Trypanosoma brucei: in vitro slender-to-stumpy differentiation of culture-adapted, monomorphic bloodstream forms

Abstract

Pleomorphic Trypanosoma brucei strains are characterized by their ability to differentiate from replicating long slender forms into non-dividing short stumpy forms in the mammalian host. The differentiation process can be efficiently induced in vitro by treatment with the membrane-permeable cAMP derivative 8-(4-chlorophenylthio)-cAMP (pCPTcAMP). In contrast, monomorphic T. brucei strains do not differentiate to stumpy forms in the host. Here, we show that exposure of monomorphic, culture-adapted T. brucei bloodstream forms to pCPTcAMP allowed their subsequent differentiation into short stumpy forms. The stumpy nature of pCPTcAMP-treated parasites was confirmed by (1) morphological change, (2) inhibition of growth and DNA synthesis, (3) cell cycle arrest in the G 1/G 0 phase, (4) expression of NADH diaphorase activity and dihydrolipoamide dehydrogenase, (5) disappearance of the small subunit of ribonucleotide reductase, (6) up-regulation of the major lysosomal membrane protein, and (7) efficient transformation into replicating procyclic insect forms after induction with citrate/ cis-aconitate. Our results indicate that the inability of monomorphic T. brucei bloodstream forms to differentiate into short stumpy forms in the host may be due to a failure in the signalling pathway rather than in the differentiation process itself. Treatment of monomorphic bloodstream trypanosomes with pCPTcAMP could be a useful method for identifying the genes involved in the slender-to-stumpy differentiation process. Index Descriptors and Abbreviations: Trypanosoma brucei; culture-adapted bloodstream forms; differentiation; cell cycle arrest; cAMP, adenosine 3 ′,5 ′-cyclic monophosphate; pCPTcAMP, 8-(4-chlorophenylthio)-cAMP; pCPTcGMP, 8-(4-chlorophenylthio)-guanosine 3 ′,5 ′-cyclic monophosphate; 4 ′,6-diamidino-2-phenylindole, DAPI; PBS, phosphate-buffered saline; HBSS, Hank’s balanced salt solution.