## 3 Results and Discussion
### Induction of the Stringent Response Underlines the Antibacterial Effect of ITC on S. dysenteriae  
The observed growth inhibition of _Shigella_ strains in the presence of ITC led us to search for the mechanisms underlying this process. As we know from our previous studies, in _E. coli_ ITCs exerted its effects via induction of the stringent response. Therefore, we evaluated cellular levels of the stringent response alarmones, ppGpp and ppPGPp, in the _S. dysenteriae_ SZ2855/97 Japan strain treated with PEITC, SFN, or serine hydroxamate, the amino acid analog that serves as a control of the stringent response induction. We also compared the observed nucleotide levels visualized by thin-layer chromatography with an untreated control (Figure 3a,b). The strong signal of both tetra- and pentaphosphate is visible in the SHX control, indicating that bacteria react to amino acid starvation by alarmone accumulation. Interestingly, both SFN and PEITC, induced the stress alarmone accumulation to an extent comparable to that induced by SHX. Apparently, similar to many bacterial strains tested so far [22], the basis of the effect of ITC-mediated growth inhibition is induction of the global stress response. The stringent response, as a general stress mechanism, is widely conserved in the Enterobacteriaceae family and _Shigella sp._ genus. The _reIA/spoT_ genes display high sequence similarity to their _E. coli_ homologues [34]. Accumulation of (p)ppGpp causes reprogramming of bacterial gene expression, shutting down such energy-consuming processes as ribosomal component synthesis, and, in general, expression of numerous household genes, while expression of genes involved in the stress response and those recognized by alternative sigma factors is activated. Such a global alteration in bacterial cellular metabolism results in growth inhibition, and importantly, leads to the downregulation of the Shiga toxin production (directly or indirectly). Another mode of action of ITCs can also involve destabilization of cellular membrane integrity, as we observed in our recent work [22] and as was suggested previously [29]. However, this phenomenon in _B. subtilis_ was not triggered by the stringent response induction. Thus, it can be concluded from the data presented here that the major mechanism underlying growth inhibition of _S. dysenteriae_ involves the alarmone accumulation. It was also suggested that ppGpp and its cofactor DKsA play a certain role in _Shigella_ virulence [35; 36], and interestingly, concentrations of the alarmones during the onset of the stringent response induced by ITC significantly exceed those observed during normal growth. Thus, the effect of (p)ppGpp accumulation mediated by ITCs is growth inhibition, which provides a promising course for additional investigation of ITCs application. Since the pathogenicity of _Shigella_ strains is strictly related to the synthesis of Stx toxin, we tested expression of the _stx_ gene in _S. dysenteriae_ SZ2855/97 Japan by qRT-PCR after the SFN/PEITC treatment (Figure 3c). We assessed the effect of both compounds at 1 \(\times\) MIC and \(\frac{1}{4}\) MIC, and found that they effectively downregulated the level of _stx_ gene expression. Namely, the fold change observed for SFN treatment was 0.16 \(\pm\) 0.01, 0.18 \(\pm\) 0.04, while for PEITC it was 0.22 \(\pm\) 0.11, 0.28 \(\pm\) 0.03 for 1 \(\times\) MIC and \(\frac{1}{4}\) MIC, respectively. However, even lower concentrations of SFN/PEITC (1/16 \(\times\) MIC) also had an inhibiting effect (0.47 \(\pm\) 0.09 and 0.83 \(\pm\) 0.79, fold level change for SFN and PEITC, respectively), although it was not as pronounced. On the other hand, mitomycin C, the SOS triggering factor, caused a significant increase in the _stx_ gene transcription (Figure 3c). The latter effect is caused by the general stress exerted by mitomycin C, as it was reported that addition of this drug had caused an increase in production and secretion of Shiga toxins through the outer membrane vesicles [37]. Although the Shiga toxin production by _S.