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The cell-envelope of Gram-negative bacteria contains endotoxic lipopolysaccharides (LPS) that are recognized by the innate immune system via Toll-Like Receptors (TLRs). The intestinal mucosal symbiont Akkermansia muciniphila is known to confer beneficial effects on the host and has a Gram-negative architecture. Here we show that A. muciniphila LPS lacks the O-polysaccharide repeating unit, with the resulting lipooligosaccharide (LOS) having unprecedented structural and signaling properties. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each containing three 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residues. The lipid A moiety appears as a mixture of differently phosphorylated and acylated species and carries either linear or branched acyl moieties. Peritoneal injection of the LOS in mice increased higher gene expression of liver TLR2 than TLR4 (100-fold) and induced high IL-10 gene expression. A. muciniphila LOS was found to signal both through TLR4 and TLR2, whereas lipid A only induced TLR2 in a human cell line. We propose that the unique structure of the A. muciniphila LOS allows interaction with TLR2, thus generating an anti-inflammatory response as to compensate for the canonical inflammatory signaling associated with LOS and TLR4, rationalizing its beneficial host interaction., (© 2024. The Author(s).)

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Lipopolysaccharides (LPS) are major players in bacterial infection through the recognition by Toll-like receptor 4 (TLR4). The LPS chemical structure, including the oligosaccharide core and the lipid A moiety, can be strongly influenced by adaptation and modulated to assure bacteria protection, evade immune surveillance, or reduce host immune responses. Deep structural understanding of TLRs signaling is essential for the modulation of the innate immune system in sepsis control and inflammation, during bacterial infection. To advance this knowledge, we have employed computational techniques to characterize the TLR4 molecular recognition of atypical LPSs from different opportunistic members of α2-Proteobacteria, including Brucella melitensis, Ochrobactrum anthropi, and Ochrobactrum intermedium, with diverse immunostimulatory activities. We contribute to unraveling the role of uncommon lipid A chemical features such as bearing very long-chain fatty acid chains, whose presence has been rarely reported, on modulating the proper heterodimerization of the TLR4 receptor complex. Moreover, we further evaluated the influence of the different oligosaccharide cores, including sugar composition and net charge, on TLR4 activation. Our studies contribute to elucidating, from the molecular and biological perspectives, the impact of the α2-Proteobacteria LPS cores and the chemical structure of the atypical lipid A for immune system evasion in opportunistic bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Polymyxin antibiotics are the last resort for treating patients in intensive care units infected with multiple-resistant Gram-negative bacteria. Due to their polycationic structure, their mode of action is based on an ionic interaction with the negatively charged lipid A portion of the lipopolysaccharide (LPS). The most prevalent polymyxin resistance mechanisms involve covalent modifications of lipid A: addition of the cationic sugar 4-amino-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN). The modified structure of lipid A has a lower net negative charge, leading to the repulsion of polymyxins and bacterial resistance to membrane disruption. Genes encoding the enzymatic systems involved in these modifications can be transferred either through chromosomes or mobile genetic elements. Therefore, new approaches to resistance diagnostics have been developed. On another note, interfering with these enzymatic systems might offer new therapeutic targets for drug discovery. This literature review focuses on diagnostic approaches based on structural changes in lipid A and on the therapeutic potential of molecules interfering with these changes., (© 2023 Vanessa Rogga et al., published by Sciendo.)

Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, cyaA , fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ptsI ) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. IMPORTANCE Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance., Competing Interests: The authors declare no conflict of interest.

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, and its active principle, lipid A, have immunostimulatory properties and thus potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, it is necessary to structurally modify LPS and lipid A to minimize toxicity while maintaining adjuvant effects for use as vaccine adjuvants. Various studies have focused on the chemical synthetic method of lipid As and their structure-activity relationship, which are reviewed in this chapter., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipid A plays an important role in the pathogenicity and antimicrobial resistance of Vibrio parahaemolyticus, but little is known about the structure and biosynthesis of lipid A in V. parahaemolyticus. In this study, lipid A species were either directly extracted or obtained by the acid hydrolysis of lipopolysaccharide from V. parahaemolyticus ATCC33846 cells and analyzed by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. Several lipid A species in V. parahaemolyticus cells were characterized, and two of these species were not connected to polysaccharides. One free lipid A species has the similar structure as the hexa-acylated lipid A in Escherichia coli, and the other is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain. Three lipid A species were isolated by the acid hydrolysis of lipopolysaccharide: the 1 st one has the similar structure as the hexa-acylated lipid A in E. coli, the 2 nd one is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain and a secondary 2-OH C12:0 acyl chain, and the 3 rd one is equal to the 2 nd species with a phosphoethanolamine modification. These results are important for understanding the biosynthesis of lipid A in V. parahaemolyticus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC 50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.

