Kirill V. Shur

Toxin-antitoxin (TA) systems are commonly found in most species of bacteria, including the causative agent of tuberculosis M. tuberculosis. TA systems play a role in the adaptation of the pathogen to stresses during host infection. Here we present the results of our study of the effect of the mutation in the vapC46 (C113G) gene using Mycolicibacterium smegmatis as a model. Expression of wild-type and mutant variants of the vapC46 gene in pKW08-MCS-Int vector was shown to increase resistance to erythromycin as well as the survival of M. smegmatis cells under short-term exposure to 0.5 mM H₂O₂. The introduction of the wild-type variant of vapC46 gene into M. smegmatis cells leads to a slowdown in growth. Expression of the mutant variant of the vapC46 gene did not affect the growth rate. However, no change in the level of RNase activity in cells carrying the vapC46 C113G mutation was observed in an in vitro model. Thus, the VapC46 toxin is involved in the regulation of the survival of M. smegmatis under oxidative stress, and the C113G mutation does not affect this function. The normal growth rate of M. smegmatis, containing the mutant vapC46 gene, along with the absence of changes in the ribonuclease activity of the VapC46 toxin, may be associated either with a change in the specificity of the mutant toxin for bacterial cell mRNA, or with the use of tRNA or rRNA as a substrate. The study of the effects of introducing a mutation into the vapC46 gene on the specificity of the VapC46 toxin is the subject of our further research.

Drug resistance (DR) in Mycobacterium tuberculosis is the main problem in fighting tuberculosis (TB). This pathogenic bacterium has several types of DR implementation: acquired and intrinsic DR. Recent studies have shown that exposure to various antibiotics activates multiple genes, including genes responsible for intrinsic DR. To date, there is evidence of the acquisition of resistance at concentrations well below the standard MICs. In this study, we aimed to investigate the mechanism of intrinsic drug cross-resistance induction by subinhibitory concentrations of antibiotics. We showed that pretreatment of M. smegmatis with low doses of antibiotics (kanamycin and ofloxacin) induced drug resistance. This effect may be caused by a change in the expression of transcriptional regulators of the mycobacterial resistome, in particular the main transcriptional regulator whiB7.

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains (including MDR, XDR, and TDR) force scientists worldwide to search for new anti-tuberculosis drugs. We have previously reported a number of imidazo[1,2-b][1,2,4,5]tetrazines – putative inhibitors of mycobacterial eukaryotic-type serine-threonine protein-kinases, active against M. tuberculosis. Whole genomic sequences of spontaneous drug-resistant M. smegmatis mutants revealed four genes possibly involved in imidazo[1,2-b][1,2,4,5]tetrazines resistance; however, the exact mechanism of resistance remain unknown. We used different approaches (construction of targeted mutants, overexpression of the wild-type (w.t.) and mutant genes, and gene-expression studies) to assess the role of the previously identified mutations. We show that mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines by increased efflux through the MmpS5-MmpL5 system, similarly to the mechanisms of resistance described for M. tuberculosis and M. abscessus. Mycobacterial MmpS5-MmpL5 transporters should be considered as an MDR-efflux system and they should be taken into account at early stages of anti-tuberculosis drug development.

Here, we report the draft genome sequences of 15 Mycobacterium tuberculosis isolates of the Beijing-B0/W-148 sublineage that carry a 7-bp insertion within the pks15 gene, which leads to the synthesis of Pks15/1 fusion protein. Pks15/1 is involved in phenolglycolipid synthesis and biofilm formation, thus potentially contributing to the B0/W-148 lineage’s enhanced virulence and drug resistance.

Although drug resistance in M. tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance in M. tuberculosis by constructing point mutations V219A, S292L in Rv1258c in the chromosome of M. tuberculosis and assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in M. tuberculosis . While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance in M. tuberculosis and address their role in affecting treatment outcome in vivo.