Development and Application of a High-Throughput Method for the Purification and Analysis of Surface Carbohydrates from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type.

IMPORTANCE: Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

Microscopy and modelling investigations on the morphology of the biofilm exopolysaccharide produced by Burkholderia multivorans strain C1576.

Bacteria form very often biofilms where they embed in a self-synthesized matrix exhibiting a gel-like appearance. Matrices offer several advantages, including defence against external threats and the easiness of intercellular communication. In infections, biofilm formation enhances bacteria resistance against antimicrobials, causing serious clinical problems for patients' treatments. Biofilm matrices are composed of proteins, extracellular DNA, and polysaccharides, the latter being the major responsible for matrix architecture. The repeating unit of the biofilm polysaccharide synthesized by Burkholderia multivorans strain C1576 contains two mannoses and two sequentially linked rhamnoses, one of them 50 % methylated on C-3. Rhamnose, a 6-deoxysugar, has lower polarity than other common monosaccharides and its methylation further reduces polarity. This suggests a possible role of this polysaccharide in the biofilm matrix; in fact, computer modelling and atomic force microscopy studies evidenced intra- and inter-molecular non-polar interactions both within polysaccharides and with aliphatic molecules. In this paper, the polysaccharide three-dimensional morphology was investigated using atomic force microscopy in both solid and solution states. Independent evidence of the polymer conformation was obtained by transmission electron microscopy which confirmed the formation of globular compact structures. Finally, data from computer dynamic simulations were used to model the three-dimensional structure.

The Effect of Mushroom Culture Filtrates on the Inhibition of Mycotoxins Produced by Aspergillus flavus and Aspergillus carbonarius.

Two of the mycotoxins of greatest agroeconomic significance are aflatoxin B1 (AFB1), and ochratoxin A (OTA). It has been reported that extracts from some wood-decaying mushrooms, such as Lentinula edodes and Trametes versicolor showed the ability to inhibit AFB1 or OTA biosynthesis. Therefore, in our study, a wide screening of 42 isolates of different ligninolytic mushrooms was assayed for their ability to inhibit the synthesis of OTA in Aspergillus carbonarius and AFB1 in Aspergillus flavus, in order to find a metabolite that can simultaneously inhibit both mycotoxins. The results showed that four isolates produce metabolites able to inhibit the synthesis of OTA, and 11 isolates produced metabolites that inhibited AFB1 by >50%. Two strains, the Trametes versicolor strain TV117 and the Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. Preliminary results suggest that the mechanism of efficacy of the S. commune rough and semipurified polysaccharides could be analogous to that found previously for Tramesan®, by enhancing the antioxidant response in the target fungal cells. The overall results indicate that S. commune's polysaccharide(s) could be a potential agent(s) in biological control and/or a useful component of the integrated strategies able to control mycotoxin synthesis.

The Bep gene cluster in Burkholderia cenocepacia H111 codes for a water-insoluble exopolysaccharide essential for biofilm formation.

Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.

The Human Milk Microbiota Produces Potential Therapeutic Biomolecules and Shapes the Intestinal Microbiota of Infants.

Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four Levilactobacillus brevis strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six Lactiplantibacillus plantarum strains by PCR analysis and in vitro antibacterial studies. 1H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain, Limosilactobacillus fermentum MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel Lactobacillus strains expressing specific functional biomolecules.

Genetic and Structural Variation in the O-Antigen of Salmonella enterica Serovar Typhimurium Isolates Causing Bloodstream Infections in the Democratic Republic of the Congo.

Salmonella enterica serovar Typhimurium causes a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. No licensed vaccine is available, but O-antigen-based candidates are in development, as the O-antigen moiety of lipopolysaccharides is the principal target of protective immunity. The vaccines under development are designed based on isolates with O-antigen O-acetylated at position C-2 of abequose, giving the O:5 antigen. Serotyping data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without O:5. The importance and distribution of this loss of O:5 antigen in the population as well as the genetic mechanism responsible for the loss and chemical characteristics of the O-antigen are poorly understood. In this study, we Illumina whole-genome sequenced 354 Salmonella Typhimurium isolates from the DRC, which were isolated between 2002 and 2017. We used genomics and phylogenetics combined with chemical approaches (1H nuclear magnetic resonance [NMR], high-performance anion-exchange chromatography with pulsed amperometric detection [HPAEC-PAD], high-performance liquid chromatography-PAD [HPLC-PAD], and HPLC-size exclusion chromatography [HPLC-SEC]) to characterize the O-antigen features within the bacterial population. We observed convergent evolution toward the loss of the O:5 epitope predominantly caused by recombination events in a single gene, the O-acetyltransferase gene oafA. In addition, we observe further O-antigen variations, including O-acetylation of the rhamnose residue, different levels of glucosylation, and the absence of O-antigen repeating units. Large recombination events underlying O-antigen variation were resolved using long-read MinION sequencing. Our study suggests evolutionary pressure toward O-antigen variants in a region where invasive disease by Salmonella Typhimurium is highly endemic. This needs to be taken into account when developing O-antigen-based vaccines, as it might impact the breadth of coverage in such regions. IMPORTANCE The bacterium Salmonella Typhimurium forms a devastating burden in sub-Saharan Africa by causing invasive bloodstream infections. Additionally, Salmonella Typhimurium presents high levels of antimicrobial resistance, jeopardizing treatment. No licensed vaccine is available, but candidates are in development, with lipopolysaccharides being the principal target of protective immunity. The vaccines under development are designed based on the O:5 antigen variant of bacterial lipopolysaccharides. Data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without this O:5 antigen. We studied this loss of O:5 antigen in the population at the genetic and chemical levels. We genome sequenced 354 isolates from the DRC and used advanced bioinformatics and chemical methods to characterize the lipopolysaccharide features within the bacterial population. Our results suggest evolutionary pressure toward O-antigen variants. This needs to be taken into account when developing vaccines, as it might impact vaccine coverage.