The lipopolysaccharide (LPS) is a characteristic molecule of the outer leaflet of the Gram-negative bacterial outer membrane, which consists of lipid A, core oligosaccharide, and O antigen. The lipid A is embedded in outer membrane and provides an efficient permeability barrier, which is particularly important to reduce the permeability of antibiotics, toxic cationic metals, and antimicrobial peptides. LPS, an important modulator of innate immune responses ranging from localized inflammation to disseminated sepsis, displays a high level of structural and functional heterogeneity, which arise due to regulated differences in the acylation of the lipid A and the incorporation of non-stoichiometric modifications in lipid A and the core oligosaccharide. This review focuses on the current mechanistic understanding of the synthesis and assembly of the lipid A molecule and its most salient non-stoichiometric modifications.

Lipopolysaccharides (LPS) are the main components of the external leaflet of the Gram-negative outer membrane and consist of three different moieties: lipid A, core oligosaccharide, and O-polysaccharide. The lipid A is a glucosamine disaccharide with different levels of acylation and phosphorylation, beside carrying, in certain cases, additional substituents on the sugar backbone. It is also the main immunostimulatory part of the LPS, as its recognition by the host immune system represents a fundamental event for detection of perilous microorganisms. Moreover, an uncontrolled immune response caused by a large amount of circulating LPS can lead to dramatic outcomes for human health, such as septic shock. The immunostimulant properties of an LPS incredibly vary depending on lipid A chemical structure, and for this reason, natural and synthetic variants of the lipid A are under study to develop new drugs that mimic or antagonise its natural effects. Here, we review past and recent findings on the lipid A as an antibiotic target and immune-therapeutic molecule, with a special attention on the crucial role of the chemical structure and its exploitation for conceiving novel strategies for treatment of several immune-related pathologies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 E T (=CECT 7113 T = LMG 23376 T ), a mesophilic bacterium isolated from cockles ( Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues.

Gram-negative bacterial cell surface component lipopolysaccharide (LPS) and its active principle, lipid A, exhibit immunostimulatory effects and have the potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, LPS and lipid A must be structurally modified to minimize their toxic effects while maintaining their adjuvant effect for application as vaccine adjuvants. In the field of chemical ecology research, various biological phenomena occurring among organisms are considered molecular interactions. Recently, the hypothesis has been proposed that LPS and lipid A mediate bacterial-host chemical ecology to regulate various host biological phenomena, mainly immunity. Parasitic and symbiotic bacteria inhabiting the host are predicted to possess low-toxicity immunomodulators due to the chemical structural changes of their LPS caused by co-evolution with the host. Studies on the chemical synthesis and functional evaluation of their lipid As have been developed to test this hypothesis and to apply them to low-toxicity and safe adjuvants.

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium ( Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups ( n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram-negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O-antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC-MS, MALDI and NMR techniques. The results revealed a novel repeat of the O-antigen structure of the LPS, [→4)-β-d-GlcpNAcA-(1→4)-β-d-GlcpNAc3NAlaA-(1→3)-α-d-FucpNAc4NR-(1→], (R=acetylated 60 %), and a bis-phosphorylated hexa-acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins., (© The Author(s) (2020). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function 1,2 . LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics 3,4 . Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains 5 , our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter 6-9 , our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme 10-12 . In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli., (Copyright © 2018. Published by Elsevier B.V.)

