Entamoeba histolytica: a novel cysteine protease and an adhesin form the 112kDa surface protein

Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using mono clonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles, mAbAdh inh ibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We c loned a 3587 bp DNA fragment (Eh112) with two open reading frames (ORFs) se parated by a 188 bp non-coding region. The ORF at the 5' end (Ehcp112) enco des a protein with a cysteine protease active site, a transmembranal segmen t and an RGD motif. The second ORF (Ehadh112) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylatio n sites. Northern blot, primer extension and Southern blot experiments reve aled that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and E hadh112 genes were expressed in bacteria. The recombinant peptides presente d protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides coul d be joined by covalent or strong electrostatic forces, which are not broke n during phagocytosis.