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

James E Cassat; Neal D Hammer; J Preston Campbell; Meredith A Benson; Daniel S Perrien; Lara N Mrak; Mark S Smeltzer; Victor J Torres; Eric P Skaar

doi:10.1016/j.chom.2013.05.003

. Author manuscript; available in PMC: 2014 Jun 12.

Published in final edited form as: Cell Host Microbe. 2013 Jun 12;13(6):759–772. doi: 10.1016/j.chom.2013.05.003

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

James E Cassat ¹, Neal D Hammer ², J Preston Campbell ³, Meredith A Benson ⁴, Daniel S Perrien ^5,⁶, Lara N Mrak ⁷, Mark S Smeltzer ⁷, Victor J Torres ⁴, Eric P Skaar ^2,^*

PMCID: PMC3721972 NIHMSID: NIHMS484604 PMID: 23768499

Summary

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. Micro-computed 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.

Introduction

Osteomyelitis is one of the most common invasive manifestations of Staphylococcus aureus infection, and S. aureus is the most frequent etiologic agent of musculoskeletal infection (Lew and Waldvogel, 2004). In children, staphylococcal osteomyelitis accounts for approximately 2.5 out of every 1,000 hospital admissions, with annual incidence estimates ranging from 1 in 5,000 to 1 in 10,000 (Gerber et al., 2009; Weichert et al., 2008). Among adults, osteomyelitis frequently complicates such comorbidities as open fractures, contiguous soft tissue infections, and diabetes (Lew and Waldvogel, 1997). However, in the era of community-acquired methicillin-resistant S. aureus (CA-MRSA), osteomyelitis now most frequently occurs in patients who have no appreciable risk factors for invasive staphylococcal infection.

Treatment of osteomyelitis is complex and typically involves one or more surgical debridements followed by prolonged antimicrobial therapy. Even with an appropriate antimicrobial regimen, patients suffering from staphylococcal osteomyelitis frequently experience serious, life-threatening complications such as septicemia, deep venous thrombosis, and pathologic fractures (Belthur et al., 2012; Gonzalez et al., 2006). Such complications are related to the ability of S. aureus to incite significant bone destruction, trigger surrounding soft tissue inflammation, and ultimately disseminate via the bloodstream. Bone destruction during osteomyelitis also confounds therapy by limiting antimicrobial penetration to the infectious focus. For this reason, prolonged antimicrobial treatment is required for osteomyelitis. An enhanced understanding of the mechanisms that promote bone destruction during osteomyelitis could facilitate development of therapies that limit bone destruction, thus improving the efficacy of current antimicrobials while reducing adverse outcomes. However, little is known regarding the specific bacterial factors that promote bone destruction during osteomyelitis.

Previous in vitro studies have investigated the interaction between S. aureus and the cells responsible for bone remodeling, osteoblasts and osteoclasts. S. aureus is capable of invading and persisting within osteoblasts (Ellington et al., 1999; Hudson et al., 1995). The interaction of S. aureus and osteoblasts leads to physiologic alterations that favor bone resorption by two primary mechanisms. First, S. aureus-infected osteoblasts incite osteoclastogenesis through increased expression of pro-inflammatory cytokines and the critical osteoclast-activating molecule receptor activator of NF-κB ligand (RANK-L) (Claro et al., 2013; Marriott et al., 2004; Somayaji et al., 2008). Second, S. aureus invasion of osteoblasts results in cell death (Alexander et al., 2003; Young et al., 2011). However, the specific staphylococcal factors that trigger osteoblast cell death or induce osteoclastogenesis are unknown. Furthermore, it has yet to be determined if these in vitro phenomena are responsible for pathogen-mediated bone destruction in vivo.

In this manuscript, a murine model of osteomyelitis is established to investigate staphylococcal factors contributing to the pathogenesis of osteomyelitis. Through the use of high-resolution micro-computed tomography (microCT), global proteomic analyses, and cell culture assays, specific staphylococcal virulence factors that contribute to pathologic bone remodeling and intraosseous bacterial survival are identified. Additionally, a significant role is identified for the secreted protease aureolysin in shaping the staphylococcal virulence repertoire during invasive infection. The tools developed in this work are readily adaptable to a variety of bacterial species, allowing for detailed analyses of the interaction between bacterial pathogens and bone.

Results

Establishment of a murine model of osteomyelitis for the study of pathologic bone remodeling and intraosseous bacterial survival

In order to interrogate the mechanisms of pathologic bone remodeling and intraosseous bacterial survival during osteomyelitis, a murine model was created. Derivatives of the USA300 type strain LAC were used as wild type (WT) for all infections, as USA300 type clinical isolates are common causes of musculoskeletal infections in both adults and children (Carrillo-Marquez et al., 2009; Peyrani et al., 2012). To induce osteomyelitis, 1×10⁶ colony forming units (CFU) of S. aureus are inoculated into the intramedullary canal of murine femurs following the creation of a 1 mm unicortical bone defect. Infected femurs exhibit peak bacterial burdens by day 4 post-infection (data not shown), and display profound changes in bone remodeling surrounding the inoculation site by day 14 post-infection. In addition to progressive cortical bone destruction at the inoculation site, infected femurs display marked new bone formation peripheral to the infectious focus (Figure 1A). In contrast, mice mock-infected with sterile PBS experience healing of the surgically induced cortical bone defect by day 14 post-infection (Figure 1B). To quantify both cortical bone destruction and peripheral new bone formation, imaging analysis algorithms were created and applied to images of both infected and mock-infected femurs. Infected femurs sustain significantly more cortical bone destruction and peripheral new bone formation than mock-infected femurs (Figure 1C and 1D). Quantitative imaging analyses underscored the magnitude of changes in bone remodeling during osteomyelitis, as infected femurs lost approximately 10–20% of total cortical volume near the infectious focus, while peripheral new bone formation accounted for bone growth of approximately 30–50% of the entire original cortical volume (data not shown). Histopathologic examination confirmed microCT findings, while also demonstrating substantial abscess formation throughout the bone marrow of infected femurs (Figure 1E). In contrast, the bone marrow architecture of mock-infected femurs remains intact (Figure 1F). Collectively, these results establish a murine model of staphylococcal osteomyelitis amenable to imaging analysis of pathologic bone remodeling, and demonstrate that S. aureus infection incites profound changes in bone turnover.

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.

S. aureus secreted virulence factors contribute to the pathogenesis of osteomyelitis

Based on the distinct zones of cortical bone destruction and peripheral new bone formation surrounding the inoculation site in infected murine femurs, we hypothesized that secreted staphylococcal factors trigger concentration-dependent changes in bone remodeling and enhance bacterial survival in the bone. To test this hypothesis, mice were infected with a S. aureus strain rendered exoprotein-deficient by virtue of mutations in the global regulatory loci agr and sae (Δagr/sae). SDS-PAGE analysis of concentrated culture supernatants confirmed that in comparison to WT, Δagr/sae has a severely limited exoprotein profile, with inactivation of either agr (Δagr) or sae (Δsae) alone having more modest effects (Supplemental Figure 1). To investigate the contribution of the agr and sae loci to the pathogenesis of S. aureus osteomyelitis, groups of mice were infected with either WT or Δagr/sae. At 14 days post-infection, infected femurs were harvested and either subjected to microCT imaging or processed to quantify bacterial burdens. Femurs infected with Δagr/sae have significantly less cortical bone destruction and peripheral new bone formation when compared to WT (Figure 2A – D). Additionally, femurs infected with LAC Δagr/sae have significantly lower bacterial burdens than LAC-infected femurs (Figure 2E). Together, these results reveal that the agr and sae regulatory loci are critical to the pathogenesis of S. aureus osteomyelitis and suggest that Agr- or Sae-regulated virulence factors contribute to pathologic bone remodeling and intraosseous bacterial survival.

Groups of mice were subjected to experimental osteomyelitis via inoculation of WT or Δ*agr/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 Δ*agr/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 (Δ*agr/sae*) or 10 (WT) per group. *** denotes p<0.001 as calculated by Student’s t-test. See also Supplementary Figure 1.

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

In order to further delineate specific S. aureus virulence factors that \contribute to pathologic bone remodeling and intraosseous bacterial survival during osteomyelitis, mice were infected with strains inactivated for either agr (Δagr) or sae (Δsae) and compared to WT-infected mice. At 14 days post-infection, femurs were harvested and subjected to microCT imaging or processed to quantify bacterial burdens. Murine femurs infected with Δsae sustain significantly less cortical bone destruction and new bone formation than WT-infected femurs (Figure 3A – F). Moreover, Δsae-infected femurs have a significant decrease in bacterial burdens when compared to WT-infected femurs, showing a similar reduction to that observed in Δagr/sae-infected femurs (Figure 3G). Conversely, murine femurs infected with Δagr exhibit no significant change in bacterial burdens relative to WT-infected femurs, but sustain significantly less cortical bone destruction (Supplemental Figure 2). Collectively, these studies define the sae and agr regulatory loci as contributors to pathologic bone remodeling during S. aureus osteomyelitis, with sae also significantly impacting intraosseous bacterial survival.

Groups of mice were subjected to experimental osteomyelitis via inoculation of WT or Δ*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 Δ*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=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 Δ*sae*-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.

Sae-regulated secreted virulence factors are cytotoxic to osteoblasts

In vivo studies revealed a critical role for the sae regulatory locus in the pathogenesis of S. aureus osteomyelitis. In order to investigate the mechanism by which Sae-regulated virulence factors perturb bone remodeling, a cytotoxicity assay was developed using murine (MC3T3) and human (Saos-2) osteoblastic cells. Incubation of MC3T3 or Saos-2 cell monolayers with concentrated culture supernatant from WT S. aureus results in potent cytotoxicity (Figure 4A and 4B). Cytotoxicity is observed as early as 2 hours after the addition of culture supernatant (data not shown). Incubation of MC3T3 cell monolayers with varying amounts of unconcentrated culture supernatant revealed that cytotoxicity is dose-dependent (Figure 4C). Inactivation of the sae locus leads to a complete loss of cytotoxicity in both murine and human osteoblastic cells following intoxication with concentrated supernatant (Figure 4A and 4B). However, when osteoblast monolayers are incubated with unconcentrated culture supernatant from Δsae, a significant increase in cytotoxicity relative to concentrated supernatant preparations is observed (Figure 4A). Importantly, the enhanced cytotoxicity of unconcentrated Δsae supernatant was not explained simply by differences in protein abundance in the concentrated versus unconcentrated samples, as roughly equivalent final protein concentrations were added to cell monolayers (data not shown). Rather, as supernatant concentration required three additional hours of sample processing, an alternative explanation is that loss of cytotoxicity in Δsae concentrated culture supernatant is due to increased degradation of osteolytic factors over time. Together, these results reveal that secreted S. aureus factors are dose-dependently cytotoxic to both human and murine osteoblastic cells and suggest that these osteolytic factors experience enhanced degradation in the absence of Sae.

