Trypanosoma cruzi: ultrastructural changes produced by an anti-trypanosomal factor from Pseudomonas fluorescens.

Trypomastigotes and amastigotes of Trypanosoma cruzi exhibited distinct ultrastructural alterations when treated with an extracellular lytic substance (anti-trypanosomal factor) produced by Pseudomonas fluorescens. Marked swelling of the parasites and detachment of the plasma membrane from the subjacent cytoplasm were observed after 15 min of treatment. After 3 hr, the nucleus was extensively damaged, the kinetoplast was indistinguishable, the mitochondrion was markedly swollen, the cytoplasm was disrupted, and the plasma membrane showed extensive blebbing and focal loss of subpellicular microtubules. These changes were progressive, as shown by the occurrence of parasite ghosts after 10 hr. Amastigotes exhibited an extremely swollen mitochondrion with disrupted internal structure, widening of the perinuclear space, and blebbing of the external nuclear membrane. The kinetoplast, however, remained clearly discernible. The drugs used today in controlling Chagas' disease are toxic. Therefore, there is a need for new anti-trypanosomal agents such as the Pseudomonas fluorescens antibiotics. The observations described in this study indicate the potential chemotherapeutic usefulness of these compounds for this disease.

Lysis of Trypanosoma cruzi by Pseudomonas fluorescens.

Trypomastigotes of Trypanosoma cruzi isolated from the blood of infected mice were lysed within 24 h by an extracellular substance produced by Pseudomonas fluorescens. Isolation of the anti-trypanosomal factor (ATF) was accomplished by growth of the organisms in a defined medium, extracellular secretion by the sedimented cells, sterilization by filtration, lyophilization, dialysis, and gel filtration. Chromatographic separation with Sephadex G-25 and G-200 disclosed the occurrence of three active fractions. ATF-I(1) exhibited a molecular weight higher than 440,000. ATF-II and ATF-III were considerably smaller (molecular weights approximately 1,355 and 1,060, respectively). The lytic substance contained protein and lipopolysaccharide, was resistant to heat and freezing, was not proteolytic or hemolytic, and was not inhibited by trypsin but was suppressed by pronase.

Creatine phosphokinase isoenzymes and Trypanosoma cruzi infections.

1. Substantial increases in total creatine phosphokinase (CPK) and in isoenzymes from heart (CPK-MB) and skeletal muscle (CPK-MM) were observed during acute infections with the House 510 and House 11 strains of Trypanosoma cruzi. 2. In infections with the reticulotropic Tulahuen strain total CPK levels were lower and the isoenzyme pattern was essentially normal. 3. Gamma-glutamyl transpeptidase was considerably increased in the Tulahuen but not in the House 510 and House 11 infections. 4. These findings are useful in assessing tissue damage during T. cruzi infections and they also demonstrate differences between myotropic and reticulotropic strains which may aid in their taxonomic classification.

Isolation of Trypanosoma cruzi from the blood of infected mice by column chromatography.

Trypomastogotes of three strains of Trypanosoma cruzi were isolated from the blood of infected mice employing lymphoprep for separation of the red blood cells and a column of DEAE cellulose for removal of white cells and platelets. An average recovery of 45 to 58 percent of actively motile, infective organisms, free of contaminating blood cells was obtained. Protein and carbohydrate assays of the separated organisms revealed significant differences between the Tulahuen, a reticulotropic strain, and the House 510 and House 11, two myotropic strains of this parasitic species. The present procedure should provide sufficient parasites for physiological and biochemical studies; it has also served to indicate particular strain characteristics which may aid in a taxonomic classification of these organisms.