A novel function of Mycobacterium tuberculosis chaperonin paralog GroEL1 in copper homeostasis.

Among the two GroEL paralogs in Mycobacterium tuberculosis, GroEL1 and GroEL2, GroEL1 has a characteristic histidine-rich C terminus. Since histidine richness is likely to be involved in metal binding, we attempted to decipher the role of GroEL1 in chelating metals and the consequence on M. tuberculosis physiology. Isothermal titration calorimetry showed that GroEL1 binds copper and other metals. Mycobacterial viability assay, redox balance, and DNA protection assay concluded that GroEL1 protects from copper stress in vitro. Solution X-ray scattering and constrained modeling of GroEL1 -/+ copper ions showed reorientation of the apical domain as seen in functional assembly. We conclude that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and consequent functional divergence of chaperonins.

Quantitative secretome analysis unravels new secreted proteins in Amphotericin B resistant Leishmania donovani.

Leishmaniasis is second most neglected disease after malaria and seems to be a worldwide concern because of increased drug resistance and non-availability of approved vaccine. The underlying molecular mechanism of drug resistance (Amp B) in Leishmania parasites still remains elusive. Herein, the present study investigated differentially expressed secreted proteins of Amphotericin B sensitive (S) and resistant (R) isolate of Leishmania donovani by using label free quantitative LC-MS/MS approach. A total of 406 differentially expressed secreted proteins were found between sensitive (S) and resistant (R) isolate. Among 406 proteins, 32 were significantly up regulated (>2.0 fold) while 22 were down regulated (<0.5 fold) in resistant isolate of L. donovani. Further, differentially expressed proteins were classified into 11 various biological processes. Interestingly, identified up regulated proteins in resistant parasites were dominated in carbohydrate metabolism, stress response, transporters and proteolysis. Western blot and enzymatic activity of identified proteins validate our proteomic findings. Finally, our study demonstrated some new secreted proteins associated with Amp B resistance which provides a basis for further investigations to understand the role of proteins in L. donovani. BIOLOGICAL SIGNIFICANCE: Although great advances have been achieved in the diagnosis and treatment of leishmaniasis, still drug resistance is major hurdle in control of disease. Present study will enhance the deeper understanding of altered metabolic pathways involved in Amp B resistance mechanism and provide possible new proteins which can be potential candidate either for exploring as new drug target or vaccine. Protein-protein interactions highlighted the up-regulated metabolic pathways in resistant parasites which further unravel the adaptive mechanism of parasites.

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

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.