DOI: 10.1002/chem.201403923

Review

& Lipopolysaccharides

Chemistry of Lipid A: At the Heart of Innate Immunity Antonio Molinaro,*[a] Otto Holst,[b] Flaviana Di Lorenzo,[a] Maire Callaghan,[c] Alessandra Nurisso,[d] Gerardino D’Errico,[a] Alla Zamyatina,[e] Francesco Peri,[f] Rita Berisio,[g] Roman Jerala,[h] Jesffls Jimnez-Barbero,[i] Alba Silipo,[a] and Sonsoles Martn-Santamara*[i, j] In memory of Gerd Dçring who recently passed away, a friend, a colleague and a giant in the research on cystic fibrosis

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Review Abstract: In many Gram-negative bacteria, lipopolysaccharide (LPS) and its lipid A moiety are pivotal for bacterial survival. Depending on its structure, lipid A carries the toxic properties of the LPS and acts as a potent elicitor of the host innate immune system via the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) receptor complex. It often causes a wide variety of biological effects ranging from a remarkable enhancement of the resistance to the infection to an uncontrolled and massive immune response resulting in sepsis and septic shock. Since the bioactivity of

lipid A is strongly influenced by its primary structure, a broad range of chemical syntheses of lipid A derivatives have made an enormous contribution to the characterization of lipid A bioactivity, providing novel pharmacological targets for the development of new biomedical therapies. Here, we describe and discuss the chemical aspects regarding lipid A and its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level.

1. Introduction

cell surface. The remaining space constitutes integral membrane proteins such as porins, which serve as channels for the entrance and the exit of hydrophilic small molecules.[1] The LPS is an indispensable macromolecule for the growth and survival of many but not all Gram-negative bacteria, for the correct assembly of the OM and the right positioning of porins.[2] A low fluidity of the highly ordered structure of the LPS monolayer helps bacteria to resist both hydrophobic and hydrophilic antimicrobial compounds.[3] Furthermore, owing to its external location, LPS is involved in interactions with other biological systems, particularly in host-bacterium interactions including recognition, adhesion, and colonization, as well as in virulence, tolerance for commensal bacteria, and symbiosis.[4] Once toxic LPS is released, it plays a key role in the pathogenesis of Gram-negative infections in plant, animal and human hosts, in which it triggers the activation of both innate and adaptive immune systems.[2] The LPS is built up according to a common structural principle. It is composed of three distinct domains (Figure 1) covalently linked to each other which are genetically, biosynthetically, biologically and chemically distinct: a glycolipid portion termed lipid A, a glycan, and between them a core oligosaccharide (Figure 1).[2] The glycan usually is the O-specific polysaccharide (or O-antigen), but may be a capsular polysaccharide or, in enterobacteria, the enterobacterial common antigen. The core region contains at least one residue of 3-deoxy-dmanno-oct-2-ulosonic acid (Kdo), which is considered as a marker of LPS. Lipid A is embedded in the outer leaflet and anchors the LPS macromolecule in the OM through electrostatic and hydrophobic interactions, with the carbohydrate chain oriented outwards.[5] The complete LPS comprising all three regions is termed S-form (smooth form) LPS whereas that in mutants lacking the glycan is called R-form (rough form) LPS, also referred to as lipo-oligosaccharide (LOS).[6] Lipid A has crucial functions of protection and defense.[7, 8] The immunoelicitation power of toxic LPS principally resides in the lipid A, which, in mammals acts as a potent stimulator of the innate immune system by a Toll-like receptor, the TLR4, or, as recently discovered, by intracellular receptors.[9a, b] This leads to a significant enhancement of the resistance to infection and is beneficial to the host. However, an uncontrolled and massive immune response caused by circulation of a large amount of such LPS results in more severe symptoms of sepsis and, in the

The Gram-negative bacterial cell envelope comprises as a characteristic feature two membranes, that is, the cytoplasmic (CM) and the outer membranes (OM). In most but not all Gram-negative bacteria, the outer leaflet of the OM is composed of lipopolysaccharide (LPS) molecules, which cover up to 75 % of the [a] Prof. A. Molinaro, Dr. F. Di Lorenzo, Dr. G. D’Errico, Dr. A. Silipo Department of Chemical Sciences University of Naples Federico II via Cinthia 4, 80126 Napoli (Italy) E-mail: [email protected] [b] Prof. O. Holst Division of Structural Biochemistry Research Center Borstel, Leibniz-Center for Medicine and Biosciences Parkallee 4a/c, 23845 Borstel (Germany) [c] Dr. M. Callaghan Centre of Microbial Host Interactions (CMHI) Department of Science, ITT-Dublin Dublin 24 (Ireland) [d] Dr. A. Nurisso School of Pharmaceutical Sciences University of Geneva, University of Lausanne 30, Quai Ernest-Ansermet, 1211 Geneva (Switzerland) [e] Dr. A. Zamyatina Department of Chemistry University of Natural Resources and Life Sciences Muthgasse 18, 1190 Vienna (Austria) [f] Prof. F. Peri Department of Biotechnology and Biosciences University of Milano-Bicocca Piazza della Scienza 2, 20126 Milano (Italy) [g] Dr. R. Berisio Institute of Biostructures and Bioimaging National Research Council Via Mezzocannone 16, 80134 Naples (Italy) [h] Prof. R. Jerala National Institute of Chemistry Hajdrihova 19, Ljubljana (Slovenia) [i] Prof. J. Jimnez-Barbero, Prof. S. Martn-Santamara Department of Chemical and Physical Biology Centre for Biological Research, CIB-CSIC Ramiro de Maeztu 9, 28040 Madrid (Spain) E-mail: [email protected] [j] Prof. S. Martn-Santamara Department of Chemistry Faculty of Pharmacy, Universidad CEU San Pablo Urbanizacion Monteprincipe, 28668-Boadilla del Monte (Spain)

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Review Molecular Patterns (PAMPs). The receptors that recognize PAMPs are called Pattern Recognition Receptors (PRR). The recognition of microbial PAMPs allows the host to unequivocally signal the presence of infection. LPS, lipoproteins, and small peptidoglycan fragments as the structural motifs not present in the eukaryotic cells, are therefore most appropriate as the PAMPs and represent molecular signatures for a given bacterial pathogen class.[15, 16] TLR4 is the signal transducing receptor triggered by the LPS, and lipid A is the minimal structural moiety required to activate TLR4 signaling pathway in conjunction with a soluble TLR4 co-receptor protein MD-2 (myeloid differentiation factor 2), which directly and physically binds to LPS (Figures 2 and 7).[17, 18] The activation of the TLR4/MD-2 complex triggers the biosynthesis of inflammatory cytokines such as TNF-a, IL-1, and IL-6 acting as endogenous mediators of the infection, as well as the superoxide anion (O2), hydroxyl radicals (COH), nitric oxide and antimicrobial peptides. As previously mentioned, low and balanced concentrations of these mediators and soluble immune response modulators lead a resulting inflammation that is one of the most important and ubiquitous

Figure 1. Scheme of the general chemical structure of enterobacterial LPS possessing phosphorylated core regions. S-LPSs are composed of three distinct regions termed lipid A, core and glycan, e.g., the O-antigen. Lipid A is rather conserved among bacteria belonging to the same genus, whereas the core oligosaccharide and O-antigen regions are more variable. The third (terminal) Kdo residue is usually present in small, non-stoichiometric amounts. The polysaccharide moiety acts as the antigenic determinant of the LPS macromolecule. In case of R-LPS or LOS, in which the O-antigen is absent, the antigenic properties are embedded in the core oligosaccharide region.

worst case, septic shock and multi-organ failure. As indicated above, the bioactivity of lipid A, including the capacity to interact and activate receptor(s) of the immune system, is strongly influenced by its primary structure.[10, 11] Likewise, in plant pathogenic bacteria, lipid A has the same role as a potent stimulus for the innate immune system even though not so much is known about its receptor(s) and the overall molecular mechanism of plant innate immunity elicitation. So, it can be concluded that toxic lipid A is certainly one of the most potent immunostimulatory molecules of eukaryotic cells originating from bacteria and can be regarded as an inspirative tool for vaccines, immuno-stimulant, -adjuvant or -suppressive agent. The first lipid A structure has been approached by Westphal and Lderitz in 1954 and was finally confirmed in 1983.[12] Since that time, an increasing number of novel lipid A variants have been found in various bacteria and characterized in detail, due to a large extent to the development of improved procedures for their extraction and purification and modern methods and instruments for structural analysis. This article reviews all aspects of lipid A chemistry and immunology with a particular focus on its immunochemical properties.

