Novel small molecule modulators of quorum sensing in avian pathogenic Escherichia coli (APEC)

Figures & data

Figure 1. A) High-throughput screening for growth inhibition against APEC O78. 4,122 compounds did not impact the growth of APEC O78 (no elevated OD). B) Screening of the SMs for their effect on the AI-2 activity of APEC O78. The Vibrio harveyi BB170 AI-2 bioluminescent indicator bacteria was used to screen the cell-free culture supernatant of APEC O78. APEC cell-free culture supernatant was prepared from cultures grown in the presence of 100 μM of small molecules. Sixty nine compounds inhibited ≥ 75% of the AI-2 activity of APEC O78. AI-2 bioluminescence indicator assay was repeated four times for these 69 compounds and 10 compounds that showed highest AI-2 inhibition were selected for further studies.

Table 1. Effect of the selected SMs on the AI-2 activity of different APEC serotypes.

	% Inhibtion
Serotypes	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	Sources
O78	84	92	78	75	83	92	94	98	79	84	[62]
O2	94	96	54	76	89	93	83	94	51	89	[63]
O1	76	64	65	63	46	93	47	41	19	64	[4]
O8	20	20	39	0	18	79	2	18	20	0	[63]
O15	13	27	0	46	45	97	36	25	0	22	[63]
O18	37	43	8	54	61	95	48	54	14	35	[63]
O35	48	55	37	60	68	96	62	57	36	47	[63]
O109	65	57	0	67	69	60	67	75	66	57	[63]
O115	64	52	35	58	63	99	45	44	44	78	[63]

Inhibition of AI-2 activity was calculated by comparing bioluminescence inhibition from SM treated APEC O78 culture with that of the DMSO treated control

Figure 2. Effect of the selected AI-2 inhibitors on; A) biofilm formation of APEC O78. Biofilm formation was assessed using crystal violet assay by measuring the OD₅₅₀. All of the compounds significantly inhibited the biofilm formation except C1 in comparison to the DMSO treated control (PC). Two independent experiments were conducted with triplicate wells in each experiment and the average OD₅₅₀ (black bars) and inhibition % (red dots) are shown. B) motility of APEC O78. Motility was assessed by measuring the halo on a semisolid agar. Except C9 and C10, all compounds resulted in complete inhibition (did not form detectable motility halos) in comparison to the DMSO treated control. *Significant difference between AI-2 inhibitors treated compared to DMSO treated control (P < 0.05).

Figure 3. Cytotoxicity (A) and hemolytic activity (B) of the selected AI-2 inhibitors. Cytotoxicity was assessed using Caco-2 and HD-11 cells and hemolytic activity was determined using sheep and chicken RBCs. 100 μM of each compound was used in both assays. Most of the compounds showed significantly less cytotoxicity and hemolytic activity (P ≤ 0.05) compared to DMSO treated control. Two independent experiments were conducted with triplicate wells in each experiment and the average is shown. *Significant difference between AI-2 inhibitors treated wells compared to 10X LDH (cytotoxicity assay control) or Triton X-100 (hemolytic activity assay control).

Figure 4. Effect of the AI-2 inhibitors on the intracellular survival of different APEC O78, O2, and O1 in HD-11 (A) and THP-1 (B) cells. Cells were infected with APEC strains at MOI = 100, treated for 6 h with 1 µL (100 µM) of each compound and the internalized bacteria were determined. Two independent experiments were conducted with triplicate wells in each experiment and the average is shown. *Significant difference between AI-2 inhibitors treated cells (P < 0.05) compared to DMSO treated control. **Significant difference between AI-2 inhibitors treated cells (P < 0.001) compared to DMSO treated control.

Figure 5. Effect of the AI-2 inhibitors on G. mellonella larvae infected with APEC O78; A) survival rate of the treated larvae, B) bacterial load in dead larvae at different time points (24, 48, 72 h) and in live larvae at 72 h, and C) toxic effect of each compound on G. mellonella larvae. The larvae were treated with the AI-2 inhibitors (12.5 µg/larva) and inoculated with APEC O78 (8.5 µL ~ 4.25 × 10⁴ CFU), survival monitored for 72 h. The results of two independent experiments were averaged. Significant difference between AI-2 inhibitors treated compared to DMSO treated control at 48 h^a and 72 h^b (P < 0.05).

Table 2. Real Time PCR Primers used in this study.

