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Summary: Mammalian target of rapamycin (mTOR) is a central regulator of growth and immunity of host cells. It's involvement in cancer and tuberculosis is well documented but least explored in Leishmania donovani invasion of host cells. Therefore, in the present study, we aimed to investigate the role of mTOR in M2 macrophage polarization for Leishmania survival. We observed that Leishmania infection activated host mTOR pathway characterized by phosphorylation of mTOR, 70S6K and 4‐EBP1. Inhibition of mTOR resulted in decreased parasite load and percent infectivity. Moreover, Leishmania infection triggered cell proliferation as was evidenced by increased expression of cyclin A and p‐RPS6. mTOR activation during Leishmania infection resulted in reduced expression of M1 macrophage markers (eg, ROS, NO, iNOS, NOX‐1, IL‐12, IL‐1β and TNF‐α), and increased expression of M2 macrophage markers (eg, arginase‐1, IL‐10, TGF‐β, CD206 and CD163). Furthermore, we observed that in case of Leishmania infection, mTOR inhibition increased the translocation of NF‐κB to nucleus and deactivation of STAT‐3. Eventually, we observed that inhibition of M2 macrophage polarization reduced Leishmania survival inside macrophages. Therefore, our findings suggest that mTOR plays a crucial role in regulation of M2 macrophage polarization and direct the innate immune homeostasis towards parasite survival inside host. [ABSTRACT FROM AUTHOR]

The transformation of Leishmania donovani from a promastigote to an amastigote during mammalian host infection displays the immense adaptability of the parasite to survival under stress. Induction of translation initiation factor 2-alpha (eIF2α) phosphorylation by stress-specific eIF2α kinases is the basic stress-perceiving signal in eukaryotes to counter stress. Here, we demonstrate that elevated temperature and acidic pH induce the phosphorylation of Leishmania donovani eIF2α (LdeIF2α). In vitro inhibition experiments suggest that interference of LdeIF2α phosphorylation under conditions of elevated temperature and acidic pH debilitates parasite differentiation and reduces parasite viability (P < 0.05). Furthermore, inhibition of LdeIF2α phosphorylation significantly reduced the infection rate (P < 0.05), emphasizing its deciding role in successful invasion and infection establishment. Notably, our findings suggested the phosphorylation of LdeIF2α under H 2 O 2 -induced oxidative stress. Inhibition of H 2 O 2 -induced LdeIF2α phosphorylation hampered antioxidant balance by impaired redox homeostasis gene expression, resulting in increased reactive oxygen species accumulation (P < 0.05) and finally leading to decreased parasite viability (P < 0.05). Interestingly, exposure to sodium antimony glucamate and amphotericin B induces LdeIF2α phosphorylation, indicating its possible contribution to protection against antileishmanial drugs in common use. Overall, the results strongly suggest that stress-induced LdeIF2α phosphorylation is a necessary event for the parasite life cycle under stressed conditions for survival., (Copyright © 2016 American Society for Microbiology.)

Regulation of macrophage PCD plays an important role in pathogenesis of leishmaniasis. However, the precise involvement of any parasite molecule in this process remains uncertain. In the current study, in silico wide analysis demonstrated that genes in the Leishmania donovani genome are highly enriched for CpG motifs, with sequence frequency of 8.7%. Here, we show that unmethylated species-specific CpG motifs in LdDNA significantly (P = 0.01) delay macrophage PCD by endosomal interaction with TLR9 via the adaptor protein MyD88. Importantly, LdDNA triggered high levels of luciferase activity (P = 0.001) under NF-κB-dependent transcription in HEK-TLR9 cells. Furthermore, the activation of caspases in macrophages was inhibited (P = 0.001) in the presence of LdDNA. Notably, the delay of PCD was mediated by modulation of the antiapoptotic proteins, Mcl-1 and Bfl-1, and impairment of loss of Δψm in macrophages through the neutralization of oxidative and nitrosative stress. The inhibition of caspase activation and up-regulation of Mcl-1 by LdDNA were TLR9 dependent. Analysis of the targets of LdDNA identified an early activation of the TLR9-dependent PI3K/Akt and SFK pathways, which were required for the observation of the antiapoptotic effects in macrophages. Moreover, we demonstrate that LdDNA modulates the TLR9-IκB-α pathway by promoting the tyrosine phosphorylation of TLR9 and the TLR9-mediated recruitment of Syk kinase. The results have identified a novel, TLR9-dependent antiapoptotic function of LdDNA, which will provide new opportunities for discovering and evaluating molecular targets for drug and vaccine designing against VL., (© Society for Leukocyte Biology.)