Pagps Response To Outer Membrane Stress

Published Date: 02 Nov 2017

Outer membrane (OM) 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 is an OM enzyme that acts to transfer a palmitate group from phospholipids to lipid A’s proximal glucosamine unit. PagP activity simultaneously attenuates host innate immune signaling through TLR4 and protects the cell against end products of the TLR4 pathway, cationic antimicrobial peptides. PagP’s contribution to the OM stress response was previously attributed solely to its subtle modification of lipid A; however, new evidence suggests that PagP may also function in a signaling capacity.

The Gram-negative cell envelope: Structural overview

The Gram-negative cell envelope is a unique and complex cellular structure that is composed of an inner membrane (IM) and outer membrane (OM) separated by a gelatinous region called the periplasmic space [1] The inner membrane (IM) 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 of Gram-negative bacteria, which envelops the organism, providing structural support and anchoring OM lipoproteins [2]. Outer membrane of Gram negative bacteria is unique asymmetric bilayer and its inner leaflet comprises of phospholipids, including phosphatidylethanolamine, phoshatidylglycerol and cardiolipin whereas the outer leaflet is composed predominantly of lipopolysaccharide (LPS) [1]. LPS is a tripartite molecule consisting of the hydrophobic lipid A region, the interconnecting core oligosaccharide, and the distal O-antigen polysaccharide [3] (Fig. 1(A)).

Lipid A is a polyacylated disaccharide of glucosamine that makes up outer monolayer of the outer membrane of Gram negative cell envelope. Under normal circumstances, only lipid A and the innermost Kdo sugars of the core oligosaccharide are essential in most Gram-negative bacteria, although an intact inner core is required to maintain the OM permeability barrier. While the outer core and O-antigen polysaccharide are not essential, they are important in evading the membrane attack complex of serum complement [3].

The OM permeability barrier:

LPS has phosphates 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 chain of lipid A helps in lowering the fluidity of outer membrane [4]. This tight architecture because of lateral interactions among LPS and low fluidity makes outer membrane highly impermeable to hydrophobic compounds in comparison to regular phospholipid bilayer [5].

When cationic antimicrobial peptides (CAMPS) attacked bacteria, displaces the Mg2+ ions that are use in bridging adjacent LPS. As a result, negatively charged LPS begins to repel with each other thereby allowing the phospholipid to migrate from inner to outer leaflet. Rafts of phospholipid now mix with the LPS and creates the localize symmetry. This breach in asymmetric composition of OM allows the hydrophobic antibiotics and detergents to enter freely into the periplasm [6] (Fig 1(B)).

The Gram-negative response to envelope stress

Bacteria come across with numerous hostile conditions in nature and their host [7]. To survive in such environment of the host and to infect them successfully, they are provided with number of signaling networks, including alternative extracytoplasmic function sigma factor σE and two component systems like CpxAR, PhoPQ etc. Bacteria activate the σE pathway in response to the stresses that interfere with outer membrane protein biogenesis [8]. Anti sigma factor RseA and its periplasmic counterpart RseB render σE to be inactive in the normal condition [9]. In the presence of extracytoplasmic stress activates the protease activity of DegS and cleave RseA on cytoplasmic site and release σE into the cytoplasm. Activated σE causes the transcriptional activation of set of genes involved in outer membrane protein and outer membrane biogenesis [8].

Two component system CpxAR responds to stresses that adversely affect the assembly of surface molecule, OMPs folding [10]. Stress causes the CpxA to get rid of its periplasmic inhibitor CpxP and triggers the autophosphorylation of conserved histidine residue on cytoplasmic domain. Phosphorylated CpxP then transfer phosphate group to the response regulator CpxR and ultimately causes the transcription of genes in Cpx regulon [9].

PhoPQ two component systems consist 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 during its repressed state [6]. Cationic antimicrobial peptides (CAMPs) displace the Mg2+ form PhoQ [11] and promotes phosphorylation of PhoP. PhoP then regulates the transcription of genes related to lipid A structure, resistance to antimicrobial peptides, phagosome alteration [12].

