Increasing outer membrane complexity: the case of the lipopolysaccharide lipid A from marine Cellulophaga pacifica.

Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.

Atomic-Level Dissection of DC-SIGN Recognition of Bacteroides vulgatus LPS Epitopes.

The evaluation of Bacteroides vulgatus mpk (BVMPK) lipopolysaccharide (LPS) recognition by DC-SIGN, a key lectin in mediating immune homeostasis, has been here performed. A fine chemical dissection of BVMPK LPS components, attained by synthetic chemistry combined to spectroscopic, biophysical, and computational techniques, allowed to finely map the LPS epitopes recognized by DC-SIGN. Our findings reveal BVMPK's role in immune modulation via DC-SIGN, targeting both the LPS O-antigen and the core oligosaccharide. Furthermore, when framed within medical chemistry or drug design, our results could lead to the development of tailored molecules to benefit the hosts dealing with inflammatory diseases.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

The chemistry of gut microbiome-derived lipopolysaccharides impacts on the occurrence of food allergy in the pediatric age.

Introduction: Food allergy (FA) in children is a major health concern. A better definition of the pathogenesis of the disease could facilitate effective preventive and therapeutic measures. Gut microbiome alterations could modulate the occurrence of FA, although the mechanisms involved in this phenomenon are poorly characterized. Gut bacteria release signaling byproducts from their cell wall, such as lipopolysaccharides (LPSs), which can act locally and systemically, modulating the immune system function. Methods: In the current study gut microbiome-derived LPS isolated from fecal samples of FA and healthy children was chemically characterized providing insights into the carbohydrate and lipid composition as well as into the LPS macromolecular nature. In addition, by means of a chemical/MALDI-TOF MS and MS/MS approach we elucidated the gut microbiome-derived lipid A mass spectral profile directly on fecal samples. Finally, we evaluated the pro-allergic and pro-tolerogenic potential of these fecal LPS and lipid A by harnessing peripheral blood mononuclear cells from healthy donors. Results: By analyzing fecal samples, we have identified different gut microbiome-derived LPS chemical features comparing FA children and healthy controls. We also have provided evidence on a different immunoregulatory action elicited by LPS on peripheral blood mononuclear cells collected from healthy donors suggesting that LPS from healthy individuals could be able to protect against the occurrence of FA, while LPS from children affected by FA could promote the allergic response. Discussion: Altogether these data highlight the relevance of gut microbiome-derived LPSs as potential biomarkers for FA and as a target of intervention to limit the disease burden.

Lipopolysaccharides from Ralstonia solanacearum induce a broad metabolomic response in Solanum lycopersicum.

Ralstonia solanacearum, one of the most destructive crop pathogens worldwide, causes bacterial wilt disease in a wide range of host plants. The major component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPS), has been shown to function as elicitors of plant defense leading to the activation of signaling and defense pathways in several plant species. LPS from a R. solanacearum strain virulent on tomato (LPSR. sol.), were purified, chemically characterized, and structurally elucidated. The lipid A moiety consisted of tetra- to hexa-acylated bis-phosphorylated disaccharide backbone, also decorated by aminoarabinose residues in minor species, while the O-polysaccharide chain consisted of either linear tetrasaccharide or branched pentasaccharide repeating units containing α-L-rhamnose, N-acetyl-ß-D-glucosamine, and ß-L-xylose. These properties might be associated with the evasion of host surveillance, aiding the establishment of the infection. Using untargeted metabolomics, the effect of LPSR. sol. elicitation on the metabolome of Solanum lycopersicum leaves was investigated across three incubation time intervals with the application of UHPLC-MS for metabolic profiling. The results revealed the production of oxylipins, e.g., trihydroxy octadecenoic acid and trihydroxy octadecadienoic acid, as well as several hydroxycinnamic acid amide derivatives, e.g., coumaroyl tyramine and feruloyl tyramine, as phytochemicals that exhibit a positive correlation to LPSR. sol. treatment. Although the chemical properties of these metabolite classes have been studied, the functional roles of these compounds have not been fully elucidated. Overall, the results suggest that the features of the LPSR. sol. chemotype aid in limiting or attenuating the full deployment of small molecular host defenses and contribute to the understanding of the perturbation and reprogramming of host metabolism during biotic immune responses.

Bacterial lipids drive compartmentalization on the nanoscale.