**(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.

Analysis of the Sae exoproteome identifies aureolysin as a significant determinant of the S. aureus secreted virulence repertoire

Osteoblast cytotoxicity assays suggested that S. aureus secretes osteolytic factors that experience enhanced degradation in the absence of Sae. To identify Sae-regulated exoproteins that elicit osteoblast cell death and contribute to the pathogenesis of S. aureus osteomyelitis, the Sae exoproteome was determined. Multidimensional protein identification technology (MudPIT) analyses were performed on concentrated culture supernatants from WT and Δsae. Spectral counts were determined and used as a measure of protein abundance. Inactivation of sae leads to a significant decrease in the abundance of 49 proteins, including secreted virulence factors such as cytotoxins (Hla, LukA, LukB, HlgA, HlgC, PVL), immunomodulatory molecules (CHIPS, Sbi, Fprl1 inhibitory protein), and exoenzymes (SplA-F, Nuc) (Table 1). A supplemental file containing all proteins that change in abundance upon inactivation of sae is also included in the supplementary data (Supplementary Table 1). Thirty-one proteins significantly increase in abundance upon inactivation of sae, and of these the secreted protease aureolysin is most altered, increasing 8-fold (Table 1). Based on the observation that inactivation of sae leads to decreased abundance of osteolytic factors (Figure 4A) and globally altered exoprotein patterns (Supplemental Figure 1), we hypothesized that aureolysin activity might significantly modify the Sae exoproteome. Therefore, the exoproteome of a strain inactivated for both sae and aur (Δsae/aur) was determined. Concomitant inactivation of sae and aur leads to profound changes in the exoproteome, with 230 proteins significantly increasing in abundance, and 51 proteins significantly decreasing in abundance relative to WT (Table 2). Of the 269 proteins that decrease in abundance upon inactivation of sae, 225 (83%) increase in abundance upon concomitant inactivation of aur, with 190 (84%) of these proteins either returning to or exceeding WT protein levels. (Supplementary Table 2). Thus, the Sae exoproteome is significantly impacted by the activity of aureolysin, with the majority of Sae-regulated exoproteins experiencing aureolysin-dependent changes in abundance. These findings support a model whereby the Sae-regulated protease aureolysin remodels the staphylococcal exoproteome to modulate virulence.

Table 1. Proteins altered in abundance upon inactivation of sae.

Table 2. Proteins altered in abundance upon concomitant inactivation of sae and aur.

