A secreted bacterial protease tailors the Staphylococcus aureus virulence repertoire to modulate bone remodeling during osteomyelitis

Abstract

Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection. Pathogen-induced bone destruction limits antimicrobial penetration to the infectious focus and compromises treatment of osteomyelitis. To investigate mechanisms of S. aureus-induced bone destruction, we developed a murine model of osteomyelitis. Microcomputed tomography of infected femurs revealed that S. aureus triggers profound alterations in bone turnover. The bacterial regulatory locus sae was found to be critical for osteomyelitis pathogenesis, as Sae-regulated factors promote pathologic bone remodeling and intraosseous bacterial survival. Exoproteome analyses revealed the Sae-regulated protease aureolysin as a major determinant of the S. aureus secretome and identified the phenol-soluble modulins as aureolysin-degraded, osteolytic peptides that trigger osteoblast cell death and bone destruction. These studies establish a murine model for pathogen-induced bone remodeling, define Sae as critical for osteomyelitis pathogenesis, and identify protease-dependent exoproteome remodeling as a major determinant of the staphylococcal virulence repertoire.

Figure 1. S. aureus triggers pathologic bone remodeling during osteomyelitis

Groups of mice were subjected to experimental osteomyelitis via intramedullary inoculation of either S. aureus strain LAC (WT) or an equivalent volume of sterile PBS (mock). Femurs were harvested at 14 days post-inoculation and subjected to microCT analysis. (A) Antero-posterior (left) and lateral (right) views of a S. aureus-infected femur at 14 days post-inoculation. Asterisk denotes inoculation site and surrounding cortical bone destruction. Arrowheads denote peripheral new bone formation. (B) Antero-posterior (left) and lateral (right) views of a mock-infected femur at 14 days post-inoculation. Asterisk denotes inoculation site. (C and D) MicroCT imaging analysis of cortical bone destruction (C) and new bone formation (D) in infected femurs at day 14 post-inoculation. N=4 mice per group. Error bars denote standard error of the mean (SEM). * denotes p<0.05 and ** denotes p<0.01 relative to WT infection as calculated by Student’s t-test. (E and F) Modified H&E-phloxine-orange G-stained sections of representative WT-infected (E) or mock-infected (F) femurs at low (left) or high (right) magnification. High magnification images are centered over the inoculation site, and represent different tissue depths from the same femurs in low-magnification images. Arrows denote inoculation site, arrowheads denote peripheral new bone formation, and asterisks denote abscesses.

Figure 2. S. aureus secreted virulence factors contribute to pathologic bone remodeling and intraosseous bacterial survival during osteomyelitis

Groups of mice were subjected to experimental osteomyelitis via inoculation of WT or Δagr/sae. Femurs were harvested at 14 days post-inoculation and subjected to microCT imaging analysis or processed for enumeration of bacterial burdens. (A and B) Antero-posterior views of WT (A) or Δagr/sae (B) infected femurs at 14 days post-inoculation. (C and D) MicroCT imaging analysis of cortical bone destruction (C) and new bone formation (D) in infected femurs at day 14 post-inoculation. N=4 mice per group. Error bars denote SEM. * denotes p<0.05 relative to WT as calculated by Student’s t-test. (E) CFU recovery per mg of bone tissue at day 14 post-inoculation. N=9 (Δagr/sae) or 10 (WT) per group. *** denotes p<0.001 as calculated by Student’s t-test. See also Supplementary Figure 1.

Figure 3. The sae regulatory locus contributes to pathologic bone remodeling and intraosseous bacterial survival during S. aureus osteomyelitis

Groups of mice were subjected to experimental osteomyelitis via inoculation of WT or Δsae. Femurs were harvested at 14 days post-inoculation and subjected to microCT imaging analysis or processed for enumeration of bacterial burdens. (A and B) Antero-posterior views of WT (A) or Δsae (B) infected femurs at 14 days post-inoculation. (C and D) MicroCT imaging analysis of cortical bone destruction (C) and new bone formation (D) in infected femurs at day 14 post-inoculation. N=5 mice per group. Error bars denote SEM. *** denotes p<0.001 relative to WT as calculated by Student’s t-test. (E and F) Modified H&E-phloxine-orange G-stained sections of representative WT-infected (E) or Δsae-infected (F) femurs at low magnification. Arrows denote inoculation site and arrowheads denote peripheral new bone formation. (G) CFU recovery per mg of bone tissue at day 14 post-inoculation. N=10 per group. *** denotes p<0.001 as calculated by Student’s t-test. See also Supplementary Figure 2.

Figure 4. Sae-regulated secreted virulence factors are dose-dependently cytotoxic to osteoblasts

(A and B) MC3T3 murine osteoblastic cells (A) or Saos-2 human osteoblastic cells (B) were seeded into 96-well plates at 5,000 cells per well or 10,000 cells per well, respectively. After 24 hours, growth media were replaced, and 40 μl of concentrated culture supernatant, 160 μl of unconcentrated supernatant, or an equivalent volume of sterile RPMI supplemented with 1% casamino acids (control) were added to cell monolayers. Osteoblast viability was assessed 23 hours later using the Promega CellTiter 96^® AQueous One kit, and results are expressed as percent of control. N=10 per group and results are representative of at least three independent experiments. Error bars denote SEM. *** denotes p-value < 0.001 as calculated by Student’s t-test. (C) MC3T3 cells were seeded into 96-well plates at a density of 2,500 cells per well. Twenty-four hours after seeding, growth media were replaced and varying amounts of unconcentrated culture supernatant were added to cell monolayers. Cell viability was assessed as above. N=10 per group. Error bars denote SEM.

Figure 5. Alpha-type phenol soluble modulins are aureolysin-processed peptides that trigger osteoblast cell death in vitro and contribute to cortical bone destruction in vivo

(A) MC3T3 cells (5,000 cells per well) were incubated with 40 μl of concentrated culture supernatant from the indicated strains or an equivalent volume of sterile RPMI supplemented with 1% casamino acids (control). Osteoblast viability was assessed 23 hours later using the Promega CellTiter 96^® AQueous One kit, and results are expressed as percent of control. N=10 per group and results are representative of at least three independent experiments. Error bars denote SEM. *** denotes p-value < 0.001 as calculated by Student’s t-test. (B and C) Groups of mice were subjected to experimental osteomyelitis via inoculation of WT, Δsae, or Δsae/aur. Femurs were harvested at 14 days post-inoculation and processed for bacterial recovery (n=10 per group) (B) or subjected to microCT imaging analysis of cortical bone destruction (n=5 per group) (C). Error bars denote SEM. * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001 as calculated by Student’s t-test. (D and E) MC3T3 (D) or Saos-2 (E) cell monolayers were incubated with 40 μl of concentrated culture supernatant from the indicated strains or an equivalent volume of sterile RPMI supplemented with 1% casamino acids (control). Osteoblast viability was determined 23 hours later as above. Error bars denote SEM. *** denotes p<0.001 as calculated by Student’s t-test. (F) Groups of mice (n=10 per group) were subjected to experimental osteomyelitis via inoculation of WT or Δpsmα1-4. Femurs were harvested at 14 days post-inoculation and subjected to microCT imaging analysis of cortical bone destruction. Error bars denote SEM. * denotes p<0.05 relative to WT as calculated by Student’s t-test. See also Supplementary Figure 3.