PhoPQ two component systems also regulate the outer membrane β-barrel enzyme involve 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 there by converting hexa acylated lipid A to hepta acylated lipid A [13]. This seemingly minor modification neutralizes the challenge of various cationic antimicrobial peptide and attenuate host innate immune signaling through TLR4 [14], and has also been reported to activate the σE response in E. coli [15].

Lipid A palmitoylation and bacterial pathogenesis

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 [3]. 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-kB into the nucleus, where it initiates the transcription of pro-inflammatory cytokines [16]. Although effective in clearing local infections, during severe sepsis dysregulation of the innate immune response can lead to endotoxic shock, which is often fatal [17].

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-kB than un-palmitoylated lipid A [18]. Due to their partial activation of innate immunity, modified structures of lipid A are being tested as immune adjuvants [19].

Activation of innate immunity through TLR4 results in the production of cationic antimicrobial peptides (CAMPs). The interaction of CAMPS with the OM induces an amphipathic secondary structure that allows them to penetrate the OM, and insert into the IM. CAMPs are lethal to bacteria through their disruption of the electrochemical potential across the IM [20]. The absence of pagP significantly increases the susceptibility of bacteria to killing by CAMPs. PagP’s ability to protect against CAMPs is attributed to the decrease in membrane fluidity predicted to result from the palmitoylation of lipid A [21].

PagP’s ability to attenuate signaling through TLR4 and protect bacteria from its consequences clearly indicate the importance of the enzyme in pathogenesis, which is reflected in PagP’s narrow distribution amongst primarily pathogenic organisms [22]. Homologs of pagP are important for virulence in the respiratory pathogens L. pneumophila and B. bronchiseptica. Furthermore, homologs of pagP are present in the highly virulent Yersiniae, as well as in pathogens of insects and plants [23-27].

Enzymology of lipid A palmitoylation

Outer membrane acyltransferase PagP catalyze the transfer of palmitate chain from the sn-1 position of phospholipid to the hydroxyl group of R-3 hydroxymyristate chain at position 2 of lipid A [13] (Fig. 2(B). The structure of PagP is determined by both solution nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography to reveal an 8-stranded antiparallel b-barrel enzyme preceded by 19- residue N terminal amphipathic a-helix [28]. Recent research indicates that a-helix acts as a post secondary clamp which helps in stabilizing PagP in membrane once folding and docking with membranes are complete [29] PagP is heat-stable 161 amino acid b-barrel enzyme whose barrel axis is tilted by 25 degree with respect to membrane normal [30] (Fig. 2(A)). Like most b-barrel enzyme, PagP lacks cysteine residues and the enzyme's extracellular loops are long, while its periplasmic loops are short [28]

PagP has an unusual interior with the lower half facing the periplasm is hydrophilic, filled with polar side chains and upper half facing the outer leaflet is hydrophobic and devoid of water molecules. The upper hydrophobic region consists of lipid binding pocket, which is specific in its ability to select the saturated 16 carbon fatty acid chain of all the fatty acid present in the membrane [28]. This pocket is called hydrocarbon ruler and a single molecule of detergent helps identify its position [6]. The base of hydrocarbon ruler is lined by glycine residue, mutating this residue raises the floor of hydrocarbon ruler and select the acyl chains by a degree predictable from the expected rise in the floor, ten carbon chain being the shortest one [31].

PagP process its substrate by ternary complex mechanism. Proline residues don’t have amide proton to donate and form weakened transmembrane β-strand hydrogen bonding. These gateways produce therefore are called crenel and embrasure which provide lateral routes for diffusion of phospholipid and lipid A respectively [30].

