Broad-Spectrum Legionaminic Acid-Specific Antibodies in Pooled Human IgGs Revealed by Glycan Microarrays with Chemoenzymatically Synthesized Nonulosonosides.

The presence and the level of antibodies in human sera against bacterial glycans are indications of prior encounters with similar antigens and/or the bacteria that express them by the immune system. An increasing number of pathogenic bacteria that cause human diseases have been shown to express polysaccharides containing a bacterial nonulosonic acid called 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac2). To investigate the immune recognition of Leg5,7Ac2, which is critical for the fight against bacterial infections, a highly effective chemoenzymatic synthon strategy was applied to construct a library of α2-3/6-linked Leg5,7Ac2-glycans via their diazido-derivatives (Leg5,7diN3-glycans) formed by efficient one-pot three-enzyme (OP3E) synthetic systems from a diazido-derivative of a six-carbon monosaccharide precursor. Glycan microarray studies using this synthetic library of a Leg5,7Ac2-capped collection of diverse underlying glycan carriers and their matched sialoside counterparts revealed specific recognition of Leg5,7Ac2 by human IgG antibodies pooled from thousands of healthy donors (IVIG), suggesting prior human encounters with Leg5,7Ac2-expressing pathogenic bacteria at the population level. These biologically relevant Leg5,7Ac2-glycans and their immune recognition assays are important tools to begin elucidating their biological roles, particularly in the context of infection and host-pathogen interactions.

Specificity and modularity of flagellin nonulosonic acid glycosyltransferases.

Many bacterial flagella are specifically O-glycosylated with nonulosonic acids, including the sialic acid derivatives, pseudaminic acid or legionaminic acid. Unlike protein glycosyltransferases that are extracytoplasmic, flagellin glycosyltransferases (fGTs) act cytoplasmically with unknown donor or acceptor specificities. The recent reconstitution of fGT-based glycosylation in heterologous hosts enables analyses underpinning such specificity.

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCEBacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

K units of the K8 and K54 capsular polysaccharides produced by Acinetobacter baumannii BAL 097 and RCH52 have the same structure but contain different di-N-acyl derivatives of legionaminic acid and are linked differently.

The K8 and K54 capsular polysaccharides were isolated from Acinetobacter baumannii BAL 097 and RCH52, respectively, and studied by sugar analysis, partial acid hydrolysis and selective solvolysis with CF3CO2H in the presence of 2-methyl-1-propanol, along with 1D and 2D 1H and 13C NMR spectroscopy. The following structures of related branched tetrasaccharide repeats (K units) of the polysaccharides were established: where Leg indicates 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid and R indicates (R)-3-hydroxybutanoyl or acetyl in the ratio ~2.5:1. The sequences of the KL8 and KL54 capsule gene clusters were closely related. The difference in the acyl group at O-7 on the sidechain legionaminic acid is due to differences in two genes in the legionaminic acid biosynthesis cluster. The wzy genes encoding the K unit polymerases are also different and make different linkages between the K units, allowing the first sugar of both K units to be identified as d-GlcpNAc. The shared Gtr20 glycosyltransferase, also encoded in KL63, forms the α-l-FucpNAc-(1 → 3)-d-GlcpNAc linkage, and Gtr19 was predicted to form α-d-GalpNAc-(1 → 3)-l-FucpNAc. Gtr18 from KL8 is 75% identical to Gtr108 from KL54 and both would link the Leg derivative to d-GalpNAc. Hence the genes present at the K locus were consistent with the composition and structures of the K8 and K54 capsular polysaccharides.

A versatile de novo synthesis of legionaminic acid and 4-epi-legionaminic acid starting from d-serine.

Legionaminic acid and 4-epi-legionaminic acid are 5,7-diacetamido nonulosonic acids and are assumed to play a crucial role in the virulence of Legionella pneumophila, the causative agent of Legionnaires' disease. Moreover, they are ideal target motifs for the development of vaccines and pathogen detection. Herein, we present a versatile de novo synthesis of legionaminic acid and 4-epi-legionaminic acid. Starting from simple d-serine, the C9-backbone is built up by two CC-bond formation reactions. First, the protected d-serine motif is elongated utilizing a highly stereoselective nitroaldol reaction to give a C6-precursor of desired d-rhamno configuration. Second, an indium-mediated allylation is employed to further elongate the carbon backbone and introduce a masked α-keto acid function.

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide.

Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6.

N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea Halorubrum sp. PV6 includes an N-formylated legionaminic acid. Analysis of the Halorubrum sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii, were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2 ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac2 -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

Tannerella forsythia strains display different cell-surface nonulosonic acids: biosynthetic pathway characterization and first insight into biological implications.

Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.

Total Biosynthesis of Legionaminic Acid, a Bacterial Sialic Acid Analogue.

Legionaminic acid, Leg5,7Ac2 , a nonulosonic acid like 5-acetamido neuraminic acid (Neu5Ac, sialic acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac2 has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac2 biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over-expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac2 (≈120 mg L(-1) of culture broth) to be produced. Pure Leg5,7Ac2 could be isolated and converted into CMP-activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac2 enabling study of its role in prokaryotic and eukaryotic glycobiology.

Comprehensive analysis of flagellin glycosylation in Campylobacter jejuni NCTC 11168 reveals incorporation of legionaminic acid and its importance for host colonization.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis. It relies on several virulence factors for host colonization, including glycosylated flagella. C. jejuni NCTC 11168 modifies its flagellins with pseudaminic acid derivatives. It is also presumed to modify these proteins with legionaminic acid, although no glycopeptide evidence was available at the onset of this study. The enzyme encoded by cj1319 can be used to make legionaminic acid in vitro, but the pathway for legionaminic acid synthesis partially inferred by knockout mutagenesis in Campylobacter coli VC167 excludes Cj1319. To address this contradiction, we examined the presence of legionaminic acid in flagellin glycopeptides of wild-type (WT) C. jejuni NCTC 11168 and of a cj1319 knockout mutant. We used high-energy collision-induced dissociation to obtain amino acid sequences while also visualizing signature sugar oxonium ions. Data analysis was performed with PEAKS software, and spectra were manually inspected for glycopeptide determination and verification. We showed that legionaminic acid is present on the flagellins of C. jejuni NCTC 11168 and that flagellin glycosylation is highly heterogeneous, with up to six different sugars singly present at a given site. We found that the cj1319 mutant produces more legionaminic acid than WT, thus excluding the requirement for Cj1319 for legionaminic acid synthesis. We also showed that this mutant has enhanced chicken colonization compared with WT, which may in part be attributed to the high content of legionaminic acid on its flagella.

Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters.

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

The polar and lateral flagella from Plesiomonas shigelloides are glycosylated with legionaminic acid.

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested.

Sialyltransferases with enhanced legionaminic acid transferase activity for the preparation of analogs of sialoglycoconjugates.

Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

Structure and genetics of the O-antigen of Enterobacter cloacae C6285 containing di-N-acetyllegionaminic acid.

On mild acid degradation of the lipopolysaccharide of Enterobacter cloacae C6285, the O-polysaccharide was cleaved at residues of 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyllegionaminic acid, Leg5Ac7Ac) in the main chain. The resultant oligosaccharide and an alkali-treated lipopolysaccharide were studied by sugar analysis along with (1)H and (13)C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit of the linear O-polysaccharide was established: →4)-α-d-Galp-(1→4)-α-Legp5Ac7Ac-(2→3)-ß-d-Galp-(1→3)-ß-d-GalpNAc-(1→ The O-antigen gene cluster of E. cloacae C6285 was sequenced, the gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure.