Bacterial capsular polysaccharides with antibiofilm activity share common biophysical and electrokinetic properties.

Bacterial biofilms are surface-attached communities that are difficult to eradicate due to a high tolerance to antimicrobial agents. The use of non-biocidal surface-active compounds to prevent the initial adhesion and aggregation of bacterial pathogens is a promising alternative to antibiotic treatments and several antibiofilm compounds have been identified, including some capsular polysaccharides released by various bacteria. However, the lack of chemical and mechanistic understanding of the activity of these polymers limits their use to control biofilm formation. Here, we screen a collection of 31 purified capsular polysaccharides and first identify seven new compounds with non-biocidal activity against Escherichia coli and/or Staphylococcus aureus biofilms. We measure and theoretically interpret the electrophoretic mobility of a subset of 21 capsular polysaccharides under applied electric field conditions, and we show that active and inactive polysaccharide polymers display distinct electrokinetic properties and that all active macromolecules share high intrinsic viscosity features. Despite the lack of specific molecular motif associated with antibiofilm properties, the use of criteria including high density of electrostatic charges and permeability to fluid flow enables us to identify two additional capsular polysaccharides with broad-spectrum antibiofilm activity. Our study therefore provides insights into key biophysical properties discriminating active from inactive polysaccharides. The characterization of a distinct electrokinetic signature associated with antibiofilm activity opens new perspectives to identify or engineer non-biocidal surface-active macromolecules to control biofilm formation in medical and industrial settings.

Glycosylation of Staphylococcus aureus cell wall teichoic acid is influenced by environmental conditions.

Wall teichoic acid (WTA) are major constituents of Staphylococcus aureus (S. aureus) cell envelopes with important roles in the bacteria's physiology, resistance to antimicrobial molecules, host interaction, virulence and biofilm formation. They consist of ribitol phosphate repeat units in which the ribitol residue is substituted with D-alanine (D-Ala) and N-acetyl-D-glucosamine (GlcNAc). The complete S. aureus WTA biosynthesis pathways was recently revealed with the identification of the two glycosyltransferases, TarM and TarS, respectively responsible for the α- and ß-GlcNAc anomeric substitutions. We performed structural analyses to characterize WTAs from a panel of 24 S. aureus strains responsible for invasive infections. A majority of the S. aureus strains produced the ß-GlcNAc WTA form in accordance with the presence of the tarS gene in all strains assessed. The ß-GlcNAc anomer was preferentially expressed at the expense of the α-GlcNAc anomer when grown on stress-inducing culture medium containing high NaCl concentration. Furthermore, WTA glycosylation of the prototype S. aureus Newman strain was characterized in vivo in two different animal models, namely peritonitis and deep wound infection. While the inoculum used to infect animals produced almost exclusively α-GlcNAc WTA, a complete switch to ß-glycosylation was observed in infected kidneys, livers and muscles. Overall, our data demonstrate that S. aureus WTA glycosylation is strongly influenced by environmental conditions and suggest that ß-GlcNAc WTA may bring competitive advantage in vivo.

The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures.

Aldehyde detoxification is a process used to convert toxin into toxoid for vaccine applications. In the case of tetanus toxin (TT), formaldehyde is used to obtain the tetanus toxoid (TTd), which is used either for the tetanus vaccine or as carrier protein in conjugate vaccines. Several studies have already been conducted to better understand the exact mechanism of this detoxification. Those studies led to the identification of a number of formaldehyde-induced modifications on lab scale TTd samples. To obtain greater insights of the changes induced by formaldehyde, we used three industrial TTd batches to identify repeatable modifications in the detoxification process. Our strategy was to combine seven analytical tools to map these changes. Mass spectrometry (MS), colorimetric test and amino acid analysis (AAA) were used to study modifications on amino acids. SDS-PAGE, asymmetric flow field flow fractionation (AF4), fluorescence spectroscopy and circular dichroism (CD) were used to study formaldehyde modifications on the whole protein structure. We identified 41 formaldehyde-induced modifications across the 1315 amino acid primary sequence of TT. Of these, five modifications on lysine residues were repeatable across TTd batches. Changes in protein conformation were also observed using SDS-PAGE, AF4 and CD techniques. Each analytical tool brought a piece of information regarding formaldehyde induced-modifications, and all together, these methods provided a comprehensive overview of the structural changes that occurred with detoxification. These results could be the first step leading to site-directed TT mutagenesis studies that may enable the production of a non-toxic equivalent protein without using formaldehyde.

