title: Unmethylated CpG motifs in the L. donovani DNA regulate TLR9-dependent delay of programmed cell death in macrophages.
abstract: 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.
authors.affiliation: *Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India sushmita.de2008@gmail.com.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Das
authors.affiliation: *Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India.
authors.forename: Ayan Kumar
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: *Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India.
authors.forename: Shikha
authors.initials: S
authors.lastname: Singh
authors.affiliation: *Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Saha
authors.affiliation: *Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Ganguly
authors.affiliation: *Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D020169:Caspases / Q000276:immunology; D018899:CpG Islands / Q000276:immunology*; D019175:DNA Methylation; D016054:DNA, Protozoan / Q000276:immunology* / Q000494:pharmacology; D004789:Enzyme Activation / Q000276:immunology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007893:Leishmania donovani / Q000276:immunology*; D008264:Macrophages / Q000166:cytology / Q000276:immunology*; D008297:Male; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000276:immunology; D053594:Myeloid Differentiation Factor 88 / Q000276:immunology; D010766:Phosphorylation / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000276:immunology*; D015854:Up-Regulation / Q000276:immunology
chemical_list: D016054:DNA, Protozoan; C579095:MCL1 protein, human; C507779:MYD88 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D053594:Myeloid Differentiation Factor 88; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D020169:Caspases
keywords: PCD; PI3K/Akt pathway; Syk; apoptosis; visceral leishmaniasis