See also Supplementary Table 2

A. Proteins significantly decreased in abundance upon inactivation of sae/aur
Proteins¹	Mean LAC spectra	Mean LACΔsae/aur spectra	LAC/LACΔsaeaur²	Quasi-p value³	Description
Toxins, proteases, and exoenzymes
Q2FHS2\|Q2FHS2_STAA3	242.3333	0.6667	363.5000	0.0004	Alpha-hemolysin; SAUSA300_1058
Q2FFA3\|LUKL1_STAA3	152.6667	2.6667	57.2500	0.0048	LukA; SAUSA300_1974
Q2FE78\|Q2FE78_STAA3	53.6667	1.0000	53.6667	0.0040	Gamma-hemolysin component A; HlgA
Q2FFA2\|LUKL2_STAA3	157.6667	4.3333	36.3846	0.0112	LukB; SAUSA300_1975
Q2FFS9\|SPLA_STAA3	57.6667	2.3333	24.7143	0.0096	Serine protease SplA
Q2FFT1\|SPLC_STAA3	24.3333	1.0000	24.3333	0.0101	Serine protease SplC
Q2FFT0\|SPLB_STAA3	261.6667	12.0000	21.8056	0.0040	Serine protease SplB
Q2FFT3\|SPLE_STAA3	93.6667	4.3333	21.6154	0.0026	Serine protease SplE
Q2FK40\|Q2FK40_STAA3	19.3333	1.0000	19.3333	0.0133	Staphylocoagulase; Coa
Q2FFT4\|SPLF_STAA3	75.3333	4.0000	18.8333	0.0046	Serine protease SplF
Q2FIK5\|Q2FIK5_STAA3	12.0000	0.6667	18.0000	0.0208	Putative staphylocoagulase; SAUSA300_0773
Q2FFT2\|SPLD_STAA3	71.0000	4.3333	16.3846	0.0049	Serine protease SplD
Q2FJL8\|Q2FJL8_STAA3	4.6667	0.3333	14.0000	0.0352	Exotoxin 7; Set7
Q2FJP4\|Q2FJP4_STAA3	58.6667	5.6667	10.3529	0.0040	Putative staphylococcal enterotoxin; SAUSA300_0370
Q2FGU9\|Q2FGU9_STAA3	191.0000	20.3333	9.3934	0.0026	Panton-Valentine leukocidin, LukS-PV
Q2FKN2\|RLMH_STAA3	3.0000	0.3333	9.0000	0.0454	Exotoxin; SAUSA300_0398
Q2FGV0\|Q2FGV0_STAA3	600.0000	88.6667	6.7669	0.0049	Panton-Valentine leukocidin, LukF-PV
Q2FE77\|Q2FE77_STAA3	19.0000	3.3333	5.7000	0.0125	Gamma-hemolysin component C; HlgC
Q2FIK2\|Q2FIK2_STAA3	216.6667	43.0000	5.0388	0.0016	Thermonuclease; Nuc
Q2FJL3\|Q2FJL3_STAA3	3.3333	0.6667	5.0000	0.0269	Exotoxin; SAUSA300_0401
Q2FJL6\|Q2FJL6_STAA3	6.0000	1.3333	4.5000	0.0425	Exotoxin; SAUSA300_0398
Q2FJV7\|Q2FJV7_STAA3	67.0000	20.3333	3.2951	0.0173	5-nucleotidase, lipoprotein e(P4) family; SAUSA300_0307
Q2FFR8\|Q2FFR8_STAA3	26.6667	8.3333	3.2000	0.0133	Leukotoxin LukE
Q2FHS0\|Q2FHS0_STAA3	6.3333	2.0000	3.1667	0.0387	Putative exotoxin 4; SAUSA300_1060
Q2FJL9\|Q2FJL9_STAA3	3.0000	0.0000	3.0000	0.0127	Exotoxin; SAUSA300_0395
Q2FE76\|Q2FE76_STAA3	74.6667	28.0000	2.6667	0.0381	Gamma-hemolysin component B; HlgB
Q2FFR9\|Q2FFR9_STAA3	55.3333	29.6667	1.8652	0.0251	Leukotoxin LukD
Q2FJU4\|Q2FJU4_STAA3	6.3333	3.6667	1.7273	0.0454	Triacylglycerol lipase; SAUSA300_0320
Q2FKN3\|Q2FKN3_STAA3	39.3333	34.3333	1.1456	0.0154	5′-nucleotidase family protein; SAUSA300_0025
Immunomodulatory
Q2FFF7\|CHIPS_STAA3	856.0000	6.6667	128.4000	0.0016	Chemotaxis inhibitory protein; Chp
Q2FE79\|SBI_STAA3	85.6667	1.6667	51.4000	0.0070	Immunoglobulin-binding protein Sbi
Q2FHS7\|FLIPR_STAA3	10.0000	1.0000	10.0000	0.0091	FPRL1 inhibitory protein; Flr
Metabolic, replication, or cell division-associated
Q2FJ08\|Q2FJ08_STAA3	16.6667	0.3333	50.0000	0.0148	Na+/H+ antiporter; SAUSA300_0617
Q2FEA0\|Q2FEA0_STAA3	6.6667	4.6667	1.4286	0.0413	Respiratory nitrate reductase, alpha subunit; SAUSA300_2343
Regulatory
Q2FKE7\|Q2FKE7_STAA3	2.3333	0.6667	3.5000	0.0329	Staphylococcal accessory regulator SarS; SAUSA300_0114
Surface/cell-wall associated
Q2FE03\|FNBA_STAA3	19.3333	1.0000	19.3333	0.0131	Fibronectin-binding protein A; FnbA
Q2FHS5\|Q2FHS5_STAA3	31.3333	2.6667	11.7500	0.0040	Fibrinogen-binding protein; Efb
Q2FE04\|Q2FE04_STAA3	4.0000	0.6667	6.0000	0.0179	Fibronectin binding protein B; FnbB
Q2FE08\|Q2FE08_STAA3	57.0000	33.6667	1.6931	0.0454	Putative cell wall surface anchor family protein; SAUSA300_2436
Q2FG07\|ISDH_STAA3	20.0000	14.3333	1.3953	0.0454	Iron-regulated surface determinant protein H; IsdH
Q2FHV2\|ISDB_STAA3	36.6667	27.0000	1.3580	0.0199	Iron-regulated surface determinant protein B; IsdB
Q2FDL5\|Q2FDL5_STAA3	253.6667	233.3333	1.0871	0.0419	N-acetylmuramoyl-L-alanine amidase domain protein; SAUSA300_2579
Q2FJK6\|Q2FJK6_STAA3	30.3333	2.3333	13.0000	0.0036	Putative surface protein; SAUSA300_0408
Q2FF99\|Q2FF99_STAA3	12.6667	4.0000	3.1667	0.0266	Ferric hydroxamate receptor; SAUSA300_1978
Uncharacterized/Other
Q2FIT2\|Q2FIT2_STAA3	235.3333	0.3333	706.0000	0.0011	Putative lipoprotein; SAUSA300_0693
Q2FIG3\|Q2FIG3_STAA3	42.3333	3.6667	11.5455	0.0031	Ear protein; Ear
Q2FHS4\|Q2FHS4_STAA3	6.0000	0.0000	6.0000	0.0055	Putative uncharacterized protein; SAUSA300_1056
Q2FFW5\|Q2FFW5_STAA3	7.3333	3.3333	2.2000	0.0482	Putative uncharacterized protein; SAUSA300_1720
Q2FES8\|Q2FES8_STAA3	353.0000	213.6667	1.6521	0.0258	Putative uncharacterized protein; SAUSA300_2164
Q2FJJ6\|Y419_STAA3	8.6667	6.3333	1.3684	0.0289	Uncharacterized lipoprotein SAUSA300_0419; SAUSA300_0419
B. Proteins significantly increased in abundance upon inactivation of sae/aur
Proteins	Mean LAC spectra	*Mean LACΔsae/aur* spectra**	*LACΔsaeaur/LAC*⁴	Quasi-p value	Description
Toxins, proteases, and exoenzymes
Q2FG30\|Y1654_STAA3	0.6667	12.3333	18.5000	0.0101	Uncharacterized peptidase SAUSA300_1654; SAUSA300_1654
Q2FHF9\|Q2FHF9_STAA3	0.6667	10.6667	16.0000	0.0393	Peptidase, M16 family; SAUSA300_1172
Q2FJD0\|Q2FJD0_STAA3	0.6667	8.6667	13.0000	0.0302	ATP-dependent zinc metalloprotease FtsH
Q2FIM5\|CLPP_STAA3	1.6667	12.6667	7.6000	0.0189	ATP-dependent Clp protease proteolytic subunit; ClpP
Q2FJB5\|CLPC_STAA3	4.3333	25.0000	5.7692	0.0339	ATP-dependent Clp protease ATP-binding subunit ClpC
Q2FG62\|CLPX_STAA3	0.0000	3.3333	3.3333	0.0173	ATP-dependent Clp protease ATP-binding subunit ClpX
Metabolic, replication, or cell division-associated
Q2FEQ6\|RS5_STAA3	0.3333	79.6667	239.0000	0.0199	30S ribosomal protein S5; RpsE
Q2FGW9\|Q2FGW9_STAA3	1.3333	137.3333	103.0000	0.0148	DNA-binding protein HU; Hup
Q2FEP3\|RS19_STAA3	0.3333	30.6667	92.0000	0.0496	30S ribosomal protein S19; RpsS
Q2FI09\|PURL_STAA3	0.3333	25.3333	76.0000	0.0096	Phosphoribosylformylglycinamid ine synthase 2; PurL
Q2FHN5\|CARB_STAA3	0.3333	23.0000	69.0000	0.0219	Carbamoyl-phosphate synthase large chain; CarB
Q2FFJ5\|GATA_STAA3	0.3333	21.6667	65.0000	0.0073	Glutamyl-tRNA(Gln) amidotransferase subunit A; GatA
Q2FFA8\|Q2FFA8_STAA3	0.3333	16.3333	49.0000	0.0109	Phi77 ORF011-like protein, phage transcriptional repressor; SAUSA300_1969
Q2FF01\|PYRG_STAA3	0.3333	15.6667	47.0000	0.0100	CTP synthase; PyrG
Q2FEQ8\|RL15_STAA3	0.3333	15.0000	45.0000	0.0219	50S ribosomal protein L15; RplO
Q2FHK9\|PLSX_STAA3	0.3333	14.0000	42.0000	0.0155	Phosphate acyltransferase; PlsX
Q2FJC1\|PDXS_STAA3	0.3333	13.3333	40.0000	0.0314	Pyridoxal biosynthesis lyase PdxS
Q2FH13\|Q2FH13_STAA3	0.3333	12.6667	38.0000	0.0134	DegV family protein; SAUSA300_1318
Q2FHU2\|SYFB_STAA3	0.3333	11.0000	33.0000	0.0245	Phenylalanine--tRNA ligase beta subunit; PheT
Q2FG68\|Q2FG68_STAA3	0.6667	21.6667	32.5000	0.0279	Delta-aminolevulinic acid dehydratase; HemB
Q2FG72\|SYV_STAA3	0.0000	31.0000	31.0000	0.0049	Valine--tRNA ligase; ValS
Q2FHK7\|Q2FHK7_STAA3	1.3333	41.0000	30.7500	0.0244	3-oxoacyl-(Acyl-carrier-protein) reductase; FabG
Q2FG15\|Q2FG15_STAA3	0.3333	10.0000	30.0000	0.0265	Aminotransferase, class V; SAUSA300_1669
Q2FHG4\|PNP_STAA3	0.3333	10.0000	30.0000	0.0381	Polyribonucleotide nucleotidyltransferase; Pnp
Q2FJ93\|EFG_STAA3	5.0000	147.0000	29.4000	0.0376	Elongation factor G; FusA
Q2FER4\|RS11_STAA3	0.6667	19.3333	29.0000	0.0097	30S ribosomal protein S11; RpsK
Q2FI13\|Q2FI13_STAA3	0.6667	19.3333	29.0000	0.0417	Phosphoribosylaminoimidazole carboxylase, ATPase subunit; PurK
Q2FH38\|Q2FH38_STAA3	0.3333	9.3333	28.0000	0.0425	Diaminopimelate decarboxylase; LysA
Q2FJ49\|Q2FJ49_STAA3	0.3333	9.3333	28.0000	0.0468	Putative Pyridine nucleotide-disulphide oxidoreductase; SAUSA300_0576
Q2FHN3\|Q2FHN3_STAA3	0.3333	9.0000	27.0000	0.0387	Orotate phosphoribosyltransferase; PyrE
Q2FIB7\|Q2FIB7_STAA3	0.6667	18.0000	27.0000	0.0482	Glutamate dehydrogenase; GudB
Q2FG42\|Q2FG42_STAA3	0.3333	8.3333	25.0000	0.0281	Citrate synthase; GltA
Q2FKE9\|Q2FKE9_STAA3	0.3333	8.0000	24.0000	0.0301	L-lactate permease; LctP
Q2FHM2\|FMT_STAA3	0.3333	8.0000	24.0000	0.0440	Methionyl-tRNA formyltransferase; Fmt
Q2FJA1\|RL10_STAA3	0.0000	23.3333	23.3333	0.0139	50S ribosomal protein L10; RplJ
Q2FHP0\|PYRR_STAA3	0.3333	7.6667	23.0000	0.0264	Bifunctional protein PyrR
Q2FDQ2\|Q2FDQ2_STAA3	0.3333	7.6667	23.0000	0.0413	Putative AMP-binding enzyme; SAUSA300_2542
Q2FJF2\|Q2FJF2_STAA3	1.6667	37.6667	22.6000	0.0101	Methionine--tRNA ligase; MetS
Q2FGI7\|GCSPB_STAA3	1.0000	22.3333	22.3333	0.0330	Probable glycine dehydrogenase [decarboxylating] subunit 2; GcvPB