The design of cellular functions in synthetic systems, inspired by the internal partitioning of living cells, is a constantly growing research field that is paving the way to a large number of new remarkable applications. Several hierarchies of internal compartments like polymersomes, liposomes, and membranes are used to control the transport, release, and chemistry of encapsulated species. However, the experimental characterization and the comprehension of glycolipid mesostructures are far from being fully addressed. Lipid A is indeed a glycolipid and the endotoxic part of Gram-negative bacterial lipopolysaccharide; it is the moiety that is recognized by the eukaryotic receptors giving rise to the modulation of innate immunity. Herein we propose, for the first time, a combined approach based on hybrid Particle-Field (hPF) Molecular Dynamics (MD) simulations and Small Angle X-Ray Scattering (SAXS) experiments to gain a molecular picture of the complex supramolecular structures of lipopolysaccharide (LPS) and lipid A at low hydration levels. The mutual support of data from simulations and experiments allowed the unprecedented discovery of the presence of a nano-compartmentalized phase composed of liposomes of variable size and shape which can be used in synthetic biological applications.

Pushing the Boundaries of Structural Heterogeneity with the Lipid A of Marine Bacteria Cellulophaga.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

Expanding Knowledge of Methylotrophic Capacity: Structure and Properties of the Rough-Type Lipopolysaccharide from Methylobacterium extorquens and Its Role on Membrane Resistance to Methanol.

The ability of Methylobacterium extorquens to grow on methanol as the sole carbon and energy source has been the object of intense research activity. Unquestionably, the bacterial cell envelope serves as a defensive barrier against such an environmental stressor, with a decisive role played by the membrane lipidome, which is crucial for stress resistance. However, the chemistry and the function of the main constituent of the M. extorquens outer membrane, the lipopolysaccharide (LPS), is still undefined. Here, we show that M. extorquens produces a rough-type LPS with an uncommon, non-phosphorylated, and extensively O-methylated core oligosaccharide, densely substituted with negatively charged residues in the inner region, including novel monosaccharide derivatives such as O-methylated Kdo/Ko units. Lipid A is composed of a non-phosphorylated trisaccharide backbone with a distinctive, low acylation pattern; indeed, the sugar skeleton was decorated with three acyl moieties and a secondary very long chain fatty acid, in turn substituted by a 3-O-acetyl-butyrate residue. Spectroscopic, conformational, and biophysical analyses on M. extorquens LPS highlighted how structural and tridimensional features impact the molecular organization of the outer membrane. Furthermore, these chemical features also impacted and improved membrane resistance in the presence of methanol, thus regulating membrane ordering and dynamics.

Polymyxin Resistance and Heteroresistance Are Common in Clinical Isolates of Achromobacter Species and Correlate with Modifications of the Lipid A Moiety of Lipopolysaccharide.

The Achromobacter genus includes opportunistic pathogens that can cause chronic infections in immunocompromised patients, especially in people with cystic fibrosis (CF). Treatment of Achromobacter infections is complicated by antimicrobial resistance. In this study, a collection of Achromobacter clinical isolates, from CF and non-CF sources, was investigated for polymyxin B (PmB) resistance. Additionally, the effect of PmB challenge in a subset of isolates was examined and the presence of PmB-resistant subpopulations within the isolates was described. Further, chemical and mass spectrometry analyses of the lipid A of Achromobacter clinical isolates enabled the determination of the most common structures and showed that PmB challenge was associated with lipid A modifications that included the addition of glucosamine and palmitoylation and the concomitant loss of the free phosphate at the C-1 position. This study demonstrates that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying the addition of positively charged residues and additional acyl chains to lipid A can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates. IMPORTANCE Achromobacter species can cause chronic and potentially severe infections in immunocompromised patients, especially in those with cystic fibrosis. Bacteria cannot be eradicated due to Achromobacter's intrinsic multidrug resistance. We report that intrinsic resistance to polymyxin B (PmB), a last-resort antimicrobial peptide used to treat infections by multiresistant bacteria, is prevalent in Achromobacter clinical isolates; many isolates also display increased resistance upon PmB challenge. Analysis of the lipopolysaccharide lipid A moiety of several Achromobacter species reveals a penta-acylated lipid A, which in the PmB-resistant isolates was modified by the incorporation of glucosamine residues, an additional acyl chain, loss of phosphates, and hydroxylation of acyl chains, all of which can enhance PmB resistance in other bacteria. We conclude that PmB resistance, particularly in Achromobacter isolates from chronic respiratory infections, is a common phenomenon, and that Achromobacter lipid A displays modifications that may confer increased resistance to polymyxins and potentially other antimicrobial peptides.

