High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae

Abstract

Background: Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen.

Results: A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 μM and a minimal bactericidal concentration (MBC) of 3.2 μM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype.

Conclusion: The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target.

Figure 1

HTS assay. Growth of V. cholerae MO10 pG13 strain in 96- (A) and 384-well MTP (B) in the presence of test compounds and controls. (A): 12 A-B: 1% DMSO, 12C-D: 100 μM ciprofloxacin, 12 E-F: no addition of compounds, 12 G-H: sterile medium. (B): 23 A-D and 24 A-D: 1% DMSO, 23 E-H and 24 E-H: 100 μM ciprofloxacin, 23 J-M and 24 J-M: no addition of compounds, 23 M-P and 24 M-P: sterile medium. Upper panels: absorbance at 600 nm; lower panels: fluorescence (485/535 nm). Wells framed in red indicate active compounds.

Figure 2

Screening results. Summary of the initial screening results for novel antibacterial compounds. The tested compounds came from the NCH, Peptide, LOPAC, VAR, EMC and CDI collections. The shaded area highlights the activities that were defined as initial hits. The most active compound, vz0825, stemming from the VAR collection, is highlighted in red.

Figure 3

Chemical structures. Most active compounds of V. cholerae growth inhibition. Panel A: compound vz0825; Panel B: compound vz0500; Panel C: compound 1541-0004.

Figure 4

Growth determination. Growth of V. cholerae wild type strain NM06-058 (A) and the T283M exchange mutant (B) in the presence of vz0825 in media with different K⁺ and Na⁺ concentrations.

Figure 5

Sequence of KdpD from V. cholerae. Amino acids labeled in green in the regions H, N, G1, F, G2 are conserved in different species [20]. Labeled in red is threonine 283 which is exchanged by methionine in the dominant mutations of the resistant strains. Amino acids labeled in blue indicate the positions that are modified in four additional mutants (L73P, P341H, E393K and E393G).