Sonsoles Martn-Santamara obtained her Ph.D. from University Complutense of Madrid (ES, 1998) and carried out postdoctoral research with Prof. Rzepa (Imperial College, UK, 1998-2000) and then with Prof. Gago (University of Alcala, ES, 2001-2003). In 2004 she joined University CEU San Pablo in Madrid as “Ramon y Cajal” Researcher (2004-2008), where she was promoted to Assistant Professor (2008) and to Associate Professor (2011). Since June 2014, she is Tenured Scientist at the Center for Biological Research at the Scientific Research Council of Spain (CIB-CSIC). Her team’s efforts are focused on the understanding of molecular recognition processes of Toll-like receptors and human galectins by means of molecular modelling and computational chemistry. Sonsoles is currently Secretary of the Chemical Biology Division of the Royal Society of Chemistry of Spain. Antonio (Tony) Molinaro is Professor of Organic Chemistry and professor of Carbohydrate Chemistry at the Department of Chemical Sciences of University of Naples Federico II. His laboratories and his group are internationally recognized as leaders in Glycoscience. The research of prof. A. Molinaro is focused on the isolation and structural determination of carbohydrate containing molecules. In particular, in the last years his interest has been in the study of structure and role of microbial cell wall glycoconjugates in the elicitation of innate immune response in mammals and plants. Within this frame, he has acquired a recognized experience in isolation and structural determination of glycoconjugates from Gram positive and Gram negative bacteria using state of the art methodologies such as 2D NMR spectroscopy and MS spectrometry. Prof. A. Molinaro has published about 190 peer-reviewed papers in international journals, he is the Past President of European Carbohydrate Organization and currently he is the Coordinator of the Interdivisional Group of Carbohydrates of the Italian Society of Chemistry.

2. Function of Lipid A in Gram-Negative Bacteria, and Role on Innate Immunity: An Overview Toll-like receptors (TLRs) are a family of type I membrane proteins, that recognize different components of microbes, such as LPS, flagellin, lipopeptides, double-stranded RNA, singlestranded non-methylated DNA. In addition, some eukaryotic endogenous danger signals produced by the tissue injury.[13, 14] In mammalian species, there are at least ten TLRs, each having a distinct role, namely a different ligand specificity.[15, 16] The primary and essential requisite of TLRs in the activation of the innate immune response is the detection of microbial components that are constitutively expressed, highly conserved and invariant through different species, that is, essential for their survival. Such molecular targets are called Pathogen Associated Chem. Eur. J. 2014, 20, 1 – 21

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Figure 2. Schematized LPS signaling pathway. LPS binding to the TLR4/MD-2 receptor complex results in the activation of two different signaling transduction pathways. The Myd88-dependent pathway provides the recruitment of the adaptor molecules TIRAP and MyD88 to the TIR domain. This leads to the phosphorylation of IRAK, recruitment of TRAF-6 and ubiquitination of IRAK1, thus promoting the activation of NF-kB and JNK/p38, with the consequent release of pro-inflammatory cytokines. The MyD88-independent pathway results in the activation of IRF-3 which allows the transcription of genes with an interferon response element and chemokine genes.

features of the immune host defense against invading microorganisms. On the contrary, an uncontrolled and massive immune response, due to the circulation of large amount of toxic LPS, leads to severe symptoms of sepsis and septic shock. Being influenced by its primary structure, chemical variations of lipid A strongly influence its capacity to interact and activate receptors of the immune system. Moreover, it has been demonstrated that the lipid A intrinsic conformation is responsible for its agonistic and antagonistic activity.[10, 19] As a rather conserved portion of the LPS within a bacterial species, lipid A possesses a unique and archetypal structure characterized by a b-(1!6)-linked amino sugar disaccharide backbone which in most cases comprises two 2-amino-2deoxy-d-glucopyranose (d-GlcpN) residues, however, several lipid A contain one or two 2,3-diamino-2,3-dideoxy-d-glucopyranose (d-GlcpN3N) residues instead. Many lipid A d-GlcpN-containing backbones are phosphorylated at positions 1 of the proximal a-GlcpN (GlcpN I) and 4’ of the distal b-GlcpN (GlcpN II), and all can be acylated with 3-hydroxy fatty acids at positions 2 and 3 of both GlcpN or GlcpN3N residues by amide and ester linkages. The core oligosaccharide is linked to the non-reducing distal amino sugar of lipid A.[1] For example, in the lipid A from Escherichia coli (Figure 3), both GlcpN units carry (R)-3-hydroxymyristoyl groups [14:0(3-OH)] as primary acyl chains and lauroyl (12:0) and myristoyl group (14:0) as secondary fatty acids on the nonreducing sugar unit. &

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The lipid A, like that of E. coli (Figure 3), possessing a bisphosphorylated disaccharide backbone with an asymmetric (4 + 2) distribution of six acyl residues, represents the most potent agonist of innate immunity in humans.[5, 8, 20] Different primary structures with respect to this latter lipid A are less or not agonistically active. The major determinants that influence the toxicity are the number and the distribution of acyl chains, the phosphorylation pattern and the presence of further charged groups on the polar heads.[21] The degree of acylation of lipid A correlates with cytokine induction capacity as previously reported for several bacterial LPS with hexa-acylated (4 + 2) species being the most active ones.[22] In this context, lipid A species with low or absent endotoxic activity have been identified and operate as “antagonist” reducing or, in a dose-dependent manner, completely inhibiting the cytokine production otherwise induced by toxic lipid A species. It has been proposed that the inhibition of the immune cell occurs in a competitive way: the antagonistic lipid A is able to compete with toxic species for the interaction with the receptor complex on the immune cells but does not result in signaling. Variations of the primary structure of lipid A obviously dictate its physicochemical and biological behavior and namely the structure of their supramolecular aggregates. The classical antagonistic lipid A in human cells is the tetra-acyl lipid IVa (Figure 3), which, however, is an agonist for the mouse and receptors from several other species. It is a biosynthetic precursor of the agonistic E. coli hexa-acylated lipid A and differs by the ab4

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Figure 3. Heterogeneity in lipid A structures. Usually, lipid As present (subtle) chemical variations which are reflected in lipid A bioactivity. E. coli lipid A is composed of a hexa-acylated bis-phosphorylated disaccharide backbone whereas its biosynthetic precursor counterpart (lipid IVa) has a bis-phosphorylated tetraacylated form. Lipid A from Burkholderia genus is typically represented by a mixture of tetra- and penta-acylated species carrying amino-arabinose (l-Arap4N) residues on one or both phosphate groups. Lipid As from Pseudomonas aeruginosa strains are various depending on environmental- or growth conditions, indeed an environmental strain is typically penta-acylated whereas lipid As from chronic strains isolated from individuals affected by cystic fibrosis are hexaacylated or hepta-acylated carrying l-Arap4N residues on both phosphate groups. In A. tumefaciens C58 lipid A possesses a very long acyl chain that is present in almost all rizobial bacteria. Rhodobacter capsulatus possesses a lipid A consisting of a bis-phosphorylated diglucosamine backbone, carrying an aminolinked and genus-characteristic 3-oxotetradecanoic acid. Two 3-hydroxydecanoic acids in ester-linkage are present, of which that one at the distal glucosamine is substituted by dodecenoic acid. This latter is absent in Rhodobacter sphaeroides lipid A that is similar to that of R. capsulatus, except for the amidelinked hydroxytetradecanoic acidon the distal glucosamine which is, in turn, substituted by a tetradecenoic acid residue. Chem. Eur. J. 2014, 20, 1 – 21

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Review sence of both secondary fatty acids.[5, 8, 20] Strong antagonistic effects have also been demonstrated for the penta-acylated LPS lipid A from two phototropic species, Rhodobacter sphaeroides and Rhodobacter capsulatus and their chemical derivatives (Figure 3).[23] A substantial step forward has been the discovery of monophosphoryl lipid A as vaccine adjuvant, this compound has a poor toxicity and proven to be safe and effective.[24] The mechanisms of plant interaction with invading pathogens display parallels with animals.[25–27a–c] Plants have evolved and maintained competence to recognize several general elicitors which are pathogen surface molecules and that can be considered as PAMPs. They bind to plant PRR and trigger the expression of immune response genes and the production of antimicrobial compounds. LPSs act as general elicitors of plant innate immunity and can be considered as PAMPs also in plants.[27a–c] The delivery of an invading bacterium triggers the activation of a signal transduction pathway that can lead to the Hypersensitive Response (HR). This latter is a programmed cell death response, triggered by live bacteria, that is associated with plant host resistance. It is associated with a decline of the number of viable bacteria recovered in the tissue followed by rapid necrosis of plant tissue representing the final stage of resistance, when stress signals induce strong defensive responses. LPSs are able to retard or completely block the HR induced by non-pathogenic bacteria. Dow et al.,[27b,c] demonstrated that inoculation of leaves with heat-killed bacteria followed by inoculation with living bacteria prevents the HR effects. Furthermore, it was demonstrated that intact LOS from Xanthomonas campestris pv. campestrisis able to induce defense-related genes in Arabidopsis and to prevent the HR caused by nonpathogenic bacteria.[28a] Recent findings have shed light on the molecular aspect of these biological events. The lipid A moiety may be at least partially responsible for the LPS perception by Arabidopsis thaliana leading to a rapid burst of NO, a hallmark of innate immunity in animals.[29] The minimal structural requirements for the elicitor activity can be different in plants compared to mammalian hosts. In case of LOS, both lipid A and core oligosaccharide from a pathogenic bacterium can be recognized by plant receptors and are potent inducers of immune responses.[28a–c] Using synthetic O-antigen polysaccharides (oligorhamnans) it has been shown that the O-chain of LPS is recognized by Arabidopsis, and that this recognition elicits a specific gene transcription response associated with defense.[30] A single pathogen-associated compound can consequently originate multiple signals, indicating the existence of multiple receptors for several general elicitors. As in the case of mammalian pathogenic bacteria, structural variation in the lipid A region can strongly influence LPS induction of defense-related genes and recognition of LPS as a PAMP.[31] The alteration of the acylation pattern and the modification of the polar heads seem to play a central role. Thus far, the receptor for LPS in plant cells is not identified and cloning and characterization of LPS receptors in plants is currently a major goal in this area.

