Isolation of Glossina Typanolysis/Trypanoglutinin and Assessment of their use in the Interruption of Trypanosome Differentiation

Abstract

Midgut trypanolysin from Glossina morsitans morsitans was isolated by a combination of an anion-exchange and gel permeation chromatography. The trypanolysin activity was recovered in the bound fraction. The native molecular weight of trypanolysin was determined -669kDa. Analysis oftrypanolysin by SDS-PAGE recovered a single sub-unit ofMr-14kDa. The induction of trypanolysin activity by bloodmeal increased gradually reaching a peak at 72-120 h after the bloodmeal, and then decreased rapidly, with only 25% of the peak activity remaining after 192 h. Midgut homogenates from twice-fed G. m. morsitans had the highest trypanolysin activity against bloodstream-form trypanosomes followed by those once-fed and unfed. Trypanolysin was found in all Glossina species with Glossina longipennis which had the highest titre and G. m. morsitans the lowest. The minimum trypanolysin concentration that lysed trypanosomes was determined as 1.76 mg/ml. Tsetse membrane-fed with serum showed slightly higher trypanolysin activity compared to those fed on red blood cells and whole blood. However, the titre with red blood cells was the same as whole blood. Divalent cations Ca++ and Mg++ had no effect on the activity of the trypanolysin. Trypanolysin was not affected by tosylamide-2- phenylethyl chloromethyl ketone (TPCK), N-a-p-tosyl-1-lysine chloromethyl ketone (TLCK), phenyl methyl sulphonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP) and iodoacetamide. However, the activity of trypanolysin was partially inhibited by approtinin and completely by pronase and diethyl pyrocarbonate (DEPC). While trpanoagglutinin agglutinates bloodstreamfonn trypanosomes, trypanolysin lysis the trypanosomes. In contrast, while procyclic-form trypanosomes were agglutinated by trypagglutinin, they were not lysed by trypanolysin. The absence of the trypanolysis may be related to presence ofprocyclin coat on procyclic-forms. Thus trypanolysin is unlike trypanoagglutinin. All the sugars tested had no effect on trypanolysin activity. However, trypanoagglutnin was N-acetyl diglucosamine specific. It could be that trypanolysin has no specific binding sites for these sugars and is thus, trypanolysin is unlike trypanoagglutinin that has sugar specificity. Although soybean trypsin inhibitor (STI) completely inhibited trypanogglutinin activity for G. m. morsitans midgut, the same concentration had no effect on trypanolysin activity. While trypsin activates and digests trypanoagglutinin, the enzyme has no similar effects on trypanolysin. Therefore, trypanolysin was unlikely to be a trypanoagglutinin. Additionally, lipid moieties absent in trypanoagglutinin are present in trypanolysin. Based on these differences, trypanolysin is unlikely to be trypanoagglutinin or acted as one. The trypanolysin was inactivated during storage at 27° C and 4° C after 15 and 32 days, respectively. However, trypanolysin activity remained stable when stored at 0° Cup to the end of experiment ( 60 days). The trypanolysin was found to be very stable at -20, -70 and -80° C up to the 30th freeze-thaw cycle. However, the activity of the molecule progressively decreased after the 33rct, 41 st and 55th cycle at -80, -70 and -20° C, respectively, suggesting that several freezethaw cycle denatured trypanolysin. Temperature about 80° C, virtually rendered trypanolysin inactive, suggest that midgut trypanolysin, being proteinaceous are denatured by heat, just as was the case with trypanoagglutinin. Whereas, 50° C was found to be the optimum temperature for trypanolysin activity. Antibodies raised against the trypanolysin showed that G. m. morsitans, G. m. central is, G. longipennis, G. f fuscipes and G. pallidipes gave positive reactions in western blots comparing to Schistosoma calcitrans and A. aegypti which showed negative reactions. The in vitro and in vivo transformation studies using trypanolysin meal showed that trypanolysin meal fed to G. m. morsitans, G. m. central is, G. longipennis, G. f fuscipes and G. pallidipes was responsible for transformation blockage and lysis of trypanosomes. The results from this study suggest that the midgut trypanolysin play important role in the establishment of trypnosome infection in tsetse.