Transient inhibition of protein synthesis accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms

Abstract

It has been widely believed that bloodstream forms of Trypanosoma brucei must be first transformed into intermediary and/or short-stumpy forms in the bloodstream of the mammalian host before differentiation to the procyclic culture form can occur. In our recent studies, the pleomorphic T. brucei strain TREU667 was found to differentiate directly from the long-slender bloodstream form to the procyclic form in Cunningham's medium at 26°C [7]. In the present investigation, the same was found true for another pleomorphic strain of T. brucei, STIB366D. Four independent monomorphic strains of T. brucei were tested; two, #427 and EATRO164, were found capable of differentiating in vitro directly into procyclic forms, whereas the other two, TREU667/RP-56 and EATRO110, could not. There is thus no correlation between the capability of differentiating in vitro and the ability of being converted from long-slender to intermediary and short-stumpy bloodstream forms. Two additional markers for following differentiation, other than observing morphological changes, were tested. Assays for the emerging phosphoenolpyruvate carboxykinase (PEPCK) by immunoblottings worked well, with results agreeing closely with the morphological change. But immunoblottings of glycosomal phosphoglycerate kinase (gPGK) failed to demonstrate a significant decrease in the protein level upon completion of differentiation. Apparently, gPGK has a rather long half-life and is unsuitable as a marker of differentiation. When temperature was dropped from 37°C to 26°C at the starting point of in vitro differentiation, protein synthetic activity in the pleomorphic T. brucei TREU667 bloodstream form was decreased by 4-fold. When the activity was gradually brought back to and beyond the original level after a day's incubation, the profile of newly synthesized proteins was that of the procyclic form. A monomorphic variant of TREU667, RP-56, which is incapable of differentiating in vitro, has a much higher protein synthetic activity than its pleomorphic parent in the bloodstream form. This high activity and the bloodstream profile of proteins thus synthesized were unaffected by the decreased temperature in Cunningham's medium until cell death. We thus conclude that a general inhibition of protein synthesis in bloodstream forms caused by temperature drop may be among the early events triggering differentiation into the procyclic form.