Crystallographic studies of the enzymes revealed that PagP exists in two dynamically distinct states, termed R (relaxed) and T (tense). PagP’s crystal structure was determined for the protein in the more flexible R-state in the absence of substrate, and it is postulated that the R-state reflects PagP’s initial conformation (Fig. 2). T state is proposed to play role in catalysis and its structural details are unknown [32].

Asymmetric bilayer composition of outer membrane located phospholipids on inner leaflet but PagP’s active site located on outer leaflet. Therefore, phospholipids can only access PagP’s lipid binding pocket from outer membrane outer leaflet. 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 [33].

Assays of PagP using EDTA, which strips a fraction of LPS from the cell surface and promotes the migration of phospholipids into the OM outer leaflet, made it possible to rule out the possibility that lipid A palmitoylation is dependent on the PhoPQ-mediated activation of pagP expression. Although pagP is transcribed by PhoPQ in response to Mg2+-limitation, palmitoylation occurs too rapidly to be dependent on transcription, and was shown to occur independent of de novo protein synthesis [34]. Taken together, the above results suggest that when sufficient Mg2+ is available, PagP lies dormant in the OM, prepared to rapidly react to any perturbations in lipid asymmetry. 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 OM distress, PagP represents a form of first-line defense for many Gram-negative bacteria.

Evidence for signaling activity

Recently, a new role of PagP in trans-envelope signalling was elucidated in E. coli O157:H7. Merely the absence of msbB, an enzyme responsible for transferring the myristate chain to the distal glucosamine unit of lipid A, is sufficient to activate PagP in E.coli O157:H7 suggesting an intrinsic disruption to lipid asymmetry in this mutant. The presence of a permeability defect was supported by the reduced viability of cells plated on MacConkey agar, and a decrease in the minimum inhibitory concentrations of hydrophobic antibiotics [35]

It was further determined that msbB-deficiency doubles E. coli O157:H7’s susceptibility to killing by serum, suggesting defective assembly of the outer polysaccharide regions of LPS. Indeed, electrophoretic analysis of LPS isolated from the msbB mutant revealed a severe truncation in the core oligosaccharide and the complete absence of the O-antigen repeats normally present in this serotype of E. coli. Structural analysis of LPS isolated from the msbB mutant through 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 [35].

Unexpectedly, the simultaneous absence of pagP corrected truncation, which is restored by complementing the msbB-deficient mutant with pagP in trans. There is possibility that the byproduct of palmitoylation reaction, lyso phsospholipid, during the process of shuttling back into cytoplasm for reacylation, can communicate between outer membrane and cytoplasm. So, this transenvelope signalling that PagP is showing might be just an artifact. To check the relationship between the palmitoylation and signal transduction, catalytically essential serine residue was mutated. LPS analysis of pagPser77ala mutant showed the similar truncation of core. PagP, is therefore communicating with the cytoplasm in response to msbB-deficiency itself, or as a consequence of msbB-deficiency through a separate mechanism. [35]

Unpublished research from the Bishop laboratory has identified 3 amino acids that are essential for signaling. Asp 61, His 67 and Tyr 87 form a tight charge-relay network protruding from the b-barrel exterior that is buried beneath the a-helix in the enzyme’s inactive conformation, the R state (Fig. 3 (A)). Intact LPS is observed when msbB/pagP-deficient E. coli O157:H7 is complemented in trans with pagPasp61ala, pagPhis67ala, or pagPtyr87phe alleles. This result is most striking for the tyrosine mutant, in which the substitution of a single hydroxyl group with a methyl group has completely abrogated signaling (Smith A. and Bishop R., unpublished data). Although buried beneath the a-helix when the protein is inactive, NMR studies of the enzyme suggest that the helix may be mobile [36], and it is possible that Asp 61, His 67 and Tyr 87 become exposed during PagP’s transition to the T state. Due to its amphipathic nature, it is likely that the helix swings outward from PagP along the membrane-periplasm interface, exposing the putative triad and forming a docking area for an effector protein.

