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Species‐specific unmethylated CpG‐motifs in L. donovaniDNA in modulating macrophage apoptosis through TLR9 and activation of the PI3K/Akt pathway. 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 donovanigenome 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‐αpathway 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.

ABSTRACTAmphotericin B (AmB), a polyene macrolide, is now a first-line treatment of visceral leishmaniasis cases refractory to antimonials in India. AmB relapse cases and the emergence of secondary resistance have now been reported. To understand the mechanism of AmB, differentially expressed genes in AmB resistance strains were identified by a DNA microarray and real-time reverse transcriptase PCR (RT-PCR) approach. Of the many genes functionally overexpressed in the presence of AmB, the ascorbate peroxidase gene from a resistant Leishmania donovanistrain (LdAPx gene) was selected because the gene is present only in Leishmania, not in humans. Apoptosis-like cell death after exposure to AmB was investigated in a wild-type (WT) strain in which the LdAPx gene was overexpressed and in AmB-sensitive and -resistant strains. A higher percentage of apoptosis-like cell death after AmB treatment was noticed in the sensitive strain than in both the resistant isolate and the strain sensitive to LdAPx overexpression. This event is preceded by AmB-induced formation of reactive oxygen species and elevation of the cytosolic calcium level. Enhanced cytosolic calcium was found to be responsible for depolarization of the mitochondrial membrane potential and the release of cytochrome c(Cyt c) into the cytosol. The redox behavior of Cyt cshowed that it has a role in the regulation of apoptosis-like cell death by activating metacaspase- and caspase-like proteins and causing concomitant nuclear alterations, as determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA fragmentation in the resistant strain. The present study suggests that constitutive overexpression of LdAPx in the L. donovaniAmB-resistant strain prevents cells from the deleterious effect of oxidative stress, i.e., mitochondrial dysfunction and cellular death induced by AmB.