PhoPQ-Mediated Regulation Produces a More Robust Permeability Barrier in the Outer Membrane ofSalmonella entericaSerovar Typhimurium

Abstract

The PhoPQ two-component system of Salmonella enterica serovar Typhimurium produces a remodeling of the lipid A domain of the lipopolysaccharide, including the PagP-catalyzed addition of palmitoyl residue, the PmrAB-regulated addition of the cationic sugar 4-aminoarabinose and phosphoethanolamine, and the LpxOcatalyzed addition of a 2-OH group onto one of the fatty acids. By using the diffusion rates of the dyes ethidium, Nile red, and eosin Y across the outer membrane, as well as the susceptibility of cells to large, lipophilic agents, we evaluated the function of this membrane as a permeability barrier. We found that the remodeling process in PhoP-constitutive strains produces an outer membrane that serves as a very effective permeability barrier in an environment that is poor in divalent cations or that contains cationic peptides, whereas its absence in phoP null mutants produces an outer membrane severely compromised in its barrier function under these conditions. Removing combinations of the lipid A-remodeling functions from a PhoP-constitutive strain showed that the known modification reactions explain a major part of the PhoPQ-regulated changes in permeability. We believe that the increased barrier property of the remodeled bilayer is important in making the pathogen more resistant to the stresses that it encounters in the host, including attack by the cationic antimicrobial peptides. On the other hand, drug-induced killing assays suggest that the outer membrane containing unmodified lipid A may serve as a better barrier in the presence of high concentrations (e.g., 5 mM) of Mg 2؉ .Cells of gram-negative bacteria are surrounded by the outer membrane (OM), which functions primarily as a permeability barrier (35). Large, hydrophilic compounds are excluded by the narrow porin channels, and lipophilic compounds cross the unusual, asymmetric bilayer of this membrane only slowly. Our early analysis by using steroids as probes (37) showed that the diffusion across the OM bilayer is nearly 2 orders of magnitude slower than the diffusion across a typical phospholipid bilayer, such as the phospholipid bilayer that exists in the inner, cytoplasmic membrane. A recent study (11) suggested that this estimate likely needs to be adjusted somewhat downward. Nevertheless, the OM bilayer clearly functions as a formidable permeation barrier, because perturbing this bilayer results in a striking sensitization of Escherichia coli and Salmonella enterica serovar Typhimurium to various lipophilic inhibitors (44).One major factor that contributes to this barrier property is presumably the asymmetric structure of the OM bilayer, whose outer leaflet is composed nearly entirely of lipopolysaccharides (LPS) (25). (In fact, very low permeability was found in symmetric LPS bilayers assembled in the laboratory [40].) According to the lattice model for diffusion in liquids, a statistical average of rapidly fluctuating variable distances creates transient holes within the bilayer into which solutes can migrate (41). Thus, an effective memb...

LPS remodeling is an evolved survival strategy for bacteria

“…S5B). The most probable explanation for the differences in susceptibility is simply the diminished integrity of the LPS layer in the outer leaflet of the outer membrane, leading to higher permeability more readily allowing the diffusion of antibiotics (31,32). To examine this alteration in the outer membrane permeability (2), the MIC and uptake of ethidium bromide (EtBr) were measured.…”

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

“…Similarly, addition of palmitate would also stabilize the bilayer by increasing the hydrophobic and van der Waals interactions between neighboring LPS molecules. Given indirect support to this notion, the outer membrane of a Salmonella PhoP-constitutive strain is more robust than the outer membrane of a PhoP-defective mutant (10). Future research efforts will be directed to understand LpxO-mediated AP resistance.…”

“…It has been hypothesized that modification of lipid A in response to different host microenvironments helps pathogens to survive in the hostile host by camouflaging the pathogen from host immune detection, promoting antimicrobial peptide resistance and by altering outer membrane properties (3,10,11). However, direct evidence showing that bacteria remodel their lipid A in vivo is lacking.…”

PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane

“…For expression of phoQ cHAMP and pagP, cells were washed to remove glucose, back diluted, induced with 0.2% arabinose, and cultured to E-phase (∼2.5 h). Bacteria exposed to polymyxin B (PMB) (USB) were cultured to OD 600 = 0.1 in LB and treated with 1 μg/mL PMB (5,22) for an additional 1.5 h at 37°C before harvest.…”

“…Survival and proliferation within phagolysosomes require the induction of systems that promote resistance to cationic antimicrobial peptides (CAMPs), oxygen and nitrogen radicals, and other specific stresses, such as acidic pH (4). Among these sensory systems is the Salmonella Typhimurium PhoPQ virulence system, which regulates enzymes that modify lipid A to increase the OM permeability barrier to CAMP and promote bacterial resistance (1,2,4,5).…”