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.

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.

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.

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.

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.

Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling.

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

Multi-technique microscopy investigation on bacterial biofilm matrices: a study on Klebsiella pneumoniae clinical strains.

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.

Fluorescence and NMR spectroscopy together with molecular simulations reveal amphiphilic characteristics of a Burkholderia biofilm exopolysaccharide.

Biofilms are a collective mode of bacterial life in which a self-produced matrix confines cells in close proximity to each other. Biofilms confer many advantages, including protection from chemicals (including antibiotics), entrapment of useful extracellular enzymes and nutrients, as well as opportunities for efficient recycling of molecules from dead cells. Biofilm matrices are aqueous gel-like structures composed of polysaccharides, proteins, and DNA stabilized by intermolecular interactions that may include non-polar connections. Recently, polysaccharides extracted from biofilms produced by species of the Burkholderia cepacia complex were shown to possess clusters of rhamnose, a 6-deoxy sugar with non-polar characteristics. Molecular dynamics simulations are well suited to characterizing the structure and dynamics of polysaccharides, but only relatively few such studies exist of their interaction with non-polar molecules. Here we report an investigation into the hydrophobic properties of the exopolysaccharide produced by Burkholderia multivorans strain C1576. Fluorescence experiments with two hydrophobic fluorescent probes established that this polysaccharide complexes hydrophobic species, and NMR experiments confirmed these interactions. Molecular simulations to model the hydrodynamics of the polysaccharide and the interaction with guest species revealed a very flexible, amphiphilic carbohydrate chain that has frequent dynamic interactions with apolar molecules; both hexane and a long-chain fatty acid belonging to the quorum-sensing system of B. multivorans were tested. A possible role of the non-polar domains of the exopolysaccharide in facilitating the diffusion of aliphatic species toward specific targets within the biofilm aqueous matrix is proposed.

The spent culture supernatant of Pseudomonas syringae contains azelaic acid.

BACKGROUND: Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA. RESULTS: A secreted metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid called azelaic acid. Azelaic acid, which is a nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as a mobile signal that primes systemic defenses. In addition, its structure,(which is associated with fatty acid biosynthesis) is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). For these reason it could be acting as s signal molecule. Analytical and structural studies by NMR spectroscopy confirmed that in PSA spent supernatants azelaic acid was present. Quantification studies further revealed that 20 µg/L of were present and was also found in the spent supernatants of several other P. syringae pathovars. The RNAseq transcriptome study however did not determine whether azelaic acid could behave as a QS molecule. CONCLUSIONS: This study reports of the possible natural biosynthesis of azelaic acid by bacteria. The production of azelaic acid by P. syringae pathovars can be associated with plant-bacteria signaling.

Investigation on Sugar-Protein Connectivity in Salmonella O-Antigen Glycoconjugate Vaccines.

Invasive nontyphoidal Salmonella disease, for which licensed vaccines are not available, is a leading cause of bloodstream infections in Africa. The O-antigen portion of lipopolysaccharide is a good target for protective immunity. Covalent conjugation of the O-antigen to a carrier protein increases its immunogenicity and O-antigen based glycoconjugate vaccines are currently under investigation at the preclinical stage. We developed a conjugation chemistry for linking O-antigen to CRM197 carrier protein, through sequential insertion of adipic acid dihydrazide (ADH) and adipic acid bis( N-hydroxysuccinimide) ester (SIDEA) as linkers, without impacting O-antigen chain epitopes. Here the resulting sugar-protein connectivity has been investigated in detail. The core portion of the lipopolysaccharide was used as a model molecule to prepare CRM197 conjugates, making structural investigations easier. The first step of reductive amination with ADH involves the terminal 3-deoxy-d- manno-oct-2-ulosonic acid (KDO) residue of the core region. The second reaction step resulted not to be selective, as SIDEA reacted with both ADH and pyrophosphorylethanolamine (PPEtN) of the core region, independently from the pH at which the reaction was performed. Peptide mapping analysis of the deglycosylated core-CRM197 conjugates confirmed that lysine residues of CRM197 were linked to SIDEA not only through KDO-ADH but also through PPEtN. This analysis also confirmed that the conjugation chemistry is random on the protein, involving a large number of lysine residues, particularly the surface exposed ones. The method for core-CRM197 characterization was successfully extended to O-antigen-CRM197 conjugate, confirming the results obtained with the core. This study not only allowed full characterization of OAg-CRM197 conjugates, but can be applied to optimize synthesis and characterization of other OAg-based glycoconjugate vaccines. Analytical methods to investigate saccharide-protein connectivity are also of fundamental importance to study the relationship between glycoconjugate structure and immune response induced.

