Understanding the mechanism of action of protein synthesis inhibitors against Mycobacterium

Abstract

Despite the availability of various drugs to combat tuberculosis, the disease remains as a major public health concern. Every year over 9 million new tuberculosis cases and ~2 million deaths related to tuberculosis are reported. The existence of multi-drug resistant and extensively drug-resistant tuberculosis strains further complicates the effort to eradicate the disease. Therefore, novel drugs are urgently needed to combat tuberculosis. The current study focuses on evaluating the possibility of pleuromutilins as novel anti-tuberculosis drug, targeting the clinically validated target, protein synthesis complex. Initially we carried out bioinformatic analysis to compare the rRNA complex (16S, 23S, 5S rRNA and proteins associated with the complex) of 4 Mycobacterium strains namely, M. smegmatis, M. bovis BCG, M. leprae and M. tuberculosis. Except for M. smegmatis, which has two sets of rRNA genes (16S, 23S and 5S), other three strains had one set of rRNA genes. We studied the anti-mycobacterial effects of pleuromutilins using all the above strains except M. leprae. Antimycobacterial analysis of valnemulin (a pleuromutilin) showed that, it not only inhibited the growth of slow growing mycobacteria but also had bactericidal activity. To date, no literature has shown that pleuromutilin is effective against the slow growing Mycobacterium tuberculosis complex (MTC). This study provides evidence that valnemulin (a pleuromutilin) is effective against MTC. Further spontaneous mutants against valnemulin and linezolid (a known protein synthesis inhibitor) were raised to understand the mechanism of action of pleuromutilins. Sequencing of rRNA region of spontaneous mutants raised against both valnemulin and linezolid showed point mutations in 23S rRNA. These mutants were used to study the cross- resistance pattern. Despite sharing a common target, here we provide evidence that the mechanisms of resistance of linezolid and valnemulin (pleuromutilin derivative) are different and independent. This further enhances the possibility of using pleuromutilins as novel anti TB drug. Methylation of 23S rRNA confers resistance to macrolides lincosamides and streptogramin B?s (MLS). Erythromycin ribosome methyltransferase (erm) are a family of genes that encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that either monomethylate or dimethylate A2058 in 23S rRNA. A single mutation in A2058 has been shown to cause resistance to MLS. Although a single mutation has never been associated with pleuromutilin resistance, A2058 mutation found in combination with other mutations has been shown to significantly increase the minimum inhibitory concentration of tiamulin (a pleuromutilin) and valnemulin. Also, biochemical foot printing data showed that A2058 exhibit altered reactivity to chemical probes in the presence of various pleuromutilin antibiotics, showing that A2058 is involved in the binding of pleuromutilin antibiotics. We propose that erm may be partially involved in causing resistance to pleuromutilin antibiotics.