Differential expression of the Mycobacterium tuberculosis heat shock protein genes in response to drug-induced stress.

Heat shock proteins are essential in maintaining cellular protein function, especially during stress. Their influence in managing drug-induced stress in Tuberculosis is not clearly understood. AIMS: Study the expression of select genes of the DnaK/ClpB chaperone network to evaluate their role in stress response in Mycobacterium tuberculosis clinical isolates during exposure to Isoniazid (INH) and Rifampicin (RIF). METHODS: Sanger sequencing to detect drug-resistant mutations followed by Drug Susceptibility Testing and Minimum Inhibitory Concentration determination. Culturing the bacilli in vitro, exposed to 1/4, 1/2 and 1 × MIC, and RNA quantification of dnaK, dnaJ1, grpE and clpB genes by using Real-time PCR. RESULTS: Susceptible isolates showed marginal down-regulation of two genes for INH, whereas all genes under-expressed against RIF. INH-resistant isolates had distinct expression profiles for inhA-15 and katG315 mutants. RIF-resistant bacilli did not have significant differential expression. MDR isolate showed up-regulation of all the four genes, with two genes over-expressing (≥4-fold). CONCLUSIONS: We observed characteristic gene expression profiles for each isolate in response to lethal and sub-lethal doses of INH and RIF. This provides insight into the role of DnaK/ClpB chaperone network in managing drug-induced stress and facilitating resistance. Further, the knowledge could provide targets for new drugs and augmenters.

Simultaneous Detection of Drug-resistant Mutations in Mycobacterium tuberculosis and Determining their Role through In Silico Docking.

A molecular method for diagnosis of drug-resistant Tuberculosis is Multiplex allele-specific PCR (MAS-PCR), which is more time-efficient, and its accuracy is studied using DNA sequencing. Also, understanding the role of mutations, when translated to protein, in causing resistance helps in better drug designing. AIM: To study MAS-PCR in the detection of drug resistance in comparison to DNA sequencing in Mycobacterium tuberculosis, and understand the mechanism of interaction of drugs with mutant proteins. METHODS: MAS-PCR was used for the detection of drug-resistant mutations and validation was done through DNA sequencing. MAS-PCR targeted four genes, iniA for the drug Ethambutol, rpsL and rrs for Streptomycin, and gyrA for Fluoroquinolone resistance, respectively. Further, the sequence data was analysed and modeled for in silico docking to study the effect on the interaction of the anti-TB drug molecule with the target protein. RESULTS: We identified drug-resistant mutations in four out of 95 isolates with one of them carrying a mutation at codon iniA501, two at gyrA94, and one for both iniA501 and gyrA94 using MASPCR. DNA sequencing confirmed drug-resistant mutations in only two isolates, whereas two others had mutation adjacent to the target allele. Drug-protein docking showed Estimated Free Energy of Binding to be higher for Fluoroquinolone binding with GyrA D94V mutant. Both wild and mutant IniA interact with EMB but had no significant effect on binding energy. CONCLUSION: DNA sequencing-based drug resistance detection of TB is more accurate than MASPCR. Understanding the role of mutations in influencing the drug-protein interaction will help in designing effective drug alternatives.

Detection of First-Line Drug Resistance Mutations and Drug-Protein Interaction Dynamics from Tuberculosis Patients in South India.

BACKGROUND: Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing. AIMS: To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis. METHODS: Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted five loci on three genes, katG 315 and inhA -15 for the drug isoniazid (INH), and rpoB 516, 526, and 531 for rifampicin (RIF). Furthermore, the sequence data were analyzed to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking. RESULTS: We identified drug-resistant mutations in 8 out of 114 isolates with 2 of them as multidrug-resistant TB using MAS-PCR. DNA sequencing confirmed only six of these, recording a sensitivity of 85.7% and specificity of 99.3% for MAS-PCR. Molecular docking showed estimated free energy of binding (ΔG) being higher for RIF binding with RpoB S531L mutant. Codon 315 in KatG does not directly interact with INH but blocks the drug access to active site. CONCLUSIONS: We propose DNA sequencing-based drug resistance detection for TB, which is more accurate than MAS-PCR. Understanding the action of resistant mutations in disrupting the normal drug-protein interaction aids in designing effective drug alternatives.

Copy number variations burden on miRNA genes reveals layers of complexities involved in the regulation of pathways and phenotypic expression.

MicroRNAs are involved in post-transcriptional down-regulation of gene expression. Variations in miRNA genes can severely affect downstream-regulated genes and their pathways. However, population-specific burden of CNVs on miRNA genes and the complexities created towards the phenotype is not known. From a total of 44109 CNVs investigated from 1715 individuals across 12 populations using high-throughput arrays, 4007 miRNA-CNVs (∼ 9%) consisting 6542 (∼ 5%) miRNA genes with a total of 333 (∼ 5%) singleton miRNA genes were identified. We found miRNA-CNVs across the genomes of individuals showing multiple hits in many targets, co-regulated under the same pathway. This study proposes four mechanisms unraveling the many complexities in miRNA genes, targets and co-regulated miRNA genes towards establishment of phenotypic diversity.