Further enzymic characters of Trypanosoma cruzi and their evaluation for strain identification

Abstract
Starch-gel electrophoresis of 38 enzymes was attempted with extracts of Trypanosoma cruzi culture forms. 18 of the enzymes that gave discrete electrophoretic bands were selected for routine characterization of T. cruzi stocks; the enzymes were: aspartate aminotransferase (E.C. 2.6.1.1, ASAT); alanine aminotransferase (E.C.2.6.1.2, ALAT); phosphoglucomutase (E.C.2.7.5.1, PGM); glucosephosphate isomerase (E.C.5.3.1.9, GPI); malate dehydrogenase (oxaloacetate decarboxylating) (NADP+) (E.C.1.1.1.40, ME); glucose 6-phosphate dehydrogenase (E.C.1. 1.1.49, G6PD); malate dehydrogenase (E.C.1.1.1.37, MDH); aconitate hydratase (E.C.4.2.1.3, ACON); isocitrate dehydrogenase (NADP+) (E.C.1.1.1.42, ICD); alcohol dehydrogenase (NADP+) (E.C.1.1.1.2, ADH); lactate dehydrogenase (E.C.1.1.1.27, LDH); aminopeptidase (cytosol) (E.C.3.4.11.1, PEP); pyruvate kinase (E.C.2.7.1.40, PK); phosphoglycerate kinase (E.C.2.7.2.3, PGK); enolase (E.C.4.2.1.11, ENO); hexokinase (E.C.2.7.1.1, HK); mannosephosphate isomerase (E.C.5.3.1.8, MPI); and glutamate dehydrogenase (E.C. 1.4.1.2, GD). ADH (NADP+) in the genus Trypanosoma, and PGK, MPI and ENO, in T. cruzi, were apparently demonstrated for the first time. Between six and 18 enzymes were used to characterize more than 250 T. cruzi stocks, newly isolated from a wide range of sources in northern and central Brazil. All stocks were identified as belonging to T. cruzi zymodemes 1, 2 or 3, as originally defined—that is, by combination of electrophoretic patterns of ASAT, ALAT, PGM, GPI, ME and G6PD. The composite range of results with all enzymes confirmed the presence of three principal T. cruzi zymodemes, but some enzymic characters overlapped between zymodemes and others suggested subgroups within individual zymodemes. Seven (MDH, ACON, LDH, PK, PGK, ENO, HK) of the 18 enzymes did not distinguish the three zymodemes; five (ASAT, PGM, GPI, ICD, PEP) distinguished all three zymodemes; 10 (ASAT, ALAT, PGM, GPI, ME, G6PD, ICD, ADH, PEP, GD) distinguished zymodemes 1 and 2, of which seven plus MPI and eight plus MPI separated zymodemes 1 from 3 and 2 from 3 respectively. T. cruzi stocks were taken from a small area of the natural species distribution; the full range of enzymic characters within the species T. cruzi is expected to be far more complex. The epidemiological distribution of the zymodemes continued to accord with local transmission cycles and supported the hypothesis that distinct T. cruzi strains might be responsible for the enigmatic distribution of chronic Chagas's disease. Some of the difficulties in the empirical selection of new electrophoretic methods and the interpretation of results were presented, and the present and prospective significance of T. cruzi enzymic characters was discussed. Until the stability and genetic basis of T. cruzi enzymic characters are better understood it is recommended that isoenzymic profiles be confirmed routinely, both before and after stocks are used experimentally, as representative of a given zymodeme. A multiple biochemical approach to T. cruzi strain identification is recommended, using characters suitable for a numerical taxonomy.