Q2FKN9\|Q2FKN9_STAA3	2.3333	49.6667	21.2857	0.0244	Adenylosuccinate synthetase; PurA
Q2FJC8\|Q2FJC8_STAA3	4.6667	98.3333	21.0714	0.0145	Cysteine synthase; CysK
Q2FJN5\|Q2FJN5_STAA3	0.0000	21.0000	21.0000	0.0133	Alkyl hydroperoxide reductase, subunit F; AhpF
Q2FFV5\|PCKA_STAA3	0.3333	7.0000	21.0000	0.0258	Phosphoenolpyruvate carboxykinase [ATP]; PckA
Q2FGJ2\|Q2FGJ2_STAA3	1.6667	34.6667	20.8000	0.0350	Proline dipeptidase; SAUSA300_1491
Q2FK94\|ALDA_STAA3	0.3333	6.6667	20.0000	0.0499	Putative aldehyde dehydrogenase AldA
Q2FHY5\|Q2FHY5_STAA3	1.0000	19.6667	19.6667	0.0009	Dihydrolipoamide acetyltransferase; SAUSA300_0995
Q2FGI6\|GCSPA_STAA3	1.3333	25.6667	19.2500	0.0084	Probable glycine dehydrogenase [decarboxylating] subunit 1; GcvPA
Q2FEQ4\|RL6_STAA3	7.3333	133.6667	18.2273	0.0021	50S ribosomal protein L6; RplF
Q2FIM9\|Q2FIM9_STAA3	5.3333	95.3333	17.8750	0.0140	Thioredoxin reductase; TrxB
Q2FJ92\|EFTU_STAA3	6.3333	111.6667	17.6316	0.0097	Elongation factor Tu; Tuf
Q2FFN1\|GSA2_STAA3	0.6667	11.6667	17.5000	0.0155	Glutamate-1-semialdehyde 2,1-aminomutase 2; HemL2
Q2FHW6\|Q2FHW6_STAA3	3.6667	63.6667	17.3636	0.0031	Pyruvate carboxylase; Pyc
Q2FG36\|Q2FG36_STAA3	2.0000	34.3333	17.1667	0.0112	Putative NADP-dependent malic enzyme; SAUSA300_1648
Q2FI15\|FOLD_STAA3	4.6667	79.6667	17.0714	0.0091	Bifunctional protein FolD
Q2FG09\|SYY_STAA3	0.6667	11.3333	17.0000	0.0401	Tyrosine--tRNA ligase; TyrS
Q2FEI9\|Y2254_STAA3	2.6667	45.3333	17.0000	0.0414	Putative 2-hydroxyacid dehydrogenase; SAUSA300_2254
Q2FGF2\|Q2FGF2_STAA3	0.3333	5.6667	17.0000	0.0477	Phosphate starvation-induced protein, PhoH family; PhoH
Q2FEI5\|FDHL_STAA3	0.6667	11.3333	17.0000	0.0496	Putative formate dehydrogenase; SAUSA300_2258
Q2FID4\|Y844_STAA3	1.3333	22.0000	16.5000	0.0097	NADH dehydrogenase-like protein; SAUSA300_0844
Q2FG40\|KPYK_STAA3	7.0000	115.3333	16.4762	0.0021	Pyruvate kinase; Pyk
Q2FEG2\|HUTG_STAA3	0.3333	5.3333	16.0000	0.0417	Formimidoylglutamase; HutG
Q2FEN9\|RL3_STAA3	5.3333	82.0000	15.3750	0.0084	50S ribosomal protein L3; RplC
Q2FDQ3\|Q2FDQ3_STAA3	3.6667	55.6667	15.1818	0.0048	Probable malate:quinone oxidoreductase; Mqo
Q2FF70\|Q2FF70_STAA3	0.3333	5.0000	15.0000	0.0454	Acetolactate synthase; IlvB
Q2FKC6\|SODM2_STAA3	2.0000	29.6667	14.8333	0.0245	Superoxide dismutase [Mn/Fe] 2; SodM
Q2FEU6\|Q2FEU6_STAA3	0.3333	4.6667	14.0000	0.0499	Alcohol dehydrogenase, zinc- containing; SAUSA300_2146
Q2FGA8\|SYA_STAA3	0.0000	14.0000	14.0000	0.0026	Alanine--tRNA ligase; AlaS
Q2FEX8\|Q2FEX8_STAA3	1.0000	14.0000	14.0000	0.0097	Glutamine--fructose-6-phosphate aminotransferase [isomerizing]; GlmS
Q2FI08\|Q2FI08_STAA3	2.3333	32.0000	13.7143	0.0482	Amidophosphoribosyltransferas e; PurF
Q2FER5\|RPOA_STAA3	1.3333	17.3333	13.0000	0.0373	DNA-directed RNA polymerase subunit alpha; RpoA
Q2FGF8\|SYG_STAA3	0.0000	12.6667	12.6667	0.0133	Glycine--tRNA ligase; GlyQS
Q2FKD2\|Q2FKD2_STAA3	0.0000	12.6667	12.6667	0.0173	Acetoin(Diacetyl) reductase; SAUSA300_0129
Q2FFJ1\|DNLJ_STAA3	0.6667	8.3333	12.5000	0.0307	DNA ligase; LigA
Q2FH96\|GUAC_STAA3	1.0000	12.3333	12.3333	0.0390	GMP reductase; GuaC
Q2FEQ1\|RL5_STAA3	11.3333	138.3333	12.2059	0.0388	50S ribosomal protein L5; RplE
Q2FJ55\|Q2FJ55_STAA3	3.6667	44.3333	12.0909	0.0339	Phosphate acetyltransferase; Pta
Q2FH63\|TRPA_STAA3	0.0000	12.0000	12.0000	0.0393	Tryptophan synthase alpha chain; TrpA
Q2FI92\|Q2FI92_STAA3	2.0000	23.3333	11.6667	0.0203	3-oxoacyl-[acyl-carrier-protein] synthase 2; FabF
Q2FG06\|FTHS_STAA3	5.3333	61.0000	11.4375	0.0155	Formate--tetrahydrofolate ligase; Fhs
Q2FG97\|SYD_STAA3	0.0000	11.3333	11.3333	0.0228	Aspartate--tRNA ligase; AspS
Q2FDV3\|ROCA_STAA3	1.3333	15.0000	11.2500	0.0091	1-pyrroline-5-carboxylate dehydrogenase; RocA
Q2FIL7\|ENO_STAA3	12.6667	141.0000	11.1316	0.0155	Enolase; Eno
Q2FG53\|Q2FG53_STAA3	0.0000	11.0000	11.0000	0.0118	Primosomal protein DnaI; DnaI
Q2FEX1\|GLMM_STAA3	1.0000	11.0000	11.0000	0.0326	Phosphoglucosamine mutase; GlmM
Q2FHK0\|RS16_STAA3	2.0000	21.6667	10.8333	0.0021	30S ribosomal protein S16; RpsP
Q2FI66\|Q2FI66_STAA3	1.3333	14.3333	10.7500	0.0397	Enoyl-[acyl-carrier-protein] reductase [NADPH]; SAUSA300_0912
Q2FG37\|ACCD_STAA3	0.0000	10.6667	10.6667	0.0083	Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta; AccD
Q2FKP7\|SYS_STAA3	1.6667	17.6667	10.6000	0.0256	Serine--tRNA ligase; SerS
Q2FIE5\|NAGD_STAA3	0.6667	7.0000	10.5000	0.0248	Protein NagD homolog; NagD
Q2FFI7\|PUR8_STAA3	4.6667	49.0000	10.5000	0.0412	Adenylosuccinate lyase; PurB
Q2FF15\|GLYA_STAA3	6.0000	59.3333	9.8889	0.0297	Serine hydroxymethyltransferase; GlyA
Q2FJM6\|IMDH_STAA3	5.0000	48.6667	9.7333	0.0256	Inosine-5′-monophosphate dehydrogenase; GuaB
Q2FF95\|CH60_STAA3	0.0000	9.6667	9.6667	0.0114	60 kDa chaperonin; GroL
Q2FFI2\|Q2FFI2_STAA3	1.0000	9.6667	9.6667	0.0348	Nicotinate phosphoribosyltransferase; SAUSA300_1894
Q2FH53\|Q2FH53_STAA3	2.0000	19.3333	9.6667	0.0413	Oligoendopeptidase F; PepF
Q2FKQ4\|Q2FKQ4_STAA3	1.6667	16.0000	9.6000	0.0180	DNA polymerase III subunit beta; DnaN
Q2FDQ7\|LDH2_STAA3	9.6667	90.0000	9.3103	0.0373	L-lactate dehydrogenase 2; Ldh2
Q2FI81\|Q2FI81_STAA3	1.3333	12.3333	9.2500	0.0207	Tryptophan--tRNA ligase; TrpS
Q2FH92\|Q2FH92_STAA3	3.6667	33.6667	9.1818	0.0279	Transketolase; Tkt
Q2FG54\|SYT_STAA3	1.6667	15.0000	9.0000	0.0137	Threonine--tRNA ligase; ThrS
Q2FI07\|PUR5_STAA3	1.0000	9.0000	9.0000	0.0289	Phosphoribosylformylglycinamid ine cyclo-ligase; PurM
Q2FFJ7\|DAGK_STAA3	0.6667	6.0000	9.0000	0.0330	Diacylglycerol kinase; DagK
Q2FIQ8\|Q2FIQ8_STAA3	0.0000	8.6667	8.6667	0.0049	Ribonucleoside-diphosphate reductase, beta subunit; SAUSA300_0717
Q2FGK7\|Q2FGK7_STAA3	1.6667	14.3333	8.6000	0.0342	Acetyl-CoA carboxylase, biotin carboxylase; AccC
Q2FHD4\|Q2FHD4_STAA3	1.3333	11.0000	8.2500	0.0403	Glutathione peroxidase; SAUSA300_1197
Q2FGW6\|Q2FGW6_STAA3	4.3333	35.0000	8.0769	0.0279	30S ribosomal protein S1; RpsA
Q2FH64\|TRPB_STAA3	1.3333	10.6667	8.0000	0.0265	Tryptophan synthase beta chain; TrpB
Q2FJC0\|PDXT_STAA3	2.3333	18.3333	7.8571	0.0186	Glutamine amidotransferase subunit PdxT
Q2FGE4\|DNAJ_STAA3	1.0000	7.6667	7.6667	0.0454	Chaperone protein DnaJ
Q2FJA3\|RL11_STAA3	4.3333	33.0000	7.6154	0.0199	50S ribosomal protein L11; RplK
Q2FIA5\|CDR_STAA3	4.3333	32.6667	7.5385	0.0244	Coenzyme A disulfide reductase; Cdr
Q2FJ81\|Q2FJ81_STAA3	0.6667	5.0000	7.5000	0.0423	Hydrolase, haloacid dehalogenase-like family; SAUSA300_0544
Q2FDY9\|F16PC_STAA3	0.0000	7.3333	7.3333	0.0381	Fructose-1,6-bisphosphatase class 3; Fbp
Q2FER6\|RL17_STAA3	7.6667	53.0000	6.9130	0.0454	50S ribosomal protein L17; RplQ
Q2FGY6\|SYN_STAA3	2.6667	18.3333	6.8750	0.0428	Asparagine--tRNA ligase; AsnS
Q2FJC3\|SYK_STAA3	3.0000	20.3333	6.7778	0.0381	Lysine--tRNA ligase; LysS
Q2FFX3\|RISB_STAA3	0.0000	6.6667	6.6667	0.0097	6,7-dimethyl-8-ribityllumazine synthase; RibH
Q2FIB3\|G6PI_STAA3	13.6667	90.6667	6.6341	0.0145	Glucose-6-phosphate isomerase; Pgi
Q2FGM8\|Q2FGM8_STAA3	3.3333	22.0000	6.6000	0.0279	Glucose-6-phosphate 1-dehydrogenase; Zwf
Q2FEN8\|RS10_STAA3	4.0000	26.0000	6.5000	0.0234	30S ribosomal protein S10; RpsJ
Q2FG66\|HEM3_STAA3	1.6667	10.6667	6.4000	0.0264	Porphobilinogen deaminase; HemC
Q2FJE4\|SP5G_STAA3	3.0000	19.0000	6.3333	0.0227	Putative septation protein SpoVG; SpoVG
Q2FDW1\|Q2FDW1_STAA3	0.0000	6.3333	6.3333	0.0364	Hydroxymethylglutaryl-CoA reductase; SAUSA300_2483
Q2FGE3\|DNAK_STAA3	5.3333	33.6667	6.3125	0.0155	Chaperone protein DnaK
Q2FDT8\|ISAA_STAA3	58.0000	361.0000	6.2241	0.0179	Probable transglycosylase IsaA
Q2FEL9\|Q2FEL9_STAA3	1.6667	10.3333	6.2000	0.0180	Molybdopterin biosynthesis protein A; MoeA
Q2FFK7\|Q2FFK7_STAA3	2.0000	12.3333	6.1667	0.0387	Methionine aminopeptidase; Map
Q2FJ86\|Q2FJ86_STAA3	3.0000	18.3333	6.1111	0.0143	Branched-chain-amino-acid aminotransferase; IlvE
Q2FHZ6\|Q2FHZ6_STAA3	4.0000	24.3333	6.0833	0.0101	Phosphoenolpyruvate-protein phosphotransferase; PtsI
Q2FF03\|Q2FF03_STAA3	4.0000	24.3333	6.0833	0.0382	Fructose bisphosphate aldolase; Fba
Q2FHQ4\|MURD_STAA3	0.0000	6.0000	6.0000	0.0016	UDP-N-acetylmuramoylalanine--D-glutamate ligase; MurD
Q2FK52\|Q2FK52_STAA3	0.0000	6.0000	6.0000	0.0186	Oxidoreductase, Gfo/Idh/MocA family; SAUSA300_0212
Q2FGB6\|GREA_STAA3	0.0000	6.0000	6.0000	0.0283	Transcription elongation factor GreA; GreA
Q2FIF9\|Q2FIF9_STAA3	0.0000	6.0000	6.0000	0.0381	FeS assembly protein SufD
Q2FFZ7\|Q2FFZ7_STAA3	1.3333	7.6667	5.7500	0.0101	FtsK/SpoIIIE family protein; SAUSA300_1687
Q2FJ29\|SYR_STAA3	2.6667	15.3333	5.7500	0.0304	Arginine--tRNA ligase; ArgS
Q2FHJ3\|SUCC_STAA3	5.6667	32.3333	5.7059	0.0034	Succinyl-CoA ligase [ADP-forming] subunit beta; SucC
