Carbohydrate-protein interactions play a key role in human infection by E. histolytica. Killing of host cells by E. histolytica trophozoites in vitro occurs only upon direct contact (Figure 9.2), which is mediated by an amebic adhesin that recognizes N- and O-linked oligosaccharides (Ravdin and Guerrant, 1981; Petri and Ravdin, 1987; Petri, 1996). This amebic Gal/Gal/NAc lectin is a heterodimer of heavy and light subunits, which are encoded by multigene families designated hgl and lgl, respectively (Petri, 1996). Apposition of amebic and target cell plasma membranes will not lead to cytolysis if the amebic lectin is inhibited with Gal/ GalNAc, which indicates that the lectin mediates adherence and also participates in the cytolytic event. Anti-lectin monoclonal antibodies (mAb), directed against epitope 1 of the lectin heavy subunit, block cytotoxicity but not adherence, implicating the lectin in the cytotoxic as well as adherence events. Anti-lectin antibodies, which block cytotoxicity, also cause a conformational change in the lectin, which increases carbohydrate-binding capacity.

The human colonic mucin layer of the large intestine may be the first receptor encountered by the lectin (Tse and Chadee, 1992). Binding of the lectin to colonic mucins is Gal/GalNAc-inhibitable and of very high affinity (dissociation constant of 8.2 x 10~n M_1). Interaction between the trophozoites and colonic mucins appears to be a dynamic process, with trophozoites both inducing the secretion of colonic mucins and degrading them (Tse and Chadee, 1991). The mucin layer may have a paradoxical role of both protecting the host from contact-dependent cytotoxicity of the parasite by binding to and neutralizing the lectin, while at the same time serving as a site of attachment for the parasite to colonize and invade the large bowel.

During its invasion of the colon and hemato-genous spread to the liver, the trophozoite has continuous exposure to the human complement system. Virulent E. histolytica isolated from patients with invasive amebiasis activate the alternative complement pathway but are resistant to C5b-9 complexes deposited on the membrane surface (Reed and Gigli, 1990). This complement resistance is due to the presence of a C5b-9

Fig. 9.2 Killing of a human polymorphonuclear neutrophil (N) by an ameba (A). Upon establishing contact with the ameba the neutrophil undergoes membrane blebbing, and loss of granules and cytoplasmic integrity. Magnification x2000. From Petri (1996), with permission

inhibitory molecule on the amebic surface, which has been identified as the Gal/GalNAc lectin (Braga et al., 1992). The sequence of the 170 kDa subunit of the surface lectin showed limited identity with CD59, a human inhibitor of C5b-9 assembly, and the purified lectin was recognized by anti-CD59 antibodies. The lectin bound to purified human C8 and C9 and blocked assembly in the amebic membrane of the complement membrane attack complex at the steps of C8 and C9 insertion. Reconstitution of the lectin from serum-resistant into serum-sensitive amebae conferred resistance to the membrane attack complex, a direct demonstration of its C5b-9 inhibitory activity. The lectin therefore appears to be multifactorially involved in the pathogen-esis of the amebae by having a central role in adherence and host cell killing, and by allowing the amebae to evade the complement system of the host (Braga et al, 1992).

Contact-dependent killing by E. histolytica has been intensively investigated. Intracellular calcium in target cells rises approximately 20-fold within seconds of direct contact by an amebic trophozoite and is associated with membrane blebbing (Ravdin et al., 1988) and cell death occurs 5-15 minutes afterwards. Murine myeloid cells killed by E. histolytica undergo a process of death that morphologically resembles apoptosis, the programmed cell death seen with growth factor deprivation, which is associated with a nucleosomal pattern of DNA fragmentation. This apoptotic pattern of host cell death induced by the amebae was blocked with Gal/GalNAc. However, overexpression of Bcl-2, a protein that confers resistance to apoptotic death from some stimuli, did not block killing (Ragland et al., 1994).

Amebic pore-forming proteins, similar in function to pore-forming proteins of the immune system, have been reported (Leippe et al., 1994) and a purified 5 kDa amebapore has recently been shown to have cytolytic activity for nucleated cells at high concentrations (10100 ^m) (Leippe et al., 1994). Interaction with the extracellular matrix may be mediated by fibronectin, laminin and collagen receptors (Talamas-Rohana and Meza, 1989; Munoz et al., 1991). Proteolytic activities, such as the collagenase contained within electron-dense granules in the amebic cytoplasm, are also believed to be involved in damage of cells and the extracellular matrix of the host. Secreted amebic cysteine proteases cause a cytopathic effect manifest by cells being released from monolayers in vitro without cell death (McKerrow et al., 1993). The availability of DNA transfection and inducible promoter systems for E. histolytica (Nickel and Tannish, 1994; Purdy et al., 1994; Vines et al., 1995; Hamann et al., 1995; Ramakrishnan et al., 1997) should enable the in vivo validation of virulence factors such as the lectin, cysteine proteinases and amebapore in the near future.

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