Rapid generation of Shigella flexneri GMMA displaying natural or new and cross-reactive O-Antigens.

Generalized modules for membrane antigens (GMMA) are exosomes released from engineered Gram-negative bacteria and represent an attractive vaccine platform for the delivery of the O-Antigen (OAg), recognized as the key target for protective immunity against several pathogens such as Shigella. Shigella is a major cause of disease in Low- and Middle-Income countries and the development of a vaccine needs to deal with its large serotypic diversity. All S. flexneri serotypes, except serotype 6, share a conserved OAg backbone, corresponding to serotype Y. Here, a GMMA-producing S. flexneri scaffold strain displaying the OAg backbone was engineered with different OAg-modifying enzymes, either individually or in combinations. This strategy rapidly yielded GMMA displaying 12 natural serotypes and 16 novel serotypes expressing multiple epitopes combinations that do not occur in nature. Importantly, a candidate GMMA displaying a hybrid OAg elicited broadly cross-bactericidal antibodies against a large panel of S. flexneri serotypes.

Pellicle Biofilm Formation in Burkholderia cenocepacia J2315 is Epigenetically Regulated through WspH, a Hybrid Two-Component System Kinase-Response Regulator.

The chemosensory signal transduction system Wsp regulates biofilm formation and related phenotypes by influencing cyclic-di-GMP (c-di-GMP) levels in bacterial cells. This is typically achieved by activation of the diguanylate cyclase WspR, through phosphorylation of its phosphoreceiver domain. The Wsp system of Burkholderia cenocepacia J2315 is in one operon with the hybrid response regulator/histidine kinase wspH, but lacks the diguanylate cyclase wspR which is located in a different operon. The expression of wspH, the first gene in the B. cenocepacia Wsp operon as well as pellicle biofilm formation are epigenetically regulated in B. cenocepacia J2315. To investigate whether WspH regulates pellicle biofilm formation, several mutants with altered expression of wspH were constructed. Mutants with increased expression of wspH showed accelerated pellicle biofilm formation, reduced swimming motility and increased c-di-GMP levels. This was independent of WspR phosphorylation, showing that WspR is not the cognate response receiver for histidine kinase WspH. IMPORTANCE Biofilms are surface-attached or suspended aggregates of cells, that are problematic in the context of bacterial infections, as they provide protection from antibiotic treatment. Burkholderia cenocepacia can colonize the lung of immunocompromised patients and forms biofilms that increase its recalcitrance to antibiotic treatment. Pellicles are biofilms which form at an air-liquid interface to take advantage of the higher oxygen concentrations in this environment. How quickly pellicles are formed is crucial for the fitness of obligate aerobic bacteria such as B. cenocepacia. Cyclic-di-GMP (c-di-GMP) levels determine the transition between planktonic and biofilm lifestyle, and WspH controls c-di-GMP production. WspH is therefore important for the fitness of B. cenocepacia in environments with gradients in oxygen concentration, such as the human lung.

Characterisation of a new cell wall teichoic acid produced by Listeria innocua ZM39 and analysis of its biosynthesis genes.

