PagP Activation in the Outer Membrane Triggers R3 Core Oligosaccharide Truncation in the Cytoplasm of Escherichia coli O157:H7

Abstract

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagP⌬5-14 allele, which mostly eliminates a periplasmic amphipathic ␣-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.Like most enteric Gram-negative bacteria, Escherichia coli surrounds its cytoplasmic membrane with a reticulated peptidoglycan exoskeleton (murein) and an outer membrane (OM), 5 which demarcates the so-called periplasmic space. The enterobacterial OM is an asymmetric lipid bilayer in which lipopolysaccharide (LPS) exclusively lines the external leaflet, whereas phospholipids line the inner leaflet (1, 2). The asymmetric lipid organization provides a permeability barrier to hydrophobic antibiotics and detergents encountered in the natural and host environments. Although hydrophobic antibiotics can freely permeate through phospholipid bilayers, negative charges in LPS are bridged by Mg 2ϩ ions to create tight lateral packing interactions, which largely prevent permeation (3, 4). According to current models, perturbations of OM lipid asymmetry can result from the migration of phospholipids into the external leaflet to create localized rafts of phospholipid bilayers, which render bacteria susceptible to hydrophobic antibiotics (5, 6).The LPS is a tripartite molecule consisting of the hydrophobic anchor lipid A (endotoxin), the core oligosaccharide, which is divided into the inner and outer core regions, and the O-antigen polysaccharide (7). The so-called rough LPS includes only the lipid A-core and is usually distinguished from the smooth LPS ...

Extracellular Loops of Lipid A 3-O-Deacylase PagL Are Involved in Recognition of Aminoarabinose-Based Membrane Modifications inSalmonella entericaSerovar Typhimurium

“…These results suggest PagL's latency to help S. enterica to grow under specific conditions, including in the tissues of a host who has a fever, and are the first observations to suggest the physiological importance of PagL's latency. Several other outer membrane enzymes involved in the modification of lipid A, such as S. enterica LpxR and E. coli PagP, also display latency (5); LpxR-dependent lipid A deacylation in S. enterica (38) and PagP-dependent lipid A palmitoylation in E. coli (23,42) were not usually observed under normal culture conditions. These observations suggest that the latency of outer membrane enzymes is generally conserved for regulation of lipid A modifications in gram-negative bacteria.…”

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

“…I ). Whereas the heptaacyl form of lipid A has been detected in other Gram-negative bacteria ( 27 ), this study has demonstrated, for the fi rst time, that H. pylori lipid A can synthesize heptaacyl species.…”

“…Similarly, in Pseudomonas aeruginosa , palmitoylated LPS induced signifi cantly higher IL-8 response ( 29 ). In E. coli , the modifi cation enzyme, the palmitoyl transferase (PagP), catalyzes the addition of a palmitate to one of the primary linked acyl chains of lipid, resulting in a heptaacyl lipid A structure ( 27 ). The heptaacyl form observed in H. pylori 26695/hp1191::kan mutant suggest the possibility that a PagP-like enzyme may exist in H. pylori .…”

Inscribing the Perimeter of the PagP Hydrocarbon Ruler by Site-Specific Chemical Alkylation

“…Although the catalytic mechanism remains to be elucidated, PagP alternates between two dynamically distinct states likely representing latent and active conformations in the outer membrane environment (16). PagP activity is triggered by outer membrane lipid asymmetry perturbations that enable the phospholipid and lipid A substrates to each have access to the active site from the external leaflet (17)(18)(19). Phospholipid and lipid A access occurs by lateral diffusion through two gateways in the β-barrel wall known as the crenel and embrasure, respectively (20) (Figure 1A).…”