Inhibition of Shiga toxin-converting bacteriophage development by novel antioxidant compounds

Abstract

Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing Escherichia coli (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in E. coli.

Keywords: Shiga toxin-converting bacteriophage; Shiga toxin-producing Escherichia coli; antioxidants; heterocyclic compounds; oxidative stress.

Figure 1.

Structures of the selected 15 compounds.

Figure 2.

Chemical procedure for the synthesis of targeted indenoindoles.

Figure 3.

Correlations observed from NOE experiments for CM3072B.

Figure 4.

Correlations observed from NOESY experiment for THN10.

Figure 5.

NOESY interactions for AM10A.

Figure 6.

Growth of E. coli MG1655 lysogenic with Φ24_BΔstx2::cat at 37 °C in LB medium after induction with 0.5 µg/ml mitomycin C (added to the culture at time 3 h) in the absence or presence of tested compounds at indicated concentrations (added to the culture at time 0). Bacterial growth was monitored by measurement of A₆₀₀ at indicated times. Presented results are mean values from three experiments with SD indicated as error bars.

Figure 7.

Relative phage titer in cultures of E. coli MG1655 lysogenic with Φ24_BΔstx2::cat treated with 0.5 µg/ml mitomycin C (A) or 1 mM H₂O₂ (B) (inducers were added to the culture at time 3 h) in the absence (control experiments) or presence of tested compounds at indicted concentrations (added to the culture at time 0). Presented results are mean values from three experiments with SD indicated as error bars.

Figure 8.

Viability of human HEK-293 and HDFa cells in cultures treated with tested compounds at indicated concentrations for 48 h. Cell viability was tested in the MTT test. Presented results are mean values from three experiments with SD indicated as error bars. The significance of differences between control and cells treated with tested compounds was assessed by the ANOVA test. Differences were marked by asterisks (*) and considered significant when the p value was <0.05.

Figure 9.

Expression of bacterial genes coding for proteins involved in the oxidative stress response and of selected bacteriophage genes in E. coli MG1655 lysogenic with Φ24_BΔstx2::cat either non-treated (A) or after induction with 1 mM H₂O₂ (B) in the absence (control experiments) or presence of tested compounds added to final concentration of 0.2 mM. Levels of mRNAs were determined by RT-qPCR. Presented results are mean values from three experiments with SD indicated as error bars.