Extraction, Purification, and Chemical Degradation of LPS from Gut Microbiota Strains.

It is estimated that more than 500 different bacterial species colonize the human gut, and they are collectively known as the gut microbiota. Such a massive bacterial presence is now considered an additional organ of the human body, thus becoming the object of an intense and daily growing research activity. Gram-negative bacteria represent a large percentage of the gut microbiota strains. The main constituent of the outer membrane of Gram-negatives is the lipopolysaccharide (LPS). Since its first discovery, LPS has been extensively studied for its structure-dependent capability to elicit a potent immune inflammatory reaction when perceived by specific immune receptors present in our body. Therefore, traditionally, LPS, due to its peculiar chemistry, has been associated with pathogenic bacteria, and it has been extensively studied for its dangerous effects on human health. However, LPS is also expressed on the cell surface of harmless and beneficial bacteria that colonize our intestines. This necessarily implies that the LPS from harmless gut microbes is "chemically different" from that owned by pathogenic ones, hence enabling successful colonization of the intestinal tract without creating a threat to the host immune system. Deciphering the structural features of LPS from these gut bacteria is essential to improve our still scarce knowledge of how the human host lives in a harmonious relationship with its own microbiota. To this end, LPS extraction and purification are essential steps in this field of research. Yet working with gut bacteria is extremely complex for a number of reasons, one being related to the fact that they produce an array of other glycans and glycoconjugates, such as capsular polysaccharides and/or exopolysaccharides, which render the isolation and characterization of the sole LPS not at all trivial. Here, we provide a protocol that might help when dealing with LPS from gut microbial species. We describe the preliminary manipulations and checks, extraction, and purification approaches, as well as the necessary chemical manipulations that should be performed to enable the characterization of the structure of an LPS by means of techniques like nuclear magnetic resonance spectroscopy and mass spectrometry.

Dissecting Lipopolysaccharide Composition and Structure by GC-MS and MALDI Spectrometry.

Lipopolysaccharides (LPSs) are the main components of the external leaflet of the outer membrane of Gram-negative bacteria. They exert multiple functions, starting from conferring stability to the bacterial membrane to mediating the interaction of the microbe with the external environment. The composition and the structure of LPSs present tremendous diversity even within bacteria of the same species, and for this reason, the determination of the structure of these molecules is crucial because it can provide information on the motifs key for the virulence of a pathogen or that are associated to a bacterium of the commensal or beneficial microbiota. In addition, structural data disclose the effects triggered from a mutation or from the use of an antibiotic, or they can be used as tools to check the quality of adjuvants and/or medications, as vaccines, that make use of LPS.The structural study of LPSs is complex, and it can be achieved with the right combination of different techniques. In this frame, this chapter focuses on the two MS-based approaches, the gas chromatography-mass spectrometry (GC-MS) and the matrix-assisted laser desorption/ionization (MALDI).

Structural determination of the lipid A from the deep-sea bacterium Zunongwangia profunda SM-A87: a small-scale approach.

Zunongwangia profunda SM-A87 is a deep-sea sedimentary bacterium from the phylum Bacteroidetes, representing a new genus of Flavobacteriaceae. It was previously investigated for its capability of yielding high quantities of capsular polysaccharides (CPS) with interesting rheological properties, including high viscosity and tolerance to high salinities and temperatures. However, as a Gram-negative, Z. profunda SM-A87 also expresses lipopolysaccharides (LPS) as the main components of the external leaflet of its outer membrane. Here, we describe the isolation and characterization of the glycolipid part of this LPS, i.e. the lipid A, which was achieved by-passing the extraction procedure of the full LPS and by working on the ethanol precipitation product, which contained both the CPS fraction and bacterial cells. To this aim a dual approach was adopted and all analyses confirmed the isolation of Z. profunda SM-A87 lipid A that turned out to be a blend of species with high levels of heterogeneity both in the acylation and phosphorylation pattern, as well as in the hydrophilic backbone composition. Mono-phosphorylated tetra- and penta-acylated lipid A species were identified and characterized by a high content of branched, odd-numbered, and unsaturated fatty acid chains as well as, for some species, by the presence of a hybrid disaccharide backbone.

Peptidoglycan from Akkermansia muciniphila MucT: chemical structure and immunostimulatory properties of muropeptides.

