Staphylococcal protein A, Panton-Valentine leukocidin and coagulase aggravate the bone loss and bone destruction in osteomyelitis.

BACKGROUND/AIMS: Osteomyelitis is a debilitating infectious disease of the bone which is predominantly caused by Staphylococcus aureus (S. aureus). The purpose of this study was to investigate the role of the S. aureus virulence factors, i.e. protein A (SpA), Panton-Valentine leukocidin (PVL) and coagulase (Coa) on osteomyelitis. METHODS: The effect of SpA, PVL and Coa on osteoblasts was studied through the following aspects including osteoblast proliferation, apoptosis, bone formation, bone mineralization and RANK-L expression. S. aureus overexpressing PVL, SpA or Coa was constructed and used to study the role of PVL, SpA and Coa, respectively. S. aureus silencing PVL, SpA or Coa was also constructed and used for reversing verification. Osteoblast proliferation was detected by MTT tetrazolium dye reduction assay. Apoptosis was determined by Annexin V-FITC staining. The levels of pro-caspase 3, cleaved-caspase 3, pro-caspase 9 and cleaved-caspase 9 were detected by western blot. Bone formation markers including collagen I, osteopontin and osteocalcin were detected by real time RT-PCR. Alkaline phosphatase activity was measured by adding p-nitrophenyl phosphate as a phosphatase substrate. Von kossa stain and alizarin red stain were applied for determining phosphate and calcium deposition, respectively. The RANK-L expression was tested by ELISA. RESULTS: PVL, SpA and Coa inhibited osteoblast proliferation, induced osteoblast apotosis, prohibited bone formation and mineralization and upregulated RANK-L expression. CONCLUSIONS: PVL, SpA and Coa play a critical role on bone loss and bone destruction of osteomyelitis.

Expression of staphylococcal clumping factor A impedes macrophage phagocytosis.

Clumping factor A (ClfA), a fibrinogen-binding protein linked to the Staphylococcus aureus cell wall, is an important virulence factor in infection models, e.g., of septic arthritis. However, the mechanism(s) by which ClfA contributes to the virulence of the bacterium is unknown. In the present study, the impact of ClfA expression on the phagocytosis of S. aureus by macrophages was investigated using clfA-positive and clfA-negative isogenic strains. Furthermore, the possible contribution of ClfA to the proinflammatory and immunostimulatory activity of S. aureus was studied. Our results indicate that ClfA expression significantly protects S. aureus against macrophage phagocytosis. This protection does not require the presence of intact fibrinogen, a ligand for ClfA. ClfA expression by S. aureus enhanced the proliferative response of spleen cells. On the other hand, a clfA mutant strain caused more release of proinflammatory mediators by macrophages than its clfA-positive parental strain. Both the protection against phagocytosis and the enhanced immunostimulatory activity provided by ClfA expression are likely to contribute to the in vivo virulence of S. aureus.