**2. Results**

### *2.1. Mutations in MSMEG\_1380 Gene Lead to Imidazo[1,2-b][1,2,4,5]tetrazines Resistance in M. smegmatis*

The list of nonsynonymous mutations found in spontaneous drug-resistant *M. smegmatis* mutants used in this study is presented in Table 1. We were able to construct targeted *M. smegmatis* mutants harboring each mutation in genes *MSMEG\_0641*, *MSMEG\_1601*, and *MSMEG\_2087*, as well as five mutations found in *MSMEG\_1380* gene using the p2NIL/pGOAL19 suicide system [14] for homologous recombination (Table 1).

We examined theminimal inhibitory concentrations (MICs) of the four compounds on the recombinant *M. smegmatis* strains and found that mutations in *MSMEG\_1380* gene lead to elevated MICs as compared to the *w.t.* strain (4×MIC for the compound **3a**, at least 2×MIC for the compound **3c**, at least 2×MIC for the compound **3h,** and 4×MIC for the compound **3n**), while recombinants harboring mutations in genes *MSMEG\_0641*, *MSMEG\_1601*, and *MSMEG\_2087* had the same MICs as the *w.t.* strain (Table 2).


**Table 1.** Bacterial strains used in the study.

**Table 2.** Imidazo[1,2-*b*][1,2,4,5]tetrazines MICs on *M. smegmatis* strains in liquid medium.


\* The compounds were not soluble at higher concentrations; bacterial growth was observed at the stated concentrations.

Thus we have shown that only the mutations in *MSMEG\_1380* are responsible for imidazo[1,2-*b*] [1,2,4,5]tetrazines resistance in *M. smegmatis*.

### *2.2. W.t. MSMEG\_1380 Overexpression Increases M. smegmatis Susceptibility to imidazo[1,2-b][1,2,4,5]tetrazines*

In order to investigate further the role of *MSMEG\_1380* in *M. smegmatis* resistance to imidazo[1,2-*b*] [1,2,4,5]tetrazines, we cloned the *w.t. MSMEG\_1380* gene and two of its mutant variants in the tetracycline inducible plasmid pMINDKm- [15].

We used the paper-disc assay to assess the drug susceptibility of *M. smegmatis* strains to imidazo[1,2-*b*][1,2,4,5]tetrazines and found that the overexpression of the *w.t. MSMEG\_1380* gene increases *M. smegmatis* susceptibility to the tested compounds, while the overexpression of its mutant variants had no effect on the phenotype (Table 3), thus suggesting that the disruption of MSMEG\_1380 protein's function leads to the drug-resistant phenotype.


**Table 3.** Growth inhibition halos, produced by imidazo[1,2-*b*][1,2,4,5]tetrazines on *M. smegmatis* strains.

### *2.3. MSMEG\_1380 Represses the Expression of the mmpS5-mmpL5 Operon in M. smegmatis*

*MSMEG\_1380* gene lies 179 b.p. upstream the *mmpS5-mmpL5* operon (genes *MSMEG\_1381- MSMEG\_1382*) in the *M. smegmatis* genome and is transcribed in the opposite direction (Figure 2). The structure of this operon is conserved in different mycobacterial species: the *mmpS5-mmpL5* genes are controlled by a TetR-family transcriptional repressor, encoded by a gene located upstream the operon. Mutations in genes encoding the TetR-repressor, which lead to the upregulation of the *mmpS5-mmpL5* genes, are involved in *M. abscessus* resistance to the derivatives of thiacetazone [16], as well as in cross-resistance of *M. tuberculosis* to bedaquiline and clofazimine [17]. We have also identified a possible operator sequence in the 5-untranslated region of *MSMEG\_1381*, similar to the one described in [16]: 5-AAGCGGATTGACCTTATCCACTT-3.

**Figure 2.** Schematic representation of the *mmpS5-mmpL5* operon structure in *M. smegmatis* genome. The putative operator sequence is shown in red.

To test the hypothesis that resistance to imidazo[1,2-*b*][1,2,4,5]tetrazines in *M. smegmatis* has a similar origin to the ones described for *M. tuberculosis* and *M. abscessus* [16,17], we analyzed the expression of *MSMEG\_1380* gene and *mmpL5* (*MSMEG\_1382*) genes in different conditions.

All the spontaneous *M. smegmatis* mutants had increased *mmpL5* expression (54.16–80.45 times) as compared to the *w.t. M. smegmatis mc2 155* strain (Figure 3A). The overexpression of the *w.t. MSMEG\_1380* gene, cloned into the pMINDKm- plasmid led to a 7.90-fold repression of the *mmpL5* gene expression (*p* < 0.001, Figure 3B), confirming that *MSMEG\_1380* encodes the repressor of the *mmpS5-mmpL5* operon, and explaining the drug-susceptible phenotype, observed in the *MSMEG\_1380* overexpressing strain. On the contrary, the expression of *MSMEG\_1380* was upregulated in the mutant strains (Figure 3A), indicating that this transcriptional repressor is self-regulatory and that mutations lead to the loss of its function.

We also observed that the addition of subinhibitory concentrations of the compound **3a** upregulated the expression of *mmpL5* in a dose-dependent manner (Figure 3C).

**Figure 3.** Relative expression levels of *mmpS5-mmpL5* operon genes in different conditions: expression levels of *MSMEG\_1380* and *mmpL5* genes in spontaneous *M. smegmatis* imidazo[1,2-*b*][1,2,4,5] tetrazine-resistant mutants (**A**); expression levels of *MSMEG\_1380* and *mmpL5* genes in *M. smegmatis* pMINDKm-:*msmeg\_1380* (**B**); expression levels of the *mmpL5* gene after the addition of different concentrations of the compound **3a** (shown on the X-axis) (**C**). Error bars represent standard deviations from triplicates.
