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

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SummaryEhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhC-PADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.

This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30-54 % and 50-57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn 2+ -binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.

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