Trypanosoma brucei releases proteases extracellularly

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a peak at pH 8.0, but not that at pH 94, was also

1 Short Report 1 Trypanosoma brucei releases proteases extracellularly Mark Nwagwu, D. M. N. Olcenu, T. A. Olusi and R. I. Molokwu Cellular Parasitology Programme, Department of Zoology, Universi~ of Zbaah, Zbadan, NigtG

Thrombocytopaenia is a complication of trypanosomiasis (DAVIS, 1982); its biochemical basis is not clearly known but it 1sindependent of complement, kinin, antibody, adenosine diphosphate or fatty acids (DAVIS et al., 1974). Recent studies in this laboratory have shown that a factor or factors released by Typanosoma brucei induced platelet aggregation. Proteases are known potent inducers of blood coamlation and platelet aggregation. In this study we ha6 investigated whether& not active proteas& are releasedbv T. bwei as a lirst steo toward definina the b&hem&l basis of platelet a&egation in eosomiasis. T. brucei stock NITR 8/18 (a virulent stock cloned by the Nigerian Institute fo; Trypanosomiasis Research, Vom) was inoculated into rats (lo6 trvoanosomes-per r&) and at the peak of .&e‘paras%emia plasma was prepared by centrifuging heparinized whole blood (5 units heparin/ml’blood) at 16 000 g for 10 min at 26°C. Plasma from normal, uninfected rats was similarly prepared. T. brucei was obtained free of blood elements by DEAE-cellulose chromatography (LANHAM & GODFREY, 1970) and was incubated either in phosphatebuffered-saline-glucose (PSG) (LANHAM & GODFREY, 1970) or in plasma obtained from uninfected rats at a trypanosome concentration of 4X10*-4X109/ml at 4°C for 1 h. Under these conditions the trypanosomes remained actively motile? as determined by light microscopy. After incubauon, the trypanosomeswere pelleted by centrifuging at 3000 g for 10 min at 26°C and the trypanosome-affected PSG supematant flAS) and trypanosome-affected plasma (TAP) were used for determination of protease activity (WALSH & WILCOX, 1970). The protease activity of heparinized plasma of normal, uninfected rats was also determined. The results showed that T. brucei released distinct proteases, with activity at pH 5.4 and DH 8.0, into the TAS. The kinetics of the reactions catalyzeh by the proteases were different:

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the initial

rate of proteolysis was more rapid at pH 8.0 than at pH 5.4. However, a higher level of proteolysis was reached at DH 5.4 than at DH 8.0. The reaction increased l&early with increak in the concentrations of substrate (O-25 mg bovine serum albumin) and enzyme (O-0.45 mg protein). Proteolytic activity with

detected in plasma of trypanosome-infected rats. Proteolytic activity was not detected in heparinized plasma of normal, uninfected rats in the pH range 4-O-10-O.Protease activity was detected in TAP at pH 8.0, but not at pH 5.4; this suggests that the pH 5.4 protease is unstable, or undetectable, in heparinized rat plasma. A homogenate of whole T. brucei, TAS, TAP and plasma of normal, uninfected and trypanosomeinfected rats were subjected to sodium dodeql sulphate-polyacrylamidi gel electrophoresis (LAE~ LII, 1970). The results showed the presence of a polypeptide band of M, 12000in the homogenate of T. brucei as well as in TAS. TAP. and ulasma of infected rats. This band was’abseni from &asma of normal, uninfected rats. The relationship, if any, between this polypeptide band and the proteases under study has not be-enestablished. As far as we are aware this is the first report of extracellular release of protease by T. brucei. Some intracellular proteasesh&e been studied earlier (STEIGER et al.. 1980; LONSDALE-ECCLES & MPIMBAZA,

1986). It has be& suggestedthat the insect mid-gut form of T. brucei probably releasesa protease as well as a phospholipase to aid it in its passagethrough the peritrophic membrane (EVANS & ELLIS, 1983). The proteases will he characterized and their possible involvement in platelet aggregation determined. Acknowledgement. This research was supported by a grant from the Research Strengthening Group of the UNDPlWorld BankfWHO funded Special Programme in Tropical Diseases Research. References Davis. C. E. (1982). Thrombocvtooaenia: a uniform comy@ho; oi Ahan trypanokhsis. Acta Tropica, 39, Davis, C. El, Robbins, R. S., Weller, R. D. & Braude, A. I. (1974). Thrombocytopaenia in experimental trypanosomiasis. Journal of Clinical Znvestigation,53, 1359-1367. Evans, D. A. & Ellis, D. S. (1983). Recent observation on the behaviour of certain trypanosomeswithin their insect hosts. Advances in Parasitology, 22, l-42. Laemmli, U.K. (1970). Cleavage of suuctural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. La&m, S. M. & Godfrey, D. G. (1970). Isolation of +ivarian trypanosomes from .man and other animals ;~3~EAE-celltiose. Expennwntal Paraatology, 28, - . Lonsdale-Eccles, J. D. & Mpimbaza, W. N. (1986). Thiol-dependent proteases of African trypanosomes. European Jomal of Biochemisny, 155, 469-473. Steiger, R. F., Opperdoes, F. R. & Bontemps, J. (1980). &hcelIular fti&onation of T anosoma-bruiei bloodstream forms with special re ‘y4erence to hydrolases. European Joumd of Biochemisty, 105, 163-175. Walsh, K. A. & Wilcox, P. E. (1970). Serine proteases. Methodr in Enqrmologv, 19, 31-41. Received IO November 1987; accepted for publication 14 January I988