Determination of molecular size parameters and quantification of polyacrylic acid by high performance size-exclusion chromatography with triple detection.

Synthetic polyelectrolytes are a broad class of vaccine adjuvants. Among them, polyacrylic acid (PAA), a polyanionic polymer, is currently evaluated by Sanofi Pasteur. As chain length is considered to be a critical quality attribute for adjuvant properties of PAA, measurement of precise and accurate molecular size parameters is important for these polymers. In the field of synthetic polymer chemistry, methods for determination of molecular size parameters are well defined. Specifically, high performance size-exclusion chromatography (HPSEC) with multi-detection system is a method of choice. This paper describes the development of HPSEC method to well characterize and precisely quantify PAA in different adjuvant formulations. A first set of characterizations were made, with determination of dn/dc coefficient, which enabled the determination of weight- and number-average molecular weight, viscosimetric radius, and intrinsic viscosity. In-depth characterization was also made with branching study through the use of Mark-Houwink parameter determination. The quantification method was also evaluated according to validation method-like criteria: limit of detection and limit of quantification, repeatability, accuracy, and specificity with recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigen.

Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions.

Characterization and immunogenicity in mice of recombinant influenza haemagglutinins produced in Leishmania tarentolae.

The membrane displayed antigen haemagglutinin (HA) from several influenza strains were expressed in the Leishmania tarentolae system. This non-conventional expression system based on a parasite of lizards, can be readily propagated to high cell density (>10(8)cells/mL) in a simple incubator at 26°C. The genes encoding HA proteins were cloned from six influenza strains, among these being a 2009 A/H1N1 pandemic strain from swine origin, namely A/California/07/09(H1N1). Soluble HA proteins were secreted into the cell culture medium and were easily and successfully purified via a His-Tag domain fused to the proteins. The overall process could be conducted in less than 3 months and resulted in a yield of approximately 1.5-5mg of HA per liter of biofermenter culture after purification. The recombinant HA proteins expressed by L. tarentolae were characterized by dynamic light scattering and were observed to be mostly monomeric. The L. tarentolae recombinant HA proteins were immunogenic in mice at a dose of 10µg when administered twice with an oil-in-water emulsion-based adjuvant. These results suggest that the L. tarentolae expression system may be an alternative to the current egg-based vaccine production.

Development and validation of high-performance size exclusion chromatography methods to determine molecular size parameters of Haemophilus influenzae type b polysaccharides and conjugates.

Current vaccines against Haemophilus influenzae type b (Hib) consist of the polyribosyl ribitol phosphate (PRP) capsular polysaccharide chemically conjugated to a carrier protein. Among the various biological and physical analyses to be performed on these vaccines, the determination of the molecular size of the polysaccharide preparations throughout the conjugation process is particularly relevant. Comparison of results from high-performance size exclusion chromatography (HPSEC) with those routinely obtained using conventional gel permeation chromatography (CGPC) methods highlights the correlation between the two methods for determining the values of the chromatographic distribution coefficient (KD) of native and activated polysaccharides. The resulting data showed that the KD value is sufficient to characterize these polysaccharides using an HPSEC method. However, additional molecular size parameters (i.e., molar mass and hydrodynamic radius) are necessary for a reliable characterization of the tetanus conjugate (PRP-T), certainly due to the lattice-like structure of the conjugate. In practice, an absolute detection system in HPSEC composed of a low-angle light scattering detector, a viscometer, and a refractive index (RI) detector was used. As demonstrated, these HPSEC methods are rapid, accurate, and reproducible for the polysaccharides and their glycoconjugates and provide a relevant and more informative alternative to the current CGPC methods.

Genetic and structural characterization of L11 lipooligosaccharide from Neisseria meningitidis serogroup A strains.

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for alpha-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.

