## 4 Discussion
Isoniazid affects cell wall biogenesis by inhibiting enoyl reductase involved in the synthesis of mycolic acids [59]. In turn, tetracycline suppresses protein synthesis on the ribosome, preventing the interaction of aminoacyl-tRNA with the A-site of its 30S subunit [60]. Thus, ciprofloxacin suppresses replication, isoniazid suppresses cell wall synthesis, and tetracycline suppresses translation. The mechanism of action of VapC toxin, at least partially, is realized by the cleavage of 23S rRNA, which can lead to the removal of ribosomes from active protein synthesis, their deposition in a membrane-associated state, and the transition of bacterial cells into dormancy. Thus, VapC, like tetracycline, affects the translation apparatus, stopping its functioning, but ensuring preservation in an inactivated state [11]. This could potentially reduce the effectiveness of antibiotic action due to the partial vanishing of the target and the transition of cells into a dormant state; however, the absence of a positive effect of VapC overexpression on the survival of _M. smegmatis_ (Figure 1b) raised doubt about this hypothesis. The situation became somewhat clearer when we evaluated the effect of tetracycline on the survival of the AmazEF and the double deletion AvapBCAmazEF strains. Deletion of the second TA system in the AvapBC strain led not only to the cancellation of cell death but also to higher levels of _M. smegmatis_ survival compared to the wild-type cells (Figure 2). A similar level of CFU was observed in the AmazEF mutant. Apparently, the decrease in viability of the AvapBC strain under the action of tetracycline was largely due to the activity of the MazF ribonuclease, which cleaves tRNA Lys [9]. In wild-type cells, because of noncognate toxin-antitoxin interaction [61], MazF activity was partially blocked by the antitoxin VapB. In the AvapBC mutant, such interaction was impossible due to the absence of the VapB antitoxin, which led to a pronounced (two orders of magnitude) decrease in CFU value caused by MazF activity. Contrarily, the AmazEF and AVapBCAmazEF strains lacked the MazF toxin in their cells and thus exhibited viability levels three times higher. Evaluation of _M. smegmatis_ survival using the most probable number (MPN) method showed similar patterns to CFU counting. At the same time, a comparison of CFU and MPN (Table 2) revealed the highest number of dormant cells in the AvapBC mutant, which likely indicates the involvement of the MazF toxin in the formation of such forms under the action of tetracycline. It should be noted that the observed maximum proportion of non-culturable bacterial forms in the AvapBC strain does not exclude the possibility of VapC toxin's involvement in the transition to a dormant state but suggests the existence of a complex ensemble of interacting molecules in which VapC and MazF play noticeable roles. The phenotype reversion to the wild-type MPN levels in double deletion mutant (\(\Delta\)vapBC\(\Delta\)mazEF strain) supports the idea of the TA systems interaction (Table 2). It is likely that each of the VapBC and MazEF modules acts as a significant factor in the formation of dormant cells in certain specific environmental conditions. Indirect confirmation of this assumption may be detecting the activation of _vapC_ and _mazF_ gene expression under the action of tetracycline (Figure 3). The increase in the content of corresponding proteins in cells, on the one hand, contributes to the development of a general stress response and leads to the formation of a pool of dormant cells that are resistant to the action of the antibiotic. On the other hand, this leads to the death of part of the bacterial population through a mechanism not directly related to the action of the antibiotic but due to the activity of the MazF toxin. The fate of a specific cell, in this case, apparently, depends on the ratio of toxin content in it. From this perspective, the absence of a positive effect of VapC overexpression on survival under the action of tetracycline (Figure 1b) can be explained as follows.