Chemical characteristics of Staphylococcus aureus molecules that have CD14-dependent cell-stimulating activity

We have previously shown that Staphylococcus aureus contains molecules that bind CD14 and induce cellular responses. We have partially purified this activity and named the product active fraction (AF). Here, we further explore the chemical characteristics of AF. We show that 1) the activity is sensitive to base and hydrofluoric acid hydrolysis; 2) AF binds to Mono Q, indicating that it is negatively charged; 3) most activity in AF runs as a large species in aqueous buffer, but a small proportion runs with a size of <15 kDa by gel chromatography; and 4) native PAGE of AF yields a band of high apparent m.w., and SDS-PAGE yields a ladder of bands of lower apparent m.w. These data suggest that AF is a mixture of heterogeneous amphiphilic molecules that form tight micelles. These characteristics are shared by LPS, suggesting that AF has both functional and structural similarity to LPS. S. aureus cell wall has been shown to stimulate cells in a CD14-dependent fashion, and peptidoglycan (PGN) has thus been described as a ligand for membrane CD14. However, we found that the ability of S. aureus cell wall to stimulate CD14-bearing monocytes was enhanced by preincubation with soluble CD14 (sCD14). Upon treatment with sCD14, the cell-stimulating activity appeared predominantly in the supernatant, not in the insoluble PGN pellet. These findings are not consistent with a direct interaction of membrane CD14 with insoluble PGN. They suggest, rather, that the activity of cell wall may be caused by a sCD14-binding molecule that associates noncovalently with PGN.