Biology subjects

K B, A.

Publications and source records attributed to K B, A..

3 recordsLinked to original sources

bioRxiv2020

Transcription repressor protein ZBTB25 interacts with HDAC1 in macrophages infected with Mycobacterium tuberculosis, and its inhibition leads to autophagy and killing of the intracellular pathogen

Downregulation of host gene expression is one of the key strategies adopted by intracellular pathogens such as Mycobacterium tuberculosis (MTB) for their survival and subsequent pathogenesis. In a previous study, we have shown that HDAC1 levels go up in macrophages infected with MTB and it hypoacetylates histone H3 at the promoter of IL-12B gene leading to its downregulation. Here we show that after infection with MTB, the levels of the phosphorylated form of HDAC1 increase significantly in macrophages. Employing immunoprecipitation and LC-MS/MS, we found transcriptional repressor protein ZBTB25 associates with HDAC1 silencing complex along with transcriptional corepressor Sin3a. By chromatin immunoprecipitation and PCR analyses, we found that phosphorylated HDAC1, Sin3a, and ZBTB25 are recruited to the promoter of IL-12B to downregulate its expression in infected macrophages. Knocking down of ZBTB25 enhanced release of IL-12p40 from infected macrophages. Interestingly, the treatment of infected macrophages with CI994 (inhibitor of HDAC1) or dithiopyridine (inhibitor of ZBTB25) promoted the colocalization of LC3 (microtubule-associated protein 1A/1B-light chain 3, a marker for autophagy) and MTB in autophagosomes. Induction of autophagy resulted in the killing of intracellular MTB. Enhanced phosphorylation of JAK2 and STAT4 was observed in macrophages upon CI994 and dithiopyridine treatment, and inhibition of JAK2/STAT4 negated the killing of intracellular mycobacteria suggesting a possible role of these proteins in the autophagy-mediated killing of intracellular MTB.

microbiology↗

bioRxiv2020

Spatial separation of Streptomyces aerial mycelium during fermentation enhances secondary metabolite production

Conditioning of morphology is an effective technique to enhance secondary metabolite production by Streptomyces. Here we report a novel conditioning method employing glass marbles in batch cultures to enhance secondary metabolite production by Streptomyces sp. The marbles seem to spatially separate aerial and submerged mycelia in the flask which was necessary for the qualitative and quantitative enhancement of metabolite production of secondary metabolites. The method also offers shorter incubation period compared to conventional methods for effective production. Further, using a combination of this method and response surface methodology we could enhance the production of antimycobacterial molecules chrysomycin A and B significantly. ImportanceRediscovery of existing molecules and lack of techniques to induce production of secondary metabolites are the major bottlenecks associated with drug discovery of novel bioactive molecules from Streptomyces, the major source of marketable drugs today. We found a new method to increase the diversity and quantity of secondary metabolites in two Streptomyces species. This method thus enhances the chance of finding novel active principles from Streptomyces.

microbiology↗

bioRxiv2020

Acetylation of isoniazid - a novel mechanism of isoniazid resistance in Mycobacterium tuberculosis

Isoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a pro-drug which is converted into its active form by the intracellular KatG enzyme of Mycobacterium tuberculosis. The activated drug hinders cell wall biosynthesis by inhibiting InhA protein. INH resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze transfer of acetyl group to INH from acetyl CoA. HPLC and LC-MS analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. Drug susceptibility assay and confocal analysis showed that M. smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at minimum inhibitory concentrations that inhibited wildtype strains. In addition, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity.

biochemistry↗