LAUSR

The prevalence of multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis) is an increasing problem worldwide (Zumla et al., 2013; Dong et al., 2015). According to a 2014 World Health Organization (WHO) report, 480,000 individuals world-wide developed multidrug-resistant tuberculosis (MDR-TB) and more than 100 countries have cases of extensively drug-resistant tuberculosis (XDR-TB). Compared with drug-susceptible TB, MDR-TB and XDR-TB require prolonged therapeutic treatment with a combination of a number of second-line drugs (Chen et al., 2013). For patients where TB remains persistent despite prolonged therapy with second-line TB drugs, the add-on agents including bedaquiline and delamanid are recommended for salvage therapy (Günther, 2014; WHO, 2014; Shim and Jo, 2013). The mechanisms of antibiotic resistance developed by bacteria are highly diverse (Alekshun and Levy, 2007), including protection of the antibiotic target by site-of-action mutations or by chemical modification of the target/target site (e.g., methylation in the ribosomal 23S rRNA by ErmMT methyl-transferase) (Buriankova et al., 2003). The direct modification or inactivation of antibiotics by specific enzymes (e.g., acetyltransferases, phosphotransferases) can be another resistance mechanism. Alternatively, reduced permeability or increased efflux (Black et al., 2014) of the drugs can also occur, thus preventing the drug from gaining access to the target. M. tuberculosis has adopted several of these strategies to become resistant to the most widely used antibiotics. One of these is the utilization of the ATP-binding cassette (ABC) transporters, which are drug efflux pumps that lower the concentration of antibiotics within the bacterium. Indeed, ABC transporters have been divided into three classes based on phylogenetic analysis (Dassa and Bouige, 2001): (i) the classical exporters (ii) importers which are composed of hydrophobic transmembrane domains and hydrophilic nucleotide-binding domains, and (iii) the type-II ABC proteins that lack transmembrane domains (NunezSamudio and Chesneau, 2013), some of which mediate antibiotic resistance (Kerr et al., 2005; Sharkey et al., 2016). Bioinformatics analysis shows that M. tuberculosis possesses as many as 87 ABC containing proteins. Amongst these, Rv3197 is predicted to be a non-canonical ABC protein with an N-terminal extension (1–117), an ABC1 motif (118–232) and an aminoglycoside phosphotransferase (APH) motif (256–312) but lacking any transmembrane regions. The sequence characterization suggested Rv3197 might be an antibiotic transporter. In our preliminary study, we evaluated the effect of Rv3197 in antibiotic susceptibility by measuring the MICs of several antibiotics including isoniazid, rifampicin, erythromycin, streptomycin, ampicillin, chloromycetin and tetracycline by overexpression of Rv3197 in Mycobacterium smegmatis (M. smegmatis), which is commonly used in lab as a model bacteria for M. tuberculosis. Our results indicated that Rv3197 affected the susceptibility of erythromycin (Table S1). Since a biosafety level 3 lab is needed to direct study of M. tuberculosis and the amino acid sequence of Rv3197 in M. tuberculosis is equivalent to Mb3320 in M. bovis BCG, a vaccine strain with low pathogenicity, we chose M. bovis BCG as a model for studying the function of Rv3197. Quantitative real-time PCR (qRT-PCR) analysis showed that the transcription level of rv3197 increases by 3.6 fold when M. bovis BCG is treated with 6 mg/L erythromycin (Fig. 1A), showing that expression of rv3197 is erythromycin-inducible. To verify the effect of Rv3197 on erythromycin susceptibility, we first made an Mb3320-deletion mutant strain, denoted Δrv3197-BCG. This mutant strain grew at a similar rate to the wild-type strain in 7H9 medium (Fig. S1A), demonstrating that, under the conditions where no antibiotic is added, Rv3197 has no effect on cell growth. However, upon exposure to 2 mg/L of erythromycin, cell growth was reduced in Δrv3197-BCG compared to wild-type BCG. This effect was partially reversed in a complemented pMV361-rv3197/ Δrv3197-BCG strain (Fig. 1B). In the presence of 3 mg/L of erythromycin, M. smegmatis, which contains the rv3197