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3. From the Biosynthesis to the Structural Diversity of Lipid A As far as is known to date, lipid A is usually not synthesized as such but as Kdo2-lipid A. This synthesis has been comprehensively investigated in E. coli (the “Raetz Pathway”) and has been reviewed several times.[5, 32] Biosynthesis comprises nine enzymatic steps performed by the enzymes LpxA-D, LpxH, K, L, M, and WaaA, beginning with the transfer of one molecule 14:0(3-OH), provided by 14:0(3-OH)-acyl carrier protein (ACP), to position O-3 of UDP-d-GlcpNAc, a reaction that is catalyzed by LpxA (Figure 4). In E. coli, 14:0(3-OH) is the preferred substrate of this enzyme and its 3-OH group is essential. In other bacteria, the acyl chain length differs, usually between 12 and 18 carbon atoms.[33, 34] In a next step, UDP-3-O-14:0-d-GlcpNAc is N-deacylated by LpxC to yield UDP-3-O-14:0-d-GlcpN, which is followed by another acylation step, that is, the transfer of a second 14:0(3-OH) to the free amino function, catalyzed by LpxD. The product is UDP-3-O-14:0-d-GlcpN-[14:0(3-OH)] (UDP2,3-diacyl-GlcpN) (Figure 4). LpxD has very similar properties as LpxA, thus, 14:0(3-OH) is again the preferred substrate. UDP2,3-diacyl-d-GlcpN is then cleaved by the pyrophosphatase LpxH to UMP and 2,3-diacyl-d-GlcpN-a-1!P (lipid X) (Figure 4), the latter of which accumulates and is then condensed with UDP-2,3-diacyl-d-GlcpN by LpxB (the disaccharide synthase) to produce a b-(1!6)-linked d-GlcpN disaccharide which carries four 14:0(3-OH) residues at positions 2, 3, 2’, and 3’, and an alinked phosphate at O-1 (Figure 4). This disaccharide represents the next substrate that is utilized, together with ATP, by LpxK adding a phosphate group to O-4’, thus producing lipid IVa (Figure 4). With the next step, the first two sugars of the core region are transferred, prior to completing the acylation of lipid A. WaaA, the Kdo-transferase, represents in E. coli a bifunctional Kdo-transferase which synthesizes the a-Kdo-(2!4)-Kdo disaccharide linked to O-6’ of lipid IVa. Only then the last two acyl residues are introduced, namely a 12:0 (by LpxL) and a 14:0 (by LpxM) which are 3-O-linked to the C14:0(3-OH) residues at positions 2’ and 3’ of the non-reducing GlcpN, thus forming two acyloxyacyl groups. With that, the E. coli Kdo2lipid A is completed (Figure 4). All above reactions occur in the cytoplasm and at the cytoplasmic side of the inner membrane (CM). LpxK, L, M, and WaaA are integral membrane proteins,[35a–e] LpxB and LpxH are peripheral[36–38] and LpxA, C, and D are soluble ones.[39–41] The core region is also completed in the cytosol. This rough-type LPS is then shuttled through the CM to the periplasm and either transferred directly to the OM or substituted by, for example, an O-specific polysaccharide (synthesized at a different location of the CM) to form smooth-form LPS which is then transported to the OM. Whereas the latter step is currently not fully understood, it is known that the ABC transporter MsbA flips the core–lipid A molecule from the inner to the outer leaflet of the CM.[42, 43] It was indicated by bio-informatic investigations that many bacteria possess enzymes similar to LpxA-D, LpxH, K, L, M, and WaaA, suggesting that they are able to synthesize a lipid A similar to that of E. coli. Thus, lipid A biosynthesis appears to 6

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Figure 4. Schematized biosynthetic pathway of Kdo2-lipid A in E. coli. The structure and names (orange) of intermediates, as well as the names (blue) of enzymes that catalyze each reaction are highlighted. The initial reactions are represented by the acylation of UDP-GlcNAc followed by the addition of two 3-OH acyl chains to the positions 2 and 3 of this UDP-GlcNAc forming the UDP-2,3-diacyl-GlcN catalyzed by enzymes LpxA, LpxC and LpxD. Thereafter, the pyrophosphate bond of UDP-2,3-diacylglucosamine is cleaved by LpxH to form lipid X and UMP. LpxB is responsible for the subsequent condensation of one molecule of lipid X with one molecule of UDP-2,3-diacylglucosamine. LpxK is a specific kinase that phosphorylates position 4’ of the disaccharide-1-P to form lipid IVa.

be conserved, however, structural as well as molecular biology investigations have made clear that different bacterial species produce structural lipid A modifications due to both, genetic differences or also environmental influences.[21] The d-GlcpN variant d-GlcpN3N was found in lipid A of several species, like in the phototrophic genus Rhodopseudomonas (for the first time),[44] Campylobacter or Aquifex (Figure 5).[45, 46] Two enzymes, GnnA and GnnB, have been identified that synthesize UDP-dGlcpN3N from UDP-d-GlcpNAc.[47] GnnA is an oxidoreductase that produces the 3-keto-derivative which is then aminated by GnnB. The 3-amino group is also acylated by a LpxA enzyme. As mentioned above, different Gram-negative species synthesize a lipid A containing fatty acids that differ in chain length [like 10:0(3-OH) in Pseudomonas aeruginosa, 12:0(3-OH) in Neisseria gonorrhoeae, 16:0(3-OH) in Helicobacter pylori, and 18:0(3-OH) in Rhizobium meliloti][33] suggesting different LpxA and LpxD specificities in different bacteria. In some species (e.g., Porphyromonas fragilis and Bordetella pertussis), these enzymes appear to be multifunctional and transfer fatty acids of various chain lengths.[48, 49] Apart from chain length differences, Chem. Eur. J. 2014, 20, 1 – 21

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also the number and distribution of fatty acids vary in bacterial species. Fatty acid number ranges from four in F. tularensis,[50–52] Helicobacter pylori,[53, 54] and Pseudoalteromonas issachenkonii KMM 3549T,[55] to seven, as in Erwinia carotovora, Acinetobacter,[56, 57] Halomonas magadiensis,[58] Klebsiella, Salmonella enterica, and Hafnia alvei.[59, 60] Such modulation is performed by the outer membrane enzymes PagL, LpxR and PagP, the first two of which reduce the number of acyl chains, and the latter increases it.[29, 61–64] Concerning the acylation pattern, fatty acids can be attached to the disaccharide backbone either symmetrically (3 + 3, for example, Neisseria meningitides) (Figure 5) or asymmetrically (4 + 2, for example, E. coli) (Figure 3). Finally, lipid A fatty acids present less common structural features such as methyl branching, different functional and hydroxyl groups, differing chain length (up to 28 carbon atoms) and odd numbered carbon chains. A branched 2,3-dihydroxy fatty acid, 2,3-di-OH-i14:0 has been found in L. pneumophila (Figure 5),[65] while 3keto fatty acids are present in Rhodobacter sphaeroides and R. 7

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Figure 5. Structural heterogeneity of lipid A fatty acids and disaccharide backbone. In lipid A from N. meningitidis, both phosphate groups are commonly substituted by PPEtN groups. In A. pyrophilus lipid A phosphate groups are completely replaced by GalpA residues. L. pneumophila lipid A contains a very long and uncommon acyl chain as well as a GlcpN3N disaccharide backbone. Despite the very long acyl chains and the GlcpN3N disaccharide backbone, B. elkanii lipid A possesses no phosphate groups but, instead, mannose residues.

capsulatus (Figure 3).[66, 67] Furthermore, amide linked 4-keto fatty acids were found as typical components of Mesorhyzobium species, except for M. tianshanense;[68a,b] whereas esterlinked 4-keto fatty acids were identified in Pseudomonas diminuita (reclassified as Brevundimonas diminuita)[69] and Mycoplasma bullata.[70] The secondary fatty acids are more variable, comprising saturated and unsaturated acyl chains of different length. The secondary 28:0(27-OH) group present in Rhizobiaceae and Agrobacterium[71] is partially O-acylated itself with 3&

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hydroxybutanoic acid. In B. elkanii (Figure 5), two long-chain acyl groups are present, which is unusual, and either of them may be non-stoichiometrically O-acylated.[72] Other modifications concern the phosphate groups in lipid A which may either be removed or decorated. Lipid A of the tularemia bacterium Francisella tularensis lacks one of the phosphate groups at position 4’ or both phosphate groups,[50, 51] and these features appear to be responsible for a low immunological activity of the LPS of this bacterium. 8