Acetobacter pasteurianus is an acetic acid-producing Gram-negative bacterium commonly found associated with plants and plant products and widely used in the production of fermented foods, such as kefir and vinegar. Due to the acid conditions of the bacterium living habitat, uncommon structural features composing its cell envelope are expected. In the present work we have investigated the A. pasteurianus CIP103108 lipopolysaccharide (LPS) structure and immunoactivity. The structure of the lipid A and of two different O-polysaccharides was assessed. Furthermore, immunological studies with human cells showed a low immunostimulant activity of the isolated LPS, in addition to a slight capability to lower the NF-kB activation upon stimulation by toxic LPS., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The pathogenicity of Campylobacter concisus , increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen C. jejuni , the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of C. concisus .

Vaccines are one of the most important methods for preventing infectious disease. Structural modification of lipopolysaccharide (LPS) provides a strategy for the development of live attenuated vaccines, either by altering the immunogenicity or by attenuating virulence of the bacteria. This review summarizes various approaches that utilize LPS mutants as whole-cell vaccines., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Aim: To investigate the influence of auxiliary chemical substances (ACSs) and calcium hydroxide [Ca(OH) 2 ] dressings on lipopolysaccharides (LPS)/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4)., Methodology: Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); (ii) intracanal medicament: Ca(OH) 2 paste for various periods (1 h, 24 h, 7 days, 14 days and 30 days); (iii) combination of substances: (a) 2.5% NaOCl (1 h), followed by 17% EDTA (3 min) and Ca(OH) 2 (7 days); (b) 2% CHX (1 h), afterwards, 17% EDTA (3 min) followed by Ca(OH) 2 (7 days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrom (MALDI-TOF MS) whilst LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way anova with Tukey-Kramer post-hoc tests at the 5% significance level., Results: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (P ≤ .001). 5.25% NaOCl treatment led to the absence of lipid A peaks and LPS bands, whilst no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl-treated LPS did not activate TLR4 (P < .0001). As for Ca(OH) 2 , lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24 h. LPS treated with Ca(OH) 2 was a weak TLR4 activator (P < .0001). From 24 h onwards, no significant differences were found amongst the time periods tested (P > 0.05). The addition of Ca(OH) 2 for 7 days to cells treated either with 2.5% NaOCl or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR4., Conclusion: 5.25% NaOCl and Ca(OH) 2 dressings from 24 h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Burkholderia pseudomallei causes the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological "footprint." The binding strength of B. pseudomallei LPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated from B. pseudomallei under different temperatures were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes, lpxO and pagL , was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A from B. pseudomallei 1026b lpxO and pagL mutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication of lpxO and pagL mutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity., (Copyright © 2018 Norris et al.)

The Gram-negative bacterial outer membrane contains lipopolysaccharide, which potently stimulates the mammalian innate immune response. This involves a relay of specialized complexes culminating in transfer of lipopolysaccharide from CD14 to Toll-like receptor 4 (TLR4) and its co-receptor MD-2 on the cell surface, leading to activation of downstream inflammatory responses. In this study we develop computational models to trace the TLR4 cascade in near-atomic detail. We demonstrate through rigorous thermodynamic calculations that lipopolysaccharide molecules traversing the receptor cascade fall into a thermodynamic funnel. An affinity gradient for lipopolysaccharide is revealed upon extraction from aggregates or realistic bacterial outer membrane models and transfer through CD14 to the terminal TLR4/MD-2 receptor-co-receptor complex. We subsequently assemble viable CD14/TLR4/MD-2 oligomers at the plasma membrane surface, and observe lipopolysaccharide exchange between CD14 and TLR4/MD-2. Collectively, this work helps to unravel the key structural determinants governing endotoxin recognition in the TLR4 innate immune pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Detection and removal of lipopolysaccharides (LPS) from food and pharmaceutical preparations is important for their safe intake and administration to avoid septic shock. We have developed an abiotic system for reversible capture, removal, and detection of LPS in aqueous solutions. Our system comprises long C18 acyl chains tethered to Fe 3 O 4 /Au/Fe 3 O 4 nanoflowers (NFs) that act as solid supports during the separation process. The reversible LPS binding is mediated by facile hydrophobic interactions between the C18 chains and the bioactive lipid A component present on the LPS molecule. Various parameters such as pH, solvent, sonication time, NF concentration, alkane chain length, and density are optimized to achieve a maximum LPS capture efficiency. The NFs can be reused at least three times by simply breaking the NF-LPS complexes in the presence of food-grade surfactants, making the entire process safe, efficient, and scalable. The regenerated particles also serve as colorimetric labels in dot blot bioassays for simple and rapid estimation of the LPS removed.

