Authors

I Subota²; A Fekete¹; M J Müller¹; M Engstler²;  ¹ Lehrstuhl für Pharmazeutische Biologie, Biozentrum der Universität Würzburg, Germany;  ² Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum der Universität Würzburg, Germany

Discussion

The parasite Trypanosoma brucei has evolved mechanisms to guarantee persistent infection and tsetse fly transmissibility. A key in this interplay is the up to now elusive stumpy induction factor (SIF), which is parasite-derived and accumulates in a cell-density dependant manner. Once a threshold is reached, SIF triggers differentiation of the proliferating slender form to the cell-cycle arrested stumpy stage, which is preadapted for transmission by the vector.

Our study aims at identifying SIF by combining liquid chromatography (LC), untargeted metabolomics and activity bioassays using a pleomorphic reporter cell line.

In a first purification step conditioned cell culture medium containing SIF was subjected to methanolic protein precipitation extraction. The resulting deproteinated concentrate was further purified by reversed-phase solid phase extraction eluted with 10% methanol in water, which demonstrated the highly polar nature of SIF. We thus opted for hydrophilic interaction LC using an amide column in order to separate polar metabolites. Surprisingly, the active fractions contained pyruvate as a possible candidate. In fact, pyruvate can mimic SIF action, however, only at concentrations that appear unphysiological. We present data that rule out that pyruvate is SIF, but that also suggest that it might be a related compound.