Identification of a novel family of riboflavin transporters in trypanosomatids

BALCAZAR DE; BONOMI HR; PEREIRA CA; GOLDBAUM FA; CARRILLO C

Mar del Plata

Congreso; X Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2014

Sociedad Argentina de Protozoologìa

Trypanosomatids Leishmania mexicana, Trypanosoma brucei and Trypanosoma cruzi are human pathogenic parasites. The current drug treatments are highly toxic, posses limited efficacy and can generate resistance, thus there is a need to identify targets to develop new therapies. Riboflavin (Rf), an essential vitamin for all living cells, is the precursor of the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) responsible of a variety of vital cellular reactions. The aim of this work is to determine the molecular basis of the Rf metabolism in trypanosomatids. Our in silico analyses suggest that trypanosomatids are auxotrophic for Rf because no biosynthetic Rf pathways are coded in their genomes. We also identified a putative novel transporter family for this vitamin by bioinformatic studies. Moreover, these studies indicate conservation of twelve transmembrane regions and an MSF domain (for Major Facilitator Superfamily) in all members of the family. The corresponding transporters for L. mexicana, T. cruzi and T. brucei were cloned and expressed in an Rf auxothroph E. coli strain, which naturally lacks of Rf transport activities. We observed that T. cruzi and T. brucei genes were able to restore bacterial growth in low Rf media suggesting their functionality as flavin transporter. In vitro experiments with each trypanosomatid showed that the maximum density of parasites (in growth curves) increases with the concentration of Rf, FMN and FAD. Finally, overexpression of T. cruzi putative Rf transporter lowers the riboflavin dependency in culture, achieving higher number of epimastigotes per ml in Rf limited media compared to the wild-type strain. The putative Rf transporter family we identified in trypanosomatids would be essential for their survival. This transporter family is different from any other Rf transporter known to date, particularly the mammalian ones, thus we propose it as a potential target for the development of trypanocidal therapies.