Q2FHN6\|Q2FHN6_STAA3	0.0000	5.6667	5.6667	0.0021	Carbamoyl-phosphate synthase small chain; CarA
Q2FGJ3\|EFP_STAA3	2.3333	13.0000	5.5714	0.0348	Elongation factor P; Efp
Q2FIM0\|PGK_STAA3	10.0000	55.0000	5.5000	0.0215	Phosphoglycerate kinase; Pgk
Q2FFH6\|PPAC_STAA3	4.0000	21.6667	5.4167	0.0454	Probable manganese-dependent inorganic pyrophosphatase; PpaC
Q2FHI4\|HSLU_STAA3	0.0000	5.3333	5.3333	0.0069	ATP-dependent protease ATPase subunit HslU
Q2FIQ3\|MURB_STAA3	0.0000	5.3333	5.3333	0.0144	UDP-N-acetylenolpyruvoylglucosamine reductase; MurB
Q2FEP4\|RL22_STAA3	2.0000	10.6667	5.3333	0.0186	50S ribosomal protein L22; RplV
Q2FEG6\|HUTI_STAA3	0.0000	5.3333	5.3333	0.0381	Imidazolonepropionase; HutI
Q2FHA5\|Q2FHA5_STAA3	0.0000	5.3333	5.3333	0.0381	Homoserine dehydrogenase; SAUSA300_1226
Q2FGN9\|Q2FGN9_STAA3	1.3333	7.0000	5.2500	0.0152	Pseudouridine synthase; RluB
Q2FJE1\|Q2FJE1_STAA3	2.0000	10.3333	5.1667	0.0414	Ribose-phosphate pyrophosphokinase; Prs
Q2FH85\|Q2FH85_STAA3	11.6667	59.6667	5.1143	0.0155	Aconitate hydratase; AcnA
Q2FHI2\|RS2_STAA3	22.0000	110.3333	5.0152	0.0142	30S ribosomal protein S2; RpsB
Q2FEX4\|MTLD_STAA3	0.0000	5.0000	5.0000	0.0091	Mannitol-1-phosphate 5-dehydrogenase; MtlD
Q2FKP1\|RL9_STAA3	3.3333	16.6667	5.0000	0.0307	50S ribosomal protein L9; RplI
Q2FG27\|ACKA_STAA3	6.6667	33.3333	5.0000	0.0307	Acetate kinase; AckA
Q2FK14\|Q2FK14_STAA3	0.0000	5.0000	5.0000	0.0381	Alcohol dehydrogenase, zinc-containing; SAUSA300_0250
Q2FH70\|Q2FH70_STAA3	4.0000	19.3333	4.8333	0.0348	Putative glutamyl aminopeptidase; SAUSA300_1261
Q2FER0\|Q2FER0_STAA3	5.0000	24.0000	4.8000	0.0258	Adenylate kinase; Adk
Q2FG88\|TGT_STAA3	0.0000	4.6667	4.6667	0.0016	Queuine tRNA-ribosyltransferase; Tgt
Q2FJC7\|Q2FJC7_STAA3	0.0000	4.6667	4.6667	0.0031	Dihydropteroate synthase; FolP
Q2FDE9\|MNMG_STAA3	0.0000	4.6667	4.6667	0.0112	tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG
Q2FIB8\|Q2FIB8_STAA3	0.0000	4.6667	4.6667	0.0139	Ornithine aminotransferase; RocD
Q2FIE3\|DLTA_STAA3	3.0000	14.0000	4.6667	0.0381	D-alanine--poly(phosphoribitol) ligase subunit 1; DltA
Q2FFL6\|Q2FFL6_STAA3	8.0000	36.6667	4.5833	0.0251	Aminopeptidase PepS
Q2FIM1\|Q2FIM1_STAA3	91.6667	398.3333	4.3455	0.0314	Glyceraldehyde-3-phosphate dehydrogenase, type I; Gap
Q2FIN3\|HPRK_STAA3	0.0000	4.3333	4.3333	0.0003	HPr kinase/phosphorylase; HprK
Q2FEU5\|Q2FEU5_STAA3	0.0000	4.3333	4.3333	0.0101	Alcohol dehydrogenase, zinc-containing; SAUSA300_2147
Q2FFR3\|Q2FFR3_STAA3	0.0000	4.3333	4.3333	0.0176	Ferrochelatase; HemH
Q2FHA4\|Q2FHA4_STAA3	0.0000	4.3333	4.3333	0.0319	Threonine synthase; ThrC
Q2FJS4\|Q2FJS4_STAA3	0.0000	4.3333	4.3333	0.0419	NADH-dependent FMN reductase; SAUSA300_0340
Q2FH14\|Q2FH14_STAA3	1.0000	4.3333	4.3333	0.0454	Peptide methionine sulfoxide reductase MsrA
Q2FJ89\|HCHA_STAA3	8.6667	37.3333	4.3077	0.0414	Molecular chaperone Hsp31 and glyoxalase 3; HchA
Q2FEY0\|Q2FEY0_STAA3	1.3333	5.6667	4.2500	0.0495	Haloacid dehalogenase-like hydrolase; SAUSA300_2102
Q2FHI1\|EFTS_STAA3	51.6667	213.6667	4.1355	0.0205	Elongation factor Ts; Tsf
Q2FHY6\|Q2FHY6_STAA3	5.6667	23.0000	4.0588	0.0468	Pyruvate dehydrogenase E1 component, beta subunit; PdhB
Q2FI06\|Q2FI06_STAA3	0.0000	4.0000	4.0000	0.0049	Phosphoribosylglycinamide formyltransferase; PurN
Q2FE81\|GPMA_STAA3	28.3333	113.3333	4.0000	0.0279	2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; GpmA
Q2FFV8\|Q2FFV8_STAA3	0.0000	4.0000	4.0000	0.0373	Oxidoreductase, aldo/keto reductase family; SAUSA300_1728
Q2FHK3\|Q2FHK3_STAA3	1.6667	6.6667	4.0000	0.0432	Signal recognition particle receptor FtsY
Q2FHQ1\|Q2FHQ1_STAA3	1.6667	6.6667	4.0000	0.0474	Cell division protein FtsZ
Q2FI12\|PUR7_STAA3	6.0000	23.6667	3.9444	0.0454	Phosphoribosylaminoimidazole-succinocarboxamide synthase; PurC
Q2FJA4\|Q2FJA4_STAA3	3.3333	13.0000	3.9000	0.0301	Transcription antitermination protein NusG
Q2FFW0\|Q2FFW0_STAA3	6.3333	23.6667	3.7368	0.0322	Transaldolase; SAUSA300_1725
Q2FF83\|Q2FF83_STAA3	0.0000	3.6667	3.6667	0.0021	Sucrose-6-phosphate hydrolase; ScrB
Q2FGW3\|Q2FGW3_STAA3	0.0000	3.6667	3.6667	0.0040	L-asparaginase; AnsA
Q2FHN4\|PYRF_STAA3	0.0000	3.6667	3.6667	0.0097	Orotidine 5′-phosphate decarboxylase; PyrF
Q2FJ63\|Q2FJ63_STAA3	0.0000	3.6667	3.6667	0.0474	Phosphomethylpyrimidine kinase; ThiD
Q2FHU4\|Q2FHU4_STAA3	1.0000	3.3333	3.3333	0.0108	RNA methyltransferase, TrmH family; SAUSA300_1036
Q2FF23\|Q2FF23_STAA3	0.0000	3.3333	3.3333	0.0155	ATP synthase gamma chain; AtpG
Q2FE01\|Q2FE01_STAA3	0.0000	3.3333	3.3333	0.0173	Gluconate kinase; GntK
Q2FIG0\|Q2FIG0_STAA3	0.0000	3.3333	3.3333	0.0380	FeS assembly ATPase SufC
Q2FK51\|Q2FK51_STAA3	0.0000	3.3333	3.3333	0.0380	Oxidoreductase, Gfo/Idh/MocA family; SAUSA300_0213
Q2FKI2\|COPB_STAA3	0.0000	2.3333	2.3333	0.0127	Probable copper-transporting P-type ATPase B; CopB
Q2FHH9\|RRF_STAA3	3.6667	8.0000	2.1818	0.0273	Ribosome-recycling factor; Frr
Q2FIB6\|Q2FIB6_STAA3	74.3333	75.3333	1.0135	0.0219	Glycerophosphoryl diester phosphodiesterase; GlpQ
Regulatory
Q2FG02\|Q2FG02_STAA3	1.3333	25.0000	18.7500	0.0389	Catabolite control protein A; CcpA
Q2FF85\|Q2FF85_STAA3	0.6667	7.0000	10.5000	0.0497	Accessory gene regulator protein A; AgrA
Q2FIV3\|Q2FIV3_STAA3	4.3333	33.0000	7.6154	0.0145	Transcriptional regulator, MarR family; SAUSA300_0672
Q2FJ20\|SARA_STAA3	11.3333	64.6667	5.7059	0.0040	Transcriptional regulator SarA
Q2FF59\|RSBW_STAA3	2.0000	8.6667	4.3333	0.0097	Serine-protein kinase RsbW
Q2FH23\|ARLR_STAA3	0.0000	3.3333	3.3333	0.0173	Response regulator ArlR
Surface/cell-wall associated
Q2FKM6\|Q2FKM6_STAA3	49.6667	239.6667	4.8255	0.0496	Penicillin-binding protein 2′; MecA
Q2FGW1\|EBPS_STAA3	5.6667	20.0000	3.5294	0.0417	Elastin-binding protein EbpS
Uncharacterized/Other
Q2FG32\|Q2FG32_STAA3	0.3333	65.0000	195.0000	0.0133	Putative uncharacterized protein; SAUSA300_1652; Usp1
Q2FHL2\|Y1119_STAA3	0.3333	13.6667	41.0000	0.0241	Uncharacterized protein SAUSA300_1119
Q2FG28\|Y1656_STAA3	6.6667	238.6667	35.8000	0.0137	Putative universal stress protein SAUSA300_1656; Usp2
Q2FH32\|TELL_STAA3	0.6667	16.3333	24.5000	0.0091	TelA-like protein SAUSA300_1299
Q2FFP1\|Q2FFP1_STAA3	2.0000	41.3333	20.6667	0.0403	Putative uncharacterized protein; SAUSA300_1804
Q2FH61\|Q2FH61_STAA3	0.3333	6.6667	20.0000	0.0330	Methicillin resistance protein FemB
Q2FHZ8\|Q2FHZ8_STAA3	0.3333	6.3333	19.0000	0.0495	Putative uncharacterized protein; SAUSA300_0982
Q2FG98\|Q2FG98_STAA3	0.3333	6.0000	18.0000	0.0330	Putative uncharacterized protein; SAUSA300_1585
Q2FEB6\|Q2FEB6_STAA3	0.6667	10.6667	16.0000	0.0264	Putative uncharacterized protein; SAUSA300_2327
Q2FDS6\|Y2518_STAA3	0.3333	5.3333	16.0000	0.0454	Uncharacterized hydrolase; SAUSA300_2518
Q2FHG0\|Q2FHG0_STAA3	0.0000	13.3333	13.3333	0.0011	Putative uncharacterized protein; SAUSA300_1171
Q2FGK8\|Q2FGK8_STAA3	0.0000	12.6667	12.6667	0.0016	Putative uncharacterized protein; SAUSA300_1474
Q2FHF0\|Q2FHF0_STAA3	0.6667	8.3333	12.5000	0.0307	Putative uncharacterized protein; SAUSA300_1181
Q2FJH8\|Q2FJH8_STAA3	1.0000	11.0000	11.0000	0.0044	Lipoprotein; SAUSA300_0437
Q2FG00\|Q2FG00_STAA3	1.0000	7.6667	7.6667	0.0310	Putative uncharacterized protein; SAUSA300_1684
Q2FEV1\|Q2FEV1_STAA3	0.0000	7.3333	7.3333	0.0381	Putative uncharacterized protein; SAUSA300_2140
Q2FFQ3\|Q2FFQ3_STAA3	0.0000	7.0000	7.0000	0.0175	Putative uncharacterized protein; SAUSA300_1792
Q2FDL6\|Q2FDL6_STAA3	2.3333	15.3333	6.5714	0.0154	Putative phage infection protein; SAUSA300_2578
Q2FGZ5\|Q2FGZ5_STAA3	0.0000	6.3333	6.3333	0.0381	Putative uncharacterized protein; SAUSA300_1336
Q2FIF6\|Y822_STAA3	1.0000	6.0000	6.0000	0.0307	UPF0051 protein; SAUSA300_0822
Q2FJ32\|Q2FJ32_STAA3	0.0000	5.6667	5.6667	0.0131	Putative uncharacterized protein; SAUSA300_0593
Q2FIM8\|Y748_STAA3	0.0000	5.6667	5.6667	0.0279	UPF0042 nucleotide-binding protein; SAUSA300_0748
Q2FFY6\|Q2FFY6_STAA3	0.0000	5.3333	5.3333	0.0049	Putative uncharacterized protein; SAUSA300_1698
Q2FEY5\|Q2FEY5_STAA3	0.0000	5.3333	5.3333	0.0058	Putative uncharacterized protein; SAUSA300_2097
Q2FFZ5\|Y1689_STAA3	0.0000	4.6667	4.6667	0.0155	UPF0354 protein SAUSA300_1689
Q2FIA4\|Q2FIA4_STAA3	0.0000	4.3333	4.3333	0.0244	Putative uncharacterized protein; SAUSA300_0874
Q2FEI6\|Q2FEI6_STAA3	0.0000	4.0000	4.0000	0.0403	Putative uncharacterized protein; SAUSA300_2257
Q2FE49\|Q2FE49_STAA3	0.0000	3.3333	3.3333	0.0155	Putative uncharacterized protein; SAUSA300_2394
Q2FK61\|Q2FK61_STAA3	0.0000	2.3333	2.3333	0.0279	Putative lipoprotein; SAUSA300_0203