Structurally related O-specific polysaccharide (O-antigen) and lipid A components were obtained by mild acid degradation of the lipopolysaccharides (LPSs) of two strains of bacteria Pantoea agglomerans, 7604 and 8674. Studies by sugar analysis along with 1D and 2D 1 H and 13 C NMR spectroscopy enabled elucidation of the following structures of the O-polysaccharides, which differ only in the linkage configuration of a side-chain glucose residue: R=α-d-Glcp in strain 7604 or β-d-Glcp in strain 8674 Lipid A samples were studied by GC-MS and high-resolution ESI-MS and found to be represented by penta- and tetra-acyl species; lipid A of strain 8674 also included hexaacyl species. A peculiar feature of lipid A of both strains is the presence of the major cis-9-hexadecenoic (palmitoleic) acid, which has not been found in P. agglomerans strains studied earlier. The LPSs of both strains were pyrogenic, reduced the average adhesion and the index of adhesiveness and showed a relatively low level of lethal toxicity. O-antiserum against strain 7604 showed one-way cross-reactivity with the LPS of strain 8674, and O-antisera against both strains cross-reacted with LPSs of some other Р. agglomerans strains but more strains were serologically unrelated. These structural and serological data indicate immunochemical heterogeneity of Р. agglomerans strains and will find demand in classification of Р. agglomerans by O-antigens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A monophosphoryl lipid A (MPLA) derivative having the 6'-OH group substituted with an NH 2 group was synthesized and coupled with the upstream terminal tetrasaccharide of mycobacterial lipoarabinomannan (LAM) via an amide bond to create a novel type of MPLA-based fully synthetic glycoconjugate vaccine. The same tetrasaccharide was also coupled with MPLA at the 1-O-position. Immunological activities of the two synthetic conjugates were evaluated in mice and compared. Both afforded robust overall and IgG antibody responses, but intraperitoneal injection elicited responses significantly stronger than those from subcutaneous injection. It was thus speculated that MPLA conjugates might act via stimulating B1 lymphocytes present in the intrapleural and peritoneal cavities. Moreover, the 6'-N-conjugate afforded antibody titers much higher than those of the 1-O-conjugate. These results revealed not only the self-adjuvant property of MPLA conjugates to elicit robust IgG antibody responses but also the impact of MPLA structure on the immunological activity of its conjugates. It was concluded that LAM oligosaccharide-MPLA conjugates, especially 6'-N-linked, are promising candidates as antituberculosis vaccines worthy of further investigation. Additionally, the 6'-amino derivative of MPLA was proved to be a useful carrier for the development of fully synthetic carbohydrate-based conjugate vaccines.

The partial structure and immunology of the lipopolysaccharide (LPS) of Pseudomonas stutzeri KMM 226, a bacterium isolated from a seawater sample collected at a depth of 2000 m, was characterised. The O-polysaccharide was built up of disaccharide repeating units constituted by L-Rhap and D-GlcpNAc: →2)-α-L-Rhap-(1→3)-α-D-GlcpNAc-(1→. The structural analysis of the lipid A showed a mixture of different species. The major species were hexa-acylated and penta-acylated lipids A, bearing the 12:0(3-OH) in amide linkage and 10:0(3-OH) in ester linkage, while the secondary fatty acids were present only as 12:0. The presence of 12:0(2-OH) was not detected. The immunology experiments demonstrated that P. stutzeri KMM 226 LPS displayed a low ability to induce TNF-α, IL-1β, IL-6, IL-8 and IL-10 cytokine production and acted as an antagonist of hexa-acylated Escherichia coli LPS in human blood in vitro.

Background: Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited., Objectives: To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains., Methods: The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS., Results: All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype., Conclusions: The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Suberites domuncula is a marine demosponge harbouring a large bacterioflora, including commensal, opportunistic and pathogenic bacteria, among which, species of the Gram-negative genus Pseudoalteromonas were identified. The sponge-bacteria interaction mechanisms are still not fully understood. As the main component of the Gram-negative bacterial outer membrane, the lipopolysaccharide (LPS) may play a role in such a crucial relationship. Moreover, the LPS is known to be the most versatile bioactive macromolecule of Gram-negative bacteria and its lipid A structure is responsible for the immunological activity of the whole LPS on eukaryotic host cells. Here it is reported the structural characterisation of the LPS lipid A moiety isolated from the S. domuncula-associated commensal bacterium, Pseudoalteromonas sp. 2A. Chemical and MALDI mass spectrometry analyses, performed on both the LPS and the isolated lipid A as well as on the intact bacterial cells, highlighted a complex family of penta-acylated lipid A species carrying two phosphate units on the disaccharide backbone.

