The broadly conserved regulator PhoP links pathogen virulence and membrane potential in Escherichia coli

Abstract

PhoP is considered a virulence regulator despite being conserved in both pathogenic and non-pathogenic Enterobacteriaceae. While Escherichia coli strains represent non-pathogenic commensal isolates and numerous virulent pathotypes, the PhoP virulence regulator has only been studied in commensal E. coli. To better understand how conserved transcription factors contribute to virulence, we characterized PhoP in pathogenic E. coli. Deletion of phoP significantly attenuated E. coli during extraintestinal infection. This was not surprising since we demonstrated that PhoP differentially regulated the transcription of > 600 genes. In addition to survival at acidic pH and resistance to polymyxin, PhoP was required for repression of motility and oxygen-independent changes in the expression of primary dehydrogenase and terminal reductase respiratory chain components. All phenotypes have in common a reliance on an energized membrane. Thus, we hypothesized that PhoP mediates these effects by regulating genes encoding proteins that generate proton motive force. Indeed, bacteria lacking PhoP exhibited a hyperpolarized membrane and dissipation of the transmembrane electrochemical gradient increased susceptibility of the phoP mutant to acidic pH, while inhibiting respiratory generation of the proton gradient restored resistance to antimicrobial peptides independent of lipopolysaccharide modification. These findings demonstrate a connection between PhoP, virulence and the energized state of the membrane.

Fig. 1

PhoP is required for virulence, resistance to antimicrobial peptides, and survival at acidic pH in E. coli. A. Bladder colonization levels at 48 h post-transurethral inoculation following independent challenge with wild-type uropathogenic E. coli CFT073 and ΔphoP strains. B. In vivo complementation of ΔphoP with phoPQ (pGEN+phoPQ) restores colonization of female CBA/J mice at 48 h following independent challenge. C. CFU ml⁻¹ and D. percent wild-type (wt) survival for CFT073, ΔphoP, and ΔphoP+phoPQ following 45 min incubation of 10⁷ CFU logarithmic phase cells in fresh LB medium containing 10 μM FeCl₃ and 2 μg/ml polymyxin B (PB). E. CFU ml⁻¹ and F. percent wild-type (wt) survival for CFT073, ΔphoP, and ΔphoP+phoPQ at 1 h post-inoculation of buffered LB medium pH 2.5 with 10⁷ CFU of bacteria. In A. and B. black dots represent the log CFU g⁻¹ from individual mice and horizontal bars represent the median CFU g⁻¹. P-values were determined using the non-parametric Mann Whitney significance test. In C.-F. Bars represent mean values and significant differences (P < 0.01) determined by Welch’s t test are indicated with an asterisk.

Fig. 2

Whole genome view of PhoP-dependent gene expression and identification of the large PhoP regulon in pathogenic E. coli. Log₂-fold changes in gene expression between exponential phase uropathogenic E. coli CFT073 wild-type and ΔphoP were determined using microarrays and plotted as rays from the outer ring. Individual genes that have significantly decreased transcription in the absence of phoP (*) are considered directly or indirectly activated by PhoP (green) while significant increases in gene expression represent directly or indirectly PhoP-repressed genes (red). Gene expression levels that were not considered significant are colored grey. Lines in the center ring (blue) mark uropathogenic E. coli CFT073 genes absent in commensal E. coli K12 strain MG1655 that were identified using Mauve whole genome sequence alignments. The inner-most ring represents positive (purple) and negative (orange) values for GC skew (C−G)/(C+G) calculated using a 10 kb sliding-window. Local changes in base composition bias indicate recent integration sites of foreign DNA or recombination events. The values switch polarity at the origin (ori) and terminus (ter). Coordinates from the CFT073 chromosome, the Acid Fitness Island (AFI), flagellar gene operons, pathogenecity (PAI), and prophage (Φ) islands are labeled on the periphery.

Fig. 3

PhoP represses motility in pathogenic E. coli. Microarray analysis showed increased expression of 37/44 flagellar genes in the absence of phoP. A.-D. Soft agar motility plates of A. CFT073, B. ΔphoP, C. ΔphoP complemented with phoPQ or with D. phoPQ from E. coli K12 MG1655. E. Diameter of motility from triplicate experiments (* P < 0.05). F. Western blot with anti-H1 FliC antisera.

Fig. 4

Prevention of membrane hyper-polarization and lipid A modification with L-Ara-4N are necessary for PhoP-dependent antimicrobial peptide resistance phenotype. A. Ratio of red:green fluorescence for 10⁷ CFU of logarithmic phase CFT073, ΔphoP, ΔfumAΔfumBΔfumC, and ΔsdhBΔfrdA incubated in 30 μM 3′3′-diethyloxacarbocyanine iodide (DiOC₂) (black bars). Depolarized control cells were incubated in 30 μM DiOC₂ and 100 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (white bars). The green fluorescent carbocyanine dye (DiOC₂) shifts to red fluorescence when the dye self-associates at high intracellular concentrations. Bars represent mean values and significant differences determined by Welch’s t test are indicated with an asterisk. B. CFU/ml for CFT073, ΔphoP, and ΔarnT following 45 min incubation of 10⁷ CFU logarithmic phase cells in fresh LB medium containing 10 μM FeCl₃ and 2 μg ml⁻¹ polymyxin B (PB) or LB medium containing only 2 μg ml⁻¹ PB. Viable counts were determined from triplicate experiments by plating serial dilutions on LB agar.

