Surface Glycopolymers Are Crucial for In Vitro Anti-Wall Teichoic Acid IgG-Mediated Complement Activation and Opsonophagocytosis of Staphylococcus aureus.

The cell envelopes of many Gram-positive bacteria contain wall teichoic acids (WTAs). Staphylococcus aureus WTAs are composed of ribitol phosphate (RboP) or glycerol phosphate (GroP) backbones substituted with D-alanine and N-acetyl-D-glucosamine (GlcNAc) or N-acetyl-D-galactosamine (GalNAc). Two WTA glycosyltransferases, TarM and TarS, are responsible for modifying the RboP WTA with α-GlcNAc and ß-GlcNAc, respectively. We recently reported that purified human serum anti-WTA IgG specifically recognizes ß-GlcNAc of the staphylococcal RboP WTA and then facilitates complement C3 deposition and opsonophagocytosis of S. aureus laboratory strains. This prompted us to examine whether anti-WTA IgG can induce C3 deposition on a diverse set of clinical S. aureus isolates. To this end, we compared anti-WTA IgG-mediated C3 deposition and opsonophagocytosis abilities using 13 different staphylococcal strains. Of note, the majority of S. aureus strains tested was recognized by anti-WTA IgG, resulting in C3 deposition and opsonophagocytosis. A minority of strains was not recognized by anti-WTA IgG, which correlated with either extensive capsule production or an alteration in the WTA glycosylation pattern. Our results demonstrate that the presence of WTAs with TarS-mediated glycosylation with ß-GlcNAc in clinically isolated S. aureus strains is an important factor for induction of anti-WTA IgG-mediated C3 deposition and opsonophagocytosis.

Human SAP is a novel peptidoglycan recognition protein that induces complement-independent phagocytosis of Staphylococcus aureus.

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer--a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an FcγR-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain-like receptors.

Glycoepitopes of staphylococcal wall teichoic acid govern complement-mediated opsonophagocytosis via human serum antibody and mannose-binding lectin.

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or ß-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, ß-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ß-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact ß-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-ß-GlcNAc-specific WTA IgGs, anti-ß-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.

Specific serum Ig recognizing staphylococcal wall teichoic acid induces complement-mediated opsonophagocytosis against Staphylococcus aureus.

Wall teichoic acid (WTA) of Staphylococcus aureus is a major cell envelope-associated glycopolymer that is a key molecule in promoting colonization during S. aureus infection. The complement system plays a key role in the opsonization and clearance of pathogens. We recently reported that S. aureus WTA functions as a ligand of human serum mannose-binding lectin (MBL), a recognition molecule of the lectin complement pathway. Intriguingly, serum MBL in adults does not bind to WTA because of an inhibitory effect of serum anti-WTA-IgG. In this study, serum anti-WTA-IgG was purified to homogeneity using a purified S. aureus WTA-coupled affinity column to examine the biological function of human anti-WTA-IgG. The purified anti-WTA-IgG contained the IgG2 subclass as a major component and specifically induced C4 and C3 deposition on the S. aureus surface in the anti-WTA-IgG-depleted serum, but not in C1q-deficient serum. Furthermore, the anti-WTA-IgG-dependent C3 deposition induced phagocytosis of S. aureus cells by human polymorphonuclear leukocytes. These results demonstrate that serum anti-WTA-IgG is a real trigger for the induction of classical complement-dependent opsonophagocytosis against S. aureus. Our results also support the fact that a lack of the lectin complement pathway in MBL-deficient adults is compensated by Ag-specific, Ab-mediated adaptive immunity.

Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells.

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca2(+) release from intracellular Ca2(+) stores and anti-PGN-IgG dependent Ca2(+) mobilization is controlled via a phospholipase Cγ-2-mediated pathway.

Intradermal immunization with wall teichoic acid (WTA) elicits and augments an anti-WTA IgG response that protects mice from methicillin-resistant Staphylococcus aureus infection independent of mannose-binding lectin status.

The objectives of this study were to investigate the immune response to intradermal immunization with wall teichoic acid (WTA) and the effect of MBL deficiency in a murine model of infection with methicillin-resistant Staphylococcus aureus (MRSA). WTA is a bacterial cell wall component that is implicated in invasive infection. We tested susceptibility to MRSA infection in wild type (WT) and MBL deficient mice using two strains of MRSA: MW2, a community-associated MRSA (CA-MRSA); and COL, a healthcare-associated MRSA (HA-MRSA). We also performed in vitro assays to investigate the effects of anti-WTA IgG containing murine serum on complement activation and bacterial growth in whole blood. We found that MBL knockout (KO) mice are relatively resistant to a specific MRSA strain, MW2 CA-MRSA, compared to WT mice, while both strains of mice had similar susceptibility to a different strain, COL HA-MRSA. Intradermal immunization with WTA elicited and augmented an anti-WTA IgG response in both WT and MBL KO mice. WTA immunization significantly reduced susceptibility to both MW2 CA-MRSA and COL HA-MRSA, independent of the presence of MBL. The protective mechanisms of anti-WTA IgG are mediated at least in part by complement activation and clearance of bacteria from blood. The significance of these findings is that 1) Intradermal immunization with WTA induces production of anti-WTA IgG; and 2) This anti-WTA IgG response protects from infection with both MW2 CA-MRSA and COL HA-MRSA even in the absence of MBL, the deficiency of which is common in humans.