Open in a new tab

UniprotKB nomenclature (www.uniprot.org)

Ratio of mean LAC spectra divided by mean LACΔsae/aur spectra. If mean spectra equaled zero, a value of one was used to calculate ratio.

False discovery rate-corrected p value as calculated by QuasiTel program.

⁴

Ratio of mean LACΔsae/aur spectra divided by mean LAC spectra. If mean spectra equaled zero, a value of one was used to calculate ratio.

Inactivation of aureolysin in the Δsae background restores osteoblast cytotoxicity and increases cortical bone destruction

Exoproteome analyses revealed that the majority of proteins that decrease in abundance upon inactivation of sae do so in an aureolysin-dependent manner. We therefore hypothesized that the lack of osteoblast cytotoxicity in concentrated supernatant preparations from Δsae was due to aureolysin-mediated degradation of osteolytic exoproteins. To test this hypothesis, concentrated culture supernatants were prepared from Δsae and Δsae/aur, and each preparation was tested for the ability to induce cell death in murine or human osteoblasts. Inactivation of aur in the Δsae background restores cytotoxicity to levels equivalent to WT in both murine (Figure 5A) and human (data not shown) osteoblasts. To investigate whether the increased osteoblast cytotoxicity observed with Δsae/aur correlates to increased bone destruction during osteomyelitis, groups of mice were infected with WT, Δsae, or Δsae/aur. At 14 days post-infection, femurs were harvested and imaged by microCT or processed to quantify bacterial burdens. Concomitant inactivation of aur does not lead to a significant increase in bacterial recovery from infected femurs relative to inactivation of sae alone (Figure 5B). Despite this, inactivation of aur in the Δsae background leads to a significant increase in cortical bone destruction (Figure 5C). Taken together, these results demonstrate that inactivation of Sae causes increased abundance of aureolysin, which decreases levels of osteolytic factors and thereby significantly reduces cortical bone destruction. Thus, modulation of the S. aureus virulence repertoire by a secreted protease impacts pathogenesis.

**(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, Δ*sae*, or Δ*sae*/*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 Δ*psmα1-4*. 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.

Alpha-type phenol soluble modulins are cytotoxic to murine and human osteoblasticcells and contribute to pathogen-induced bone destruction during osteomyelitis

Exoproteome analyses revealed proteins with altered abundance upon inactivation of sae or concomitant inactivation of sae and aur. As cytotoxicity was observed with concentrated supernatants from WT and Δsae/aur, but not Δsae, the exoproteomes were analyzed to identify proteins that were detected only in cytotoxic supernatant preparations. The alpha-type phenol-soluble modulins (PSMs) PSMα1 and PSMα2 were present only in the exoproteomes of WT and Δsae/aur, but not in the exoproteome of Δsae (Supplementary table 2). Similarly, PSMα4 is present at lower levels in the exoproteome of Δsae as compared to WT and Δsae/aur. The alpha-type PSMs are cytolytic peptides that lyse neutrophils, provoke chemotaxis, and contribute to CA-MRSA virulence (Wang et al., 2007). Additionally, alpha-type PSMs undergo processing by secreted proteases, including aureolysin (Gonzalez et al., 2012; Zielinska et al., 2011). However, the role of PSMs in the pathogenesis of osteomyelitis has not been previously investigated. To test the hypothesis that alpha-type PSMs are responsible for osteoblast cytotoxicity, genes encoding the alpha-type PSMs were inactivated in the WT background and concentrated culture supernatant was prepared from this strain. Inactivation of the alpha-type PSMs (Δpsmα1–4) leads to a complete loss of cytotoxicity towards both murine and human osteoblastic cells (Figure 5D and 5E), indicating that these peptides are responsible for the cytotoxicity observed with concentrated culture supernatant from WT S. aureus. To directly test the ability of PSMs to trigger osteoblast cell death, PSMα1, PSMα2, PSMα3, and PSMα4 were synthesized and added directly to MC3T3 or Saos-2 monolayers. PSMα1, PSMα2, and PSMα3 are each dose-dependently cytotoxic to murine and human osteoblasts, with PSMα2 being the most potent (Supplementary Figure 3A and 3B). In contrast, PSMα4 does not elicit cytotoxicity at concentrations up to 100 μg/ml. To investigate the impact of aureolysin-mediated processing of PSMs on osteoblast cytotoxicity, PSMα2 and PSMα3 were incubated with purified aureolysin prior to intoxication of MC3T3 monolayers. We were unable to test the effect of aureolysin processing on PSMα1-mediated cytotoxicity due to loss of activity of this peptide after incubation at 37°C (data not shown). Pre-treatment of PSMα2 and PSMα3 with aureolysin, but not heat-inactivated aureolysin or buffer control, significantly limits osteoblast cytotoxicity (Supplementary Figure 3C). In order to test the contribution of alpha-type PSMs to pathogen-induced bone destruction during osteomyelitis, groups of mice were infected with WT or Δpsmα1–4 and femurs were harvested for microCT analysis on day 14 post-infection. Inactivation of psmα1–4 significantly limits cortical bone destruction in infected femurs (30% decrease in cortical bone destruction, p=0.0156 relative to WT), demonstrating that osteoblast cytotoxicity induced by the alpha-type PSMs correlates to increased pathogen-induced bone destruction during osteomyelitis (Figure 5F). Collectively, these data identify the alpha-type PSMs as Sae-regulated osteolytic exoproteins that are targeted by aureolysin and contribute to the morbidity of osteomyelitis. These findings confirm that protease-mediated exoproteome remodeling impacts S. aureus pathogenesis during invasive infection.

Discussion

Osteomyelitis is a common and debilitating manifestation of invasive S. aureus disease. 1Treatment of osteomyelitis is compromised by pathogen-induced bone destruction, which destroys the vascular architecture of infected bone and limits antimicrobial penetration to the infectious focus. Therapies aimed at limiting bone destruction during osteomyelitis could enhance traditional antimicrobial therapy, while reducing pathologic sequelae. In order to investigate the mechanisms by which bacterial pathogens induce bone destruction during osteomyelitis, a murine model of bone infection was developed, and microCT analyses of infected femurs were used to quantify pathologic bone remodeling. These tools revealed that S. aureus induces profound changes in bone remodeling, in part through the production of osteolytic exoproteins that modulate osteoblast proliferation. Surprisingly, S. aureus infection also leads to profound new bone formation, suggesting that bacterial modulation of bone remodeling involves complex changes in bone physiology. A crucial role for the S. aureus regulatory locus sae is demonstrated for both pathologic bone remodeling and intraosseous bacterial survival. The animal model of osteomyelitis described here demonstrates quantifiable differences in intraosseous bacterial survival and pathologic bone remodeling between isogenic bacterial strains, making it a valuable tool both for study of osteomyelitis pathogenesis and for the development of new therapies. Furthermore, the surgical techniques and ancillary studies associated with this animal model are easily adaptable to other bacterial species, enabling detailed study of host-pathogen interactions using a variety of bone pathogens. By modeling the interaction between bacterial pathogens and host cells comprising the skeletal system, these tools provide the opportunity to discover the mechanisms underlying bone turnover in response to infectious and inflammatory stimuli.