Fig. 5

Influence of the transmembrane proton motive force on E. coli susceptibility to antimicrobial peptides. A. Viable counts of CFT073 (black bars) and ΔphoP (white bars) following 45 min incubation in 2 μg ml⁻¹ PB with and without 50 μM m-chlorophenyl carbonyl cyanide hydrozone (CCCP) or 0.1% sodium azide (NaN₃). B. CFT073, ΔphoP, and ΔphoP+phoPQ cells grown to OD₆₀₀ = 0.8 were diluted 1:10 (input) and pre-treated with CCCP or NaN₃ for 1 h. Following pre-treatment, cells were washed with LB medium and incubated in fresh LB medium (mock) or LB containing 2 μg/ml PB for 45 min. C. CFT073, ΔphoP, and ΔphoP+phoPQ were grown and incubated in PB alone or PB containing CCCP or NaN₃ as described in (A) or in PB containing 0.5 mM EDTA or 10 mM MgCl₂. D. CFT073, ΔphoP, ΔarnT, ΔpagP, and ΔarnTΔpagP were incubated in LB medium containing 10 μM FeCl₃ and 2 μg ml⁻¹ PB or LB medium containing 0.5 mM EDTA, 2 μg ml⁻¹ PB and CCCP or NaN₃. Bacteria treated with EDTA were pre-cultured in LB medium with 0.5 mM EDTA. Viable counts in A.–D. were determined from triplicate experiments by plating serial dilutions on LB agar.

Fig. 6

PhoP-mediated control of membrane potential is required for E. coli resistance to acidic pH. CFT073 (black bars) and ΔphoP (white bars) were cultured to OD₆₀₀ = 0.8 were diluted and incubated in buffered LB medium at pH 7 and pH 5 with and without CCCP and NaN₃ for 1 h. Viable counts were determined from triplicate experiments by plating serial dilutions on LB agar.

Fig. 7

PhoP represses prophage induction. A. CFT073 and B. ΔphoP supernatants prepared from cells cultured in LB medium to OD₆₀₀ = 1.0 were mixed in molten top agar containing >10⁹ CFU E. coli strain C and poured onto TCMG agar plates to visualize plaques. C. Growth of CFT073 (□), ΔphoP (○), and complemented mutant (Δ) in LB medium containing 5 μg ml⁻¹ mitomycin C (MMC). CFT073 (■) and ΔphoP (●) growth in LB medium alone is shown for comparison. D. Growth of MG1655, which lacks a functional prophage, in the presence (■) and absence of MMC (●). E. Burst size (no. plaques CFU⁻¹) for CFT073, ΔphoP, and MG1655 cultured in LB or LB medium containing 5 μg ml⁻¹ MMC. F. CFT073 genes homologous to lambda cI were amplified by PCR from E. coli C lysate (CI) following incubation with plaques isolated from MC induced CFT073 supernatant. Genomic DNA purified from CFT073 (+) and E. coli C (−) were included as controls.

Fig. 8

The PhoP regulon controls energy transformations and membrane potential as one protective counter-measure against mammalian host defenses. A. PhoPQ is not activated. Proton motive force (μH⁺), consisting of a gradient of protons (ΔpH) and charge (Δψ), across the cytoplasmic membrane (CM), is generated by respiration using electron transfer systems. Protons are pumped into the periplasm (P) by electrogenic respiratory chain components concomitant with the re-oxidation of NAD⁺ from NADH produced by the tricarboxylic acid (TCA) cycle. The transmembrane potential (inside-negative) allows protons to diffuse into the cytoplasm, with the [H⁺] gradient, to drive processes including ATP synthesis and rotation of the flagellum and motility. B. In response to antimicrobial activities of host phagocytes, PhoPQ is activated by acidification and cationic antimicrobial peptides (AMP) that have traversed the bacterial outer membrane (OM). PhoP-mediated induction (green arrow and text) of acid resistance responses, non-electrogenic respiratory chain components that do not pump protons, active import of cations, and efflux of Cl⁻ create a reversed membrane potential (inside positive) and reduce the bactericidal effects of AMPs and inorganic acid by reducing their ability to disrupt the CM or acidify the cytoplasm. Activated PhoP represses (red arrow and text) the transcription of flagellar and prophage genes, expression of the latter can be induced by oxidative DNA damage caused by Fenton chemistry during assault by the phagocyte NADPH oxidase.