The outer membrane of Gram-negative bacteria is not a static structure, but can be remodeled in response to environmental conditions that allow bacteria to survive and function in hostile conditions. PagP, an outer membrane enzyme, plays a crucial role in its remodeling by transferring a palmitate chain from phospholipids to the proximal glucosamine unit of lipid A. This modification protects bacteria from host immune defenses by reinforcing the outer membrane permeability barrier and attenuating the host immune signalling. A new role of PagP in signal transduction in response to outer membrane stress was recently conceptualized in addition to its previous role as palmitoyltransferase.
The Gram-negative cell envelope and permeability barrier
The Gram-negative cell envelope is a unique and complex cellular structure that is composed of an inner membrane and outer membrane separated by a gelatinous region called the periplasmic space  The inner membrane is a phospholipid bilayer involved in selective nutrient uptake, protein translocation, lipid biosynthesis and oxidative phosphorylation. The periplasmic space contains soluble proteins and the peptidoglycan exoskeleton, which provides the structural framework for the cell and an anchor for certain outer membrane lipoproteins .
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The outer membrane of Gram negative bacteria is a unique asymmetric bilayer and its inner leaflet comprises phospholipids, including phosphatidylethanolamine, phoshatidylglycerol and cardiolipin whereas the outer leaflet is composed predominantly of lipopolysaccharide (LPS) . LPS comprises of three parts, proximal lipid A anchor, distal O-antigen polysaccharide and core oligosachcharides connecting them (Fig. 1(A)). Lipid A is phosphoglycolipid that is polyacylated by saturated fatty acid chains and makes up the outer monolayer of the Gram negative cell envelope .
LPS has phosphate groups and acidic sugars that make it negatively charged. These negatively charged molecules are bridge by divalent cations, mainly Mg2+, to stabilize electrostatic repulsion between adjacent molecules. Six or seven saturated acyl chains of lipid A help in lowering the fluidity of the outer membrane . These tight lateral interactions and reduced fluidity makes the outer membrane highly impermeable to hydrophobic compounds in comparison with a regular phospholipid bilayer .
The Gram-negative response to envelope stress
Bacteria encounter with numerous hostile conditions in nature and their hosts . To survive in the host environment and to cause infection enteric bacteria like E. coli have evolved a number of signaling networks, including the alternative extracytoplasmic function sigma factor σE and the two component systems such as CpxAR and PhoPQ. Bacteria activate the σE pathway in response to the stresses that interfere with outer membrane protein biogenesis . Anti-sigma factor RseA and its periplasmic counterpart RseB render σE inactive in the normal condition . Extracytoplasmic stress activates the protease activity of DegS, which cleaves RseA on a cytoplasmic site and thus releases σE into the cytoplasm. Activated σE causes the transcriptional activation of a set of genes involved in outer membrane biogenesis .
The two component system CpxAR responds to stresses that adversely affect the assembly of surface molecules like pili and certain outer membrane proteins . These environmental cues provide the stimulus for dissociation of periplasmic inhibitor CpxP from CpxA and trigger the autophosphorylation of a conserved histidine residue on its cytoplasmic domain. Phosphorylated CpxP then transfers phosphoryl groups to the response regulator CpxR, which ultimately causes the transcription of genes involved in envelope protein maintenance .
The PhoPQ two component system consists of PhoQ as the membrane bound sensor histidine kinase and PhoP as the cognate response regulator. PhoQ binds to divalent cations like Mg2+ via its periplasmic domain in its repressed state . Cationic antimicrobial peptides displace the Mg2+ from PhoQ  and promotes phosphorylation of PhoP. PhoP then regulates the transcription of genes related to lipid A structure, resistance to antimicrobial peptides and phagosome alteration .
The PhoPQ two component system also regulates the outer membrane β-barrel enzyme involved in lipid A modification called PagP. Of the many enzymes involved in the modification of lipid A, PagP is the only known enzyme that is located in the OM of E. coli. It catalyzes the transfer of palmitate from phospholipid to lipid A thereby converting hexa acylated lipid A to hepta acylated lipid A . This seemingly minor modification neutralizes the challenge of various cationic antimicrobial peptide and attenuate host innate immune signaling through TLR4 , and one study reported that PagP can activate the σE response in E. coli but only under abnormal growth conditions .
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Integral outer membrane protein of gram negative bacteria discover so far shows β barrel architect. PagP being an outer membrane protein is no exception, and has 8 strands to transverse the outer membrane and short N- terminus amphipathic α-helix on the periplasmic side . Recent research indicates that the ï¡-helix acts as a post assembly clamp which helps in stabilizing PagP in the membrane once folding is complete . Unlike other outer proteins, its barrel axis is at an angle of 25° with respect to membrane normal and tilted barrel is supported by the aromatic residues. . PagP exists in two dynamically distinct states, termed R (relaxed) which facilitate the substrate entry and T (tense) which play role in catalysis .
