Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-ManpN3NA.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-ß-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The ß-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Chemical Synthesis of a Densely Functionalized Aminodisaccharide from Fusobacterium nucleatum ATCC 23726 O-Antigen.

Fusobacterium nucleatum, a colorectal-cancer-associated oncomicrobe, can trigger or accelerate numerous pathologies. We report the first synthesis of a conjugation-ready disaccharide containing six amino groups from F. nucleatum ATCC 23726 O-antigen. Rare 2,3-diamido-d-glucuronic acid amide and 2-acetamido-4-amino-d-fucose were synthesized from d-glucosamine through configuration inversion, nucleophilic substitution, C6 oxidation, and C6 deoxygenation. A judicious choice of protecting groups and reaction conditions enabled the selective installation of N-acetyl, N-propanoyl, N-formyl, and carboxamido groups.

Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.

A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM197 lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM197 lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections. IMPORTANCE: The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

Hybrid Materials Obtained by Immobilization of Biosynthesized Ag Nanoparticles with Antioxidant and Antimicrobial Activity.

Ag nanoparticles (AgNPs) were biosynthesized using sage (Salvia officinalis L.) extract. The obtained nanoparticles were supported on SBA-15 mesoporous silica (S), before and after immobilization of 10% TiO2 (Degussa-P25, STp; commercial rutile, STr; and silica synthesized from Ti butoxide, STb). The formation of AgNPs was confirmed by X-ray diffraction. The plasmon resonance effect, evidenced by UV-Vis spectra, was preserved after immobilization only for the sample supported on STb. The immobilization and dispersion properties of AgNPs on supports were evidenced by TEM microscopy, energy-dispersive X-rays, dynamic light scattering, photoluminescence and FT-IR spectroscopy. The antioxidant activity of the supported samples significantly exceeded that of the sage extract or AgNPs. Antimicrobial tests were carried out, in conditions of darkness and white light, on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Higher antimicrobial activity was evident for SAg and STbAg samples. White light increased antibacterial activity in the case of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In the first case, antibacterial activity increased for both supported and unsupported AgNPs, while in the second one, the activity increased only for SAg and STbAg samples. The proposed antibacterial mechanism shows the effect of AgNPs and Ag+ ions on bacteria in dark and light conditions.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

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

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

Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii. Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, ∆2903 and ∆lpsB, that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii. Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly. IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Chemical synthesis of 6-deoxy-D-talose containing a tetrasaccharide repeating unit of the O-specific polysaccharide from Enterobacter cloacae G3422 in the form of its 2-aminoethyl glycoside.

Chemical synthesis of the tetrasaccharide repeating unit of the O-specific polysaccharide from Enterobacter cloacae G3422 is reported. The synthesis of the target tetrasaccharide is achieved through a convergent [2 + 2]-block strategy. The conjugation ready target oligosaccharide is attractive for further glycoconjugate formation with a suitable aglycon. Synthesis of the challenging 6-deoxy-L-talose moiety is reported using two different approaches and the obvious difficulties are discussed.

Genomic surveillance of STEC/EHEC infections in Germany 2020 to 2022 permits insight into virulence gene profiles and novel O-antigen gene clusters.

Shiga toxin-producing E. coli (STEC), including the subgroup of enterohemorrhagic E. coli (EHEC), are important bacterial pathogens which cause diarrhea and the severe clinical manifestation hemolytic uremic syndrome (HUS). Genomic surveillance of STEC/EHEC is a state-of-the-art tool to identify infection clusters and to extract markers of circulating clinical strains, such as their virulence and resistance profile for risk assessment and implementation of infection prevention measures. The aim of the study was characterization of the clinical STEC population in Germany for establishment of a reference data set. To that end, from 2020 to 2022 1257 STEC isolates, including 39 of known HUS association, were analyzed and lead to a classification of 30.4 % into 129 infection clusters. Major serogroups in all clinical STEC analyzed were O26, O146, O91, O157, O103, and O145; and in HUS-associated strains were O26, O145, O157, O111, and O80. stx1 was less frequently and stx2 or a combination of stx, eaeA and ehxA were more frequently found in HUS-associated strains. Predominant stx gene subtypes in all STEC strains were stx1a (24 %) and stx2a (21 %) and in HUS-associated strains were mainly stx2a (69 %) and the combination of stx1a and stx2a (12.8 %). Furthermore, two novel O-antigen gene clusters (RKI6 and RKI7) and strains of serovars O45:H2 and O80:H2 showing multidrug resistance were detected. In conclusion, the implemented surveillance tools now allow to comprehensively define the population of clinical STEC strains including those associated with the severe disease manifestation HUS reaching a new surveillance level in Germany.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Specific labeling of newly synthesized lipopolysaccharide via metabolic incorporation of azido-galactose.

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-ß-d-Fucf-(1→3)-ß-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-ß-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

Lipopolysaccharides of Herbaspirillum species and their relevance for bacterium-host interactions.

The lipopolysaccharides of Herbaspirillum lusitanum P6-12T (HlP6-12T) and H. frisingense GSF30T (HfGSF30T) was isolated by phenol-water extraction from bacterial cells and was characterized using chemical analysis and SDS-PAGE. It was shown that these bacteria produce LPSs that differ in their physicochemical properties and macromolecular organization. In this paper, the lipid A structure of the HlP6-12T LPS, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To prove the effect of the size of micelles on their bioavailability, we examined the activity of both LPSs toward the morphology of wheat seedlings. Analysis of the HlP6-12T and HfGSF30T genomes showed no significant differences between the operons that encode proteins involved in the biosynthesis of the lipids A and core oligosaccharides. The difference may be due to the composition of the O-antigen operon. HfGSF30T has two copies of the rfb operon, with the main one divided into two fragments. In contrast, the HlP6-12T genome contains only a single rfb-containing operon, and the other O-antigen operons are not comparable at all. The integrity of O-antigen-related genes may also affect LPS variability of. Specifically, we have observed a hairpin structure in the middle of the O-antigen glycosyltransferase gene, which led to the division of the gene into two fragments, resulting in incorrect protein synthesis and potential abnormalities in O-antigen production.