## 2 Results and Discussion
### The SFN and PEITC Treatment Controls Expression of Virulence Factors of V. cholerae  
Rehydration and antibiotic therapy remain the first line of treatment of cholera infections. This strategy is targeted to cure the symptoms but remains blind to the serious problem, namely, the toxicignicity of bacteria due to CT production, which, in consequence, leads to severe health complications after the therapy and slows the patient convalescence. To gain insight into the potency of ITC to reduce the pathogenicity of _V. cholera_, we performed a quantitative RT-qPCR analysis of virulence genes expression (Figure 4a). The virulence of _V. cholerae_ is maintained by several factors where the ToxT, ToxR, and ToxS regulatory proteins remain crucial for _ctx_AB genes expression called then the \"ToxR regulon\" after the first identified positive regulator [50]. Thus, the following genes were chosen according to their role in _V. cholerae_ pathogenicity: _toxT_, _toxR_, _toxS_, encoding regulatory proteins; _ctxB_ the CT beta subunit; _tcpH_, _tcpP_, and _tcpA_ as involved in toxin coregulated pilus synthesis. Moreover, because of described significant role of (p)ppGpp alarmone in effective transcription from ToxR regulon, we decided to study the genes expression pattern also in amino acid starved cells during SHX treatment. Our results indicated that a high level of (p)ppGpp, elevated due to starvation signal from RelA activity, resulted in a general decrease in the expression of tested genes in SHX-treated cells. This downregulation was observed in SFN and PEITC challenged cultures (Figure 4a). The strong and statistically significant downregulation of _toxT_, the main toxin synthesis regulator, was noticed in a relatively short time, 15 min after the addition of compounds, namely \(-3.1\), \(-7.6\), and \(-25\) fold change for SFN, PEITC, and SHX, respectively. The prolonged treatment (60 min) resulted in a less pronounced effect, especially for PEITC and SHX. Expression of _toxS_ and _toxR_ was downregulated efficiently by SHX and SFN, while PEITC reduced the expression of the _toxS_ gene only. Notably and importantly, for the prospective treatment of cholera with ITC, the toxin gene expression was reduced significantly by PEITC and SHX already 15 min after addition of compounds, while for SFN, the effective downregulation of _ctxAB_ gene expression required longer (60 min) exposure. The expression of genes involved in the synthesis of coregulated pilus was inhibited most effectively by SHX and SFN after longer treatment and by PEITC earlier. The Western blot analysis was used to confirm the downregulation of CT synthesis. Figure 4b shows that treatment of _V. cholerae_ with SFN or PEITC resulted in significant impairment of toxin synthesis after 20 h (80% decrease compared to the control). Comparable results were observed for cells treated with SHX, where CT production decreased by 60%. In conclusion, expression of the genes involved in _V. cholerae_ virulence is downregulated by all tested compounds-ITC and amino acid starvation is induced by SHX. This is expected to reduce the pathogenicity of the bacteria. It was suggested by others that a high basal level of cellular (p)ppGpp positively correlates with toxin synthesis and _V. cholerae_ virulency [51]. Nevertheless, even _relA_ deficiency was not sufficient to reduce _V. cholerae_ motility, biofilm formation abilities and pathogenicity in the murine model. This apparent discrepancy can be, however, explained as [PERSON] and [PERSON] [52] described distinguished differences in the effect of alarmone accumulation on gene expression when it happens as a natural process responding to environmental nutrient availability and growth rate, and in \"sudden burst\" scenario in response to harsh stress conditions. Thus, we could conclude that ITC treatment leads not only to growth inhibition but also affects the virulence genes expression through the rapid elevation of cellular (p)ppGpp level. Figure 4: The expression of _V. cholerae_ virulence factors is decreased by ITC treatment.