The structure of lipid A from lipopolysaccharide of Phyllobacterium trifolii PETP02 T , a nitrogen-fixing symbiotic bacterium, was studied. It was found that the lipid A backbone was composed of two 2,3-diamino-2,3-dideoxy-D-glucose (GlcpN3N) residues connected by a β-(1 → 6) glycosidic linkage, substituted by galacturonic acid (GalpA) at position C-1 and partly decorated by a phosphate residue at C-4' of the non-reducing GlcpN3N. Both diaminosugars were symmetrically substituted by 3-hydroxy fatty acids (14:0(3-OH) and 16:0(3-OH)). Ester-linked secondary acyl residues [i.e. 19:0cyc and 28:0(27-OH) or 28:0(27-4:0(3-OMe))] were located in the distal part of lipid A. A high similarity between the lipid A of P. trifolii and Mesorhizobium was observed and discussed from the perspective of the genetic context of both genomes.

Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates. IMPORTANCE There is an urgent need to develop effective vaccines against infectious diseases that continue to be major causes of morbidity and mortality worldwide. Making effective vaccines requires selecting an adjuvant to strengthen an appropriate and protective immune response. This work describes a practical method, bacterial enzymatic combinatorial chemistry (BECC), for generating functionally diverse molecules for adjuvant use. These molecules were analyzed in cell culture for their ability to initiate immune stimulatory activity. Several of the assays described herein show promising in vitro cytokine production and costimulatory molecule expression results, suggesting that the BECC molecules may be useful in future vaccine preparations., (Copyright © 2017 Gregg et al.)

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure-activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host-range specificity. This revealed an intriguing difference in LPS O-chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.

Vitreoscilla filiformis is a Gram-negative bacterium isolated from spa waters and described for its beneficial effects on the skin. We characterized the detailed structure of its lipopolysaccharide (LPS) lipid A moiety, an active component of the bacterium that contributes to the observed skin activation properties. Two different batches differing in postculture cell recovery were tested. Chemical analyses and mass spectra, obtained before and after mild-alkali treatments, revealed that these lipids A share the common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxydecanoic acid in an amide linkage. Short-chain FAs, hydroxydecanoic and dodecanoic acid, were found in a 2:1 ratio. The two lipid A structures differed by the relative amount of the hexa-acyl molecular species and phosphoethanolamine substitution of the phosphate groups. The two V. filiformis LPS batches induced variable interleukin-6 and TNF-α secretion by stimulated myelomonocytic THP-1 cells, without any difference in reactive oxygen species production or activation of caspase 3/7. Other different well-known highly purified LPS samples were characterized structurally and used as standards. The structural data obtained in this work explain the low inflammatory response observed for V. filiformis LPS and the previously demonstrated beneficial effects on the skin., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Rhizobium bacteria live in soil and plant environments, are capable of inducing symbiotic nodules on legumes, invade these nodules, and develop into bacteroids that fix atmospheric nitrogen into ammonia. Rhizobial lipopolysaccharide (LPS) is anchored in the bacterial outer membrane through a specialized lipid A containing a very long-chain fatty acid (VLCFA). VLCFA function for rhizobial growth in soil and plant environments is not well understood. Two genes, acpXL and lpxXL, encoding acyl carrier protein and acyltransferase, are among the six genes required for biosynthesis and transfer of VLCFA to lipid A. Rhizobium leguminosarum mutant strains acpXL, acpXL - /lpxXL - , and lpxXL - were examined for LPS structure, viability, and symbiosis. Mutations in acpXL and lpxXL abolished VLCFA attachment to lipid A. The acpXL mutant transferred a shorter acyl chain instead of VLCFA. Strains without lpxXL neither added VLCFA nor a shorter acyl chain. In all strains isolated from nodule bacteria, lipid A had longer acyl chains compared with laboratory-cultured bacteria, whereas mutant strains displayed altered membrane properties, modified cationic peptide sensitivity, and diminished levels of cyclic β-glucans. In pea nodules, mutant bacteroids were atypically formed and nitrogen fixation and senescence were affected. The role of VLCFA for rhizobial environmental fitness is discussed.

