Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein. Staphylococcus aureus is a leading cause of severe bacterial infections in both hospital and community settings. Due to its increasing resistance to antibiotics, development of additional therapeutic strategies like vaccination is required to control this pathogen. Vaccination attempts against S. aureus have not been successful so far and an important reason may be the pathogen's elaborate repertoire of molecules that dampen the immune response. These evasion molecules not only suppress natural immunity but also hamper the current attempts to create effective vaccines. In this paper, we describe a novel mechanism by which S. aureus can prevent uptake by phagocytic immune cells. We discover that the secreted S. aureus protein Extracellular fibrinogen binding protein (Efb) generates a ‘capsule’-like shield around the bacterial surface through a dual interaction with the plasma proteins complement C3b and fibrinogen. The Efb-dependent fibrinogen shield masks important opsonic molecules like C3b and antibodies from binding to phagocyte receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of this anti-phagocytic shield.

Significance: Neutrophils are among the first immune cells to migrate to the site of infection and clear invading bacteria. They store large amounts of neutrophil serine proteases (NSPs) that play key roles in immune defense. Unfortunately, NSPs also contribute to tissue destruction in a variety of inflammatory disorders. In this study we discover that the pathogenic bacterium Staphylococcus aureus secretes a family of highly potent and specific NSP inhibitors that promote the pathogenicity of this bacterium in vivo. From crystallography experiments, we conclude that these proteins constitute a unique class of NSP inhibitors, which can be used to design novel treatment strategies against excessive NSP activity. Furthermore, this study significantly increases our understanding of the complex nature of S. aureus infections.

Significance: Antibodies are crucial for the immune response against bacteria. To drive bacterial killing, antibodies should bind to the bacterial cell and induce the complement reaction. This requires target-bound IgGs to form hexameric IgG platforms that are kept together by noncovalent Fc-Fc interactions. Interestingly, pathogenic bacteria produce IgG-binding molecules that bind specifically to the Fc region needed for hexamerization. Here we demonstrate that staphylococcal protein A (SpA) fromStaphylococcus aureusspecifically blocks formation of IgG hexamers and downstream activation of complement. Furthermore, we show that IgG3 antibodies (which are not recognized by SpA) have superior capacity to activate complement and induce killing ofS. aureusby human phagocytes. These insights provide a crucial rationale for optimizing antibody therapies againstS. aureus.

The immune system kills bacteria by the formation of lytic membrane attack complexes (MACs), triggered when complement enzymes cleave C5. At present, it is not understood how the MAC perturbs the composite cell envelope of Gram‐negative bacteria. Here, we show that the role of C5 convertase enzymes in MAC assembly extends beyond the cleavage of C5 into the MAC precursor C5b. Although purified MAC complexes generated from preassembled C5b6 perforate artificial lipid membranes and mammalian cells, these components lack bactericidal activity. In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium, MACs need to be assembled locally by the C5 convertase enzymes. Our data indicate that C5b6 rapidly loses the capacity to form bactericidal pores; therefore, bacterial killing requires both in situ conversion of C5 and immediate insertion of C5b67 into the membrane. Using flow cytometry and atomic force microscopy, we show that local assembly of C5b6 at the bacterial surface is required for the efficient insertion of MAC pores into bacterial membranes. These studies provide basic molecular insights into MAC assembly and bacterial killing by the immune system.