Poly-N-Acetylglucosamine Expression by Wild-Type Yersinia pestis Is Maximal at Mammalian, Not Flea, Temperatures

Numerous bacteria, including Yersinia pestis, express the poly-N-acetylglucosamine (PNAG) surface carbohydrate, a major component of biofilms often associated with a specific appearance of colonies on Congo red agar. Biofilm formation and PNAG synthesis by Y. pestis have been reported to be maximal at 21 to 28°C or “flea temperatures,” facilitating the regurgitation of Y. pestis into a mammalian host during feeding, but production is diminished at 37°C and thus presumed to be decreased during mammalian infection. Most studies of PNAG expression and biofilm formation by Y. pestis have used a low-virulence derivative of strain KIM, designated KIM6+, that lacks the pCD1 virulence plasmid, and an isogenic mutant without the pigmentation locus, which contains the hemin storage genes that encode PNAG biosynthetic proteins. Using confocal microscopy, fluorescence-activated cell sorter analysis and growth on Congo red agar, we confirmed prior findings regarding PNAG production with the KIM6+ strain. However, we found that fully virulent wild-type (WT) strains KIM and CO92 had maximal PNAG expression at 37°C, with lower PNAG production at 28°C both in broth medium and on Congo red agar plates. Notably, the typical dark colony morphology appearing on Congo red agar was maintained at 28°C, indicating that this phenotype is not associated with PNAG expression in WT Y. pestis. Extracts of WT sylvatic Y. pestis strains from the Russian Federation confirmed the maximal expression of PNAG at 37°C. PNAG production by WT Y. pestis is maximal at mammalian and not insect vector temperatures, suggesting that this factor may have a role during mammalian infection

Synthesis and Evaluation of a Conjugate Vaccine Composed of Staphylococcus aureus Poly-N-Acetyl-Glucosamine and Clumping Factor A

The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA) and deacetylated poly-N-β-(1–6)-acetyl-glucosamine (dPNAG). Mice immunized with 1, 5 or 10 µg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA) goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to augment the OPK and protective activities of antibodies to the PNAG cell surface polysaccharide

A Poly-N-Acetylglucosamine−Shiga Toxin Broad-Spectrum Conjugate Vaccine for Shiga Toxin-Producing Escherichia coli

ABSTRACT Many pathogens produce the β-(1−6)-linked poly-N-acetylglucosamine (PNAG) surface polysaccharide that is being developed as a broadly protective antimicrobial vaccine. However, it is unknown whether systemically injected PNAG vaccines or antibodies would provide protective immunity against pathogens confined to the gastrointestinal tract such as Shiga toxin (Stx)-producing Escherichia coli (STEC), an important group of gastrointestinal (GI) pathogens for which effective immunotherapeutics are lacking. To ascertain whether systemic IgG antibody to PNAG impacts this infectious situation, a vaccine consisting of a synthetic nonamer of nonacetylated PNAG, 9GlcNH2, conjugated to the Shiga toxin 1b subunit (9GlcNH2-Stx1b) was produced. Rabbit antibodies raised to the conjugate vaccine were tested for bacterial killing and toxin neutralization in vitro and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including E. coli O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups

USA300 and USA500 Clonal Lineages of Staphylococcus aureus Do Not Produce a Capsular Polysaccharide Due to Conserved Mutations in the cap5 Locus

ABSTRACT The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP− USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates

Extended-spectrum antibodies protective against carbapenemase-producing Enterobacteriaceae

International audienc

Vaccination of yearling horses against poly-N-acetyl glucosamine fails to protect against infection with Streptococcus equi subspecies equi.

Strangles is a common disease of horses with worldwide distribution caused by the bacterium Streptococcus equi subspecies equi (SEE). Although vaccines against strangles are available commercially, these products have limitations in safety and efficacy. The microbial surface antigen β 1→6 poly-N-acetylglucosamine (PNAG) is expressed by SEE. Here we show that intramuscular (IM) injection alone or a combination of IM plus intranasal (IN) immunization generated antibodies to PNAG that functioned to deposit complement and mediate opsonophagocytic killing of SEE ex vivo. However, immunization strategies targeting PNAG either by either IM only injection or a combination of IM and IN immunizations failed to protect yearling horses against infection following contact with infected horses in an experimental setting. We speculate that a protective vaccine against strangles will require additional components, such as those targeting SEE enzymes that degrade or inactivate equine IgG