Staphylococcus aureus protein A plays a critical role in mediating bone destruction and bone loss in osteomyelitis

Abstract

Staphylococcus aureus is the most frequent causative organism of osteomyelitis. It is characterised by widespread bone loss and bone destruction. Previously we demonstrated that S. aureus protein A (SpA) is capable of binding to tumour necrosis factor receptor-1 expressed on pre-osteoblastic cells, which results in signal generation that leads to cell apoptosis resulting in bone loss. In the current report we demonstrate that upon S. aureus binding to osteoblasts it also inhibits de novo bone formation by preventing expression of key markers of osteoblast growth and division such as alkaline phosphatase, collagen type I, osteocalcin, osteopontin and osteocalcin. In addition, S. aureus induces secretion of soluble RANKL from osteoblasts which in turn recruits and activates the bone resorbing cells, osteoclasts. A strain of S. aureus defective in SpA failed to affect osteoblast growth or proliferation and most importantly failed to recruit or activate osteoclasts. These results suggest that S. aureus SpA binding to osteoblasts provides multiple coordinated signals that accounts for bone loss and bone destruction seen in osteomyelitis cases. A better understanding of the mechanisms through which S. aureus leads to bone infection may improve treatment or lead to the development of better therapeutic agents to treat this notoriously difficult disease.

Figure 1. Staphylococcus aureus prevents osteoblast growth and proliferation over a 21 day period.

Osteoblasts (5×10⁵ cells/ml) were preincubated with either control buffer (——) or formaldehyde fixed S. aureus Newman (-----), Newman Δspa (⋅⋅⋅⋅⋅), or Newman spa (pCU1spa⁺) (— ⋅ —). (A) Following days 7, 14 and 21 osteoblasts were removed by trypsinization and proliferation was determined by counting cells on a haemocytometer. (B) Osteoblasts were also visualised using an inverted bright field microscope (Leica, DMIL) at x200 magnification., *P<0.0001, n = 5.

Figure 2. Staphylococcus aureus prevents expression of osteogenic markers.

Osteoblasts (5×10⁵ cells/well) were incubated with either control buffer (——) or formaldehyde fixed S. aureus Newman (-----), Newman Δspa (⋅⋅⋅⋅⋅), or Newman spa (pCU1spa ⁺) (— ⋅ —) for a period of 21 days. (A) Osteoblasts were lysed with 1 ml of lysis buffer containing a substrate for alkaline phosphatase (0.1 M Na acetate, 2% Triton X-100 and 10 mM p-nitrophenol phosphate) and incubated in the dark for 1 hour at 37°C. RNA was isolated and reverse transcribed to cDNA. Analysis of bone formation marker expression was carried out specifically for the early bone marker (B) collagen type I, (C) osteopontin and (D) osteocalcin. *0.01, **0.05, ***0.0001, n = 3.

Figure 3. Staphylococcus aureus induces expression of soluble RANKL.

Osteoblasts (1×10⁶ cells/well) were incubated with either control buffer, S. aureus Newman, Newman Δspa or Newman spa (pCU1spa ⁺) for 24 hrs. Media from uninfected and infected osteoblasts was removed and centrifuged at x 10,000g for 2 minutes. Soluble RANKL was detected using an ELISA kit. *P<0.01, **P<0.001, n = 3

Figure 4. Staphylococcus aureus induces the migration of pre-osteoclasts.

Osteoblasts were cultured for 4 days, in the presence and absence of S. aureus, before inserting hanging cell migration chamber inserts containing serum starved pre-osteoclasts. Following 18 hour incubation, membranes from the migration chamber were removed and fixed in 4% formaldehyde. The membrane was then stained with haematoxylin (A) and counted from 5 random fields of view (B). The average cell count was established as indicative of the total cell migration. *P<0.05, **P<0.001 n = 3.

Figure 5. Staphylococcus aureus inhibits osteoclastogenesis.

Pre-osteoclasts were seeded (2×10⁴ cells/well) in 12-well tissue culture plates and cultured for 21 days. Conditioned media from osteoblasts (4 days), with either control buffer (——) or formaldehyde fixed S. aureus Newman (-----), Newman Δspa (⋅⋅⋅⋅⋅), or Newman spa (pCU1spa ⁺) (— ⋅ —) was then transferred to the pre-osteoclasts. Osteoclastogenesis was determined by measuring tartarate resistant alkaline phosphatase (TRAP). *P<0.05, **P<0.001 n = 5.