Polymyxin B resistance and biofilm formation in Vibrio cholerae are controlled by the response regulator CarR

Abstract

Two-component systems play important roles in the physiology of many bacterial pathogens. Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression of vps (Vibrio polysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of the almEFG genes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of the almEFG operon. Similarly to a carR mutant, strains lacking almE, almF, and almG exhibited enhanced polymyxin B sensitivity. We also observed that strains lacking almE or the almEFG operon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance in V. cholerae.

FIG 1

Regulation of almEFG expression. (A) Relative expression of almE, almF, and almG mRNA levels measured via qRT-PCR in A1552 wild type and the ΔcarR strain harboring vector only (pBAD/Myc-His C) or the complementation plasmid pcarR. Data are normalized to the recA expression via the Pfaffl method, with the expression of the wild type set to 1.0. The graph represents the mean expression of two independent experiments performed in triplicate. Statistical significance was determined with Student's t test; asterisks indicate P values of <0.01. Error bars represent standard deviations. (B) CarR binds to the almEFG promoter region. Mobility shift assays performed with the almEFG promoter region and _VICalm_FAM, with different concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 μM) of the response regulator CarR. (C) DNA binding by CarR is specific to the almEFG regulatory region. Lane 1, free fluorescent probe _VICalm_FAM (0.005 μM); lane 2, fluorescent probe _VICalm_FAM (0.005 μM) plus 0.8 μM CarR; lane 3, fluorescent probe _VICalm_FAM (0.005 μM) plus 0.8 μM CarR and 56× unlabeled probe alm; lane 4, fluorescent probe _VICalm_FAM (0.005 μM) plus 0.8 μM CarR and 56× cyaA_Cy3 unspecific probe. (D) Schematic representation of the reporter fragments (F1 to F4) used to analyze the expression of the almEFG operon. The coordinates correspond to the position with respect to the annotated start codon. Rectangles and arrows represent the structural genes. (E) Expression of various almEFGp-lux reporter fragments (F1 to F4) in A1552 wild-type and ΔcarR strains shown in relative luminescence units (RLU; counts min⁻¹ ml⁻¹/OD₆₀₀). The graph represents the mean expression of two independent experiments performed with four replicates. Statistical significance was determined using a one-way ANOVA and Dunnett's multiple-comparison test; asterisks indicate P values of <0.01. Error bars represent standard deviations.

FIG 2

Polymyxin B sensitivity of wild-type, carR, and almEFG strains. (A) Polymyxin B MIC assays of wild-type and mutant (ΔcarR, ΔalmE, ΔalmF, ΔalmG, ΔalmEFG, and ΔcarR ΔalmEFG) strains in A1552 and C6706 genetic backgrounds. (B) Polymyxin B MIC assay of A1552 wild-type and mutant strains harboring vector only or complementation plasmids. (C) Polymyxin B killing assays of A1552 wild-type and ΔcarR and ΔalmEFG strains grown in the presence or absence of Ca²⁺. % survival = (CFU_{PMB treatment}/CFU_{no treatment}) × 100. Statistical significance was determined with Student's t test; asterisks indicate P values of <0.05. Error bars represent standard deviations. (D) Polymyxin B MIC assays of A1552 wild-type, ΔcarR, and ΔalmEFG strains grown in the presence or absence of Ca²⁺. Arrows indicate the MIC (μg/ml) on Etest gradient polymyxin B strips; MIC values are shown below the images. Assays were carried out with at least 2 biological replicates and 2 technical replicates.

FIG 3

Intestinal colonization of wild-type, carR, and almEFG strains. Wild type (A1552 or C6706) was coinoculated with ΔcarR, ΔalmE, ΔalmF, ΔalmG, and ΔalmEFG mutants at a ratio of ∼1:1 into infant mice. The number of bacteria per intestine was determined 20 to 22 h postinoculation. The competitive index (CI) was determined as the output ratio of mutant to wild type divided by the input ratio of mutant to wild type. Each dot represents data from an individual mouse. Statistical analysis was performed using Student's two-tailed t test; the asterisk indicates a P value of <0.05.

FIG 4

Biofilm formation of alm mutants and complemented strains. (A) Three-dimensional view of biofilms formed by A1552 wild type and ΔcarR, ΔalmE, ΔalmF, ΔalmG, and ΔalmEFG mutants after 24 h and 48 h. (B) Biofilms formed by A1552 wild type harboring the empty vector and alm deletion strains harboring empty vector or respective complementation plasmids. Biofilms were grown in flow cells for 30 h and stained with Syto-9 prior to confocal imaging. (C) Biofilms formed by wild-type C6706 and ΔalmE and ΔalmEFG strains in the C6706 genetic background after 24 h. Bars, 40 μm.

FIG 5

Analysis of vpsL expression in alm mutants. The expression of a vpsLp-lux transcriptional fusion was determined in wild-type, ΔcarR, ΔalmE, ΔalmF, ΔalmG, and ΔalmEFG strains. The data represent the mean expression (relative luminescence units [RLU]) of four replicates from two independent experiments. The negative control, A1552 wild type harboring vector only, reflects the background luminescence obtained from the promoterless pBBRlux plasmid. Statistical significance was determined using a one-way ANOVA and Dunnett's multiple-comparison test. Asterisks indicate P values of <0.01. Error bars represent standard deviations.