Development of a high-resolution magic-angle spinning nuclear magnetic resonance identity assay of the capsular polysaccharide from Haemophilus influenzae type b present in cetavlon precipitate.

We describe the use of high-resolution magic-angle spinning nuclear magnetic resonance to control the identity of the capsular polysaccharide from Haemophilus influenzae type b (Hib) present in the cetavlon precipitate. This step is one of the earliest in the purification of this polysaccharide, which is further used in the production of Hib polysaccharide-protein conjugate vaccine. The effects of sample procedure and magnetic field strength have been investigated. Since this assay is rapid and simple, it may represent a useful technique for characterization of polysaccharides present in complex and insoluble matrices. Moreover, it allows a rapid evaluation of the structure of the produced polysaccharides very early on during the production process and is as such an essential analytical tool before starting the purification process.

The teichoic acid (C-polysaccharide) synthesized by Streptococcus pneumoniae serotype 5 has a specific structure.

The teichoic acid synthesized by Streptococcus pneumoniae serotype 5, also known as pneumococcal common antigen (C-polysaccharide), was purified. On the basis of compositional analysis, HPAEC-PAD analysis, MALDI-TOF mass spectrometry and NMR spectroscopy, made on the native polysaccharide and on the dephosphorylated repeating unit, the following structure is proposed: [structure: see text]. This C-polysaccharide (C-PS), differs from those previously described by the replacement of Glc by Gal in its repeating unit structure.

High affinity binding of long-chain polysialic acid to antibody, and modulation by divalent cations and polyamines.

Long-chain polysialic acid (PSA) is expressed on the vertebrate neural cell adhesion molecule (NCAM) during neuronal plasticity. Its structural similarity to the capsular PSAs of some pathogenic bacteria has hampered the development of polysaccharide vaccines against meningitis. The antibodies formed during immunization require a long epitope for binding, and cross-react with host tissue PSA. The nature of the epitope and possible external effectors involved are still unclear. We have evaluated the interaction of PSA with its antibody mAb735 by surface plasmon resonance. The influences of PSA chain length, pH, temperature, ionic environment, and polyamines were also determined. The antibody binding affinity was found to dramatically increase with PSA chain length. A sub-nanomolar dissociation constant (K(D)=8.5 x 10(-10)M) was obtained for the binding of very long chain native MenB polysaccharides (approximately 200 Neu5Ac-residues). Colominic acid from Escherichia coli K1 (approximately 100 residues) and shorter polymers exhibited progressively weaker affinities. The antibody also bound tightly (K(D) approximately 5 x 10(-9)M) to polysialylated glycopeptides from human embryonal brain. The effects of pH and ionic strength suggested that the interaction is largely electrostatic. Ca2+ and Mn2+ ions promoted the observed surface plasmon resonance response in a concentration dependent fashion. Spermine increased the response in a similar way. Our results suggest that divalent cations and polyamines may play significant role in the regulation of the PSA epitope presentation in vivo.

Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates.

A method, using high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD), has been developed to determine the concentrations of Streptococcus pneumoniae capsular polysaccharides and polysaccharide conjugates used in formulating a conjugate vaccine for the prevention of pneumococcal infections. In an effort to determine optimum hydrolysis conditions for the analysis, pneumococcal polysaccharides were subjected to three different hydrolysis methods: trifluoroacetic acid (TFA) hydrolysis, methanolysis followed by TFA hydrolysis, or hydrofluoric acid (HF) hydrolysis followed by TFA hydrolysis. For quantification purposes, best results were obtained by methanolysis followed by TFA hydrolysis for uronic acid-containing polysaccharides, and by TFA hydrolysis for all the others. For the quantification of all the polysaccharides (from native to conjugated forms), a monosaccharide reference mixture (Rha, Gal and GlcA) hydrolyzed along with the samples can be used as standards for routine analysis. This is much more convenient than to hydrolyze a well-characterized reference polysaccharide (necessary standard only for type 1 capsular polysaccharide). This method is rapid, very sensitive (less than 10 microg of polysaccharide is required), and may replace advantageously the currently used colorimetric assays used to determine polysaccharides content. Moreover, it can be readily adapted for use with other bacterial polysaccharide preparations as well.

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.