Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: a study from Eastern India.

BACKGROUND: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method. AIM: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. METHODS: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. RESULTS: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). LIMITATIONS: The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. CONCLUSION: The study indicates the existence of rifampicin drug resistance in Eastern India.

A Mycobacterium leprae Hsp65 Mutant as a Candidate for Mitigating Lupus Aggravation in Mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins havebeen observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F1 mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K409A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K409A pep synthetic peptides, which cover residues 352–371, are presented. Peptides had immunomodulatory effects similar to that observedwith their respective proteins on survival and the combined administration of K409A+Leader pep or K409A pep+WT showedthat the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352–371 region. The number of interactions observed for WT is much higher than for Hsp65 K409A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F1 mice were inoculated with Hsp60 or K409A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K409A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment ofautoimmune diseases.