PagP has usual flexible extracellular loops but has an unusual centre with the lower periplasmic-exposed half being hydrophilic, and upper half being hydrophobic. The upper hydrophobic region consists of a lipid binding pocket called the hydrocarbon ruler and a single molecule of detergent helps identify its position. PagP selects the saturated 16 carbon fatty acid palmitate chain among all the fatty acids present in the membrane lipid pool and transfer it to lipid A . The depth of the hydrocarbon ruler can be modified to select shorter acyl chains by mutating the glycine 88 residue that lies on the base of hydrocarbon ruler. Gly88 substitution generated by a combination of site directed mutagenesis and chemical alkylation have generated modified PagP enzymes that transfer acyl chain in single carbon increments from C16 to C10 
Mitigation of outer membrane stress by Palmitoylation
Asymmetric bilayer composition of outer membrane confine phospholipids on inner leaflet but PagP's active site is located on outer leaflet. Therefore, phospholipids can only access PagP's lipid binding pocket from outer membrane outer leaflet. As long as phospholipid remain on the inner leaflet PagP remains in dormant condition. It seems that PagP doesn't play any role in migrating phospholipids from inner to outer leaflet but simply respond to the externally mediated perturbation in the outer membrane asymmetry by palmitoylating the lipid A .
When cationic antimicrobial peptides attack bacteria, the Mg2+ ions bridging adjacent LPS molecules are displaced. As a result, negatively charged LPS begins to repel each other, thereby allowing phospholipids to migrate from the inner to the outer leaflet. This breach in the asymmetric distribution of outer membrane phospholipids allow the hydrophobic antibiotics and detergents to enter freely into the periplasm . This stress condition of outer membrane makes phospholipids to be available in the outer membrane along with LPS and both this substrate can now access to the active site of PagP.
It has been shown that Phospholipid and lipid A get access to the PagP's lipid binding pocket through lateral diffusion. PagP possess some proline residues which can't participate in hydrogen bonding because they don't have amide proton to donate and display weakened hydrogen bonding in the adjacent strands. Gateways so formed are called crenel and embrasure, which provide lateral routes for entry and egress of phospholipid and lipid A respectively .
PagP mitigate the outer membrane stress cause by the attack of antimicrobial peptides by palmitoylating the lipid A. Extra acyl chain present as a result of palmitoyl transferase lower the fluidity of outer membrane and restore the outer membrane permeability barrier. PagP specifically use the palmitate chain from sn-1 position of phospholipid to transfer it to the hydroxyl group of R-3 hydroxymyristate chain at position 2 of lipid A.
Normally, pagP is transcribed by PhoPQ in response to Mg2+-limitation. When cells are treated with EDTA, which strips a fraction of LPS from the cell surface and promotes the migration of phospholipids into the OM outer leaflet, palmitoylation occurs too rapidly to be dependent on transcription, and was shown to occur independent of de novo protein synthesis . PagP is unique in its ability to modify lipid A independent of de novo protein or LPS biosynthesis in E. coli. Due to its location and ability to react immediately to outer membrane distress, PagP represents a form of first-line defense for many Gram-negative bacteria.
Lipid A palmitoylation eases the survival of bacteria in host
Lipid A is highly immunogenic in mammalian cells. Picomolar levels of the molecule are sufficient to activate the innate immune response via the TLR4 signaling pathway . Signal transduction occurs through the mammalian LPS receptor, a complex of TLR4, MD2, and CD14 that assembles on the surface of a number of cell types, including macrophages and dendritic cells. Signaling through TLR4 ultimately leads to the translocation of NF-ï«B into the nucleus, where it initiates the transcription of pro-inflammatory cytokines . Activation of innate immunity through TLR4 results in the production of cationic antimicrobial peptides. The interaction of cationic antimicrobial peptides with the outer membrane induces an amphipathic secondary structure that allows them to penetrate the outer membrane, and then insert into the inner membrane. Cationic antimicrobial peptides are lethal to bacteria through their disruption of the electrochemical potential across the inner membrane .
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Human TLR4 is highly sensitive to the structure of lipid A, and modifications to lipid A's acylation pattern can attenuate its ability to activate mammalian innate immunity through the TLR4 pathway. Palmitoylated lipid A was observed to be 30-fold less active in stimulating the activity of NF-ï«B than un-palmitoylated lipid A . On the other hand, decrease in membrane fluidity resulted from the palmitoylation of lipid A protect bacteria against cationic antimicrobial peptides . PagP's ability to attenuate signaling through TLR4 and protect bacteria from immune effectors eases the ability of bacteria to establish their infection in their host. PagP's narrow distribution amongst primarily pathogenic organisms like Salmonella enterica, L. pneumophila, B. bronchiseptica, Yersiniae pestis clearly indicate the importance of the enzyme in pathogenesis 
Due to their partial activation of innate immunity, modified structures of lipid A are being used as immune adjuvants . The Glaxo Smith Kline Cervarix vaccine used in human populations includes the monophosphoryl lipid A adjuvant that depends on palmitate incorporated by PagP.
