Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae.

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

A heptavalent O-antigen bioconjugate vaccine exhibits differential functional antibody responses against diverse Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat due to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting seven predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Further, sera from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with O-antigen antibodies' ability to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.

Cataloging natural sialic acids and other nonulosonic acids (NulOs), and their representation using the Symbol Nomenclature for Glycans.

Nonulosonic acids or non-2-ulosonic acids (NulOs) are an ancient family of 2-ketoaldonic acids (α-ketoaldonic acids) with a 9-carbon backbone. In nature, these monosaccharides occur either in a 3-deoxy form (referred to as "sialic acids") or in a 3,9-dideoxy "sialic-acid-like" form. The former sialic acids are most common in the deuterostome lineage, including vertebrates, and mimicked by some of their pathogens. The latter sialic-acid-like molecules are found in bacteria and archaea. NulOs are often prominently positioned at the outermost tips of cell surface glycans, and have many key roles in evolution, biology and disease. The diversity of stereochemistry and structural modifications among the NulOs contributes to more than 90 sialic acid forms and 50 sialic-acid-like variants described thus far in nature. This paper reports the curation of these diverse naturally occurring NulOs at the NCBI sialic acid page (https://www.ncbi.nlm.nih.gov/glycans/sialic.html) as part of the NCBI-Glycans initiative. This includes external links to relevant Carbohydrate Structure Databases. As the amino and hydroxyl groups of these monosaccharides are extensively derivatized by various substituents in nature, the Symbol Nomenclature For Glycans (SNFG) rules have been expanded to represent this natural diversity. These developments help illustrate the natural diversity of sialic acids and related NulOs, and enable their systematic representation in publications and online resources.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ß-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.

Characterizing the N- and O-linked glycans of the PGF-CTERM sorting domain-containing S-layer protein of Methanoculleus marisnigri.

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-ß-GlcNAc3NGaAN-4-ß-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide ß-GlcNAc3NAcAN-4-ß-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-ß-Ribf-3-α-Rha2OMe-3-α-Rha - 2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Bipartite rgp Locus Diversity in Streptococcus thermophilus Corresponds to Backbone and Side Chain Differences of Its Rhamnose-Containing Cell Wall Polysaccharide.

The rhamnose-glucose polysaccharide (Rgp) of Streptococcus thermophilus represents a major cell wall component, and the gene cluster responsible for its biosynthesis (termed rgp) has recently been identified. Significant genetic diversity among these loci has previously been reported, with five distinct rgp genotypes identified (designated rgp1 through -5). In the present study, two additional genotypes were identified (designated rgp6 and rgp7) through comparative analysis of the rgp loci of 78 Streptococcus thermophilus genomes. The rgp locus of a given S. thermophilus strain encoded the biosynthetic machinery for a rhamnan-rich backbone and a variable side chain component, the latter being associated with the highly specific interactions with many bacteriophages that infect this species. The chemical structure of the Rgp from three S. thermophilus strains, representing the rgp2, -3, and -4 genotypes, was elucidated, and based on bioinformatic and biochemical analyses we propose a model for Rgp biosynthesis in dairy streptococci. Furthermore, we exploited the genetic diversity within the S. thermophilus bipartite rgp locus to develop a two-step multiplex PCR system to classify strains based on gene content associated with the biosynthesis of the variable side chain structure as well as the rhamnan backbone. IMPORTANCE Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species. Detailed analysis of S. thermophilus rgp loci has revealed an unprecedented level of genetic diversity, particularly within the glycosyltransferase-encoding gene content of a given locus. Our study shows that this genetic diversity reflects the biochemical structure(s) of S. thermophilus Rgp. As such, we harnessed the genetic diversity of S. thermophilus rgp loci to develop a two-step multiplex PCR method for the classification of strain collections and, ultimately, the formation of phage-robust rational starter sets.

Brussowvirus SW13 Requires a Cell Surface-Associated Polysaccharide To Recognize Its Streptococcus thermophilus Host.

