Figure 5: PSMα peptides do not activate TLR2 directly but are required for the release of lipoproteins from S. aureus.

Synthetic PSMs do not stimulate IL-8 secretion by untransfected or TLR2-transfected HEK cells (a). PSMs do not affect the capacity of S. aureus USA300 cell lysates to induce IL-8 in HEK-TLR2 (b) but are required for efficient release of the major lipoprotein SitC with a C-terminal His tag as shown by western blotting of equal amounts of culture filtrates of USA300 wild-type, Δα, and Δα,β,hld (c, Supplementary Fig. 7). Inactivation of Agr or absence of PSMs leads to drastically altered Coomassie Blue-stained exoprotein patterns and reduced protein content in culture filtrates (d,e). Amounts of cytoplasmic proteins are also strongly reduced in USA300 PSM and Agr mutant exoproteomes compared with the wild-type strain (f). In contrast, signal peptide-bearing secreted proteins are not affected by deletion of PSMα peptides but the amount of this group of proteins is reduced in culture filtrates of the Agr mutant, probably because Agr controls expression of many major extracellular proteins. Overall amounts of most S. aureus lipoproteins and the lipoprotein SitC are reduced in culture filtrates of Agr and PSM mutants (g,h). Data in (a), (b) and (f) represent means +/− s.e.m. of at least three independent experiments. ***P<0.001, ****P<0.0001, significantly different for HEK versus HEK-TLR2 (a) and mutants versus USA300 wild-type (b,f) as calculated by Student’s t test.