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.

Differential utilization of metabolites and metabolic plasticity can confer adaptation to cancer cells under metabolic stress. Glutamine is one of the vital and versatile nutrients that cancer cells consume avidly for their proliferation, and therefore mechanisms related to glutamine metabolism have been identified as targets. Recently, sestrin2 (SESN2), a stress-inducible protein, has been reported to regulate survival in glutamine-depleted cancer cells; based on this, we explored if SESN2 could regulate glutamine metabolism during glucose starvation. This report highlights the role of SESN2 in the regulation of glutamine-dependent activation of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) under glucose scarcity in liver cancer cells (HepG2). We demonstrate that down-regulation of SESN2 induces a decrease in the levels of intracellular glutamine and PGC-1α under glucose deprivation, concomitant with a decline in cell survival, but no effect was observed on the invasive or migration potential of the cells. Under similar metabolic conditions, SESN2 forms a complex with c-Jun N-terminal kinase (JNK) and forkhead box protein O1 (FOXO1). Absence of SESN2 or inhibition of JNK reduces nuclear translocation of FOXO1, consequently causing transcriptional inhibition of PGC-1α. Notably, our observations demonstrate a reduction in cell viability under high glutamine and low glucose conditions during SESN2 down-regulation that could be rescued on JNK inhibition. To recover from acetaminophen-induced mitochondrial damage, SESN2 was crucial for glutamine-mediated activation of PGC-1α in HepG2 cells. Collectively, we demonstrate a novel role of SESN2 in mediating activation of PGC-1α by modulating glutamine metabolism that facilitates cancer cell survival under glucose-limited metabolic conditions.

The parasite causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.