Listeria innocua is genetically closely related to the foodborne human pathogen Listeria monocytogenes. However, as most L. innocua strains are non-pathogenic, it has been proposed as a surrogate organism for determining the efficacy of antimicrobial strategies against L. monocytogenes. Teichoic acids are one of the three major cell wall components of Listeria, along with the peptidoglycan backbone and cell wall-associated proteins. The polymeric teichoic acids make up the majority of cell wall carbohydrates; the type of teichoic acids directly attached to the peptidoglycan are termed wall teichoic acids (WTAs). WTAs play vital physiological roles, are important virulence factors, antigenic determinants, and phage-binding ligands. The structures of the various WTAs of L. monocytogenes are well known, whereas those of L. innocua are not. In the present study, the WTA structure of L. innocua ZM39 was determined mainly by 1D and 2D NMR spectroscopy and it was found to be the following: [→4)-[α-D-GlcpNAc-(1→3)]-ß-D-GlcpNAc-(1→4)-D-Rbo-(1P→]n This structure is new with respect to all currently known Listeria WTAs and it shares structural similarities with type II WTA serovar 6a. In addition, the genome of strain L. innocua ZM39 was sequenced and the majority of putative WTA synthesis genes were identified.

Multifrequency STD NMR Unveils the Interactions of Antibiotics With Burkholderia multivorans Biofilm Exopolysaccharide.

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

Lyophilized alginate-based microspheres containing Lactobacillus fermentum D12, an exopolysaccharides producer, contribute to the strain's functionality in vitro.

Lactobacillus (Limosilactobacillus) fermentum D12 is an exopolysaccharide (EPS) producing strain whose genome contains a putative eps operon. Whole-genome analysis of D12 was performed to disclose the essential genes correlated with activation of precursor molecules, elongation and export of the polysaccharide chain, and regulation of EPS synthesis. These included the genes required for EPS biosynthesis such as epsA, B, C, D and E, also gt, wzx, and wzy and those involved in the activation of the precursor molecules galE, galT and galU. Both the biosynthesis and export mechanism of EPS were proposed based on functional annotation. When grown on MRS broth with an additional 2% w/v glucose, L. fermentum D12 secreted up to 200 mg/L of a mixture of EPSs, whose porous structure was visualized by scanning electron microscopy (SEM). Structural information obtained by 1HNMR spectroscopy together with composition and linkage analyses, suggested the presence of at least two different EPSs, a branched heteropolysaccharide containing t-Glcp and 2,6-linked Galf, and glycogen. Since recent reports showed that polysaccharides facilitate the probiotic-host interactions, we at first sought to evaluate the functional potential of L. fermentum D12. Strain D12 survived simulated gastrointestinal tract (GIT) conditions, exhibited antibacterial activity against enteropathogenic bacteria, adhered to Caco-2 cells in vitro, and as such showed potential for in vivo functionality. The EPS crude extract positively influenced D12 strain capacity to survive during freeze-drying and to adhere to extracellular matrix (ECM) proteins but did not interfere Caco-2 and mucin adherence when added at concentrations of 0.2, 0.5, and 1.0 mg/mL. Since the viable bacterial count of free D12 cells was 3 logarithmic units lower after the exposure to simulated GIT conditions than the initial count, the bacterial cells had been loaded into alginate for viability improvement. Microspheres of D12 cells, which were previously analyzed at SEM, significantly influenced their survival during freeze-drying and in simulated GIT conditions. Furthermore, the addition of the prebiotic substrates mannitol and lactulose improved the viability of L. fermentum D12 in freeze-dried alginate microspheres during 1-year storage at 4 °C compared to the control.

The development and characterization of an E. coli O25B bioconjugate vaccine.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

Oligosaccharides Derived from Tramesan: Their Structure and Activity on Mycotoxin Inhibition in Aspergillus flavus and Aspergillus carbonarius.

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental problems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, "green", approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no significant effect on mycotoxin inhibition; heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins.

Effect of O-Antigen Chain Length Regulation on the Immunogenicity of Shigella and Salmonella Generalized Modules for Membrane Antigens (GMMA).

Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, Shigella and Salmonella GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.

The biofilm of Burkholderia cenocepacia H111 contains an exopolysaccharide composed of l-rhamnose and l-mannose: Structural characterization and molecular modelling.

Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic fibrosis patients. B. cenocepacia accounts for the majority of the clinical isolates, comprising the most virulent and transmissible strains. The capacity to form biofilms is among the many virulence determinants of B. cenocepacia, a characteristic that confers enhanced tolerance to some antibiotics, desiccation, oxidizing agents, and host defenses. Exopolysaccharides are a major component of biofilm matrices, particularly providing mechanical stability to biofilms. Recently, a water-insoluble exopolysaccharide produced by B. cenocepacia H111 in biofilm was characterized. In the present study, a water-soluble exopolysaccharide was extracted from B. cenocepacia H111 biofilm, and its structure was determined by GLC-MS, NMR and ESI-MS. The repeating unit is a linear rhamno-tetrasaccharide with 50% replacement of a 3-α-L-Rha with a α-3-L-Man. [2)-α-L-Rhap-(1→3)-α-L-[Rhap or Manp]-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→]n Molecular modelling was used to obtain information about local structural motifs which could give information about the polysaccharide conformation.

