Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis

Jayeeta Giri,Supriya Srivastav,Moumita Basu,Shreyasi Palit,Purnima Gupta,Anindita Ukil

doi:10.1074/jbc.m115.672873

Abstract

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.

Highlights

Leishmania donovani, an obligate intracellular parasite, is the causative agent of the fatal visceral leishmaniasis [1]
Because apoptosis could be one such means used by macrophages to eliminate intracellular infection, we wanted to see the effect of L. donovani infection on actinomycin D-induced macrophage apoptosis
A similar trend was observed in the absence of actinomycin D where cells infected with Leishmania showed very little extent of apoptosis (9.3 Ϯ 0.8, 6.3 Ϯ 0.6, 5.2 Ϯ 0.4, 6.9 Ϯ 0.6, and 7.5 Ϯ 0.7% annexin V-positive cells at 2, 4, 6, 12, and 24 h after infection) in bone marrow-derived macrophages (BMDM) (Fig. 1A) and (12.1 Ϯ 0.9, 7.8 Ϯ 0.8, 6.2 Ϯ 0.6, 7.9 Ϯ 0.7, and 8.3 Ϯ 0.8% annexin V-positive cells at 2, 4, 6, 12, and 24 h after infection) in RAW macrophages (Fig. 1B)

Summary

Introduction

Leishmania donovani, an obligate intracellular parasite, is the causative agent of the fatal visceral leishmaniasis [1]. Expression of caspase 3, the main effector caspase involved in apoptotic signaling cascade, was found to be significantly reduced (maximum 63.4 and 72.8% reduction in RAW and BMDM, respectively, at 6 h post-infection, p Ͻ 0.001, as compared with uninfected cells after actinomycin D treatment) (Fig. 1C).

Results

Conclusion