Genomic and functional evaluation of exopolysaccharide produced by Liquorilactobacillus mali t6-52: technological implications.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

The Astounding World of Glycans from Giant Viruses.

Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed.

Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans.

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a ß-l-rhamnosyltransferase; domain 2 is an α-l-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α-l-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α-l-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N-glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α-l-Rha-(1→2)-ß-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

Rhamnolipid Self-Aggregation in Aqueous Media: A Long Journey toward the Definition of Structure-Property Relationships.

The need to protect human and environmental health and avoid the widespread use of substances obtained from nonrenewable sources is steering research toward the discovery and development of new molecules characterized by high biocompatibility and biodegradability. Due to their very widespread use, a class of substances for which this need is particularly urgent is that of surfactants. In this respect, an attractive and promising alternative to commonly used synthetic surfactants is represented by so-called biosurfactants, amphiphiles naturally derived from microorganisms. One of the best-known families of biosurfactants is that of rhamnolipids, which are glycolipids with a headgroup formed by one or two rhamnose units. Great scientific and technological effort has been devoted to optimization of their production processes, as well as their physicochemical characterization. However, a conclusive structure-function relationship is far from being defined. In this review, we aim to move a step forward in this direction, by presenting a comprehensive and unified discussion of physicochemical properties of rhamnolipids as a function of solution conditions and rhamnolipid structure. We also discuss still unresolved issues that deserve further investigation in the future, to allow the replacement of conventional surfactants with rhamnolipids.

Structural Determination and Chemical Synthesis of the N-Glycan from the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Asparagine-linked protein glycosylations (N-glycosylations) are one of the most abundant post-translational modifications and are essential for various biological phenomena. Herein, we describe the isolation, structural determination, and chemical synthesis of the N-glycan from the hyperthermophilic archaeon Thermococcus kodakarensis. The N-glycan from the organism possesses a unique structure including myo-inositol, which has not been found in previously characterized N-glycans. In this structure, myo-inositol is highly glycosylated and linked with a disaccharide unit through a phosphodiester. The straightforward synthesis of this glycan was accomplished through diastereoselective phosphorylation and phosphodiester construction by SN 2 coupling. Considering the early divergence of hyperthermophilic organisms in evolution, this study can be expected to open the door to approaching the primitive function of glycan modification at the molecular level.

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.

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.

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis.

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing ß-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.

Structure of the O-Antigen and the Lipid†A from the Lipopolysaccharide of Fusobacterium nucleatum ATCC 51191.

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)-ß-d-GlcpNAcA-(1→4)-ß-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.

Structure of the chlorovirus PBCV-1 major capsid glycoprotein determined by combining crystallographic and carbohydrate molecular modeling approaches.

The glycans of the major capsid protein (Vp54) of Paramecium bursaria chlorella virus (PBCV-1) were recently described and found to be unusual. This prompted a reexamination of the previously reported Vp54 X-ray structure. A detailed description of the complete glycoprotein was achieved by combining crystallographic data with molecular modeling. The crystallographic data identified most of the monosaccharides located close to the protein backbone, but failed to detect those further from the glycosylation sites. Molecular modeling complemented this model by adding the missing monosaccharides and examined the conformational preference of the whole molecule, alone or within the crystallographic environment. Thus, combining X-ray crystallography with carbohydrate molecular modeling resulted in determining the complete glycosylated structure of a glycoprotein. In this case, it is the chlorovirus PBCV-1 major capsid protein.

Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid†A.

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.

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases.

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a ß-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a ß-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.

Carbohydrate-based adjuvants.

Carbohydrate adjuvants are safe and biocompatible compounds usable as sustained delivery systems and stimulants of ongoing humoral and cellular immune responses, being especially suitable for the development of vaccines against intracellular pathogens where alum is useless. The development of new adjuvants is difficult and expensive, however, in the last two years, seven new carbohydrate-based adjuvants have been patented, also there are twelve ongoing clinical trials of vaccines that contain carbohydrate-based adjuvants, as well as numerous publications on their mechanism of action and safety. More research is necessary to improve the existent adjuvants and develop innovative ones.

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O-Antigen.

The lipopolysaccharide (LPS) O-antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant-microbe interaction was investigated. The analyses disclosed the presence of two O-antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4-α-l-rhamnose linked to a 3-α-d-fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino-acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4-amino-4-deoxy-3-O-methyl-d-fucose and (2'R,3'R,4'S)-N-methyl-3',4'-dihydroxy-3'-methyl-5'-oxoproline). Differently from the O-antigens of LPSs from other pathogenic Gram-negative bacteria, these two O-antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.

Structure of the N-glycans from the chlorovirus NE-JV-1.

Results from recent studies are breaking the paradigm that all viruses depend on their host machinery to glycosylate their proteins. Chloroviruses encode several genes involved in glycan biosynthesis and some of their capsid proteins are decorated with N-linked oligosaccharides with unique features. Here we describe the elucidation of the N-glycan structure of an unusual chlorovirus, NE-JV-1, that belongs to the Pbi group. The host for NE-JV-1 is the zoochlorella Micractinium conductrix. Spectroscopic analyses established that this N-glycan consists of a core region that is conserved in all of the chloroviruses. The one difference is that the residue 3OMe-L-rhamnose is acetylated at the O-2 position in a non-stoichiometric fashion.

N-Linked Glycans of Chloroviruses Sharing a Core Architecture without Precedent.

N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a ß-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.

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).

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.

Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans.

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.

Structural characterisation of the oligosaccharide from Moraxella bovoculi type strain 237 (ATCC BAA-1259) lipooligosaccharide.

The Gram-negative bacterium Moraxella bovoculi is associated with infectious bovine keratoconjunctivitis (IBK), colloquially known as 'pink-eye'. IBK is an extremely contagious ocular disease of cattle. We report here the structure of the oligosaccharide derived from the lipooligosaccharide from M. bovoculi type strain 237 (also known as ATCC BAA-1259T). GLC-MS and correlation NMR analysis of the oligosaccharide revealed 5 sugar residues, with a notable central branched 3,4,6-α-D-Glcp. An additional α-D-Manp was present ~30% on the sub-terminal α-D-Manp of the 4-linked branch. This oligosaccharide structure was consistent with other members of the Moraxellaceae where no heptose was present and 5-linked Kdo was directly attached to the central 3,4,6-α-D-Glcp.