**3. Discussion**

Deorphaning phenotypic screening hits, that is determining their mechanism of action and/or resistance, is a key part in the early-stage anti-TB drug development [18]. Here we determine the mechanism of *M. smegmatis* imidazo[1,2-*b*][1,2,4,5]tetrazines resistance based on the previously obtained whole-genome sequencing data for 12 spontaneous mutants with cross-resistance to four compounds [9,13].

The construction of targeted mutants showed that only mutations in *MSMEG\_1380* are responsible for drug resistance. In *M. smegmatis*, *MSMEG\_1380* encodes a TetR-family transcriptional repressor, which controls the *mmpS5-mmpL5* operon, encoding transmembrane transporters, conserved throughout mycobacterial species. Mutations occurring in *MSMEG\_1380* led to the upregulation of the *mmpS5-mmpL5* operon and increased efflux of the drug-candidates from the cells, similarly to the mechanisms described for *M. tuberculosis* and *M. abscessus* [16,17]. Overexpression of the *w.t. MSMEG\_1380* led to the repression of the *mmpS5-mmpL5* operon and expectedly to an increased drug susceptibility phenotype. Interestingly, we observed a dose-dependent upregulation of the *mmpS5-mmpL5* operon upon the addition of one of the compounds, which may indicate the ability of this compound to bind to the MSMEG\_1380 protein, inhibiting its affinity to the operator sequence; however, this needs to be examined in vitro in future studies.

The tested compounds showed activity as ESTPK inhibitors [9,11]; however, we have not observed any mutations in ESTPK genes. One or more ESTPKs might still be the biotargets of imidazo[1,2-*b*][1,2,4,5]tetrazines but determining them by spontaneous mutagenesis might be difficult: some of the ESTPKs may fulfill the functions of others in the situation when they might be inhibited [8], and there is a possibility that more than one mutation might be required.

The primary biological role of the MmpS5-MmpL5 system consists in siderophore transport, which is crucial for *M. tuberculosis* survival under low-iron conditions within macrophages [19]. Yet, this efflux system has also shown itself to be an important factor of drug resistance: besides the mentioned efflux-mediated resistance to thiacetazone derivatives, bedaquiline and clofazimine [16,17], it has also been reported to provide*M. tuberculosis*resistance to azoles [20]. We can expect that*M. tuberculosis* strains resistant to bedaquiline and clofazimine might also be resistant to imidazo[1,2-*b*][1,2,4,5]tetrazines; however, a 36% mismatch in the amino acid sequences of the MmpL5 proteins in *M. smegmatis* and *M. tuberculosis* may affect the drug-specificity of the transporter, and this should be additionally examined in future studies. Still, the MmpS5-MmpL5-mediated resistance mechanism needs to be considered during early stages of anti-TB drug development, and convenient in silico and in vitro test-systems for rapid analysis should be developed.

### **4. Materials and Methods**

### *4.1. Bacterial Strains and Growth Conditions*

*M. smegmatis* strains described in this study are presented in Table 1. Middlebrook 7H9 medium (Himedia, India) supplemented with OADC (Himedia, India), 0.1% Tween-80 (v/v), and 0.4% glycerol (v/v) was used as liquid medium, while the M290 Soyabean Casein Digest Agar (Himedia, India) was used as solid medium. *Escherichia coli* DH5α was used for plasmids propagation. Cultures in liquid medium were incubated in the Multitron incubator shaker (Infors HT, Switzerland) at 37 ◦C and 250 rpm.

### *4.2. Targeted M. smegmatis mutants' Construction*

Targeted *M. smegmatis mc2 155* mutants were constructed by homologous recombination using the p2NIL/pGOAL19 suicide vector system [14]. Briefly, genes*MSMEG\_0641, MSMEG\_1380, MSMEG\_1601*, and *MSMEG\_2087* with adjacent 1-1,5 kb fragments were amplified from genomic DNA, isolated from respective mutants by phenol-chloroform/isoamyl alcohol extraction after enzymatic cell lysis [21], with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, USA) using the following primers, picked with primer-BLAST [22]: pN\_0641\_f 5- TTTTCTGCAGCCAACAACGATCCAGATGTCCGT-3 and pN\_0641\_r 5- TTTTAAGCTTCAATGGCGGCGTCTTCATTCTG-3 for *MSMEG\_0641*; pN\_1380\_f 5- TTTTAAGCTTGTACTACTCGCTGGTGGCGTC-3 and pN\_1380\_r 5- TTTTGGATCCTGCTGCACGTG TTCGGTGTC-3 for *MSMEG\_1380*; pN\_1601\_f 5- CCCATGACGGGCATCATCAACC-3 and pN\_1601\_r 5- TTTTTTAATTAACGACGATCAGCACGTCCACAC-3 for *MSMEG\_1601*; pN\_2087\_f 5- TTTTAAGCTTCCAGAAGGTCACCAGCGATCTG-3 and pN\_2087\_r. The amplified products were digested with respective restriction enzymes (Thermo Scientific, USA) and ligated in the p2NIL plasmid. The cassette from pGOAL19 was subsequently cloned in the obtained plasmids at the *Pac*I restriction site. The plasmids were electroporated in *M. smegmatis mc2 155* cells as described in [23] and plated on M290 plates supplemented with kanamycin (50 μg/mL), hygromycin (50 μg/mL), and X-Gal (50 μg/mL); blue single-crossover colonies were selected. Blue colonies were grown overnight in liquid 7H9 medium with ADC, and serial 10-fold dilutions were plated on M290 plates supplemented with X-Gal (50 μg/mL) and sucrose (2% w/v); white double-crossover colonies were selected and tested for Km susceptibility. Target genes were then Sanger-sequenced for a final confirmation of the mutation.