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Review Phosphate groups can be also completely replaced, for examcepacia suggested that in addition to conferring resistance to ple, by GalA residues, as in the hyperthermophilic bacterium A. both polymyxin B and mellitin, l-Arap4N addition plays a role pyrophilus[46] (Figure 5) and in the rhizobacterium Azospirillum in the export and assembly of LPS at the outer membrane.[82] lipoferum which has an unusual a-(1,1)-galA lipid A modificaWell known examples of lipid A species substituted by ltion;[73, 74] similarly, lipid A of C. crescentus has an a-GalpA-(1! Arap4N are from S. enterica and E. coli, where the synthesis is performed by the transferase ArnT.[83] For optimal activity of 4)-b-GlcpN3N-(1!6)-a-GlcpN3N-(1!1)-a-GalpA tetrasaccharide backbone,[75] whereas rhizobacteria M. huakuii[74] possesses a bthis enzyme, the presence of a 3’-acyloxyacyl group is needed.[84] In a few bacteria, for example, Klebsiella pneumoGlcpN3N-(1!6)-a-GlcpN3N-(1!1)-a-GalpA trisaccharide backbone. Finally, other rizobial strains such as Azorhizobium cauliniae, the 1-phosphate is also substituted.[85] There are also siminodans, possess GlcpN3N-based disaccharide backbone decolar enzymes that transfer either d-GlcpN, d-GalpN and d-Manp rated at position C-1 with an a-GlcpA.[76] As the glycoside linkin Bordetella pertussis (to both, 1- and 4’-phosphate),[86] F. noviage is more resistant than the ester phosphate linkage the cida (to the 1-phosphate)[87] and to the 4’-phosphate of F. novipresence of uronic acids may contribute to the membrane stacida[88] respectively. Lipid A of Neisseria meningitides carries two bility under non-canonical physico-chemical environmental PPEtN groups (Figure 5),[89] Bordetella pertussis and B. bronchiconditions. Lipid A of B. elkanii contains no negatively charged septica, the causes of whooping cough in humans, have free groups and carries a single mannose residue at the reducing non-acylated GlcN substituents on both phosphate groups.[90] end and a mannose disaccharide on the non-reducing GlcN3N With regard to changes in lipid A structure due to environresidue (Figure 5).[72] In lipid A of Rhizobium etli, the phosphate mental influences, growth temperature plays a significant role. This phenomenon has been intensively investigated for examgroup at position 4’ of the distal GlcN II is replaced with GalA ple, in Yersinia pestis,[91] in which a toxic hexa-acyl lipid A (toand the proximal GlcN I is either devoid of the 1-phosphate [77] gether with penta- and tetra-acyl) is synthesized at low(er) group or is oxidized into 2-amino-2-deoxygluconic acid. The temperature (20–28 8C) (Figure 6). At 37 8C (mimicking condilatter component is present also in lipid A of Rhizobium legumitions in the human host), the toxic form is not synthesized at nosarum bv. phaseoli and Rhizobium sp. Sin-1[78, 79] but has not been reported in any non-rhizobial LPS. all, and also the penta-acyl type is present only in minor Decorations of both lipid A phosphate groups occur in variamounts (Figure 6). ous bacteria. Indeed, phosphate groups (P) can be non-stoiSuch a change from toxic to non-toxic lipid A may play an chiometrically substituted by other P, EtN, P-EtN, P-methyl, P-4important role in overcoming the human defense mechanisms. amino-4-deoxy-l-arabinopyranose (P-l-Arap4N), P-d-GalpN, PModification of lipid A by palmitoylation has been demonstratd-Manp, P-heptopyranose (P-Hepp),[36] considerable as evolued in bacterial species as S. enterica, E. coli, L. pneumophila, B. bronchiseptica, Y. pseudotuberculosis. Introduction of palmitate tionary modification utilized by the bacterium to make possi(16:0) is correlated to the presence of antimicrobial peptides ble the life under different habitat. Charged groups allow bacand is activated by low concentration of Mg2 + .[92] teria to modulate the net surface charge and may vary considerably depending on growth conditions (pH, temperature, antibiotic, antimicrobial peptides etc.). The abundant presence of 4. Characterization of Lipid A Structure anionic groups in the lipid A-core region helps the connection of LPS molecules to each other through their association with The structural elucidation of lipid A is of pivotal importance for divalent cations (Mg2 + and Ca2 + ). This phenomenon contribthe comprehension of the biological properties of lipid A/LPS, utes to the remarkable stability of the outer membrane and to including the agonistic/antagonistic action on the host innate a significant reduction in membrane permeability, resulting in immune response. The amphiphilic nature of the LPS, given by an efficient protective barrier. Such negatively charged groups the presence of hydrophilic and hydrophobic regions, makes can be selectively targeted by cationic antimicrobial peptides; the structural study a challenge, due to the tendency to form however, several bacteria decorate their lipid A with positively micelles with low solubility in any solvent system. This problem charged l-Arap4N or EtN, which shield the negative charges of the lipid A and confers resistance to antimicrobial peptides. Lipid l-Arap4N modifications play a special role in P. aeruginosa and a group of related species known as the Burkholderia cepacia complex, which are the dominant Gram-negative opportunistic pathogens that infect the respiratory tract in cystic fibrosis patients. In the case of Burkholderia lipid A, that consists of a GlcpN disaccharide backbone, two phosphate groups non-stoichiometrically substituted with l-Arap4N, and four or five acyl chains (Figure 3),[80] the presence of l-Arap4N is absolutely essential for bacterial viability, making its synthesis an Figure 6. Structures of the main lipid A variants isolated from Yersinia pestis KIM5 grown optimal target to develop new antibiotics.[81] Further- in liquid culture at 26 8C (right) and 37 8C (left). Polar substituents of the phosphate more, data from l-Arap4N mutant strains of B. ceno- groups are not shown. Chem. Eur. J. 2014, 20, 1 – 21

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Review can be overcome by chemically splitting the glycolipid portion of lipid A from the rest of the heteropoly- or oligosaccharide portion. Thus, lipid A can be selectively obtained as a sediment after mild acid hydrolysis of the LPS, a procedure that exploits the acid-labile ketosidic linkage of Kdo, particularly sensitive to these acid conditions. As stated above, despite the general structural conservation within a bacterial species, lipid A has considerable structural microheterogeneity and consists of a mixture of intrinsically heterogeneous species. Such microheterogeneity may depend on various factors including bacterial adaptation to environmental stress, external stimuli, or incomplete biosynthesis. The state-of-art approach for the structural characterization implies a careful and combined use of chemical analysis, mass spectrometry (MS) and NMR spectroscopy, both on intact and selectively degraded lipid A preparations. In particular, MS allows characterization of the lipid A species present in the fraction, including a determination of the differing numbers and nature of acyl residues and polar heads, and their distribution on the disaccharide backbone. The study of acyl chain distribution on the sugar backbone may also require partial degradation to locate the acyloxyacyl moieties. A general and easy methodology combines ammonium hydroxide hydrolysis and MALDI MS.[93] The procedure exploits the lower stability, under mild alkaline conditions, of acyl and acyloxyacyl esters with respect to that of acyloxyacyl amides, left unaffected after the treatment. The partially degraded lipid A species obtained are then analyzed by MALDI MS. We and other colleagues have described new and immediate approaches for lipid A analysis, based on electrospray ionization (ESI).[94–97] Recently, by using a hybrid linear ion trap/orbitrap mass spectrometer (LTQ/orbitrap) for MS and MS/MS analysis,[94a,b] the distribution of primary and secondary acyl residues of intact lipid A was inferred by analysis of the ESI spectra measured in positive and negative mode. The analysis of these data allowed an unequivocal assignment of the fatty acid distribution.[95] The elucidation of lipid A species directly from intact LPS without the need for any chemical manipulations have been also proposed;[96] MALDI tandem time-of-flight (TOF/TOF) MS of lipid A facilitated the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. This approach gave information on: the molecular masses of the whole LPS, the relative intensities between the different species in the sample, and structural information about the different components of the intact R-type LPS.[97] MS spectrometry is extensively used to gain information about phosphorylation sites, and on their substitution with glycosyl or non-glycosyl neutral or charged residues. The use of NMR spectroscopy furnishes additional and complementary information on the saccharide backbone, nature of sugar and phosphate residues (anomeric configuration, attachment points, sequence of sugar residues, acylation and phosphorylation sites, and other substituents present on the polar heads and/or on the acyl chains). The amphiphilic nature of the lipid A and its intrinsic microheterogeneity makes the exe&

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cution of good NMR experiments a complex task since it is hard to find a good solvent system. Mixture of deuterated solvents can be used, like chloroform/methanol or chloroform/ methanol/water; moreover, lipid A often reveals good solubility in DMSO at relatively high temperature (around 40 8C).

