Assessment of autophagy inLeishmaniaparasites

Abstract

AbstractLeishmaniasis is a neglected tropical disease caused by numerous species ofLeishmaniaparasites, includingLeishmania major.The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown thatLeishmaniamodulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy inLeishmaniais inconclusive because of the limited tools available to study theLeishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy inLeishmania major. Transmission electron microscopy (TEM) allowed us to visualizeLeishmaniaautophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments inLeishmania majorby detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation inLeishmaniaparasites that could provide insights into developing improved therapeutic targets against leishmaniasis.