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.

Reactivating Immunity Primed by Acellular Pertussis Vaccines in the Absence of Circulating Antibodies: Enhanced Bacterial Control by TLR9 Rather Than TLR4 Agonist-Including Formulation.

Pertussis is still observed in many countries despite of high vaccine coverage. Acellular pertussis (aP) vaccination is widely implemented in many countries as primary series in infants and as boosters in school-entry/adolescents/adults (including pregnant women in some). One novel strategy to improve the reactivation of aP-vaccine primed immunity could be to include genetically- detoxified pertussis toxin and novel adjuvants in aP vaccine boosters. Their preclinical evaluation is not straightforward, as it requires mimicking the human situation where T and B memory cells may persist longer than vaccine-induced circulating antibodies. Toward this objective, we developed a novel murine model including two consecutive adoptive transfers of the memory cells induced by priming and boosting, respectively. Using this model, we assessed the capacity of three novel aP vaccine candidates including genetically-detoxified pertussis toxin, pertactin, filamentous hemagglutinin, and fimbriae adsorbed to aluminum hydroxide, supplemented-or not-with Toll-Like-Receptor 4 or 9 agonists (TLR4A, TLR9A), to reactivate aP vaccine-induced immune memory and protection, reflected by bacterial clearance. In the conventional murine immunization model, TLR4A- and TLR9A-containing aP formulations induced similar aP-specific IgG antibody responses and protection against bacterial lung colonization as current aP vaccines, despite IL-5 down-modulation by both TLR4A and TLR9A and IL-17 up-modulation by TLR4A. In the absence of serum antibodies at time of boosting or exposure, TLR4A- and TLR9A-containing formulations both enhanced vaccine antibody recall compared to current aP formulations. Unexpectedly, however, protection was only increased by the TLR9A-containing vaccine, through both earlier bacterial control and accelerated clearance. This suggests that TLR9A-containing aP vaccines may better reactivate aP vaccine-primed pertussis memory and enhance protection than current or TLR4A-adjuvanted aP vaccines.

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.

Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development.

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

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.