Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria

Abstract

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

FIG. 1

Abilities of four strains of S. aureus to become internalized by osteoblasts. Strains NCTC6571, 8325-4, LS-1, and FRI326 were cocultured with osteoblasts at an MOI of 300:1. The results are from one representative experiment of at least three; data are the means and standard deviations of three replicate cultures. The abilities of S. aureus strains to become internalized by osteoblasts were compared to that of strain 8325-4 using Student's t test. ∗, P < 0.05; ∗∗∗, P < 0.001.

FIG. 2

Numbers of bacteria associated (adherent and internalized) with osteoblasts for strains 8325-4 and LS-1 at an MOI of 300:1. The results are from one representative experiment of at least three; data are the means and standard deviations of three replicate cultures. Comparison of the data using Student's t test revealed no significant difference between the two strains.

FIG. 3

Abilities of S. aureus strains 8325-4 and LS-1 to bind to fibronectin. The results shown are for 10⁷ bacteria and are representative of one of at least three experiments; data are the means and standard deviations of four replicate wells. Comparison of the data using Student's t test revealed no significant difference between the two strains.

FIG. 4

Numbers of bacteria associated with osteoblasts for strains 8325-4 and LS-1 and their respective isogenic mutants DU5883 and LS-1(FnBP-). Cocultures were performed at an MOI of 300:1. The results are from one representative experiment of at least three; data are the means and standard deviations of three replicate cultures. Student's t test was used to compare differences between the isogenic mutant and its wild-type strain. ∗∗∗, P < 0.001.

FIG. 5

Abilities of S. aureus strains 8325-4, and LS-1 and their respective isogenic mutants DU5883 and LS-1(FnBP-) to become internalized by osteoblasts. Cocultures were performed at an MOI of 300:1. The results are from one representative experiment of at least three; data are the means and standard deviations of three replicate cultures. Student's t test was used to compare differences between the isogenic mutant and its wild-type strain. ∗∗, P < 0.01, ∗∗∗, P < 0.001.

FIG. 6

Kinetics of S. aureus internalization by osteoblasts. S. aureus strains 8325-4 and LS-1, at an MOI of 300:1, were cocultured in the presence of osteoblasts for between 30 min and 3 h. The results are from one representative experiment of at least three.

FIG. 7

Abilities of S. aureus strains 8325-4 and LS-1 to grow and/or survive in the intracellular environment of the osteoblast, determined by the numbers of bacteria recovered over a 6-h time period. The results are from one representative experiment of at least three; data are the means and standard deviations of three replicate cultures. Comparison of the data for each bacterial strain using Student's t test revealed no significant difference between the numbers of bacteria recovered at the different time points.