5. LPS Interaction with the TLR4/MD-2 Complex TLR4 is a transmembrane protein composed of 22 extracellular leucine-rich repeats (LRRs), a transmembrane domain, and the cytosolic Toll/IL-1 receptor domain (TIR domain) that is essential for TLR signaling and is conserved among members of the Toll receptor family.[98] Indeed, TIR domain dimerizes in response to the dimerization of the TLR4 ectodomain providing a platform for the recruitment of the downstream adaptor proteins that guide formation of the signaling complex (Figures 2 and 7). TLR4 is the only TLR that is able to mediate transduction through pathways utilizing either MyD88 or TRIF adapter (Figure 2). Activation of each of those two signaling pathways leads to different effectors, for example, NF-kB or AP-1 transcription factors for the MyD88 signaling pathway of TLR4 and IFN-b through the TRIF mediated pathway (Figure 2). The selectivity between the two pathways is to a degree still unclear. Cell activation by LPS affects expression of more than 1000 genes, and is therefore one of the most potent stimulants of the immune system.[99] Mice deficient in TLR4 or MD-2 are resistant to LPS. The genetic defect of LPS resistant C3H/HeJ mice has been mapped to the missense point mutation within the TIR domain of TLR4 in a seminal paper by Poltorak et al.,[100] although the role of TLR4 in LPS recognition has been also proposed by Medzhitov and Janeway.[101] Cellular recognition of LPS is complex, since LPS forms large aggregates, although a monomeric LPS/lipid A moiety directly bind to the MD-2 as a coreceptor of TLR4.[102a,b] LPS aggregates therefore have to be monomerized from aggregates or from the bacterial membrane by action of LPS binding protein (LPB) and, in case of S-form LPS,[102c] CD14, which can exist in the soluble form or as a protein anchored to the membrane via the GPI moiety. Both LBP and CD14 are required for the sensitive response to very low concentrations of LPS, although they are dispensable at high concentrations of LPS.[103a,b] The crystal structures of both human and mouse TLR4/MD-2 complexes have shown that the acyl chains of the LPS molecules are embedded in the hydrophobic cavity of MD-2.[104, 105] MD-2 binds to the N-terminal segment of the TLR4 ectodomain.[105] Dimerization of the TLR4 is mediated by interactions between MD-2 and TLR4 and interactions between the lipid A moiety bound to the MD-2 and TLR4 ectodomain.[104, 107] Therefore TLR4 only recognizes lipid A, when it is bound to the MD2. LPS binding induces the close proximity of the C-termini of the extracellular domains of TLR4, a finding which suggests that dimerization of the extracellular domains induces dimerization of the intracellular TIR domains and so initiates signaling (Figure 7).[104, 108, 109] The conserved structural features of lipid A in the crystal structures with human and mouse TLR4/MD-2 in10

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Review chain mediates inter-molecular interactions in the TLR4 dimer (TLR4-TLR4*, Figure 7).[104, 109] In detail, methylene groups of the sixth acyl chain of lipid A, which are solvent exposed in the binary complex with MD-2, mediate hydrophobic interactions with the hydrophobic binding site of TLR4* in the ternary complex. This binding site comprises two phenylalanine side chains (F440 and F463 in hTLR4),[107] all residues essential for receptor activation. Upon lipid A binding, MD-2 experiences a local conformational change, which involves the side chain of F126 and its immediate neighbors.[18, 104] The two phosphate groups (1 and 4’) are also important for the agonistic activity of lipid A because deletion of either phosphate group reduces the endotoxic activity.[110] Indeed, in both LPS complexes with both mouse and human TLR4/MD-2, the 4’phosphate group is located near the primary interface of TLR4/ MD-2 whereas the 1-phosphate group is located near the dimerization interface of TLR4/MD-2 (Figure 7). In contrast to hexa-acylated lipid A forms, tetra-acylated lipid IVa acts as a weak agonist to mouse TLR4/MD-2, but as an antagonist of human TLR4/MD-2. The crystal structures of human and mouse TLR4/MD-2 in complex with tetra-acylated lipid IVa provided the structural basis of this species-specific agonistic or antagonistic activities.[18, 104, 105] In human MD-2 complex with lipid Figure 7. X-ray structure of the TLR4/MD-2/LPS complex (PDB ID 3FXI). A) Front view. B) Top view. C) Detail of the IVa the GlcpN-P backbone of lipiE. coli LPS bound to the TLR4/MD-2 system. d IVa was shifted upward and rotated by about 1808, in comparison to hexa-acylated LPS.[18, 104, 105] This binding mode, denoted dicate a conserved recognition mechanism of hexa-acylated lipid A.[104, 109] As previously mentioned, the hexa-acylated E. coli as “antagonistic”, resulted in a completely different arrangement of the acyl chains, compared with the agonistic binding lipid A acts as a potent agonist for all mammalian cells. The of LPS (Figure 8).[18, 104, 105] On the other hand, lipid IVa occupies binding mode is denoted as “agonistic” binding, since it is responsible for TLR4/MD-2 dimerization and activation. Indeed, the same conformational space as LPS on mouse MD-2 five of the six acyl chains of hexa-acylated LPS are buried (Figure 8), with the 1-phosphate group near the dimerization inside the MD-2 cavity, whereas the sixth acyl chain lies on the interface.[104] This different orientation of lipid IVa when bound surface of MD-2 exposed to the solvent (R2 chain, Figure 7). Toto mouse MD-2 is ascribed to a patch of charged residues in gether with the hydrophobic residues of MD-2, the R2 acyl Chem. Eur. J. 2014, 20, 1 – 21

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Review transmission electron microscopy,[115] and analytical ultracentrifugation.[115] Liposomes are prepared for dynamic light scattering, electron paramagnetic resonance,[116a,b] and pulsed-gradient spin-echo NMR experiments.[116a] Both lamellar lyotropic liquid crystals and liposomes are usually formed by symmetric bilayers. Figure 8. A) Superimposition of the docked human MD-2 (blue)/lipid IVa (cyan) on the human MD-2 (red)/lipid IVa In other studies, solid-supported (yellow) co-crystal structure (PDB ID 2E59). B) Superimposition of the docked murine TLR4 (white)/MD-2 (green)/ monolayers, for atomic force milipid IVa (yellow) complex on the human TLR4 (purple)/MD-2(pink)/lipid A(cyan) complex isolated from the cocrystal structure (PDB ID 3FXI). C) A zoomed-in view near the MD-2 pocket entrance highlights the presence of croscopy,[117, 118] quartz crystal miionic interactions between lipid IVa and murine TLR4, fundamental for dimerization and responsiveness. crobalance with dissipation,[117] or multilayers, for specular and off-specular neutron scattering, FT-infrared spectroscopy,[119] the region of phosphate binding and explains the agonistic effect of lipid IVa in mouse.[104, 105] are employed. All of these systems are not completely representative of the asymmetric bilayer to which lipid A/LPSoccurs Variability of the acylation pattern of the lipid A therefore in nature. strongly affects the response of TLR4/MD-2. The very long Lipid A may adopt different aggregate structures in vitro: lachain of the LPS from Brucella strongly decreases cell activation mellar (L), hexagonal (HI and HII), and cubic (Q).[115] Despite the through TLR4/MD-2,[111] while some other bacteria exploit the decreased responsiveness against hypoacylated lipid A, by fact that non-lamellar phases are not commonly present in varying its acylation pattern to evade the immune response.[112] vivo, they play a key role as morphological elements required Since the role of lipid A in immune recognition was first deto support the dynamic organization of cellular membrane sysfined over 15 years ago[112] and the TLR4 interaction with LPS tems.[10] The stability range and the morphological features of [100, 113] subsequently described each kind of supramolecular aggregate derive from a delicate it has been clearly demonstrated balance of hydrophobic, electrostatic and steric interactions. that this relatively small molecule orchestrates a diversity of The extent of the hydrophobic interaction among neighborcellular responses with significant consequences for both the ing lipid A molecules depends on the number and length of host and pathogen alike. Detailed structural analysis of any puthe acyl chains linked to the disaccharide backbone. These acyl rified lipid A should therefore accompany biological studies to chains are saturated and their ordered parallel arrangement fully define the structure function relationship. The use of synwithin the inner core, with a large predominance of all-trans thetic lipid A variants also serve to further characterize the bioconformation, induces a stiffening of the bilayer. The common logical role of each structural component (see below). More direction of the acyl chains does not have to be necessarily importantly, elucidating the signals for lipid A modification perpendicular to the bilayer surface. Indeed, various experiholds the promising potential for new therapies aimed at limitmental results have indicated a possible tilting of the ing pathogen survival in a hostile host environment. chains.[116b, 118, 119] The chain arrangement is influenced by the number of acyl chains per disaccharide unit or by the presence 6. Physicochemical of the unusually longer fatty acid. With the increased temperaAspects of Lipid A ture, the acyl chains tend to assume less ordered and more dyThe outer membrane of Gram-negative bacteria is an asymnamic conformations, with the introduction of gauche conmetric bilayer resulting from the back-to-back coupling of an formers. This leads to a transition of their self-organization LPS leaflet with an analogue structure formed by phospholifrom the parallel alignment to a much more disordered arpids. The general architecture of this bilayer resembles that of rangement (fluid state, La).[114] This process is cooperative, and typical phospholipid membranes. However, its physicochemical a relatively well-defined temperature can be detected. The properties present some peculiarities connected to the LPS transition temperature for lipid A is about 45 8C, and decreases self-aggregating behavior, which in turn is finely tuned by its in the presence of polysaccharide moieties (as in LPS).[114] Howmolecular structure. Many studies have been devoted to invesever, the transition is much smoother than the similar transitigating the lipid A self-aggregating behavior. In most of them, tion observed for glycerophospholipids.[114, 116a,b] supramolecular aggregates prepared in vitro from purified The reason why in the case of lipid A and LPS the transition lipid A samples have been used. This approach allows a deis so smooth, could be the polydispersity of chain length in tailed and unambiguous analysis of the molecular determithe LOS molecule, and their different positioning with respect nants of the aggregation process. The morphology of the to the saccharidic backbone. The apolar inner region of LPS lipid A assemblies is usually optimized for the experimental dispersions presents a fluidity lower (i.e., an order of the acyl technique to be employed. Aqueous dispersions of lyotropic chain higher) than that of unsaturated phospholipids, but liquid crystals are prepared for calorimetry,[114] spectrofluorimehigher (order lower) than that of saturated ones.[114, 116a, 119] [114] [114] [115] try, infrared spectroscopy, small-angle X-ray scattering, &

