Abstract
The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (ΔAtg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ΔAtg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ΔAtg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ΔAtg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.
Highlights
Autophagy is an intracellular degradation system that delivers cellular constituents including cytosolic proteins and damaged organelles to the lysosome for digestion by enzymes[1,2]
Further confirmation of enhancement of autophagy is shown by an increase in Atg8-II expression levels on western blots of extracts derived from parasites at different days of culture (Fig. 1c)
We used several stress inducers like H2O2, which spontaneously generates hydroxyl ions in physiological conditions[19], carbonyl cyanide m-chlorophenylhydrazone (CCCP)[20], a respiratory chain uncoupler that generates reactive oxygen species (ROS), and potassium antimonyl tartrate (PAT), a drug used against Leishmania parasite that acts through generation of ROS21
Summary
Autophagy is an intracellular degradation system that delivers cellular constituents including cytosolic proteins and damaged organelles to the lysosome for digestion by enzymes[1,2]. Analysis of autophagic processes known to help remodeling of cells has attracted considerable attention because of the influence of autophagy in different cellular functions[1,2]. Official journal of the Cell Death Differentiation Association. Giri and Shaha Cell Death and Disease (2019)10:808 or autophagy-related proteins have been elegantly shown in several studies[6,7]. These Atg proteins are intimately associated with the regulation of macroautophagy ( referred to as autophagy), and the requirement of a functional Atg12–Atg[5] conjugation system for Atg8dependent autophagy in L. major has been demonstrated[6,7,8,9]. The consequences of the absence of Atg[8] protein on the formation of autophagosomes, response to drugs, and infectivity are not known
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