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis.Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.

Lipopolysaccharides (LPS) are major components of the external membrane of most Gram-negative bacteria, providing them with an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region (PS) linked to a hydrophobic one, termed lipid A. The LPS polysaccharide moiety is divided into the core oligosaccharide (OS) and O-chain repetitive elements. Depending on their individual variable fine structures, LPS may be potent immunomodulators. The lipid A structure is a key determinant for LPS activity. However, the presence of the core region, or at least of the highly charged 3-deoxy-d-manno-oct-2-ulosonic acid molecules, is also important for preserving the native lipid A conformation within individual LPS molecules. We describe herein four rapid and practical micromethods for LPS, lipid A, and core OS structural analyses. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids enabling the characterization of their substitution position in the lipid A backbone, to be determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The third one is a microscale procedure for the mass spectra screening of LPS, lipid A, and PS using triethylamine and citric acid. The fourth method is a chromatography procedure for Rough-type LPS on thin-layer-chromatography. These methods were developed to be coupled to mass-spectrometry (e.g., MALDI-MS) but can also be used with other analytical techniques (e.g., chromatography). Examples are given with reference to two major human pathogens: Bordetella pertussis and Pseudomonas aeruginosa; to one porcine pathogen: Actinobacillus pleuropneumoniae; and to commercial samples of Salmonella Minnesota Re595 LPS.

Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of lptA slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated lpxL1 mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of lpxP, an enzyme known to add a secondary 9-hexadecenoic acid to the 2' acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.

Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The bacterial membrane provides a target for antimicrobial peptides. There are two groups of bacteria that have characteristically different surface membranes. One is the Gram-negative bacteria that have an outer membrane rich in lipopolysaccharide. Several antimicrobials have been found to inhibit the synthesis of this lipid, and it is expected that more will be developed. In addition, antimicrobial peptides can bind to the outer membrane of Gram-negative bacteria and block passage of solutes between the periplasm and the cell exterior, resulting in bacterial toxicity. In Gram-positive bacteria, the major bacterial lipid component, phosphatidylglycerol can be chemically modified by bacterial enzymes to convert the lipid from anionic to cationic or zwitterionic form. This process leads to increased levels of resistance of the bacteria against polycationic antimicrobial agents. Inhibitors of this enzyme would provide protection against the development of bacterial resistance. There are antimicrobial agents that directly target a component of bacterial cytoplasmic membranes that can act on both Gram-negative as well as Gram-positive bacteria. Many of these are cyclic peptides with a rigid binding site capable of binding a lipid component. This binding targets antimicrobial agents to bacteria, rather than being toxic to host cells. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The lipopolysaccharides (LPS) of the seven strains of Pantoea agglomerans were isolated and chemically identified. It was established that the investigated strains characterized by different relative output of LPS from 5.2 to 14.0 % by dry weight of bacteria. LPS were characterized quite high content of carbohydrates - from 22 to 54 % 2-keto-3-deoxyoctonic acid (KDO) - from 0.39 to 2.22 % and heptose - from 3.3 to 14.00 %. Fatty acids, containing in the chain of 12 to 16 carbon atoms were identified. Lipids A of all tested LPS were characterized by predominant 3-OH-C14:0 acid from 31.7 to 39.3 % depending on the strain. Since all the studied strains of P. agglomerans were sensitive to polymyxin B, it can be concluded that the LPS do not contain in the structure of lipid A, such a substitute as 4-amino-4-deoxy-L-arabinose. One of the ways of changes in the functional and biological properties of LPS is the chemical modification. As modifiers were used complexes of germanium and tin. In the study of serological activity and toxicity of modified LPS it was found that some of them lost both serological and toxic activity. It was revealed that all investigated P. agglomerans LPS decreased the median adhesion and the index of the adhesiveness. The higher concentration of P. agglomerans LPS in the reaction mixture, the less interactions between surface structures of red blood cells and E. coli cells.