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Review Electrostatic interactions between the negative charges of the phosphate groups present on the saccharide moiety and the divalent cations present in the aqueous environment (mainly Ca2 + and Mg2 + ) contributes to crosslinking of the lipid A molecules, further stabilizing their assembly and enhancing their compactness.[120] This is reflected in their low permeability and high resistance to external chemical agents.[121, 122] Steric repulsions could arise among the polysaccharide chains linked to the lipid A.[117] Although these chains do not participate in the bilayer, they indirectly influence its structure. Long hydrated polysaccharide chains, protruding in the external aqueous medium, exert a significant repulsion among them, thus destabilizing the bilayer which could evolve toward the formation of micelles, cubic or hexagonal structures.[116b] From this viewpoint, the LPS behavior is similar to that observed for other amphiphiles presenting a polymeric hydrophilic head group, for example, the polyethoxylated surfactants.[116a,b] In addition, the polysaccharide chains can be phosphorylated and, in the presence of divalent cations, could form an extremely compact crosslinked layer covering the lipid membrane.[120] The co-aggregation of lipid A (or LPS) with phospholipids (e.g., PEtN) has been also investigated.[116a,b] This subject is of great relevance for two reasons. First, in the external leaflet of the bacterial outer membrane, a low fraction of phospholipids is always present. Second, the interaction of LPS (or their small aggregates) with phospholipid membranes is fundamental to the cellular activation mechanism against bacterial infection.[123] The lipid A leaflet is stabilized by the inclusion of small amounts of phospholipids, which appear to be randomly distributed among the lipid A molecules.[116a] However, when the phospholipid fraction increases, the membrane is destabilized and evidence of segregation is observed, indicating the coexistence of fluid disorder and gel [lipid A (or LPS)enriched]-like micron-sized domains.[124, 125] A variety of biophysical and biochemical techniques have been used to deduce the interaction of lipid A and analogues with different molecular receptors. Binding affinities and stoichiometries have been deduced using different techniques, with special emphasis on the use of isothermal calorimetry (ITC) and surface plasmon resonance methods (SPR), while NMR experiments have been widely employed to elucidate the structure of lipid A moieties belonging to different species, as well as many LPS molecules. However, not many groups have performed in-depth studies of the conformational features of these molecules and the evaluation of their molecular recognition features in different environments. The first seminal study of the conformational features of lipid A by NMR was performed by Wang and Hollingsworth[126] using a mixed solvent composed of pyridine, 37 % deuterium chloride in deuterium oxide, methanol, and chloroform in the ratio of 1:1:2:10, respectively. The results indicated that the molecule adopted a major conformation in which the planes of the two pyranose rings were orthogonal. A major gauche– gauche conformation around the C5C6 bond was proposed, together with an exo-anomeric conformation around the glycosidic torsion angle and a negative gauche Y angle. The authors Chem. Eur. J. 2014, 20, 1 – 21

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claimed that, in fact, this was the only geometry that permits the constituent fatty acid chains to be parallel and so attain the closest packed configuration. Further progress was performed years later.[127a] The authors employed synthetically pure molecules, including regiospecific 13 C-labeled ones. They were able to carry out a detailed NMRbased conformational of a single molecule of the tetra-acyl lipid IVa and, more interesting, to deduce the characteristic supramolecular assembly of lipid A in aqueous SDS micelles. The comparison of the results with those obtained for a biologically inactive precursor-type analogue with four shorter acyl chains, demonstrated the key importance of hydrophobic interactions for maintaining the conformation of such molecules. The shorter chain analogue was much more flexible than that the bacterial lipid A. This fact could be responsible for the lack of both endotoxic and antagonistic activities, due to the expected large entropic penalty that would arise upon binding to the LPS receptors. Different experiments with monoclonal antibodies supported this hypothesis, since the lipid A monoclonal antibody mAb A6[127b] has been proven to recognize the hydrophilic part of lipid A and its biosynthetic precursor, but did not bind to the short-chain analogue. The major conclusion is the importance of hydrophobic interactions to maintain the active conformation of lipid A. In fact, the tetra-acyl precursor displays also two conformations in SDS, similar to those observed in DMSO solution for lipid A.[128] For structural studies, TRNOESY, together with STD NMR experiments, have also been employed to characterize the binding of LPS to melittin, as a model of a membrane active amphiphilic peptide. The determined interaction affinity was in the micromolar range, as demonstrated by using ITC measurements, driven by a positive entropy gain. The NMR data facilitated the characterization of the LPS-bound conformation of the peptide, indicating that the C-terminus adopted a helical structure. The authors hypothesized that the helical conformation of melittin, at its C-terminus, could be a key element for recognizing LPS at the outer membrane.[129] Additional examples on the interaction of a designed peptide with LPS have been reported by the same group. For instance, the complex of a designed amphipathic peptide, H2N-YVKLWRMIKFIR-CONH2 (YW12D) with endotoxin has been determined by using TRNOESY.[130] The conformation of the isolated peptide was highly flexible, but underwent a striking stabilization in the presence of LPS. The obtained structure presented two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges, surrounded by aromatic and/ or aliphatic residues. ITC data demonstrated that the interaction is in the micromolar range and suggested that the YW12D peptide interacts with two molecules of lipid A. The epitope mapping of lipid A interaction with the innate immune receptor CD14 was elucidated at high resolution using NMR methods (HSQC techniques form the ligand and receptor point of view’s) and employing a wise combination of 13 15 C, N-labeled Kdo2-lipid A, and dephosphorylated Kdo2– lipid A together with labelled and unlabeled soluble CD14. This strategy allowed the identification of two adjacent areas comprising the lower portions of the sugar head group and upper 13

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Review 8. Synthetic Lipid A Derivatives

half of three acyl chains (specifically I, III, and V), which are spatially proximal to the 1- and 4’-phosphate ends. Additionally, a differential dynamic behavior was deduced, suggesting a key role for dynamics in lipid A recognition by CD14.[131] In fact, in the complexed Kdo2–lipid A, intermediate or slow exchange on the NMR chemical shift time scale for many of the affected resonances was observed, suggesting that the ligand in the bound state still shows considerable flexibility. Moreover, the essential role of phosphate groups in Kdo2–lipid A for the binding process was further confirmed by NMR using a dephosphorylated Kdo2–lipid A compound. In this case, no binding of the dephosphorylated compound to CD14 was observed.

8.1. Lipid A analogues for elucidation of structure-activity relationships and molecular basis of TLR4 activation Since the first chemical synthesis of E. coli lipid A by Shiba and Kusumoto,[135] substantial efforts have been invested in the synthesis of both natural lipid A structures and analogues thereof. Initially, long before the structural basis for LPS recognition by TLR4/MD-2 has been disclosed in the pioneering Xray studies, lipid A analogues based on the structure of hexaacylated E. coli lipid A and tetra-acylated lipid IVa, have been designed and synthesized with the aim of exploring structure– activity relationships. Determinants of endotoxicity such as length, distribution and chemical nature of fatty chains and negatively charged phosphate groups have been sequentially altered. Thus, as shown in Figure 9, (R)- b-hydroxyalkanoylchains have been exchanged to (S)-configured isomers which did not significantly influence the endotoxic properties of E. coli-type lipid A, whereas the anti-endotoxic activity of the biosynthetic precursor analogue having (S)-b-hydroxyalkanoyl chains (1) was more potent.[136] Shortening of the lipid chain length was shown to have a profound impact on the activity. Synthetic analogue of hepta-acylated S. typhimurium lipid A with shorter acyl chains was found to be 100-fold more potent than its natural counterpart.[137] The short-chain analogue 2 of lipid IVa having C10C14C10C14 acylation pattern still maintained antagonistic activity, whereas the LPS inhibiting activity of 4  C10 acylated analogue was entirely abolished.[138–139] Altering acylation pattern (number and distribution of fatty acids attached to C-2 and C-3 of reducing and non-reducing GlcN moieties) together with the subtle shortening of acyl chains in lipid A analogues 3 and 4 resulted in an overall switch of activities. It has been postulated that the distribution of lipid chains across the diglucosamine backbone of lipid A, which regulates the molecular conformation of the molecule, profoundly influences the binding mode by TLR4/MD-2 complex.[140] Because of extreme liability of the anomeric phosphate functionality and, consequently, substantial loss of the materials during the synthesis, a variety of acidic functional groups have been tested as surrogates for the anomeric phosphate group for the sake of simplification of synthetic procedures. Thus, chemically stable analogues of E. coli lipid A in which the glycosidically linked phosphate group is replaced for phosphonooxyethyl- (5), carboxymethyl- (6),[141, 142] and pyranocarboxylic acid groups[143] (7) have been prepared and evaluated for their ability to initiate pro-inflammatory signaling. It has been shown that the acidic functional groups, but not necessarily phosphate groups, are essential for the manifestation of biological activities. To increase the hydrolytic stability of lipid chains, the analogues where in ester-linked lipid chains were exchanged by the ether-type groups have been constructed.[144, 145] The antagonistic activities of tetra-acylated 3,3’-alkyl ether derivatives 8 were not very much different from the corresponding 3,3’-alkanoyl counterparts, however, the agonistic potentials of hexaacylated ether analogues were diminished.

