Although drug resistance in Mycobacterium tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in the possible role of efflux in causing drug resistance. Previously, efflux genes have been shown to be 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. We constructed point mutations V219A and S292L in Rv1258c in the chromosome of M. tuberculosis and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed M. tuberculosis Rv1258c mutants to different tuberculosis drugs was assessed using conventional drug susceptibility testing in 7H11 agar in the presence and absence of efflux pump inhibitor piperine. A C14-labeled PZA uptake experiment was performed to demonstrate higher efflux activity in the M. tuberculosis Rv1258c mutants. Interestingly, the V219A and 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 low-level drug 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. The 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 resistance, 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 .

The magnetization dynamics of the triangular lattice of Ising spin chains is investigated in the framework of a two-dimensional model. The rigid chains are assumed to interact with the nearest-neighboring chains, an external magnetic field, and a heat reservoir that causes the chains to change their states randomly with time. A probability of a single spin-flip process is assumed in a Glauber-type form. This technique allows describing properly the steps in the magnetization curves observed in Ca 3 Co 2 O 6 and their dependence on a magnetic-field sweep rate and temperature. A formation of domain walls due to the nonequilibrium magnetization process and a significant role of their motion are shown.Ising spin chains packed into a two-dimensional ͑2D͒ frustrated lattice demonstrate a complex magnetic behavior due to a combination of a low dimensionality and frustration. There are few groups of compounds in which the triangular lattice is formed by antiferromagnetic ͑AFM͒ Ising spin chains, e.g., CsCoCl 3 and CsCoBr 3 ͑Ref. 1͒ or ferromagnetic ͑FM͒ ones such as Ca 3 Co 2 O 6 . 2,3 A spin-chain system Sr 5 Rh 4 O 12 discovered recently has a complex magnetic structure of chains. 4 An interaction between the nearestneighboring chains in the lattice is of the AFM type and much weaker than the intrachain one. However, it causes the frustration and a large variety of magnetic structures. [1][2][3][4][5][6] A steplike magnetization curve in Ca 3 Co 2 O 6 has drawn recently considerable attention. 3,5-13 The number of the steps depends on a sweep rate of the external magnetic field and temperature. 3,8,10 Two steps become apparent in the temperature range from 12 to 24 K. 10 The first step takes place at the zero magnetic field. Then a plateau at about 1/3 of the full magnetization stretches up to the magnetic field of 3.6 T where the second step to the saturated FM state occurs. At least four equidistant steps are clearly visible below 12 K at a moderate magnetic-field sweep rate. 3,8 At an extremely low sweep rate the magnetization curve becomes close to the two-step shape. Similar to that is observed at high temperatures. 8 A response to alternating magnetic fields and its dependence on the temperature and magnetic field were investigated carefully in Ref. 7. The experimental results show that there exist two characteristic time scales of the magnetization dynamics. The first is of order of 1 s and the second reaches a few hours.A crystal structure of Ca 3 Co 2 O 6 consists of Co 2 O 6 chains running along the c axis. The Ca ions are situated between them and are not involved in magnetic interactions. The chains are made up of alternating face-sharing CoO 6 trigonal prisms and CoO 6 octahedra. The crystalline electric field splits the energy level of Co 3+ ions into the high-spin ͑S =2͒ and low-spin ͑S =0͒ states. The chains form triangular lattice in the ab plane that is perpendicular to the chains. An in-chain exchange interaction between high-spin cobalt ions through the octahedra with low-spin cobalt ions is ferroma...

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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.