Leishmania donovani induced Unfolded Protein Response delays host cell apoptosis in PERK dependent manner

Abstract

Background

Endoplasmic reticulum (ER) stress generated unfolded stress response (UPR) is a basic survival mechanism which protects cell under unfavourable conditions. Leishmania parasite modulates host macrophages in various ways to ensure its survival. Modulation of PI3K-Akt pathway in delayed apoptotic induction of host; enables parasite to stabilize the infection for further propagation.

Methodology

Infected RAW macrophages were exposed to campothecin or thagsigargin and phosphorylation status of PERK, Akt, BAD and Cyt-C was determined through western blotting using phospho specific antibody. Expression at transcriptional level for cIAP1 &2, ATF4, CHOP, ATF3, HO-1 and sXBP1 was determined using real time PCR. For inhibition studies, RAW macrophages were pre-treated with PERK inhibitor GSK2606414 before infection.

Findings

Our studies in RAW macrophages showed that induction of host UPR against L. donovani infection activates Akt mediated pathway which delays apoptotic induction of the host. Moreover, Leishmania infection results in phosphorylation and activation of host PERK enzyme and increased transcription of genes of inhibitor of apoptosis gene family (cIAP) mRNA. In our inhibition studies, we found that inhibition of infection induced PERK phosphorylation under apoptotic inducers reduces the Akt phosphorylation and fails to activate further downstream molecules involved in protection against apoptosis. Also, inhibition of PERK phosphorylation under oxidative exposure leads to increased Nitric Oxide production. Simultaneously, decreased transcription of cIAP mRNA upon PERK phosphorylation fates the host cell towards apoptosis hence decreased infection rate.

Conclusion

Overall the findings from the study suggests that Leishmania modulated host UPR and PERK phosphorylation delays apoptotic induction in host macrophage, hence supports parasite invasion at early stages of infection.

Author summary

Visceral Leishmaniasis or Kala-azar is one of the severe tropical neglected parasitic diseases caused by Leishmania donovani in Indian subcontinent. Modulation of host in terms of delayed apoptotic induction is one of the aspects which favours disease establishment; however the mechanism is not clearly understood yet. In the present study, we tried to explore the connection between L. donovani infection induced UPR in host with delayed onset of apoptosis. We found that L. donovani infection phosphorylates the PERK and Akt molecule in host along with delayed apoptosis. Simultaneously, the levels of cellular IAP (cIAP1 & 2) genes were also up-regulated in infected macrophages. To assess the involvement of PERK in delayed apoptosis of host, we inhibited the phosphorylation of PERK under the exposure to apoptotic inducers. We found that PERK inhibition decreased the Akt phosphorylation and fails to activate other associated downstream molecules involved in delayed apoptosis of host. Also, a significant reduction in cIAP levels was observed. Under oxidative exposure, inhibition of PERK phosphorylation debilitates infected RAW cell’s ability to maintain redox homeostasis leading to higher nitric oxide production. Altogether, L. donovani infection modulates host apoptosis in a PERK dependent manner and favours infection.