Influence of Bacterial Biofilm Polysaccharide Structure on Interactions with Antimicrobial Peptides: A Study on Klebsiella pneumoniae.

Balneotherapy is a clinically effective complementary approach in the treatment of low-grade inflammation- and stress-related pathologies. The biological mechanisms by which immersion in mineral-medicinal water and the application of mud alleviate symptoms of several pathologies are still not completely understood, but it is known that neuroendocrine and immunological responses­including both humoral and cell-mediated immunity­to balneotherapy are involved in these mechanisms of effectiveness; leading to anti-inflammatory, analgesic, antioxidant, chondroprotective, and anabolic effects together with neuroendocrine-immune regulation in different conditions. Hormesis can play a critical role in all these biological effects and mechanisms of effectiveness. The hormetic effects of balneotherapy can be related to non-specific factors such as heat­which induces the heat shock response, and therefore the synthesis and release of heat shock proteins­and also to specific biochemical components such as hydrogen sulfide (H2S) in sulfurous water and radon in radioactive water. Results from several investigations suggest that the beneficial effects of balneotherapy and hydrotherapy are consistent with the concept of hormesis, and thus support a role for hormesis in hydrothermal treatments.

Biofilms produced by Burkholderia cenocepacia: influence of media and solid supports on composition of matrix exopolysaccharides.

Bacteria usually grow forming biofilms, which are communities of cells embedded in a self-produced dynamic polymeric matrix, characterized by a complex three-dimensional structure. The matrix holds cells together and above a surface, and eventually releases them, resulting in colonization of other surfaces. Although exopolysaccharides (EPOLs) are important components of the matrix, determination of their structure is usually performed on samples produced in non-biofilm conditions, or indirectly through genetic studies. Among the Burkholderia cepacia complex species, Burkholderia cenocepacia is an important pathogen in cystic fibrosis (CF) patients and is generally more aggressive than other species. In the present investigation, B. cenocepacia strain BTS2, a CF isolate, was grown in biofilm mode on glass slides and cellulose membranes, using five growth media, one of which mimics the nutritional content of CF sputum. The structure of the matrix EPOLs was determined by 1H-NMR spectroscopy, while visualization of the biofilms on glass slides was obtained by means of confocal laser microscopy, phase-contrast microscopy and atomic force microscopy. The results confirmed that the type of EPOLs biosynthesized depends both on the medium used and on the type of support, and showed that mucoid conditions do not always lead to significant biofilm production, while bacteria in a non-mucoid state can still form biofilm containing EPOLs.

Limosilactobacillus fermentum strains MC1 and D12: Functional properties and exopolysaccharides characterization.

Lactic acid bacteria (LAB) produce a broad spectrum of exopolysaccharides (EPSs), commonly used as texturizers in food products. Due to their potential contribution to LAB probiotic properties, like adhesion to human epithelial cells and competitive exclusion of pathogens from human intestinal epithelial cells, this study was focussed on the structural and functional characterization of the EPSs produced by two Limosilactobacillus fermentum strains - MC1, originating from mother's milk, and D12, autochthonous from Croatian smoked fresh cheese. Whole-genome sequencing and functional annotation of both L. fermentum strains by RAST server revealed the genes involved in EPS production and transport, with some differences in functionally related genes. EPSs were extracted from the cell surface of both bacterial strains and purified by size-exclusion chromatography. Structural characterization of the EPSs, achieved by chemical analyses and 1D and 2D NMR spectroscopy, showed that both strains produce an identical mixture of three different EPSs containing galactofuranose and glucopyranose residues. However, a comparison of the functional properties showed that the MC1 strain adhered better to the Caco-2 cell line and exhibited stronger antimicrobial effect against Salmonella enterica serovar Typhimurium FP1 than the D12 strain, which may be attributed to the potential bacteriocin activity of the MC1 strain.

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.

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.

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.

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.

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.