Objective: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans., Methods: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS., Results: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS., Conclusion: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with l-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Planctomycete bacteria possess many unusual cellular properties, contributing to a cell plan long considered to be unique among the bacteria. However, data from recent studies are more consistent with a modified Gram-negative cell plan. A key feature of the Gram-negative plan is the presence of an outer membrane (OM), for which lipopolysaccharide (LPS) is a signature molecule. Despite genomic evidence for an OM in planctomycetes, no biochemical verification has been reported. We attempted to detect and characterize LPS in the planctomycete Gemmata obscuriglobus. We obtained direct evidence for LPS and lipid A using electrophoresis and differential staining. Gas chromatography-mass spectrometry (GC-MS) compositional analysis of LPS extracts identified eight different 3-hydroxy fatty acids (3-HOFAs), 2-keto 3-deoxy-d-manno-octulosonic acid (Kdo), glucosamine, and hexose and heptose sugars, a chemical profile unique to Gram-negative LPS. Combined with molecular/structural information collected from matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS analysis of putative intact lipid A, these data led us to propose a heterogeneous hexa-acylated lipid A structure (multiple-lipid A species). We also confirmed previous reports of G. obscuriglobus whole-cell fatty acid (FA) and sterol compositions and detected a novel polyunsaturated FA (PUFA). Our confirmation of LPS, and by implication an OM, in G. obscuriglobus raises the possibility that other planctomycetes possess an OM. The pursuit of this question, together with studies of the structural connections between planctomycete LPS and peptidoglycans, will shed more light on what appears to be a planctomycete variation on the Gram-negative cell plan., Importance: Bacterial species are classified as Gram positive or negative based on their cell envelope structure. For 25 years, the envelope of planctomycete bacteria has been considered a unique exception, as it lacks peptidoglycan and an outer membrane (OM). However, the very recent detection of peptidoglycan in planctomycete species has provided evidence for a more conventional cell wall and raised questions about other elements of the cell envelope. Here, we report direct evidence of lipopolysaccharide in the planctomycete G. obscuriglobus, suggesting the presence of an OM and supporting the proposal that the planctomycete cell envelope is an extension of the canonical Gram-negative plan. This interpretation features a convoluted cytoplasmic membrane and expanded periplasmic space, the functions of which provide an intriguing avenue for future investigation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The structure and the immunology of the lipopolysaccharide (LPS) of Pseudomonas entomophila, an entomopathogenic bacterium isolated from the fruit fly Drosophila melanogaster, was characterized. The O-antigen portion was established and resulted to be built up of a repetitive unit constituted by four monosaccharide residues, all L configured, all deoxy at C-6 and with an acetamido function at C-2: →3)-α-l-FucNAc-(1→4)-α-l-FucNAc-(1→3)-α-l-FucNAc-(1→3)-β-l-QuiNAc-(1→ The structural analysis of lipid A, showed a mixture of different species. The diphosphorylated glucosamine backbone carries six fatty acids consistent with the composition C10:0 3(OH), C12:0 2(OH) and C12:0 3(OH), whereas other species differs by the number of phosphates and/or of fatty acids. The immunology experiments demonstrated that the LPS structure of P. entomophila displayed a low ability to engage the TLR4-mediated signaling correlated to a significant antagonistic activity toward hexa-acylated LPS structures., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Escherichia coli BL21 (DE3) and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS). LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004) carrying plasmid pQK004 (pagL and lpxE) produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL) as vaccine adjuvants.

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4-amino-4-deoxy-β-L-arabinose (β-L-Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro-inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β-L-Ara4N at the anomeric phosphate and its Ara4N-free counterpart. The double glycosyl phosphodiester was assembled by triazolyl-tris-(pyrrolidinyl)phosphonium-assisted coupling of the β-L-Ara4N H-phosphonate to α-lactol of β(1→6) diglucosamine, pentaacylated with (R)-(3)-acyloxyacyl- and Alloc-protected (R)-(3)-hydroxyacyl residues. The intermediate 1,1'-glycosyl-H-phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β-L-Ara4N-substituted Burkholderia Lipid A. The β-L-Ara4N modification significantly enhanced the pro-inflammatory innate immune signaling of otherwise non-endotoxic Burkholderia Lipid A., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)