Four bacteriophage-insensitive mutants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following challenge with Brussowvirus SW13. The BIMs displayed an altered sedimentation phenotype. Whole-genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family 2 glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated rhamnose-glucose polymer (Rgp) biosynthesis locus (designated the rgp gene cluster here). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis polysaccharide pellicle (PSP) initiator WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescence labeling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide that is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. IMPORTANCE Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and, in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process, which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements that specify the surface receptor(s) is apparent. Our research identifies a single gene that is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a community-acquired pathogen causing life-threatening infections in healthy individuals. The fact that a dose as little as 50 bacteria is lethal to mice illustrates the dramatic increase of virulence associated with hvKp strains compared with classical K. pneumoniae (cKp) strains, which require lethal doses greater than 107 bacteria. Until recently, these virulent strains were mostly antibiotic-susceptible. However, multidrug-resistant (MDR) hvKp strains have been emerging, spawning a new generation of hypervirulent "superbugs." The mechanisms of hypervirulence are not fully defined, but overproduction of capsular polysaccharide significantly impedes host clearance, resulting in increased pathogenicity of hvKp strains. While there are more than 80 serotypes of K. pneumoniae, the K1 and K2 serotypes cause the vast majority of hypervirulent infections. Therefore, a glycoconjugate vaccine targeting these 2 serotypes could significantly reduce hvKp infection. Conventionally, glycoconjugate vaccines are manufactured using intricate chemical methodologies to covalently attach purified polysaccharides to carrier proteins, which is widely considered to be technically challenging. Here we report on the recombinant production and analytical characterization of bioconjugate vaccines, enzymatically produced in glycoengineered Escherichia coli cells, against the 2 predominant hypervirulent K. pneumoniae serotypes, K1 and K2. The K. pneumoniae bioconjugates are immunogenic and efficacious, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816. This preclinical study constitutes a key step toward preventing further global dissemination of hypervirulent MDR hvKp strains.

Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus.

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-ß-Man-(1-4)-[ß-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-ß-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

Trehalose-deficient Acinetobacter baumannii exhibits reduced virulence by losing capsular polysaccharide and altering membrane integrity.

Acinetobacter baumannii has become a leading cause of bacterial nosocomial infections, in part, due to its ability to resist desiccation, disinfection and antibiotics. Several factors contribute to the tenacity and virulence of this pathogen, including production of a broad range of surface glycoconjugates, secretory systems and efflux pumps. We became interested in examining the importance of trehalose in A. baumannii after comparing intact bacterial cells by high-resolution magic angle spinning nuclear magnetic resonance and by noting high levels of this disaccharide, obscuring all other resonances in the spectrum. Since this was observed under normal growth conditions, we speculated that trehalose must serve additional functions beyond osmolyte homeostasis. Using the virulent isolate A. baumannii AB5075 and mutants in the trehalose synthesis pathway, osmoregulatory trehalose synthesis proteins A and B (△otsA and △otsB), we found that the trehalose-deficient △otsA showed increased sensitivity to desiccation, colistin, serum complement and peripheral blood mononuclear cells, while trehalose-6-phosphate producing △otsB behaved similar to the wild-type. The △otsA mutant also demonstrated increased membrane permeability and loss of capsular polysaccharide. These findings demonstrate that trehalose deficiency leads to loss of virulence in A. baumannii AB5075.

The capsular polysaccharides of Pasteurella multocida serotypes B and E: Structural, genetic and serological comparisons.

We describe the structural characterization of the capsular polysaccharides (CPSs) of Pasteurella multocida serotypes B and E. CPS was isolated following organic solvent precipitation of the supernatant from flask grown cells. Structural analysis utilizing nuclear magnetic resonance spectroscopy enabled the determination of the CPS structures and revealed significant structural similarities between the two serotypes, but also provided an explanation for the serological distinction. This observation was extended by the development of polyclonal sera to the glycoconjugate of serotype B CPS that corroborated the structural likenesses and differences. Finally, identification of these structures enabled a more comprehensive interrogation of the genetic loci and prediction of roles for some of the encoded proteins in repeat unit biosynthesis.

The CWPS Rubik's cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains.

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A-C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galf and phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain.

A cell wall-associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface.

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage-insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage-insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage-insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface-associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno-polysaccharide in both ST64987 and phage-insensitive mutant BIM3. In addition, a second cell wall-associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage-resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild-type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection.

Comparative Study of Immunogenic Properties of Purified Capsular Polysaccharides from Streptococcus suis Serotypes 3, 7, 8, and 9: the Serotype 3 Polysaccharide Induces an Opsonizing IgG Response.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, which are nonimmunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C-C motif chemokine ligand 3 (CCL3), partially via Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethally infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth.

Pseudomonas aeruginosa is an opportunistic pathogen that is inherently resistant to many antibiotics and represents an increasing threat due to the emergence of drug-resistant strains. There is a pressing need to develop innovative antimicrobials against this pathogen. In this study, we identified the O-specific antigen (OSA) of P. aeruginosa serotype O6 as a novel target for therapeutic intervention. Binding of monoclonal antibodies and antigen-binding fragments therefrom to O6 OSA leads to rapid outer membrane destabilization and inhibition of cell growth. The antimicrobial effect correlated directly with antibody affinity. Antibody binding to the O antigen of a second lipopolysaccharide (LPS) type present in P. aeruginosa or to the LPS core did not affect cell viability. Atomic force microscopy showed that antibody binding to OSA resulted in early flagellum loss, formation of membrane blebs, and eventually complete outer membrane loss. We hypothesize that antibody binding to OSA disrupts a key interaction in the P. aeruginosa outer membrane.

