No abstract

SummaryHere, 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 immunouorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region. The ORF at the 58 end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.

One of the fundamental questions of the biology of Entamoeba histolytica directly related to the understanding of human amebiasis concerns the nature of the factors that determine the virulence of the parasite. The initiation of invasive amebiasis may result from the rupture of a host-parasite equilibrium that is maintained while E. histolytica is restricted to a commensal phase. No specific host factor has been shown to play a decisive role in the establishment of intestinal or liver lesions in those countries in which invasive amebiasis represents a common and important health problem. For these reasons, the emphasis of recent investigators has concentrated on the study of parasite virulence factors (1).The degree of virulence of cultured E. histolytica varies according to the strain (2, 3) and culture condition (4). The factors responsible for these variations remain obscure. Despite a large amount of information on the subject, ultrastructural (5) and biochemical (6) studies have not been able to demonstrate differences that could explain the variable degree of virulence. Certain cell surface properties appear to characterize pathogenic strains: adhesion to epithelial cells (7), susceptibility to agglutinate with concanavalin A (8), ability to produce lytic effect on cultured cells (9-1 1), and phagocytosis oferythrocytes (3,12). Recently, a correlation between collagenase production and virulence has been found (13).Traditionally, erythrophagocytosis has been the main laboratory criterion to identify pathogenic amebas (14, 15). Furthermore, a correlation between the rate of erythrocyte (RBC) ~ phagocytosis and virulence of various amebic strains has been found (3, 12). The results have been obtained with strains isolated directly from amebic patients and cultured for a long time, which therefore could differ in more than one property. Our aim was to isolate, from a virulent and phagocytic strain, a nonphagocytic clone and then ask how the virulence has changed. The reduction of phagocytosis was matched by a dramatic loss in virulence. Furthermore, virulent revertants isolated by serial passage through * Supported in part by grants SEIT-SEP, PCSABNA-002065, PCSAXNA