To identify osteolytic exoproteins that contribute to pathologic bone remodeling during osteomyelitis, global exoproteome analyses were performed. Exoproteome analysis revealed a crucial role for Sae in the expression of multiple classes of virulence factors, consistent with previous reports that Sae is required for virulence in other animal models of staphylococcal infection (Goerke et al., 2005; Liang et al., 2006; Nygaard et al., 2010; Voyich et al., 2009; Xiong et al., 2006). Unexpectedly, the secreted protease aureolysin was found to be a significant determinant of the S. aureus exoproteome. Inactivation of Sae leads to a significant increase in aureolysin abundance, consistent with previous reports demonstrating that aureolysin is repressed by Sae (Nygaard et al., 2010; Rogasch et al., 2006). Thus, in additional to transcriptional regulation by Sae, our results suggest an additional tier of regulation of the S. aureus virulence repertoire, whereby secreted protein abundance is tailored by the expression of a staphylococcal protease. These data corroborate findings from a previous study in which inactivation of all extracellular proteases led to changes in the S. aureus proteome (Zielinska et al., 2012). The benefit of such post-translational processing of virulence factors is unknown, but it is possible that this strategy allows S. aureus to combat the innate immune system while limiting antigenic accessibility to the adaptive immune response. An alternative explanation is that aureolysin-mediated processing of the secreted virulence repertoire allows S. aureus to modulate virulence factor production in response to host microenvironments. Staphylococcal abscesses are largely devoid of zinc, and thus a zinc-dependent protease such as aureolysin might allow for tailoring of the S. aureus secretome according to nutrient availability (Corbin et al., 2008). Aureolysin activity is also dependent on calcium, and therefore fluctuating calcium availability at infectious foci may also modulate the secreted virulence repertoire (Arvidson, 1973). Finally, post-translational regulation of secreted proteins by aureolysin might be a mechanism to govern the temporal and spatial abundance of specific toxins, allowing for more rapid changes in protein turnover and localization than can be achieved through transcriptional regulation alone.

The alpha-type PSMs were identified as osteolytic peptides that incite osteoblast cell death and contribute to pathogen-induced bone destruction during osteomyelitis. These results confirm that aureolysin-mediated processing of secreted virulence factors shapes the staphylococcal virulence repertoire. As aureolysin is known to activate other extracellular proteases, it is possible that PSMs are also degraded by a second, aureolysin-activated secreted protease in the absence of Sae (Nickerson et al., 2007). However, previous studies demonstrated that aureolysin is capable of degrading PSMs in the absence of the S. aureus global regulator, SarA (Zielinska et al., 2011). PSM production is also dependent on an intact agr locus, and therefore the decreased cortical bone destruction observed in Δagr-infected femurs may be a reflection of the absence of PSMs (Wang et al., 2007). Interestingly, as USA300 lineage strains of S. aureus are characterized by enhanced production of PSMs relative to other S. aureus clinical strain lineages (Li et al., 2009), our results may partially explain the increased morbidity associated with USA300 strains in patients with osteomyelitis (Carrillo-Marquez et al., 2009; Gonzalez et al., 2006).

Inactivation of aureolysin in the Δsae background did not fully restore bone destruction to wild type levels or significantly impact intraosseous bacterial survival. Similarly, inactivation of the alpha-type PSMs failed to affect pathogenesis to the same extent as inactivation of sae. It is therefore likely that additional Sae-regulated factors that impact the pathogenesis of osteomyelitis are yet to be discovered. Exoproteome analyses revealed that Sae affects the abundance of several classes of virulence factors, including factors that combat the innate immune system, cytotoxins, and degradative exoenzymes. In addition to secreted virulence factors, inactivation of Sae significantly decreased the abundance of cell surface proteins such as fibronectin- and fibrinogen-binding proteins. Therefore, the virulence defect of Δsae may also reflect deficiencies in host-tissue binding, biofilm formation, or invasion of host cells. By defining the Sae exoproteome and aureolysin-mediated processing of secreted virulence factors, this work provides a foundation for the development of immunotherapeutics that limit pathologic bone remodeling during osteomyelitis, thereby enhancing the ability to treat staphylococcal musculoskeletal infection.

Experimental procedures

Ethics statement

All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Vanderbilt University. All experiments were performed according to NIH guidelines, the Animal Welfare Act, and U.S. Federal law.

Bacterial Strains and Growth Conditions

All experiments were conducted with derivatives of the USA300 type S. aureus clinical isolate LAC (WT). Bacterial strains were routinely grown on tryptic soy broth (TSB) solidified with 1.5% agar at 37°C or in TSB with shaking at 180 rpm, unless otherwise indicated. TSB was supplemented with 10 μg/ml erythromycin where indicated. Erythromycin-sensitive LAC (AH1263), Δsae, Δagr, Δagr/sae, FPR3757 Δsae/aur, and Newman Δpsmα1–4 were previously described (Beenken et al., 2010; Benson et al., 2012; Boles et al., 2010; Kaito et al., 2011; Mrak et al., 2012). Strain Δsae/aur was created by bacteriophage phi-85 mediated transduction of the aur::erm mutation from FPR3757 Δsae/aur into the LAC Δsae background as previously described (Mazmanian et al., 2003). Strain Δpsmα1–4 was created by bacteriophage phi-85 mediated transduction of the psmα1–4::erm mutation from Newman Δpsmα1–4 into LAC. Successful transduction was confirmed by targeted PCR, hemolysis pattern, and exoprotein pattern (data not shown).

Murine Model of Osteomyelitis

Bacterial inocula were prepared by 1:100 subculture of overnight TSB cultures followed by growth at 37°C and 180 rpm shaking for 3 hours. Bacteria were collected by centrifugation, washed with phosphate-buffered saline (PBS), and resuspended to a concentration of 1×10⁶ colony forming units (CFU) in 2 μl PBS. Prior to surgery, 7–8 week C57BL/6J mice received 0.1 mg/kg buprenorphine via subcutaneous injection. Anesthesia was accomplished with isoflurane. The left hindlimb was shaved and disinfected, and a small incision was made overlying the lateral aspect of the left femur. The femur was exposed with blunt dissection, and a 1 mm diameter unicortical bone defect was created at the mid-femur by trephination with a 21-gauge PrecisionGlide needle (Becton Dickinson, Franklin Lakes, NJ). Bacterial inocula (1×10⁶ CFU in 2 μl) were delivered through the bone defect into the intramedullary canal. Muscle fasciae and skin were then closed with suture, and mice were recovered from anesthesia. Buprenorphine was administered every 12 hours for 72 hours postoperatively, and then as needed thereafter. Infection was allowed to proceed for 14 days, at which time mice were euthanized and the left femur was removed and subjected to either microCT or processed for CFU enumeration. For CFU enumeration, femurs were separated from surrounding soft tissue, frozen in liquid nitrogen, and homogenized in a BioSpec Biopulverizer (Biospec Products, Bartlesville, OK). Homogenized bone was weighed and subsequently resuspended in 1 ml PBS prior to sonication in an ice water bath for 30 min. Sonicated homogenates were vortexed for 60 sec and then serially diluted and plated on TSB solidified with 1.5% agar. Differences in CFU counts from groups of mice were analyzed using Student’s t-test.

Micro-computed tomography

Analysis of cortical bone destruction and new bone formation was determined by microCT imaging using a μCT50 (Scanco Medical, AG, Brüttisellen, Switzerland) and the manufacturer’s analytical software. Axial images of each femur were acquired with 5.0 μm voxels at 70 kV, 200 μA, 2000 projections per rotation, and an integration time of 350 msec in a 10.24 mm field-of-view. Each imaging scan comprised 1635 slices (8.125 mm) of the length of the femur, centered on the inoculation site as visualized in the scout-view radiographs. For analysis of cortical bone destruction, a volume of interest (VOI) including only the original cortical bone and any destruction was selected by drawing inclusive contours on the periosteal surface and excluding contours on the endosteal surface. Volume of cortical bone destruction was determined by segmenting the image with a lower threshold of 0 and an upper threshold of 595 mg HA/ccm, sigma 1.3 and support 1, to exclude bone in the analysis. To measure new bone volume, an inclusive contour was placed around the outer perimeter of the bone and an excluding contour was drawn along the pre-existing periosteal surface. Bone was segmented from non-mineralized tissues in the VOI with a lower threshold of 400 mg HA/ccm, sigma 1.3 and support 2. The direct voxel counting method was used for all reported calculations in each analysis. Differences in cortical bone destruction and peripheral new bone formation were analyzed using Student’s t-test.

Histopathology

After dissection, femurs were placed in neutral-buffered formalin. After 72 hours of formalin fixation at 4°C, samples were rinsed with deionized water and stored in 70% ethanol at 4°C. Femurs were subsequently decalcified in 20% EDTA pH 7.4 at 4°C for 4 days. Decalcified samples were then dehydrated and embedded in paraffin. Serial 4 μm paraffin sections were taken with a Leica 2255RM microtome on Leica Superfrost glass slides. Paraffin sections were cleared with Histo-Clear, then dehydrated and stained with a modified H&E-phloxine-orange G stain. With this stain, bone tissue stains yellow to orange in color, osteoclasts stain a vibrant pink, mesenchymal cells are blue, and bacterial biofilm stains pink to bright red.

Osteoblast cytotoxicity assays

MC3T3-E1 and Saos-2 cells were obtained from the American Type Culture Collection (ATCC) and propagated according to ATCC recommendations. Cells were grown at 37°C and 5% CO₂ with replacement of media every 2 or 3 days. For cytotoxicity assays, cells were seeded into 96-well tissue culture grade plates at a density of 2,500 or 5,000 cells per well for MC3T3 cells, or 10,000 cells per well for Saos-2 cells. After 24 hours, growth media was removed and replaced with media containing various amounts of concentrated S. aureus culture supernatant, or an equivalent volume of sterile RPMI. For some experiments, varying amounts of unconcentrated, 0.22 μm filter-sterilized culture supernatant were added to cell monolayers. Monolayers were incubated for an additional 23 hours prior to replacement of growth media and assessment of cell viability with the CellTiter 96^® AQueous One kit (Promega, Madison, WI) per manufacturer’s instructions. To test the ability of alpha-type PSMs to induce osteoblast cytotoxicity, PSMα1, PSMα2, PSMα3, and PSMα4 were synthesized at >90% purity by AAPPTec (Louisville, KY). Purified peptides were resuspended in DMSO and added at varying concentrations to osteoblast monolayers prior to assessment of cell viability as above. To test the impact of aureolysin activity on PSM cytotoxicity, purified aureolysin (Biocentrum, Krakow, Poland) was incubated with PSMα2 and PSMα3 for 8 hours at 37°C at a ratio of 1 μg aureolysin per 2 μg PSM. Aureolysin-processed PSMs were then added to MC3T3 cell monolayers at 100 μg/ml final concentration, and cytotoxicity was assessed as above. As a control, aureolysin was heat-inactivated for 30 minutes at 65°C prior to prior to incubation with PSMs and assessment of osteoblast cytotoxicity.

