Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners.

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after ß-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella.

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.

Deletion of pilA, a Minor Pilin-Like Gene, from Xanthomonas citri subsp. citri Influences Bacterial Physiology and Pathogenesis.

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process.

Characterization of lipid A profiles from Shigella flexneri variant X lipopolysaccharide.

RATIONALE: In developing countries, Shigella flexneri (Sf) is the major causative agent of the endemic shigellosis (bacillary dysentery) responsible annually for one million fatalities mostly among infants. Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. Since lipid A is essential for the viability of the Gram-negative bacteria, it is subject to extensive chemical studies with new analytical techniques. METHODS: Lipid A was released by mild acid hydrolysis from the lipopolysaccharide which was obtained via the phenol/water extraction, purified and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and matrix-assisted laser desorption/ionization laser-induced dissociation tandem mass spectrometry (MALDI-LID-MS/MS). RESULTS: A detailed structural study of the whole lipid A obtained from S. flexneri variant X was carried out for the first time. Thus, we have shown that lipid A is a heterogeneous mixture having different numbers of acylated and phosphoethanolamine groups attached to the diglucosamine backbone. Furthermore, we found in the phenol phase an unusual hepta-acylated lipid A species, although the abundance was very low. CONCLUSIONS: MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.

Structural characterization and metal biosorptive activity of the major polysaccharide produced by Pseudomonas veronii 2E.

Pseudomonas veronii 2E, an autochthonous bacterium isolated from sediments associated to a high-polluted watershed, produces a complex matrix of exopolymers with carbohydrates as main components. In this work, four polysaccharides were isolated from the extracellular material. The major acidic polysaccharide named EPO2, was purified and its structure was elucidated using Matrix-assisted laser desorption/ionization and Electrospray ionization mass spectrometry, Infrared spectroscopy, Nuclear magnetic resonance spectroscopy and chemical treatments. This heteropolysaccharide consists in an α(1-4) glucan substituted with N-Acetylglucosamine residues and with a branching α-D-GlcpA-(1-3)-L-Fucp disaccharide. The biosorption capacity of EPO2 and of the whole exopolysaccharide to Pb(II), Zn(II), Cu(II) and Fe(II) was evaluated. EPO2 showed a remarkable sorption capacity for Fe(II) with an efficiency of 70% and for Zn(II) 39%. When the whole exopolysaccharide fraction was tested it showed a significantly lower metal sorption ability than purified EPO2 suggesting the involvement of the distinct acidic branching disaccharide in this interaction.

Infrared spectroscopy with multivariate analysis to interrogate the interaction of whole cells and secreted soluble exopolimeric substances of Pseudomonas veronii 2E with Cd(II), Cu(II) and Zn(II).

Extracellular polymeric substances (EPS) are bacterial products associated to cell wall or secreted to the liquid media that form the framework of microbial mats. These EPS contain functional groups as carboxyl, amino, hydroxyl, phosphate and sulfhydryl, able to interact with cations. Thus, EPS may be considered natural detoxifying compounds of metal polluted waters and wastewaters. In this work Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) in combination with multivariate analysis (Principal Component Analysis-PCA-) were used to study the interaction of Cd(II), Cu(II) and Zn(II) and Pseudomonas veronii 2E cells, including bound EPS and cell wall, and its different soluble EPS fractions, previously characterized as Cd(II) ligands of moderate strength. Amino groups present in exopolysaccharide fraction were responsible for Zn(II) and Cu(II) complexation, while carboxylates chelated Cd(II). In lipopolysaccharide fraction, phosphoryl and carboxyl sites were involved in Cd(II) and Cu(II) binding, while Zn(II) interacted with amino groups. Similar results were obtained from cells. These studies confirmed that FTIR-PCA is a rapid analytical tool to provide valuable information regarding the functional groups in biomolecules related to metal interaction. Moreover, a discrimination and identification of functional groups present in both EPS and cells that interacted with Cd(II), Zn(II) and Cu(II) was demonstrated.

Bordetella bronchiseptica Glycosyltransferase Core Mutants Trigger Changes in Lipid A Structure.

Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-D-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2', O-acylated with C14:0(3-O-C10:0(3-OH) in C-3' and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4', both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane. Graphical Abstract.

New Features in the Lipid A Structure of Brucella suis and Brucella abortus Lipopolysaccharide.

Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract ᅟ.

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated.

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive ß-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc5-8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. SIGNIFICANCE: A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp.

Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance.

We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides.

Cyclic beta-1,2-glucans (CbetaG) are osmolyte homopolysaccharides with a cyclic beta-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CbetaG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked beta-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a beta-1,2-glucan phosphorylase present at the CbetaG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the beta-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this "length-controlling" phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.

A matrix-assisted laser desorption/ionization mass spectrometry approach to the lipid A from Mesorhizobium loti.

The isolation, purification and analysis of the lipid A obtained from Mesorhizobium loti Ayac 1 BII strain is presented. Analysis of the carbohydrate moiety after acid hydrolysis by high-pH anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD) showed the presence of glucosamine and galacturonic acid as the only sugar components. Gas chromatographic (GC) and GC/mass spectrometric (MS) analysis of the fatty acids revealed the presence of 3-OH-C12:0; 3-OH-C13:0; 3-OH-C20:0 and 27-OH-C28:0 among the major hydroxylated species. In addition, C16:0, C17:0, C18:0 and C 20:0 were shown as main saturated fatty acids. Different polyacylated species were evidenced by thin layer chromatography of lipid A, allowing the purification of two fractions. Ultraviolet matrix-assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) MS analysis with different matrices, in the positive- and negative-ion mode, was performed. The fast moving component revealed the presence of hexa-acylated species, varying in the fatty acid composition. Species containing three 3-OH fatty acids and a 27-OH-C28:0 fatty acid were observed. Individual ions within this family differ by +/-14 mass units. The slow moving component was enriched mainly in penta-acylated species. Among them, three subgroups were detected: the major one compatible with the sugar core bearing two 3-OH 20:0 fatty acids, a 3-OH 13:0 or a 3-OH 12:0 fatty acid, a 27-OH 28:0 fatty acid and one saturated fatty acid. Each signal differs in a C18:0 acyl unit from the corresponding hexa-acylated family. On the other hand, a subgroup bearing one 3-OH 20:0 fatty acid, one 27-OH 28:0 fatty acid and two non-polar fatty acids was shown. A minor subgroup compatible with structures containing two hydroxylated and three non-polar fatty acids was also detected. The results obtained showed that nor-harmane was an excellent matrix for charged lipid A structural studies in both, positive and negative ion modes.