Sialic acids and sialidases play important roles in cellular interactions and modulate the recognition of pathogenic microbes by mammalian host cells. Protozoan parasites of the genus Trypanosoma express a unique sialic acid-metabolizing enzyme. This enzyme, named trans-sialidase (TS), catalyzes the transfer of sialic acids from host glycoconjugates to acceptor molecules of the parasite plasma membrane. In African trypanosomes, the agents of sleeping sickness, TS is found only in forms developing within the insect vector, and the enzyme sialylates the major surface protein. In Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America, TS is expressed both in the insect and mammalian forms of the parasite. The T. cruzi enzyme has been biochemically characterized, and the gene encoding the enzyme has been cloned. The enzyme sialylates abundant mucin-like molecules present on the surface of the parasite. Several lines of evidence suggest that TS and sialic acid acceptors on the surface of T. cruzi participate in host-parasite interactions and mediate the initial stages of the trypanosomes' invasion of host cells.

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The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze oneelectron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase.

The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvum sporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion-and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvum infection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains ␣-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and ␣-N-acetylgalactosaminespecific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions.Cryptosporidium parvum, an intestinal Apicomplexan parasite, is a significant cause of diarrheal disease worldwide (15,17). In immunocompetent individuals, the disease is usually self-limiting, but it may be chronic and life threatening in immunocompromised patients such as those with AIDS. Recently, the parasite has gained notoriety as the causative agent of numerous outbreaks of waterborne diarrheal disease. There is currently no effective specific therapy approved for disease caused by this parasite.Infection is initiated by ingestion of oocysts, which undergo excystation to release sporozoites. Attachment of sporozoites to epithelial cells and subsequent invasion of the host cell membrane are crucial primary steps in the pathogenesis of cryptosporidiosis. The ultrastructural aspects of attachment and invasion have been characterized in detail (10,24,33,34). Sporozoites attach to host cells by their anterior pole. Attachment is followed by invagination of the host cell plasma membrane, which extends along the surface of the sporozoite and eventually completely surrounds it, leading to formation of a parasitophorus vacuole where the parasite undergoes further development in a unique intracellular but extracytoplasmic location.Using in vitro models of sporozoite attachment to epithelial cells, we previously showed that attachment was dose and time dependent and was influenced by pH, divalent cations, and the degree of differentiation of host cells (20,22). Further, attachment could be inhibited by polyclo...

The human pathogen Trypanosoma cruzi (Y strain) contains a neuraminidase activity that varies widely in the different developmental stages of the parasite. The specific neuraminidase activity of infective trypomastigotes obtained from tissue culture and from the bloodstream of infected mice is 7 to 15 times higher than that of the acellular culture forms. Amastigotes were devoid of enzyme activity. The enzyme has a pH optimum of 6.0 to 6.5. Live trypanosomes released sialic acid from human erythrocytes and plasma glycoproteins. Several sialyl compounds were hydrolyzed by the parasite, but the best substrate was the protein orosomucoid. Erythrocytes from infected mice with T. cruzi parasitemia were agglutinated by peanut lectin and the hemagglutination titer was correlated with the degree of parasitemia.

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SummsryTrypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage. Bound enzyme sheds into the extracellular milieu in a soluble form. Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T. cruzi-host interactions, like cell adhesion and complement resistance. However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood. To begin to study the role the enzyme may play in vivo, T. cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein.A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality. Maximum enhancement was achieved with 1-2-h priming. Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence. The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occurred when the sites of trans-sialidase sensitization and parasite inoculation were physically separate. Rather, virulence enhancement seemed to depend on inflammatory cells, since priming with trans-sialidase had no significant effect in severe combined immunodeficiency mice, which lack functional T and B lymphocytes. However, antibody response to T. cruzi in the transsialidase-primed BALB/c mice was the same as in the control animals. Virulence enhancement was specific for the trans-sialidase because it did not occur in mice primed with Newcastle virus sialidase, which has the same substrate specificity as the T. cruzi enzyme, or with the sialidase from the bacterium Vibrio cholerae, whose substrate specificity is broader than the trypanosome sialidase. Furthermore, no enhancement of virulence occurred after sensitization with another adhesion protein (penetrin) purified from T. cruzi trypomastigotes and engineered bacteria, nor with bacterial lipopolysaccharide. The virulence-promoting activity of soluble trans-sialidase in the mouse model may be physiologically relevant because it was achieved with tiny doses, ,ol-2 #g/kg, raising the possibility that neutralization of the enzyme with specific probes could impair the development of Chagas' disease. In fact, a monoclonal antibody specific for the tandem repeat in the trans-sialidase COOH terminus enhanced infection of BALB/c mice, in agreement with earlier experiments in vitro, whereas antibodies against an amino acid sequence in the Cys region had the opposite effect.