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

Figures & data

Table 1. Primers used in the real-time PCR assay.

Primer name	Sequence (5′→3′)
pF_OxyR pR_OxyR	GCAGGTAGCGGGATCACTTT GCACGGCAGATAAACAACCC
pF_KatG pR_KatG	GCTCTGCCTGTTCTGGAGAA CACACCAGCCAGCACTATGA
pF_AhpC pR_AhpC	GCTGGAGCGTCTTCTTCTTCT TAGTGGTCAGCAACGTCACC
pF_CysD pR_CysD	ATTTGCCCGTTGTAGTTGTGC TACTCTTTCCGTGACCGCTTC
pF_SodA pR_SodA	CTGCCAGAATTTGCCAACCTG GTACGGTTTTCTTGTCTGCTGG
pF_SoxR pR_SoxR	AAACAGCTTTCGTCCCAATGG TACATCCGTCCAGTTCGTCAC
pF_SoxS pR_SoxS	GCTGGGAGTGCGATCAAACT GCAATGGACCTGGGTTATGTGT
pF_16SrRNA pR_16SrRNA	CCTTACGACCAGGGCTACAC TTATGAGGTCCGCTTGCTCT
pF_Φ24B_N pR_ Φ24B_N	AGGCGTTTCGTGAGTACCTT TTACACCGCCCTACTCTAAGC
pF_Φ24B_cI pR_Φ24B_cI	TGCTGTCTCCTTTCACACGA GCGATGGGTGGCTCAAAATT
pF_Φ24B_cII pR_Φ24B_cII	TGATCGCGCAGAAACTGATTTAC GACAGCCAATCATCTTTGCCA
pF_Φ24B_Q pR_Φ24B_Q	GGGAGTGAGGCTTGAGATGG TACAGAGGTTCTCCCTCCCG

Table 2. Differences in OD values resulting from comparison of bacterial growth (E. coli strain MG1655 lysogenic for Φ24_B) in control culture (DMSO) and cultures carried out with the analyzed compounds. Each experiment was conducted during 9 h, in the presence or absence of mitomycin C (MITC).

		Effects on the lysogenic strain growth^a
		50 µM		100 µM		200 µM
No	Compound	–MITC	+ MITC	–MITC	+ MITC	–MITC	+ MITC
1	BZ23	–12	9	–12	5	–22	12
2	BZ26	7	52	–1	48	–2	>100
3	BZ102	–1	12	–22	2	–25	8
4	BZ105	–23	–16	–20	–29	30	–44
5	BZ106	–2	–11	–7	–68	–97	–96
6	BZ86	1	29	–2	24	–18	15
7	BZ70	0	41	–3	54	–7	40
8	BZ83	12	12	7	–2	–7	–18
9	BZ64	0	26	0	12	–18	11
10	BZ96	1	10	–1	15	–7	–2
11	BZ89	–2	8	2	–6	2	–36
12	IA011C	14	83	18	>100	16	>100
13	CM092	6	>100	6	>100	–11	>100
14	CM022G	1	48	4	48	–1	68
15	CM032D	3	17	6	49	0	65
16	CM2071F	4	>100	4	>100	–7	>100
17	CM3186B	2	65	9	72	7	>100
18	CM3141B	9	23	12	57	8	>100
19	BZA15	8	>100	17	>100	15	>100
20	AR02	–1	4	–3	20	–8	41
21	AR09	1	10	2	17	–9	45
22	BZA23	4	8	3	7	–5	76
23	CM3116A	0	18	–1	22	–9	20
24	CM3159A	4	27	5	26	–1	42
25	CM3146B	–3	4	–4	11	–14	40
26	CM3129A	0	21	5	28	–30	3
27	MF1	2	15	0	41	–1	>100
28	MF27A	–2	9	–2	25	–6	68
29	THN10	3	21	–1	32	–2	>100
30	THN6C	11	20	9	51	16	>100
31	CM3072B	3	10	3	3	2	76
32	CM3116C	5	1	3	3	–4	77
33	MF5	–1	44	–1	97	–7	>100
34	CM3159B	–1	30	1	68	0	>100
35	AR27	0	–4	–4	–1	–14	69
36	MQ4	3	–23	–2	–18	–15	45
37	MQ8	7	–8	–9	–10	–11	18
38	BZA37	10	–6	9	10	2	75
39	CM4017A	0	3	–6	19	–11	38
40	CM3130B	–6	23	–17	22	–26	29
41	CM4016A	–1	6	–4	18	–11	58
42	CM3112B	0	40	–15	1	–26	12
43	SiA5	3	25	–14	25	–17	29
44	CM032E	–2	19	–4	13	–12	20
45	MF4	–2	4	–2	41	–10	91
46	MF6	–2	1	–14	13	–13	92

^aPresented results are expressed as percent values above (numbers) or under (numbers with minus sign) the control value which is assumed as 100%. SD was below 20% for each point, and it is not shown for clarity of presentation. Compounds selected for further analyses are underlined.

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.

Table 3. 2 D ¹H–¹³C HMBC correlations for AM10A (DMSO, 500.13 MHz).

			HMBC [J(C,H)]
Atom	^13C	^1H	^1J	^2J	^3J	^4J
1	145.1			2-H,	3-H	4-H
2	124.8	8.04	2-H
3	136.1	7.99	3-H
4	128.9	8.28	4-H
4a	148.8			4-H	3-H, 4b-OH	2-H
4b	94.6				4-H, 4b-OH, 1’-H,	3-H, 9b-OH
5a	164.9				7-H,1’-H	8-H
6	36.7	2.10	6-H	7-H	8-H
7	21.9	1.84	7-H	6-H, 8-H
8	24.2	2.41-2.77	8-H	7-H	6-H
9	188.7				7-H	6-H
9a	104.5				6-H, 8-H, 9b-OH
9b	83.3			9b-OH	4b-OH
10	192.8				9b-OH	2-H
10a	126.4				2-H, 4-H	3-H
1’	44.9	4.61	1’-H	CH3

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.