Molecular Basis for Preferential Protective Efficacy of Antibodies Directed to the Poorly Acetylated Form of Staphylococcal Poly- N -Acetyl-β-(1-6)-Glucosamine

Abstract

Poly-N-acetyl-glucosamine (PNAG) is a staphylococcal surface polysaccharide influencing biofilm formation that is also under investigation for its vaccine potential. Antibodies that bind to PNAG with either low (<15%) or high (>90%) levels of acetate are superior at opsonic and protective activity compared with antibodies that bind to PNAG with only high levels (>70%) of acetate. PNAG is synthesized by four proteins encoded within the intercellular adhesin (ica) locus icaADBC. In Staphylococcus epidermidis, icaB encodes a deacetylase needed for the surface retention of PNAG and optimal biofilm formation. In this study, we confirmed that icaB plays a similar role in Staphylococcus aureus and found that an icaB mutant of S. aureus expressed significantly less surface-associated PNAG, was highly susceptible to antibody-independent opsonic killing that could not be enhanced with antibody raised against deacetylated PNAG (dPNAG), and had reduced survival capacity in a murine model of bacteremia. In contrast, an icaB-overexpressing strain produced primarily surface-associated PNAG, was more susceptible to opsonophagocytosis with antibody to dPNAG, and had increased survival in a murine bacteremia model. The highly acetylated secreted PNAG was more effective at blocking opsonic killing mediated by a human monoclonal antibody (mAb) to native PNAG than it was at blocking killing mediated by a human mAb to dPNAG, which by itself was a more effective opsonin. Retention of dPNAG on the surface of S. aureus is key to increased survival during bacteremia and also provides a molecular mechanism explaining the superior opsonic and protective activity of antibody to dPNAG.Staphylococcus aureus and coagulase-negative staphylococci are the most frequent causes of nosocomial bloodstream infections (43). A critical virulence determinant in such infections is the production of a high-molecular-weight polymer of ␤-1-6-linked N-acetyl-glucosamine (PNAG) that is involved in adherence to polymeric substrates, bacterial intercellular adhesion, biofilm formation, and protection against antibodyindependent opsonic killing (3,4,15,26,40). Proteins encoded by the icaADBC genes of the intercellular adhesin (ica) locus synthesize PNAG (6, 10-12, 30, 31). IcaA is a trans-membrane glucosyltransferase and can synthesize short PNAG polymers in vitro using UDP-N-acetyl-glucosamine as a substrate (10). IcaD increases the biosynthetic efficiency of IcaA (10). IcaC is also a transmembrane protein and appears to be involved in linking short polymers to make longer oligomers of PNAG (10). In its mature form, PNAG, also referred to by some researchers as polysaccharide intercellular adhesin (PIA) (12,44), is a mixture of polymers such that about 10 to 20% of the amino groups are not acetylated, which could give some of the polymers within the polysaccharide complex a net positive charge (16,27). It is not known whether PNAG consists of a minority of highly deacetylated molecules mixed in with a majority of highly acetylated molecules or if there is a rang...

Structural Basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine in Gram-positive Bacteria

“…First, it is an extracellular protein (26), which eliminates the need for membrane permeable drugs. Second, it is responsible for PNAG de-N-acetylation, which is required for biofilm formation and virulence in animal models (26,27). Our recent study of IcaB was the first functional characterization of its metal-and length-dependent de-N-acetylation activity (32).…”

“…IcaB is an extracellular protein that is responsible for the partial de-N-acetylation of PNAG (26), a requirement for surface retention of the polymer. Furthermore, ⌬icaB strains of S. epidermidis and S. aureus are unable to de-N-acetylate PNAG, do not form biofilms in vitro, and have highly attenuated virulence in a murine model for an indwelling medical device-related infection and bacteremia, respectively (26,27). IcaB is a member of the family 4 carbohydrate esterases (CE4s) and has sequence homology (22% identity) to the N-terminal de-N-acetylase domain of Escherichia coli PgaB (PgaB ) (28).…”

Análise fenotípica e genotípica da virulência de Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina

“…Entre estes fatores está a produção de um mucopolissacarídeo extracelular ("slime"), que parece ajudar na aderência e colonização do microrganismo ao epitélio glandular mamário. A habilidade dos S. aureus aderirem à superfície do epitélio tem sido associado à produção de biofilmes, que são descritos como aglomerações de células embebidas em matriz heterogênea extracelular, resultando em estruturas tridimensionais com características fisiológicas específicas (Cerca et al 2007, Gad et al 2009). …”

Active and Passive Immunization Against Staphylococcus aureus Periprosthetic Osteomyelitis in Rats

“…In other studies, investigators immunized mice, rabbits and goats with either native PNAG, containing >90% acetate substituents on the amino groups in the glucosamine molecule, or dPNAG, wherein the acetate substitution is <20%, conjugated to diphtheria toxoid (20). This study and another (22) indicated that antibodies to dPNAG, but not native PNAG, are opsonic and provide protection against experimental S. aureus infection. Additional studies using synthetic oligosaccharides of PNAG and dPNAG conjugated to carrier proteins confirmed the need to use only non-acetylated glucosamines for vaccination to achieve protective immunity (14,21), most likely via opsonic killing of bacteria.…”

“…PNAG, also known as intercellular adhesion polysaccharide (20), is a surface polymer produced by both S. aureus and S. epidermis, and many other pathogens (21). PNAG promotes biofilm formation and enhances staphylococcal virulence in mouse infection models (12,22). In other studies, investigators immunized mice, rabbits and goats with either native PNAG, containing >90% acetate substituents on the amino groups in the glucosamine molecule, or dPNAG, wherein the acetate substitution is <20%, conjugated to diphtheria toxoid (20).…”