Recent issues of the biochemistry and molecular biology of Trypanosoma cruzi.

Abstract

Recent issues of the biochemistry and molecu-lar biology of Trypanosoma cruzi investigated byprestigious Brazilian research groups and discussedin the present meeting in commemoration of the90th anniversary of the recognition of Chagas dis-ease, center on glycoconjugates, their structure andfunction; differential gene expression in develop-mental stages of the parasite; organization and ex-pression of multigene families encoding virulencefactors; and the epidemiology, biochemistry andevolution of different strains of the parasite usingrRNA sequences.Almeida and coworkers have studied the com-plex structure of mucin-like glycoproteins frominfective forms of T. cruzi and advanced in theknowledge of their immunological properties.These molecules are densely packed over the en-tire surface of trypomastigotes and contain O-gly-cosidically-linked oligosaccharides which are ter-minally substituted by numerous sialic acid units.Many of the biological properties of these GPI-anchored mucins have been related to the presenceof these acidic residues. Two major features of T.cruzi mucins emerged from studies by differentgroups: (a) in contrast with mammalian mucins,the O-linked oligosaccharide chains of T. cruzimucins are bound via an internal unit of N-acetylglucosamine; (b) sialic acid units are addedto the oligosaccharide chains via a trans-sialidasereaction which uses sialylated donors from themedium. Mucins from epimastigotes andmetacyclic forms differ from those of cell culturetrypomastigotes in their apparent molecular mass,susceptibility to proteolysis and fine glycosidicstructure. They also show peculiar characteristicsin the lipidic moiety of the GPI anchors which arerelated with some of their immunological proper-ties. A striking feature of the infective trypomas-tigote mucins is the presence of terminal α-galactopyranosyl units in their oligosaccharideswhich are targets of lytic antibodies isolated frompatients with chronic Chagas disease. It is highlyrelevant that the successful chemotherapy in somechagasic patients can be monitored by the gradualdecrease in the lytic antibody titers. These anti-bodies are now being detected by extremely sensi-tive chemiluminescent Elisa using very smallamounts of purified trypomastigote mucins. Re-cent studies showed that trypomastigote mucins,but not those from epimastigotes or metacyclicforms potently induce the synthesis by stimulatedmacrophages of proinflammatory cytokines (IL-12 and TNF-α) and of nitric oxide. Since theseeffects are obtained using extremely reducedamounts of mucins they appear to have physiologi-cal relevance in vivo especially since mucins maybe shed into the medium by T. cruzi infectiveforms. A most careful study is presently being car-ried out by Almeida et al. to correlate minimumstructural requirements with immunobiologicalactivity using powerful methods of purificationand characterization of the mucins and their GPImoieties. The precise role of these components ofT. cruzi in the pathophysiology of experimental andhuman infection by this parasite may thus soon beunrevealed.Metacyclogenesis in T. cruzi is the centralproject of Goldenberg and coworkers and it hasbeen addressed using differential gene expression.A chemically defined medium was developed thatmimetizes the triatomine urine so that metacyclicforms obtained from either of them show compa-rable biological properties.To isolate and charac-terize the genes specifically expressed in the ter-minal stages of differentiation as well as duringthe process of metacyclogenesis, a technique called“representation of differential expression” (RDE)has been developed. The method is based on theamplification by PCR of specific gene sequencesisolated by subtractive hybridization, using oligo-nucleotides representing the mini-exon and poly-A sequences as primers for the PCR reaction, thus