Cell density triggers slender to stumpy differentiation of Trypanosoma brucei bloodstream forms in culture

Differentiation from replicating slender forms to non-dividing stumpy bloodstream forms of T. brucei limits the parasite population size in the mammalian host in addition to and independently of the antibody response. Using a culture system for pleomorphic strains of T. brucei we show that slender forms very efficiently differentiate to stumpy forms in vitro and that the induction of differentiation is correlated to cell density. Differentiation in the host and in culture were compared using a battery of markers including cell morphology and volume, cell cycle position, the kinetics of the differentiation, expression of NADH dehydrogenase (diaphorase), expression of several differentially regulated transcripts and the kinetics of transformation to replicating procyclic forms after induction with cis-aconitate. By all available criteria, differentiation in culture reflects the natural process in the mammalian host. Time course experiments reveal a very tight temporal correlation between cell cycle arrest of bloodstream forms, appearance of a stumpy differentiation marker and the competence of a bloodstream form population to initiate transformation to procyclic forms in response to cis-aconitate. Our results show that induction of bloodstream form differentiation can occur independently of host-derived cues. We suggest a density sensing mechanism which induces differentiation to the non-dividing stumpy stage and thereby enables the parasite population to autoregulate its proliferation.