Studies on compounds promoting the in vitro transformation ofTrypanosoma brucei from bloodstream to procyclic forms

Abstract

In vitro differentiation of pleomorphic blood-stream forms ofTrypanosoma brucei to procyclic culture forms occurred rapidly and at high rates at 27°C in a culture medium containing 1 mM cis-aconitate as the transformation-inducing agent. Citrate was required at a much higher concentration (10 mM) to produce a similar transformation rate. The highest percentage of transformed cells was obtained when bloodstream-form trypanosomes were treated with pronase in the absence of a feeder-cell layer. However, under these conditions, the amount of procyclic forms obtained after 72 h was lower than that obtained in the presence ofcis-aconitate. Trypsin was also capable of inducing transformation in the absence of a feeder-cell layer, but this treatment again resulted in low numbers of transformed cells. Blood-stream-form trypanosomes were incapable of taking up citrate to any significant extent and the citrate content of these stages was negligible. After 72 h of exposure to citrate (3 mM), intracellular levels of this compound remained very low (<1 nmol (10⁹ cells)⁻¹), increasing in established procyclic stages to approximately 1.7 nmol (10⁹ cells)⁻¹. These observations suggest that the tricarboxylic acid (TCA)-cycle metabolite-dependent transformation may be initiated externally to the trypanosome cell membrane. The ability of both citrate andcis-aconitate to bind calcium and, thus, to reduce the concentration of this cation in the culture medium was found not to be responsible for the triggering effect on trypanosome transformation. Citrate levels in tsetse fly body fluid were found to be far below those required to induce trypanosome transformation in vitro. Since proteolytic enzymes can effectively induce this process, these enzymatic components may be of greater physiological relevance as environmental stimuli for the differentiation process than are the TCA-cycle metabolites.