Leishmania donovani parasite requires Atg8 protein for infectivity and survival under stress

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.

Functional partnership between carbonic anhydrase and malic enzyme in promoting gluconeogenesis in Leishmania major

“…To determine the subcellular localization of LmME in L. major , LmME‐GFP expressing cells were mounted on poly l ‐lysine coated coverslips for 1 h. Attached parasites were then stained with 500 n m MitotrackerRed CMX‐Ros (Invitrogen, Carlsbad, CA, USA) in the dark for 30 min [78]. Postincubation, cells were washed in PBS to remove excess stain, air‐dried, and finally embedded in anti‐fade mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI) (VectaShield from Vector Laboratories, Burlingame, CA, USA).…”

Autophagy in Tenebrio molitor Immunity: Conserved Antimicrobial Functions in Insect Defenses

“…Certainly, the increase in Atg8 (Atg8-II) signals overtime after exposure to L. monocytogenes infection is indicative of an increasing autophagic control in the host. The involvement of Atg8 in Leishmania parasite survival while infecting macrophages in vitro suggests regulatory control of autophagy by autophagosome formation (71).…”

The ultimate fate determinants of drug induced cell-death mechanisms in Trypanosomatids

“…Dihydroxyacetone causes G2/M cell cycle arrest and thereby inhibits the growth of T. brucei bloodstream forms, thus exhibiting morphological features like an increase in vesicular structures, formation of autophagy-like vacuoles and multivesicular bodies etc ( Uzcátegui et al., 2007 ). From these studies, it is evident that autophagic vacuoles are a key hallmark of autophagy induction which are positively stained by monodansylcadaverine, and the same is prevented by PI3K inhibitors, 3-methyladenine or wortmannin ( Giri and Shaha, 2019 ). But detailed biochemical pathway governing the autophagic process in trypanosomatids is yet to be deciphered.…”

“…Hence, it is evident now that “macroautophagy” is an important process in regulating cell death in Leishmania . Mitochondrial translocation of ATG5 protein was evidenced earlier in L. major ( Williams et al., 2012 ), while mitotoxic stress during early hours of cisplatin and etoposide treatment caused mitochondrial translocation of ATG8 ( Giri and Shaha, 2019 ).…”