It is particularly interesting that the arrangement of Asp 61, His 67 and Tyr 87 in PagP is nearly super imposable on Chymotrypsin’s catalytic triad (Fig. 3(B)). Chymotrypsin is a serine protease with a catalytic triad composed of an Asp, a His and a Ser, with serine’s hydroxyl group effecting proteolysis [37]. Although PagP’s putative catalytic triad lacks a serine residue, there is no biochemical reason that its tyrosine residue 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 [22] (Fig. 3).

Pathogenic strains like E.coli O57: H7 are subjected to harsh environment. During the process of adjustment, these strains might modified themselves to combat with such environment. It might be possible that signalling ability of PagP might also get evolved during this process and might not be true to all strains. Therefore, to test whether PagP mediated signalling is a regular phenomenon or it is present particularly to this strain, laboratory strain of E.coli K-12 has been chosen as model organism. To reiterate the condition where PagP is constitutively active, imp4213 allele has been used, which restricts the ability of Imp protein to assemble LPS in outer membrane in non lethal membrane and severely compromise the outer membrane permeability barrier.

The current evidence for PagP-mediated signaling supports a model in which myristate-deficient LPS is initially glycosylated normally and transported to the OM, where it interacts with PagP to activate signaling. The discovery of a putative catalytic triad essential for core truncation hidden beneath the helix suggests that PagP undergoes a conformational change in which the α-helix swings away from the β-barrel along the OM-periplasm interface, exposing these residues and allowing a signal resulting in truncation of the core oligosaccharide to be transduced (Fig. 4). 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 [38].

Conclusion

PagP, an OM enzyme previously thought to function only as an acyltransferase, has been observed to influence cytoplasmic steps of core biosynthesis in the absence of msbB [35]. Although rare, OM 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 [39] .

Although msbB-deficiency produces a PagP-dependent truncation of the core oligosaccharide in E. coli O157:H7, the initiating conditions and terminal targets of this putative pathway in wild-type cells are unknown. Is there an epistatic interaction between msbB, of which both plasmid-borne and chromosomal copies are present in E. coli O157:H7, and pagP [40]? It seems equally plausible that myristate-deficient lipid A produced in the absence of msbB simply destabilizes the OM in a way to which PagP responds.

PagP’s palmitoyltransferase activity is constitutive in the absence of msbB, and although it has been established that core truncation is not dependent on this activity, are PagP’s palmitoyltransferase and signaling functions activated in tandem? If that is the case, then why is core truncation not a consequence of EDTA treatment in wild-type cells? Two explanations are possible: i) while the perturbation to lipid asymmetry following EDTA treatment activates palmitoylation, it is not sufficient to activate signaling; and ii) that core truncation is an artefact of constitutive signaling by PagP in the the msbB-deficient background. Although the cellular function of core truncation in msbB-deficient E. coli O157:H7 is unknown, in S. Flexneri, reducing the length of the O-antigen polysaccharide is thought to enhance virulence by facilitating interacting of the type-III secretion system with host cells [41].

Efforts to determine the role of putative catalytic triad and downstream targets of this putative pathway are now shifted to E.coli K-12. Pathogenic strains like E.coli O57: H7 are subjected to harsh environment. During the process of adjustment, these strains might modify themselves to combat with such environment. It might be possible that signalling ability of PagP might also get evolved during this process and might not be true to all strains. Therefore, to test whether PagP mediated signalling is a regular phenomenon or it is present particularly to this strain, laboratory strain of E.coli (E.coli K-12) has been chosen as a model organism. To reiterate the condition where PagP is constitutively active, imp4213 alle has been used, which restricts the ability of Imp protein to assemble LPS in outer membrane in non lethal membrane and severely compromise the outer membrane permeability barrier [42]. Signalling deficient mutanst has been incorporated in this experiment and using the Rna-seq and comparing the gene expressions of Signalling deficient and the wild-type will elucidate the downstream target of this putative pathway. The identification of downstream target of PagP mediated signalling will provide the fertile ground of research.