New role of PagP as an apical sensory transducer
Recently, a new role of PagP in trans-envelope signalling was elucidated in E. coli O157:H7. Deficiency of myristate chain in distal glucosamine unit of lipid A, due to the deletion of msbB, was sufficient to breach the outer membrane asymmetry in E.coli O157:H7 and activate PagP. msbB deficient mutants also showed increases susceptibility to the serum treatment, suggesting that O-antigen region of lipopolysaccharide might be missing. Further analysis of lipopolysaccharide isolated from msbB mutant by electrophoresis, supports the absence of O-antigen repeats normally present in this serotype of E. coli. Nature of the core truncation was revealed by mass spectroscopy, gas chromatography of alditol acetates, and NMR spectroscopy, indicated that truncation was occurring at the level of the first outer core glucose residue, which is added in the cytoplasm by WaaG . Presence of smooth lipopolysaccharide when pagP is absent and truncated lipopolysaccharide when pagP is present supports the role of PagP in core truncation.
Lipopolysaccharides are known to play crucial role in folding of outer membrane protein  and there is the possibility that truncation is an artifact of producing an atypical lipid A species that doesn't support OM protein folding. Similarly, the lyso-phospholipid, bi-product of PagP catalysis shuttle back to the cytoplasm for re-acylation, and PagP could communicate truncation during this process. However, a catalytically inactive pagPser77ala allele was equally capable of producing truncation, indicating that PagP's palmitoyltransferase activity, and therefore the production of an atypical lipid A species, is not responsible for truncation. Therefore, PagP is communicating with the cytoplasm through separate mechanism via different domain of PagP. 
The periplasmic side of PagP if inspected closely contains 3 amino acids i.e. Asp 61, His 67 and Tyr 87 which form a tight charge-relay network protruding from the β-barrel exterior that is buried beneath the α-helix in the enzyme's inactive conformation, the R state. When PagP gets activated and transform to the T state, it is possible that the helix being mobile might change its position  and Asp 61, His 67 and Tyr 87 become exposed. This expose proteolytic triad might cleave lipoprotein and release it into the periplasm to initiate the signal transduction.
Evidence of these residues to work together as a catalytic triad becomes stronger because of their spatial arrangement. Asp61, His 67 and Tyr 87 residues of PagP has nearly superimposable arrangement with the catalytic triad of Chymotrypsin (Asp 102, His 57, Ser 195). Chymotrypsin, a serine protease, contains serine residue which serves as a nucleophile and affects the catalysis with its hydroxyl group. Catalytic triad of PagP contains rather than tyrosine instead of serine but there is no any biochemical reason that hydroxyl group of tyrosin could not function proteolytically. In fact, the additional resonance stabilization afforded by tyrosine's phenyl ring actually reduces its pKa relative to serine, theoretically improving tyrosine's ability to proteolytically cleave a target protein. Furthermore, while Asp 61 and His 67 are highly conserved amongst homologs of pagP from 8 genera, Tyr 87 is absolutely conserved .
The novice PagP mediated uncover so far in E.coli O157:H7 can be summarize as, myristate deficient lipid A somehow glycosylated and transport to the outer membrane and causes the perturbation of outer membrane symmetry which activates PagP. This activation causes the helix to swing outward towards outer membrane-periplasm interface and exposes the catalytic triad. This expose catalytic triad somehow helps in transducing and the signal to affect the cytoplasmic events of R3 core biosynthesis. Therefore, It seems reasonable to assume that that signal transduction is occurring because of outer membrane stress.
PagP, an outer membrane enzyme, is a probe for outer membrane asymmetry. Aberrant migration of phospholipid to the outer leaflet, due to the outer membrane stress, is sense by PagP. Activated PagP grasps palmitate chain from phospholipid and provides extra acyl chain to the lipid A. This hepta acylated lipid A of restores the permeability in the outer membrane and also helps in weakening the host immune signalling that arise in response to lipid A.
PagP, previously thought to function only as an acyltransferase, has been observed to influence cytoplasmic steps of core biosynthesis in the absence of msbB . Although rare, outer membrane enzymes able to communicate with the cytoplasm have been reported. For example, the ferric citrate receptor, FecA, mediates the synthesis of components of the iron uptake system in response to extracellular ligand binding  .
The investigation of this novel pathway has been shifted to the laboratory strain of E.coli, which will give the clear picture of whether PagP mediated signalling is regular phenomenon or is specific to E.coli O157:H7. The discovery of putative catalytic triad on the periplasmic face of PagP might provide a clear insight in to the domains involve in signalling. The potential that SlyB, the only PhoPQ-controlled lipoprotein common to both E. coli and S. typhimurium, may interact with PagP to mediate signaling is currently being investigated . Since PagP mediate signaling is appearing because of outer membrane stress, its link to other outer membrane stress response systems is also plausible.