Supplementary Material for: The TLR2 Antagonist Staphylococcal Superantigen-Like Protein 3 Acts as a Virulence Factor to Promote Bacterial Pathogenicity in vivo

Toll-like receptor (TLR) signaling is important in the initiation of immune responses and subsequent instigation of adaptive immunity. TLR2 recognizes bacterial lipoproteins and plays a central role in the host defense against bacterial infections, including those caused by <i>Staphylococcus aureus</i>. Many studies have demonstrated the importance of TLR2 in murine <i>S. aureus</i> infection. <i>S. aureus</i> evades TLR2 activation by secreting two proteins, staphylococcal superantigen-like protein 3 (SSL3) and 4 (SSL4). In this study, we demonstrate that antibodies against SSL3 and SSL4 are found in healthy individuals, indicating that humans are exposed to these proteins during <i>S. aureus</i> colonization or infection. To investigate the TLR2-antagonistic properties of SSL3 and SSL4, we compared the infection with wild-type and SSL3/4 knockout <i>S. aureus</i> strains in an intravenous murine infection model. Direct evaluation of the contribution of SSL3/4 to infection pathogenesis was hindered by the fact that the SSLs were not expressed in the murine system. To circumvent this limitation, an SSL3-overproducing strain (pLukM-SSL3) was generated, resulting in constitutive expression of SSL3. pLukM-SSL3 exhibited increased virulence compared to the parental strain in a murine model that was found to be TLR2 dependent. Altogether, these data indicate that SSL3 contributes to <i>S. aureus</i> virulence in vivo.