7. Chemical Synthesis of Lipid A After lipid A first structural characterization,[12] considerable attention has been devoted to the development of efficient approaches towards the synthesis of homogeneous molecular species possessing endotoxic activity. The early attempts for lipid A chemical synthesis were aimed at the production of lipid IVa (Figure 3) which is considered the first man-made endotoxic compound.[132] It was obtained through a multi-step methodology, as described in several publications,[132, 133a,c] in which the previously synthesized and protected b(1!6) glucosamine backbone was then N- and O-acylated, respectively, in one step.[132, 133a,c] The glycosyl phosphorylation and the subsequent final hydrogenolytic deprotection were executed at the final synthetic stage of the procedure.[132, 133a,c] Finally the enantioselective reduction of the corresponding keto ester,[132, 134] and the protection by benzylation of its hydroxyl functional groups, allowed the isolation of the optically pure (R)-3-hydroxytetradecanoic acid. Owing to the symmetrical distribution of the fatty acid chains, the lipid IVa synthetic procedure was rather simple compared to the more elaborated protocol developed for the synthesis of the full structure of the hexa-acylated E. coli lipid A (Figure 3). The most important synthetic procedure steps employed in complete E. coli lipid A structure were:[132, 133b,d] i) the introduction of acyl groups and of the protected 4’-phosphate group before the glucosamine disaccharide formation; ii) the introduction of the N-acyl group on the distal glucosamine by using 2,2,2-trichloroethoxycarbonyl (Troc) group to protect the amino group; iii) the introduction of the glycosyl phosphate group before the final deprotection.[132, 133b,d] The so-obtained lipid A showed to possess the endotoxic activity that is exhibited by E. coli lipid A. The historical synthetic work described above did not possess a high efficiency as compared to the latest published methodologies, but it represented a pivotal step in the lipid A history not only by providing evidences on the chemical structure of the lipid A but, most importantly, it definitively demonstrated that the lipid A is the endotoxic moiety of the LPS molecule. The interested reader will find a very detailed historical perspective of lipid A chemical synthesis in a fascinating publication by Kusumoto recently published as a book chapter.[132] &

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Figure 9. Synthetic lipid A analogues and lipid A mimetics developed for investigation of structure-activity relationships and molecular basis for lipid A recognition by the components of innate immune system.

ly evaluated. Biotin-derivatized compounds have also been synthesized and applied to the investigation of the mechanism of interaction of LPS with TLR4/MD-2 complex. To clarify the structural prerequisites for MD-2/TLR4-specific ligands and the molecular basis of the lipid A recognition by MD-2, conformationally restricted lipid A mimetics have been developed.[147] The flexible b-(1!6) diglucosamine backbone of native lipid A has been exchanged to a rigid b-GlcpN-(1$1)-a-GlcpN scaffold, whereas the acylation pattern of the lipid IVa remained preserved. In contrast to the species-specific activity of lipid IVa, which is known as a strong human MD-2/TLR4 antagonist and a weak mouse MD-2/TLR4 agonist, b-GlcpN-(1$1)-aGlcpN-based lipid A mimetics such as 12 exhibited pronounced antagonistic activities on both human and mouse TLR4. The

Pursuing further elucidation of the structural basis for endotoxic and antagonistic activities the group of K. Fukase introduced monosaccharide lipid A analogues containing acidic amino acid in place of the non-reducing GlcN residue.[146] Phosphoryl-serine type analogues 9 displayed antagonistic activity, whereas Asp derivatives 10 where the negative charge was provided by the carboxylic group revealed the immunostimulating activity. Immense effort has been invested in the synthesis of fluorescence- and biotin-labeled lipid A 11 as probes for investigation of LPS recognition by its innate immune receptor.[139] Thus, fluorescence labeled lipid A/lipid IVa with either BODIPY group or Alexa Fluor 568 linked to position 6’ via glycine or glutarylglucose linker, respectively, have been prepared and biologicalChem. Eur. J. 2014, 20, 1 – 21

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Review excellent species-unspecific anti-inflammatory properties were attributed to the reduced flexibility of the b,a-(1$1)-linked diglucosamine backbone, which prevented an exposure of a single lipid chain on the surface of mouse MD-2 and the ensuing TLR4/MD-2/(lipid A mimetic) homodimerization.

sistant LxpCs, recently led to the structure-based design of diacetylene derivatives with a good enhancement of potency together with a broad-spectrum LpxC-inhibitory activity.[157] The enhanced flexibility of the scaffold accounts for a broad-spectrum activity, thus avoiding the resistance arising from limited adjustment to the binding passage.

8.2. Biomedical applications: Lipid A and derivatives as source for vaccine adjuvants, and pharmacological agents

8.4. Ligands interrupting the interaction of lipid A with the hosts receptors

Considerable efforts have been made to identify natural or synthetic molecules able to interfere or modulate biological immune/inflammatory responses mediated by lipid A. On the one hand, several molecules have been reported targeting lipid A biosynthetic enzymes: mainly those involved in the biosynthesis of Kdo and lipid A. On the other hand, much attention has been recently focused on the design of chemical ligands able to interrupt the interaction of lipid A and the host receptors: CD14 and TLR4/MD-2 complex.

Some TLR ligands have endogenous origin, and they act as damage signals (damage-associated molecular patterns, or DAMPs) to alert the body of cell and tissue injury, in cases of necrosis, ischemic injury, etc.[158] Blocking TLR2 and TLR4 with antagonists may be useful in these circumstances to prevent an overactive immune response.[159, 160] There is also evidence that TLRs contribute to the development of atherosclerosis and Alzheimer’s disease through sensing of damage signals in the form of oxidized lipoproteins.[161] Many cationic antimicrobial peptides strongly bind lipid A moiety of the LPS and prevents its interaction with cellular receptors, including endogenous and natural peptide and lipopeptides but also many synthetic peptides.[162–164] Given their therapeutic potential in a variety of diseases, there is considerable interest in the development of pharmaceuticals that modulate TLR activation: TLR antagonists hold great clinical promise for the treatment of numerous inflammatory conditions and are under investigation for the treatment of viral infections, redirecting allergic helper T cell responses, and as anticancer therapeutics;[165] and some TLR agonists have also proven safety and efficacy in humans as vaccine adjuvants and are currently in use in Europe.[166–169]

8.3. Inhibitors targeting lipid A biosynthetic enzymes Inhibition of enzymes involved in the Kdo biosynthesis is a promising strategy to obtain novel antibiotic drugs, but is not without difficulties.[148, 149a] There are some published articles reporting the design and synthesis of ligands targeting, for instance, Kdo cytidyltransferase or CMP-Kdo synthetase (CKS),[149b,c] arabinose 5-phosphate isomerase (A5P isomerase), Kdo-8-phosphate synthase (Kdo8PS), and Kdo8P phosphatase. Among others, worth mentioning are the CKS inhibitors as antibiotic adjuvants of kamamycin and fosfomycin[150] and the Kdo biosynthetic intermediates with antibiotic activity such as the A5P isomerase inhibitors,[151] the Kdo8PS inhibitors,[152, 153] and the CMP-Kdo synthetase inhibitors.[154] Unfortunately, due to difficulties related to tissue distribution, metabolism, and pharmacokinetic properties, none of these compounds has progressed to clinical phases. Reported compounds inhibiting the lipid A moiety biosynthetic pathway only target two of the several enzymes involved in this route: LpxA, the enzyme which catalyzes acylation of UDP-GlcNAc; and LpxC, the enzyme which catalyzes the subsequent deacetylation reaction. Although the 3D structure of LpxA is available through crystallographic techniques,[155] thus constituting a sound starting point for the inhibitor design, no inhibitors of this enzyme have been reported to the best of our knowledge. In contrast, several LpxC inhibitors with antibiotic properties can be found in the literature, including hydroxamate and diacetylene derivatives. Regarding the former ones, the hydroxamate functional group has been used as an anchorage point to the non-catalytic Zn2 + cation present at the binding site, together with heterocyclic or LPS-like scaffolds, leading to inhibitors within the nM range. Among them, CHIR-090 is the most potent LpxC inhibitors described to date, killing both E. coli and P. aeruginosa in bacterial disk diffusion assays with an antibiotic activity comparable to ciprofloxacin.[156] However, this compound showed species-specific efficacy, raising concerns about antibiotic resistance. The structural characterization of the binding site of CHIR-090 re&

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8.5. TLR4 agonists New vaccine development by pharmaceutical companies is focused on non-infectious subunit vaccines.[167, 170] Preclinical and clinical studies conducted to date show that TLR agonists can improve currently applied anticancer vaccination protocols.[171] Among them, monophosphoryl lipid A (MPL) a detoxified component of LPS is derived from S. minnesota, and proposed to selectively target the MyD88-independent pathway, contains the lipid A moiety that ligates TLR4, and it has been incorporated into several vaccines[167] such as vaccines for hepatitis B, Fendrix,[172] cervical cancer, Cervarix173, 174] and also in the immunotherapy for melanoma.[175, 176] Synthetic TLR4 agonists have also been produced and tested. Some of these compounds, such as E6020[177] have good adjuvant activity, for instance, enhancing vaccine efficacy in an experimental model of toxic shock syndrome.[178] Another novel class of compounds, the aminoalkyl glucosaminide phosphates (AGP), as lipid A mimetics, have been developed as immunomodulators that activate TLR4[179] with good adjuvant activity[180] including the potent vaccine adjuvant RC-529,[181] and the bioisoster CRX-547, which show reduced toxicity in comparison to RC-529.[182] Also synthetic peptides that mimic the interaction between TLR4 and LPS, have 16

