Abstract:
An agglutinin with trypsin activity was purified from the midgut extracts of Glossina morsitans morsitans Westwood using a combination of ion exchange and affinity column chromatography procedures. The molecule had a native apparent molecular weight (Mr) of -65,700 and consisted of two non-covalently linked subunits; an a. subunit (-28,800 Da) and a B subunit (~35,700 Da). The B subunit was shown to have a glycosyl residue. This agglutinin had a bloodstream trypanosome agglutination titre of 64 and a trypsin specific activity of 69 .3 μmoles/min./mg protein. The molecule compared favourably with the lectin-trypsin, complex previously purified from the midgut of G. longipennis. The G. m. morsitans midgut lectin-trypsin complex was immunologically detected in midgut extracts of other Glossina species but not in the gut extracts of other haematophagous insects. The lectin-trypsin complex was also shown to be widely distributed in tsetse tissues as it was immunologically detected in the fat body, haemolymph, midgut tissues and the peritrophic membrane. Biological activity (ability to agglutinate trypanosomes) was detected only in the peritrophic membrane homogenate. In the other tissues, the molecule was detected at higher molecular weights. These results suggest that the molecule was present in these tissues either as a precursor or bound to some other molecule which affected the biological activity. The subunits of the G. m. morsitans midgut lectin-trypsin complex were shown to be synthesised by the fat body and the midgut tissues as -42,000 Da and -62,000 Da molecules, respectively. The trypsin moiety was shown to be synthesised only by the midgut tissues. It was suggested that the lectin-trypsin complex is activated after the two subunits link up in the rnidgut lumen. The synthesis of proteins by the fat body was also hown to be stimulated by the bloodmeal, which corresponded to the release of the agglutinin into the midgut. The purified agglutinin was also able to induce in vitro transformation of trypanosomes from bloodstream to procyclic forms more than the crude midgut homogenate. Increasing the concentration of the lectin-trypsin complex resulted into higher transformation rates accompanied by an increase in trypanosome mortality. Of all the tissues shown to be associated with the midgut lectin-trypsin complex, only the peritrophic membrane induced the in vitro parasite transformation, although the mortality rates of the parasites were high. It is suggested that the peritrophic membrane due to its close association with the lectintrypsin complex is involved in trypanosome tropisms and trafficking within the tsetse midgut. Jn vivo studies also showed that elevation of the midgut concentrations of the lectin-trypsin complex significantly lowered the establishment rates of midgut T b. brucei infections in G. m. morsitans. On the other hand lowering the midgut concentrations of the lectin-trypsin complex did not significantly affect the establishment of the trypanosome midgut infection in the tsetse. It was proposed that the tsetse midgut lectintrypsin complex has a dual role in tsetse. In addition to acting as an immune molecule at higher concentrations, clearing the trypanosomes from the midgut, at lower concentrations the same molecule is involved in the induction of differentiation of trypanosomes and hence facilitate the establishment of midgut infections. The lectin moiety of the molecule was shown to appear bound to the bloodstream trypanosomes as a distinct ~35,000 Da band and inclusion of the inhibitor D( +) glucosamine completely abrogated its binding. A lower distinct band of ~19,400 Da was not affected by any of the inhibitors for the complex subunits. The ~35,000 Da band was proposed to represent the glycosyl residue of the molecule and is involved in the binding to trypanosomes. It has been proposed that further work on the identification of genes encoding for the subunits of the molecule would aid in using the molecule in a novel trypanosomosis control. Through genetic manipulation, creation of tsetse strains with over-expressed genes for the subunits would have higher concentrations of the midgut lectin-trypsin complex, and therefore have lower rates of midgut infection establishment in tsetse.