ABSTRACT
An in vitro system for studying the transformation of bloodstream forms of Trypanosoma brucei brucei into procylic (midgut) forms is described. In this system, transformation of the parasites was stimulated by Glossina morsitans morsitans midgut homogenates at 27 degrees C but not at 4 degrees C. The transformation-stimulating capacity was irreversibly destroyed by heating the midgut homogenates at 60 degrees C for 1 h. A correlation was established between the transformation activity of the midgut homogenates and trypsin activity. The protease inhibitors (soybean trypsin inhibitor and N-p-tosyl-L-lysine-chloromethyl-ketone) inhibited trypsin activity and completely blocked the transformation of the parasites. Furthermore, the midgut homogenates could induce transformation only in the presence of blood. These results provide evidence for the involvement of trypsin or trypsin-like enzymes within the tsetse midgut in stimulation of the transformation of bloodstream trypanosomes.
Subject(s)
Insect Vectors/enzymology , Trypanosoma brucei brucei/growth & development , Trypsin/pharmacology , Tsetse Flies/enzymology , Animals , Hot Temperature , Insect Vectors/parasitology , Temperature , Trypanosoma brucei brucei/drug effects , Trypsin Inhibitors/pharmacology , Tsetse Flies/parasitologyABSTRACT
The ability of Trypanosoma brucei brucei to inhibit trypsin or trypsin-like enzymes in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The isolated parasites caused a concentration-dependent decrease in midgut trypsin activity. Furthermore, trypanosomes lysed by repeated freeze-thawing had a similar effect on trypsin activity. In both cases, the inhibition by either intact or lysed parasites was partial as revealed by Dixon plots. Similarly, trypanosome membrane proteins stoichiometrically inhibited trypsin activity, suggesting that the enzyme interacts specifically with a moiety on the parasite surface. The Km and Ki values obtained in this case were 35 microM and 0.18 mg/ml, respectively. These results suggest that one of the ways in which trypanosomes overcome the hostile tsetse-fly midgut barrier involves the inhibition of enzyme activity.
