Autophagic-related cell death of Trypanosoma brucei induced by bacteriocin AS-48

The parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48's ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC50 = 1–3 nM) against the three T. brucei subspecies, but it was innocuous to human cells at 104-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37 °C but not at 4 °C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasite's ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.This work was supported by Spanish grantsSAF2011-28215 (JMPV), SAF2016-80228-R (JMPV) and SAF2013-48971-C2-1-R (MM) from the Ministerio de Economía y Competitividad, and BIO1786 (JMPV) from the Junta de Andalucía and by FEDER funds from the EU to JMPV. MMG was recipient of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad: SAF2011-28215, SAF2016-80228-R and SAF2013-48971-C2-1-R. MMG was a student of the Biochemistry and Molecular Biology Ph.D. program of the University of Granada (Spain). JMB is funded by “Fondo de Investigación Sanitaria” (FIS)TRPY 1283/15. MN is granted by ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa (RICET)RD12/0018/0015.Peer reviewe