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Review and targeted against autoimmune disorders related to the release of inflammatory cytokines. Even after the determination of several crystal structures of the TLR4/MD-2/LPS complexes, several questions on the species and lipid A chemotype selectivity remain open. Thus, the study of lipid A represents multidisciplinary research aimed at the heart of the molecular mechanisms of innate immunity and inflammation.

been identified. These peptides, as a new class of TLR4 agonists, can potentially regulate cellular signal transduction pathways.[183] 8.6. TLR4 antagonists The design of LPS-mimicking antagonists of TLRs (especially TLR4) has emerged as a strategy to design potent antisepsis agents. Thus, rational modifications of glycolipid components of LPS would lead to ligands with high affinity. However, LPS mimetics usually fail to reach appropriate drug-like properties. This is illustrated by the case of Eritoran (also known as E5564), a well-tolerated, synthetic lipid A mimetic, potent as TLR4 antagonist, which reached phase III clinical trials as an antisepsis agent but failed to demonstrate sufficient efficacy in late stage human trials. Eritoran exhibits excellent TLR4 antagonistic activity, as recently demonstrated by the protection against the influenza-induced acute lung injury.[184] Promising results have been obtained from molecular modification of a d-glucose-derived hit compound, leading to new glycolipids and a benzylammonium lipid As lipid A antagonists with capacity to inhibit LPS-induced septic shock in vivo, and no apparent toxicity in vivo.[185a,b] Synthetic lipid A analogues modulating TLR4 activity also include a symmetric lipid A mimetic, formed by two glucose units linked through a (6!6’) succinic diamide linker, and two sulfates mimicking the phosphate groups, which inhibited endotoxin-stimulated TLR4 activation by inhibiting interaction of endotoxin with both receptors CD14 and MD-2.[186] This compound also exhibited weak TLR4 agonist activity, pointing at a putative development as vaccine adjuvant. In sepsis, both partial agonist and antagonist properties may be more advantageous than the more pure TLR4 antagonism. The field of TLR ligands that can be developed as TLRs modulators is of great interest. The potential for therapeutic applications is vast, thus constituting a very active and dynamic research field.

Acknowledgements All the Authors acknowledge the COST action BM 1003 “Microbial cell surface determinants of virulence as targets for new therapeutics for Cystic Fybrosis”. Also, the following funding institutions are gratefully acknowledged: Austrian Science Fund (FWF, grant P-22116). Slovenian Research Agency, Spanish MINECO (Grants CTQ2012-32025, and CTQ2011-22724), European Commission granted GLYCOPHARM ITN-project. Keywords: innate immunity · lipid A · lipid A analogues · lipopolysaccharide · TLR4/MD-2 complex [1] A. Silipo, A. Molinaro, in Endotoxins: Structure, Function and Recognition (Eds.: X. Wang, P. J. Quinn), Springer, London, New York, 2010, pp. 69 – 100. [2] C. Alexander, E. T. Rietschel, J. Endotoxin Res. 2001, 7, 167 – 202. [3] L. S. Cardoso, M. I. Araujo, A. M. Ges, L. G. Pacfico, R. R. Oliveira, S. C. Oliveira, Microb. Cell Fact. 2007, 6, 1. [4] A. Silipo, C. De Castro, R. Lanzetta, M. Parrilli, M. Molinaro, in Prokaryotic cell wall compounds structure and biochemistry (Eds.: H. Konig, C. Herald, A. Varma), Springer, Berlin (Germany), 2010, pp. 133 – 154. [5] C. R. Raetz, C. Whitfield, Annu. Rev. Biochem. 2002, 71, 635 – 700. [6] O. Lderitz, C. Galanos, H. J. Risse, E. Ruschmann, S. Schlecht, G. Schmidt, H. Schulte-Holthausen, R. Wheat, O. Westphal, J. Schlosshardt, Ann. N. Y. Acad. Sci. 1966, 133, 347 – 349. [7] C. Alexander, U. Z hringer, Trends Glycosci. Glycotechnol. 2002, 14, 69 – 86. [8] U. Zahringer, B. Lindner, E. T. Rietschel, Adv. Carbohydr. Chem. Biochem. 1994, 50, 211 – 276. [9] a) N. Kayagaki, M. T. Wong, I. B. Stowe, S. R. Ramani, L. C. Gonzalez, S. Akashi-Takamura, K. Miyake, J. Zhang, W. P. Lee, A. Muszyn´ski, L. S. Forsberg, R. W. Carlson, Science 2013, 341, 1246 – 9; b) J. Shi, Y. Zhao, Y. Wang, W. Gao, J. Ding, P. Li, L. Hu, F. Shao. Nature, 2014, in press, DOI:10.1038/nature13683. [10] K. Brandenburg, H. Mayer, M. H. Koch, J. Weckesser, E. T. Rietschel, U. Seydel, Eur. J. Biochem. 1993, 218, 555 – 563. [11] M. G. Netea, M. van Deuren, B. J. Kullberg, J. M. Cavaillon, J. W. van der Meer, Trends Immunol. 2002, 23, 135 – 139. [12] E. Th. Rietschel, O. Westphal, in Endotoxin in Health and Disease (Eds.: H. Brade, S. M. Opal, S. N. Vogel, D. C. Morrisson), Marcel Dekker, New York, 1999, pp. 1 – 30. [13] S. Akira, Curr. Top. Microbiol. Immunol. 2006, 311, 1 – 16. [14] N. J. Gay, M. Gangloff, Annu. Rev. Biochem. 2007, 76, 141 – 165. [15] R. Medzhitov, Nat. Rev. Immunol. 2001, 1, 135 – 45. [16] S. Akira, K. Takeda, Nat. Rev. Immunol. 2004, 4, 499 – 511. [17] A. Poltorak, P. Ricciardi-Castagnoli, S. Citterio, B. Beutler, Proc. Natl. Acad. Sci. USA 2000, 97, 2163 – 7. [18] U. Ohto, K. Fukase, K. Miyake, Y. Satow, Science 2007, 316, 1632 – 1634. [19] U. Seydel, M. Oikawa, K. Fukase, S. Kusumoto, K. Brandenburg, Eur. J. Biochem. 2000, 267, 3032 – 3039. [20] B. Beutler, E. T. Rietschel, Nat. Rev. Immunol. 2003, 3, 169 – 176. [21] C. R. Raetz, C. M. Reynolds, M. S. Trent, R. E. Bishop, Annu. Rev. Biochem. 2007, 76, 295 – 329. [22] R. S. Munford, A. W. Varley, PLoS Pathog. 2006, 2, e67. [23] J. R. Rose, W. J. Christ, J. R. Bristol, T. Kawata, D. P. Rossignol, Infect. Immun. 1995, 63, 833 – 839.

9. Summary and Outlook Within the last 60 years, an increasing number of lipid A variants from the most diverse strains of Gram-negative bacteria have been isolated and structurally characterized, highlighting common and uncommon features which render the LPS macromolecule, and lipid A in particular, even more attractive to organic chemists. At the same time, extensive studies have been performed aimed at the elucidation of lipid A bioactivity related to its role as the elicitor of host innate immune response, thus also becoming an important target of research for microbiologists and immunologists. Furthermore, the successful synthesis of lipid A analogues able to reproduce the toxic activities, notwithstanding the influence of contaminants, contributed to solving many fundamental synthetic problems related to the construction of complex glyco-conjugates and to the understanding of endotoxin structure–function relationships. The knowledge of the chemical and biological aspects of lipid A moiety is a key strategic point to develop novel and innovative drugs, to be used as vaccine adjuvants, anti-inflammatory (acute and chronic), anti-cancer and anti-sepsis agents Chem. Eur. J. 2014, 20, 1 – 21

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Review

REVIEW & Lipopolysaccharides

Pivotal understanding: The chemical and biochemical aspects of lipid A, the endotoxic moiety of the bacterial lipopolysaccharide (LPS) molecule is discussed, as well as its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level, which helps to understand the bioactivity of LPS.

A. Molinaro,* O. Holst, F. Di Lorenzo, M. Callaghan, A. Nurisso, G. D’Errico, A. Zamyatina, F. Peri, R. Berisio, R. Jerala, J. Jimnez-Barbero, A. Silipo, S. Martn-Santamara* && – && Chemistry of Lipid A: At the Heart of Innate Immunity

Lipid A is the endotoxic… …moiety of the bacterial lipopolysaccharide (LPS) molecule. The knowledge and the fine-tuning of its structure in synthetic experiments, is pivotal to understand the bioactivity of the entire LPS molecule. S. MartnSantamara, A. Molinaro et al. discuss the chemical and biochemical aspects of this key LPS moiety furnishing an up-to-date overview of the research in the lipid A field, which have attracted much interest in all fields of life science, in their Review article on page && ff.

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Chemistry of lipid A: at the heart of innate immunity.

In many Gram-negative bacteria, lipopolysaccharide (LPS) and its lipid A moiety are pivotal for bacterial survival. Depending on its structure, lipid ...
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