MilliporeSigma
  • Home
  • Search Results
  • Unmethylated CpG motifs in the L. donovani DNA regulate TLR9-dependent delay of programmed cell death in macrophages.

Unmethylated CpG motifs in the L. donovani DNA regulate TLR9-dependent delay of programmed cell death in macrophages.

Journal of leukocyte biology (2014-12-05)
Sushmita Das, Ayan Kumar Ghosh, Shikha Singh, Bhaskar Saha, Ashish Ganguly, Pradeep Das
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.

MATERIALS
Product Number
Brand
Product Description

Sucrose, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.9% trace metals basis
Sigma-Aldrich
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, 98%
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
DL-Cysteine, technical grade
Sigma-Aldrich
Sucrose, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.5% (GC)
Supelco
Sucrose, analytical standard, for enzymatic assay kit SCA20
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Sucrose, Grade I, suitable for plant cell culture
Sigma-Aldrich
Sucrose, ACS reagent
Sigma-Aldrich
Sucrose, meets USP testing specifications
Sigma-Aldrich
Sucrose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Magnesium chloride, BioReagent, suitable for insect cell culture, ≥97.0%
Sigma-Aldrich
Sucrose, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, Grade II, suitable for plant cell culture
Sigma-Aldrich
Sucrose, ≥99.5%
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Supelco
Sucrose, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Glycerol, FCC, FG
USP
Sucrose, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)
Sigma-Aldrich
Glycerol, ≥99.5%
Sigma-Aldrich
Glycerol, tested according to Ph. Eur., anhydrous
Millipore
Sucrose, ACS reagent, suitable for microbiology, ≥99.0%
Sigma-Aldrich
Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Supelco
Streptomycin solution, ~1 mg/mL in 1 mM EDTA, analytical standard
Sigma-Aldrich
Sucrose, puriss., meets analytical specification of Ph. Eur., BP, NF
Sigma-Aldrich
Sucrose, BioUltra, for molecular biology, ≥99.5% (HPLC)