Akkermansia muciniphila is an intestinal symbiont known to improve the gut barrier function in mice and humans. Various cell envelope components have been identified to play a critical role in the immune signaling of A. muciniphila, but the chemical composition and role of peptidoglycan (PG) remained elusive. Here, we isolated PG fragments from A. muciniphila MucT (ATCC BAA-835), analyzed their composition and evaluated their immune signaling capacity. Structurally, the PG of A. muciniphila was found to be noteworthy due of the presence of some nonacetylated glucosamine residues, which presumably stems from deacetylation of N-acetylglucosamine. Some of the N-acetylmuramic acid (MurNAc) subunits were O-acetylated. The immunological assays revealed that muropeptides released from the A. muciniphila PG could both activate the intracellular NOD1 and NOD2 receptors to a comparable extent as muropeptides from Escherichia coli BW25113. These data challenge the hypothesis that non-N-acetylattion of PG can be used as a NOD-1 evasion mechanism. Our results provide new insights into the diversity of cell envelope structures of key gut microbiota members and their role in steering host-microbiome interactions.

A Journey from Structure to Function of Bacterial Lipopolysaccharides.

Lipopolysaccharide (LPS) is a crucial constituent of the outer membrane of most Gram-negative bacteria, playing a fundamental role in the protection of bacteria from environmental stress factors, in drug resistance, in pathogenesis, and in symbiosis. During the last decades, LPS has been thoroughly dissected, and massive information on this fascinating biomolecule is now available. In this Review, we will give the reader a third millennium update of the current knowledge of LPS with key information on the inherent peculiar carbohydrate chemistry due to often puzzling sugar residues that are uniquely found on it. Then, we will drive the reader through the complex and multifarious immunological outcomes that any given LPS can raise, which is strictly dependent on its chemical structure. Further, we will argue about issues that still remain unresolved and that would represent the immediate future of LPS research. It is critical to address these points to complete our notions on LPS chemistry, functions, and roles, in turn leading to innovative ways to manipulate the processes involving such a still controversial and intriguing biomolecule.

Lipopolysaccharide lipid A: A promising molecule for new immunity-based therapies and antibiotics.

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.

Liquid-state NMR spectroscopy for complex carbohydrate structural analysis: A hitchhiker's guide.

Structural determination of carbohydrates is mostly performed by liquid-state NMR, and it is a demanding task because the NMR signals of these biomolecules explore a rather narrow range of chemical shifts, with the result that the resonances of each monosaccharide unit heavily overlap with those of others, thus muddling their punctual identification. However, the full attribution of the NMR chemical shifts brings great advantages: it discloses the nature of the constituents, the way they are interconnected, in some cases their absolute configuration, and it paves the way to other and more sophisticated analyses. The purpose of this review is to provide a practical guide into this challenging subject. It will drive through the strategy used to assign the NMR data, pinpointing the core information disclosed from each NMR experiment, and suggesting useful tricks for their interpretation, along with other resources pivotal during the study of these biomolecules.

N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

The Unusual Lipid A Structure and Immunoinhibitory Activity of LPS from Marine Bacteria Echinicola pacifica KMM 6172T and Echinicola vietnamensis KMM 6221T.

Gram-negative bacteria experiencing marine habitats are constantly exposed to stressful conditions dictating their survival and proliferation. In response to these selective pressures, marine microorganisms adapt their membrane system to ensure protection and dynamicity in order to face the highly mutable sea environments. As an integral part of the Gram-negative outer membrane, structural modifications are commonly observed in the lipopolysaccharide (LPS) molecule; these mainly involve its glycolipid portion, i.e., the lipid A, mostly with regard to fatty acid content, to counterbalance the alterations caused by chemical and physical agents. As a consequence, unusual structural chemical features are frequently encountered in the lipid A of marine bacteria. By a combination of data attained from chemical, MALDI-TOF mass spectrometry (MS), and MS/MS analyses, here, we describe the structural characterization of the lipid A isolated from two marine bacteria of the Echinicola genus, i.e., E. pacifica KMM 6172T and E. vietnamensis KMM 6221T. This study showed for both strains a complex blend of mono-phosphorylated tri- and tetra-acylated lipid A species carrying an additional sugar moiety, a d-galacturonic acid, on the glucosamine backbone. The unusual chemical structures are reflected in a molecule that only scantly activates the immune response upon its binding to the LPS innate immunity receptor, the TLR4-MD-2 complex. Strikingly, both LPS potently inhibited the toxic effects of proinflammatory Salmonella LPS on human TLR4/MD-2.