Structural analysis of the lipopolysaccharide O-antigen from Fusobacterium nucleatum strain CC 7/3 JVN3 C1 and development of a mouse monoclonal antibody specific to the O-antigen.

Fusobacterium nucleatum is becoming increasingly recognised as an emerging pathogen, gaining attention as a potential factor for exacerbating colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism; thus, we have initiated studies to examine the bacterium's surface glycochemistry. In an effort to characterise the surface carbohydrates of F. nucleatum, the aims of this study were to investigate the structure of the lipopolysaccharide (LPS) O-antigen of the cancer-associated isolate F. nucleatum strain CC 7/3 JVN3 C1 (hereafter C1) and to develop monoclonal antibodies (mAbs) to the LPS O-antigen that may be beneficial to the growing field of F. nucleatum research. In this study, we combined several technologies, including nuclear magnetic resonance (NMR) spectroscopy, to elucidate the structure of the LPS O-antigen repeat unit as -[-4-ß-Gal-3-α-FucNAc4N-4-α-NeuNAc-]-. We have previously identified this structure as the LPS O-antigen repeat unit from strain 10953. In this present study, we developed a mAb to the C1 LPS O-antigen and confirmed the mAbs cross-reactivity to the 10953 strain, thus confirming the structural identity.

The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology.

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-ß-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-ß-Rha-4-α-Glc-3-ß-Rha-4-(α-Rib-3-)ß-Rha-; and (iii) a nonreducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.

Distinct amino acid residues confer one of three UDP-sugar substrate specificities in Acinetobacter baumannii PglC phosphoglycosyltransferases.

Acinetobacter baumannii is an opportunistic human pathogen with the highest reported rates of multidrug resistance among Gram-negative pathogens. The capsular polysaccharide of A. baumannii is considered one of its most significant virulence factors providing resistance against complemented-mediated killing. Capsule synthesis in A. baumannii is usually initiated by the phosphoglycosyltransferase PglC. PglC transfers a phosphosugar from a nucleotide diphosphate-sugar to a polyprenol phosphate generating a polyprenol diphosphate-linked monosaccharide. Traditionally, PglC was thought to have stringent specificity towards UDP-N-N'-diacetylbacillosamine (UDP-diNAcBac). In this work we demonstrate that A. baumannii PglC has the ability to utilize three different UDP-sugar substrates: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) or UDP-diNAcBac. Using phylogenetic analyses, we first demonstrate that A. baumannii PglC orthologs separate into three distinct clades. Moreover, all members within a clade are predicted to have the same preference for one of the three possible sugar substrates. To experimentally determine the substrate specificity of each clade, we utilized in vivo complementation models and NMR analysis. We demonstrate that UDP-diNAcBac is accommodated by all PglC orthologs, but some orthologs evolved to utilize UDP-GlcNAc or UDP-GalNAc in a clade-dependent manner. Furthermore, we show that a single point mutation can modify the sugar specificity of a PglC ortholog specific for UDP-diNAcBac and that introduction of a non-native PglC ortholog into A. baumannii can generate a new capsule serotype. Collectively, these studies begin to explain why A. baumannii strains have such highly diverse glycan repertoires.

A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.

While protein glycosylation has been reported in several spirochetes including the syphilis bacterium Treponema pallidum and Lyme disease pathogen Borrelia burgdorferi, the pertinent glycan structures and their roles remain uncharacterized. Herein, a novel glycan with an unusual chemical composition and structure in the oral spirochete Treponema denticola, a keystone pathogen of periodontitis was reported. The identified glycan of mass 450.2 Da is composed of a monoacetylated nonulosonic acid (Non) with a novel extended N7 acyl modification, a 2-methoxy-4,5,6-trihydroxy-hexanoyl residue in which the Non has a pseudaminic acid configuration (L-glycero-L-manno) and is ß-linked to serine or threonine residues. This novel glycan modifies the flagellin proteins (FlaBs) of T. denticola by O-linkage at multiple sites near the D1 domain, a highly conserved region of bacterial flagellins that interact with Toll-like receptor 5. Furthermore, mutagenesis studies demonstrate that the glycosylation plays an essential role in the flagellar assembly and motility of T. denticola. To our knowledge, this novel glycan and its unique modification sites have not been reported previously in any bacteria.