STUDIES ON COMPOUNDS PROMOTING THE IN-VITRO TRANSFORMATION OF TRYPANOSOMA-BRUCEI FROM BLOOD-STREAM TO PROCYCLIC FORMS

In vitro differentiation of pleomorphic bloodstream forms of Trypanoso ma brucei to procyclic culture forms occurred rapidly and at high rate s at 27 degrees C in a culture medium containing 1 mM cis-aconitate as the transformation-inducing agent. Citrate was required at a much hig her concentration (10 mM) to produce a similar transformation rate. Th e highest percentage of transformed cells was obtained when bloodstrea m-form trypanosomes were treated with pronase in the absence of a feed er-cell layer. However, under these conditions, the amount of procycli c forms obtained after 72 h was lower than that obtained in the presen ce of cis-aconitate. Trypsin was also capable of inducing transformati on in the absence of a feeder-cell layer, but this treatment again res ulted in low numbers of transformed cells. Bloodstream-form trypanosom es were incapable of taking up citrate to any significant extent and t he citrate content of these stages was negligible. After 72 h of expos ure to citrate (3 mM), intracellular levels of this compound remained very low (<1 nmol (10(9) cells)(-1)), increasing in established procyc lic stages to approximately 1.7 nmol (10(9) cells)(-1). These observat ions suggest that the tricarboxylic acid (TCA)-cycle metabolite-depend ent transformation may be initiated externally to the trypanosome cell membrane. The ability of both citrate and cis-aconitate to bind calci um and, thus, to reduce the concentration of this cation in the cultur e medium was found not to be responsible for the triggering effect on trypanosome transformation. Citrate levels in tsetse fly body fluid we re found to be far below those required to induce trypanosome transfor mation in vitro. Since proteolytic enzymes can effectively induce this process, these enzymatic components may be of greater physiological r elevance as environmental stimuli for the differentiation process than are the TCA-cycle metabolites.