Supplementary Material for: <i>Staphylococcus aureus</i> Virulence Is Enhanced by Secreted Factors That Block Innate Immune Defenses

<i>Staphylococcus aureus</i> is a leading human pathogen that causes a large variety of diseases. In vitro studies have shown that <i>S. aureus</i> secretes several small proteins that block specific elements of the host innate immune system, but their role in bacterial pathogenicity is unknown. For instance, the extracellular complement-binding protein (Ecb) impairs complement activation by binding to the C3d domain of C3. Its homolog, the extracellular fibrinogen-binding protein (Efb), is known to block both complement activation and neutrophil adhesion to fibrinogen. Here, we show that targeted inactivation of the genes encoding Ecb and Efb strongly attenuates <i>S. aureus</i> virulence in a murine infection model: mice experienced significantly higher mortality rates upon intravenous infection with wild-type bacteria (79%) than with an isogenic ΔEcbΔEfb mutant (21%). In addition, Ecb and Efb are both required for staphylococcal persistence in host tissues and abscess formation in the kidneys (27% for wild-type vs. 7% for the ΔEcbΔEfb mutant). During staphylococcal pneumonia, Ecb and Efb together promote bacterial survival in the lungs (p = 0.03) and block neutrophil influx into the lungs. Thus, Ecb and Efb are essential to <i>S. aureus</i> virulence in vivo and could be attractive targets in future vaccine development efforts.