Proteomic Studies of the Biofilm Matrix including Outer Membrane Vesicles of Burkholderia multivorans C1576, a Strain of Clinical Importance for Cystic Fibrosis.

Biofilms are aggregates of microbial cells encased in a highly hydrated matrix made up of self-produced extracellular polymeric substances (EPS) which consist of polysaccharides, proteins, nucleic acids, and lipids. While biofilm matrix polysaccharides are unraveled, there is still poor knowledge about the identity and function of matrix-associated proteins. With this work, we performed a comprehensive proteomic approach to disclose the identity of proteins associated with the matrix of biofilm-growing Burkholderia multivorans C1576 reference strain, a cystic fibrosis clinical isolate. Transmission electron microscopy showed that B. multivorans C1576 also releases outer membrane vesicles (OMVs) in the biofilm matrix, as already demonstrated for other Gram-negative species. The proteomic analysis revealed that cytoplasmic and membrane-bound proteins are widely represented in the matrix, while OMVs are highly enriched in outer membrane proteins and siderophores. Our data suggest that cell lysis and OMVs production are the most important sources of proteins for the B. multivorans C1576 biofilm matrix. Of note, some of the identified proteins are lytic enzymes, siderophores, and proteins involved in reactive oxygen species (ROS) scavenging. These proteins might help B. multivorans C1576 in host tissue invasion and defense towards immune system assaults.

The Exopolysaccharide Cepacian Plays a Role in the Establishment of the Paraburkholderia phymatum - Phaseolus vulgaris Symbiosis.

Paraburkholderia phymatum is a rhizobial strain that belongs to the beta-proteobacteria, a group known to form efficient nitrogen-fixing symbioses within root nodules of several legumes, including the agriculturally important common bean. The establishment of the symbiosis requires the exchange of rhizobial and plant signals such as lipochitooligosaccharides (Nod factors), polysaccharides, and flavonoids. Inspection of the genome of the competitive rhizobium P. phymatum revealed the presence of several polysaccharide biosynthetic gene clusters. In this study, we demonstrate that bceN, a gene encoding a GDP-D-mannose 4,6-dehydratase, which is involved in the production of the exopolysaccharide cepacian, an important component of biofilms produced by closely related opportunistic pathogens of the Burkholderia cepacia complex (Bcc), is required for efficient plant colonization. Wild-type P. phymatum was shown to produce cepacian while a bceN mutant did not. Additionally, the bceN mutant produced a significantly lower amount of biofilm and formed less root nodules compared to the wild-type strain with Phaseolus vulgaris as host plant. Finally, expression of the operon containing bceN was induced by the presence of germinated P. vulgaris seeds under nitrogen limiting conditions suggesting a role of this polysaccharide in the establishment of this ecologically important symbiosis.

Determination of the capsular polysaccharide structure of the Klebsiella pneumoniae ST512 representative strain KPB-1 and assignments of the glycosyltransferases functions.

Klebsiella pneumoniae strain KPB-1 was isolated in early 2011 from the pleural fluid of an inpatient admitted at an Italian hospital. It was characterized to produce the KPC-3 carbapenemase and to belong to sequence type 512, a derivative of sequence type 258 clade II characterized by the cps-2 gene cluster. The K-antigen of K. pneumoniae KPB-1 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives and 1D and 2D NMR spectroscopy of the native polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KPB-1 capsular polysaccharide: The reactions catalysed by each glycosyltransferase in the cps-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens.

GMMA and Glycoconjugate Approaches Compared in Mice for the Development of a Vaccine against Shigella flexneri Serotype 6.

Shigella infections are one of the top causes of diarrhea throughout the world, with Shigella flexneri being predominant in developing countries. Currently, no vaccines are widely available and increasing levels of multidrug-resistance make Shigella a high priority for vaccine development. The serotype-specific O-antigen moiety of Shigella lipopolysaccharide has been recognized as a key target for protective immunity, and many O-antigen based candidate vaccines are in development. Recently, the Generalized Modules for Membrane Antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Here, these two technologies are compared for a vaccine against S. flexneri serotype 6. Genetic strategies for GMMA production, conjugation approaches for linkage of the O-antigen to CRM197 carrier protein, and a large panel of analytical methods for full vaccine characterization have been put in place. In a head-to-head immunogenicity study in mice, GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel. When formulated on Alhydrogel, GMMA and glycoconjugate elicited similar levels of persistent anti-O-antigen IgG with bactericidal activity. Glycoconjugates are a well-established bacterial vaccine approach, but can be costly, particularly when multicomponent preparations are required. With similar immunogenicity and a simpler manufacturing process, GMMA are a promising strategy for the development of a vaccine against Shigella.