Function	Genes	Gene product	Oligonucleotide sequence (5’ to 3’)	product size (bp)
AI-2 synthesis, biofilm formation, cell motility and exopolysaccharide formation	luxS	S-ribosylhomocysteinase	F: ACGAGTGCATCTGGTAAGTG R: CAATGGAAGACGTGCTGAAAG	87
	pfs	5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase	F: CGGCAGAACCGGTGTTAATAAT R: TGAAATCGGGCATCGGTAAAG	96
	hha	Hemolysin expression-modulating protein	F: GTGATCTGCGGCTGAGTAAA R: ACGTCGTTGCCAGACAAT	103
	wzb	Protein-tyrosine-phosphatase	F: TCGTTATCCCAGTGACCAAAC R: GTGTCGCAACTATGACCTGAT	114
	ompG	Outer membrane protein G precursor	F: CGGTTGGCTGTCGATGTATAA R: AGGTATATTGCAGACCCGTTTC	95
	rcsB	Transcriptional regulator RcsB	F: CAAGTACATCAAGCGCCATTTC R: CCCTTCGATATCCAGATCCAATAC	103
	motB	Flagellar motor protein B	F: AGGCTAATACGGTTGGGAATAC R: AGAATCGCCCGATGTTTAGAA	109
	flgN	Flagella synthesis chaperone protein FlgN	F: CCAGTAACCAGCCGTTATGTT R: TACGCAGGAAAGAACCCAATAC	117
	fliP	Flagellar biosynthetic protein FliP	F: AGCCATTCAGCGAAGAGAAA R: GTCTGGCAAACAACCCTAAATC	117
	cheW	Purine-binding chemotaxis protein CheW	F: CATCCACCTGGCTGAACTTA R: GTAACACGGATTGCGAACAC	106
	nlpC	Probable lipoprotein NlpC precursor	F: GCATCGTCACAACCACAAATC R: GGTTTGAACGACCAGCTACA	103
Virulence- associated genes	ompA	Membrane protein	F: TGACCGAAACGGTAGGAAAC R: GGAATACCAGTGGACCAACAA	99
	fimC	Fimbrial chaperone protein	F: GCTGGCAGGTATCCTGATATTC R: TGCCCTGCCGGATAAATTAC	99
	IucD	IucD protein	F: GTCCGGAGAAGCCTGAAATA R: GAGAAGCGGCGGAAATAAAC	114
	fyuA	Ferric yersiniabactin uptake A	F: CTTCCCTTCCGGTTCGTTAATC R: GGTACAGCCCAAACACCATATC	119
	iss	Serum survival protein	F: CGCTCTGGCAATGCTTATTAC R: GAAATGATGGGTGATGGTTTCC	100
	vat	Vacuolating autotransporter toxin	F: CTGAACCGCGTCCAGATTAT R: ACTCCACGGCAGGAAATATG	104
Cell division, DNA processing, and morphology	bolA	BolA protein	F: CAGTACTTTAGCGGAGGAACTC R: CAACCCTTCCCACTCCTTAAT	82
	mreD	Rod shape-determining protein MreD	F: CCGGTCTGAAAGAGACGTTAAT R: TTCCGCAACCTCGCATTAT	115
	csrA	Carbon storage regulator A	F: GTAACTGGACTGCTGGGATTT R: CCAGGTACGTATTGGCGTAAA	100
	murD	UDP-N-acetylmuramoylalanine- D-glutamate ligase	F: GCGTGGTTAATGCTGATGATG R: CCTGCTGATGATTCAGGTGATA	108
	lpp	Major outer membrane lipoprotein precursor	F: CGGTAATCCTGGGTTCTACTCT R: TGCTCAGCTGGTCAACTTTAG	108
Small molecules metabolism	frwC	Fructose-like-2 IIC component	F: AGCAGGGCAGCATTGTTAT R: GCAAATCAGCATGAAGGCATAG	104
	Fpr	Ferredoxin-NADP reductase	F: GTTCCTGCATCAGAGGTAGATAG R: TACAGCGATTGGCCCTTATT	128
	mtlR	Mannitol operon repressor	F: CGTCATTAACCGCCAGGAATA R: GTTCACCAAAGGGTCCAAGTA	122
	ugpC	SN-glycerol-3-phosphate transport ATP-binding protein UgpC	F: CTGATCGTGGGTAACGTAGAG R: GCAGTGTTCCTGTTTGATGAG	126
	thiH	Thiazole biosynthesis protein ThiH	F: GCATAAGTCGCCTCGTGATATG R: GACGGAATACGCCGAGTTAAAG	81
Housekeeping gene	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	F: CGGTACCGTTGAAGTGAAAGA R: ACTTCGTCCCATTTCAGGTTAG	99

Figure 6. Effect of the AI-2 inhibitors on expression of QS and virulence-associated genes; A) Heat map showing the impact of the selected AI-2 inhibitors on gene expression of QS and virulence associated genes. Effect on gene expression was assessed using 100 µM of AI-2 inhibitors. Three independent experiments were conducted and the average fold change was calculated using ∆∆ct. Fold changes ± 1.5 ≥ or ≤ 1.5 were considered differentially expressed. B) Principal component analysis (PCA) of the qRT-PCR data. Numbers above the arrows indicate the correlation (r) between the AI-2 inhibitors based on the gene expression data. The PCA analysis is based on the fold change of gene expression data.

Figure 7. Alignment and the chemical structures of the top 10 AI-2 inhibitors using 2D Tanimoto similarity scoring method. A chemical structure similarity score of 0.68 is statistically significant at the 95% confidence interval.

Mangiamele P, Nicholson B, Wannemuehler Y, et al. Complete genome sequence of the avian pathogenic Escherichia coli strain APEC O78. Genome Announc. 2013 Mar 21;1(2):e0002613. PubMed PMID: 23516182; PubMed Central PMCID: PMC3622999.

 PubMedGoogle Scholar

Rodriguez-Siek KE, Giddings CW, Doetkott C, et al. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiology. 2005 Jun;151(Pt 6):2097–2110. PubMed PMID: 15942016.

 PubMed Web of Science ®Google Scholar

Johnson TJ, Kariyawasam S, Wannemuehler Y, et al. The genome sequence of avian pathogenic Escherichia coli strain O1: K1:h7shares strong similarities with human extraintestinal pathogenic E. coli genomes. J Bacteriol. 2007 Apr;189(8):3228–3236. PubMed PMID: 17293413; PubMed Central PMCID: PMC1855855.

 PubMed Web of Science ®Google Scholar