Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Abstract

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

“…To our knowledge, our structures are the first atomic descriptions of antibodies in complex with an epitope from the Gram-positive restricted teichoic acid family of polysaccharides. Wall teichoic acid is a major component of the cell wall structure of S. aureus , 39 and along with PNAG 22 and the glycosylated SD repeats (SDRs) of adhesive factors such as ClfA, 8 is a key exposed epitope on the surface of the bacteria. Importantly, we showed by immunoradiometric assay that each S. aureus cell exposes between 20,000 and 30,000 binding sites for each of our anti-WTA antibodies (Figure 3), highlighting the abundance of the WTA epitope at the S. aureus surface.…”

“…Antibodies that bind S. aureus , and more specifically the S. aureus -specific GlcNAc modifications of wall teichoic acid and SDR-containing proteins, have been shown to comprise a significant percentage of the human serum IgG content, even from healthy normal donors. 8,33 Given S. aureus is a known human commensal that often colonizes the nostrils and skin, the anti-WTA antibody repertoire likely represents an important component of the human immune system to control S. aureus colonization and infection. 40 …”

“…Protein A-deficient S. aureus USA300 mutant strains lacking tarM or tarS were described previously. 8,33 The protein A-deficient S. aureus strain was used to prevent protein A-mediated antibody binding and enable analysis of WTA-specific antibody binding.…”

“…5 The recent increase in MDR infections has revitalized interest in antibody-based approaches to antibiotic discovery, 6 with molecules targeting both Gram-positive and Gram-negative pathogens an active area of discovery. The use of animal serum containing antibodies that target bacterial antigens was a common therapeutic approach pre-dating the development of small molecule antibiotics, 7 but technological advances in antibody discovery, including B-cell cloning from human patients, 8 has invigorated research into monoclonal antibody (mAb)-based antibiotics. Anti-infective biologics with novel mechanisms of action targeting S. aureus alpha toxin, 9,10 protective antigen of Bacillus anthracis , 11 toxins A and B from Clostridium difficile , 12 and Pseudomonas aeruginosa cell wall components 13,14 are currently approved or in clinical development.…”

“…20 In addition, CPs are an important component of several bacterial vaccines, 21 and opsonizing antibodies targeting polysaccharides have been investigated as therapeutic agents for Burkholderia dolosa 22 and S. aureus . 8,22 …”