parasite Entamoeba histolytica, which is responsible for the killin g of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphoc ytes, Inhibition of amoebapore gene expression in amoebae was obtained foll owing 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-L2 1) gene copies. Transfectants of Virulent E, histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by similar to 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viab le 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 abs cess formation in hamsters by the pAP-R2 transfectant was substantially imp aired. 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.