Preparation of concentrated S. aureus culture supernatant

To prepare concentrated culture supernatant, a single bacterial colony was inoculated into 15 ml RPMI supplemented with 1% casamino acids in a 50 ml conical tube. Cultures were grown for 15 hours at 37°C and 180 rpm shaking, after which time they were normalized to an OD600 of 3.5 and subjected to centrifugation for supernatant collection. Triplicate culture supernatants were filtered through a 0.22 μm filter, combined, and concentrated with an Amicon Ultra 3kDa nominal molecular weight limit centrifugal filter unit (Millipore, Billerica, MA) per manufacturer’s instructions. Supernatants were again filter-sterilized and used immediately for cytotoxicity assays, proteomic analysis, or SDS-PAGE analysis. Supernatants were routinely plated onto TSA supplemented with 5% sheep’s blood for confirmation of sterility and hemolysis pattern. For SDS-PAGE analysis, 30 μl of concentrated supernatant was resuspended in 4X loading buffer and boiled for 10 min. Proteins in the supernatant were resolved using 15% wt/vol SDS-PAGE, and stained with Coomassie blue prior to digital imaging.

Proteomic analysis of S. aureus exoproteins

Concentrated culture supernatants were prepared from WT, Δsae, and Δsae/aur as outlined above. Supernatants were resuspended in LDS sample buffer (Life Technologies, Carlsbad, CA) prior to resolving the proteins approximately 1 cm using a 10% Novex precast gel. Resolved proteins were subjected to in-gel tryptic digestion to recover peptides. Recovered peptides were analyzed via MudPIT (Multidimensional Protein Identification Technology) per previously published protocols (MacCoss et al., 2002; Martinez et al., 2012). Briefly, digested peptides were loaded onto a biphasic pre-column consisting of 4 cm of reversed phase (RP) material followed by 4 cm of strong cation exchange (RP) material. Once loaded, the column was placed in line with a 20 cm RP analytical column packed into a nanospray emitter tip directly coupled to a linear ion trap mass spectrometer (LTQ). A subset of peptides was eluted from the SCX material onto the RP analytical via a pulse of volatile salt, separated by an RP gradient, and then ionized directly into the mass spectrometer where both the intact masses (MS) and fragmentation patterns (MS/MS) of the peptides were collected. These peptide spectral data were searched against a protein database using Sequest (Yates et al., 1995) and the resulting identifications collated and filtered using IDPicker (Ma et al., 2009) and Scaffold (Proteome Software, Portland, OR). Relative protein abundances were evaluated via spectral counting techniques using the QuasiTel program to calculate false discovery rate-corrected p-values (Li et al., 2010).

Supplementary Material

NIHMS484604-supplement-01.pdf^{(3.7MB, pdf)}

NIHMS484604-supplement-02.xlsx^{(82.3KB, xlsx)}

NIHMS484604-supplement-03.xlsx^{(70.6KB, xlsx)}

Highlights.

S. aureus triggers profound alterations in bone remodeling during osteomyelitis.
The bacterial sae regulatory locus is critical to the pathogenesis of osteomyelitis.
Aureolysin, a Sae-regulated secreted protease, modifies the S. aureus virulence repertoire.
Aureolysin-degraded osteolytic peptides trigger osteoblast cell death and bone destruction.

Acknowledgments

We thank Lorenzo Olive, Ian Marriott, Florent Elefteriou, and Jon Schoenecker for assistance with surgical procedures. We thank the Vanderbilt Mass Spectrometry Research Center Proteomics Laboratory for assistance with exoproteome determinations. We are thankful to Dr. Chikara Kaito (The University of Tokyo) for providing Newman Δpsmα1–4. This research was supported by National Institutes of Health grants S10RR027631-01 to D.S.P., AI073843 and AI069233 to E.P.S., T32HD060554-03 to J.E.C., and AI091856 to V.J.T. V.J.T. was also supported by funds from the American Heart Association (Scientist Development Grant 09SDG2060036) and New York University School of Medicine Development Funds. M.A.B. was supported in part by an American Heart Association predoctoral fellowship (10PRE3420022). E.P.S. is a Burroughs Wellcome Fellow in the Pathogenesis of Infectious Diseases.

Footnotes

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Ellington JK, Reilly SS, Ramp WK, Smeltzer MS, Kellam JF, Hudson MC. Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts. Microbial pathogenesis. 1999;26:317–323. doi: 10.1006/mpat.1999.0272. [DOI] [PubMed] [Google Scholar]
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Goerke C, Fluckiger U, Steinhuber A, Bisanzio V, Ulrich M, Bischoff M, Patti JM, Wolz C. Role of Staphylococcus aureus global regulators sae and sigmaB in virulence gene expression during device-related infection. Infection and immunity. 2005;73:3415–3421. doi: 10.1128/IAI.73.6.3415-3421.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
Gonzalez BE, Teruya J, Mahoney DH, Jr, Hulten KG, Edwards R, Lamberth LB, Hammerman WA, Mason EO, Jr, Kaplan SL. Venous thrombosis associated with staphylococcal osteomyelitis in children. Pediatrics. 2006;117:1673–1679. doi: 10.1542/peds.2005-2009. [DOI] [PubMed] [Google Scholar]
Gonzalez DJ, Okumura CY, Hollands A, Kersten R, Akong-Moore K, Pence MA, Malone CL, Derieux J, Moore BS, Horswill AR, et al. Novel phenol-soluble modulin derivatives in community-associated methicillin-resistant Staphylococcus aureus identified through imaging mass spectrometry. The Journal of biological chemistry. 2012;287:13889–13898. doi: 10.1074/jbc.M112.349860. [DOI] [PMC free article] [PubMed] [Google Scholar]
Hudson MC, Ramp WK, Nicholson NC, Williams AS, Nousiainen MT. Internalization of Staphylococcus aureus by cultured osteoblasts. Microbial pathogenesis. 1995;19:409–419. doi: 10.1006/mpat.1995.0075. [DOI] [PubMed] [Google Scholar]
Kaito C, Saito Y, Nagano G, Ikuo M, Omae Y, Hanada Y, Han X, Kuwahara-Arai K, Hishinuma T, Baba T, et al. Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence. PLoS pathogens. 2011;7:e1001267. doi: 10.1371/journal.ppat.1001267. [DOI] [PMC free article] [PubMed] [Google Scholar]
Lew DP, Waldvogel FA. Osteomyelitis. The New England journal of medicine. 1997;336:999–1007. doi: 10.1056/NEJM199704033361406. [DOI] [PubMed] [Google Scholar]
Lew DP, Waldvogel FA. Osteomyelitis. Lancet. 2004;364:369–379. doi: 10.1016/S0140-6736(04)16727-5. [DOI] [PubMed] [Google Scholar]
Li M, Diep BA, Villaruz AE, Braughton KR, Jiang X, DeLeo FR, Chambers HF, Lu Y, Otto M. Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:5883–5888. doi: 10.1073/pnas.0900743106. [DOI] [PMC free article] [PubMed] [Google Scholar]
Li M, Gray W, Zhang H, Chung CH, Billheimer D, Yarbrough WG, Liebler DC, Shyr Y, Slebos RJ. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling. Journal of proteome research. 2010;9:4295–4305. doi: 10.1021/pr100527g. [DOI] [PMC free article] [PubMed] [Google Scholar]
Liang X, Yu C, Sun J, Liu H, Landwehr C, Holmes D, Ji Y. Inactivation of a two-component signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus. Infection and immunity. 2006;74:4655–4665. doi: 10.1128/IAI.00322-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
Ma ZQ, Dasari S, Chambers MC, Litton MD, Sobecki SM, Zimmerman LJ, Halvey PJ, Schilling B, Drake PM, Gibson BW, et al. IDPicker 2s: Improved protein assembly with high discrimination peptide identification filtering. Journal of proteome research. 2009;8:3872–3881. doi: 10.1021/pr900360j. [DOI] [PMC free article] [PubMed] [Google Scholar]
MacCoss MJ, McDonald WH, Saraf A, Sadygov R, Clark JM, Tasto JJ, Gould KL, Wolters D, Washburn M, Weiss A, et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:7900–7905. doi: 10.1073/pnas.122231399. [DOI] [PMC free article] [PubMed] [Google Scholar]
Marriott I, Gray DL, Tranguch SL, Fowler VG, Jr, Stryjewski M, Scott Levin L, Hudson MC, Bost KL. Osteoblasts express the inflammatory cytokine interleukin-6 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue. The American journal of pathology. 2004;164:1399–1406. doi: 10.1016/S0002-9440(10)63226-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
Martinez MN, Emfinger CH, Overton M, Hill S, Ramaswamy TS, Cappel DA, Wu K, Fazio S, McDonald WH, Hachey DL, et al. Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones. Journal of lipid research. 2012;53:379–389. doi: 10.1194/jlr.M019752. [DOI] [PMC free article] [PubMed] [Google Scholar]
Mazmanian SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P, Jelenska J, Joachmiak A, Missiakas DM, Schneewind O. Passage of heme-iron across the envelope of Staphylococcus aureus. Science. 2003;299:906–909. doi: 10.1126/science.1081147. [DOI] [PubMed] [Google Scholar]
Mrak LN, Zielinska AK, Beenken KE, Mrak IN, Atwood DN, Griffin LM, Lee CY, Smeltzer MS. saeRS and sarA act synergistically to repress protease production and promote biofilm formation in Staphylococcus aureus. PloS one. 2012;7:e38453. doi: 10.1371/journal.pone.0038453. [DOI] [PMC free article] [PubMed] [Google Scholar]
Nickerson NN, Prasad L, Jacob L, Delbaere LT, McGavin MJ. Activation of the SspA serine protease zymogen of Staphylococcus aureus proceeds through unique variations of a trypsinogen-like mechanism and is dependent on both autocatalytic and metalloprotease-specific processing. The journal of biologic chemistry. 2007;282:34129–34138. doi: 10.1074/jbc.M705672200. [DOI] [PubMed] [Google Scholar]
Nygaard TK, Pallister KB, Ruzevich P, Griffith S, Vuong C, Voyich JM. SaeR binds a consensus sequence within virulence gene promoters to advance USA300 pathogenesis. The Journal of infectious diseases. 2010;201:241–254. doi: 10.1086/649570. [DOI] [PMC free article] [PubMed] [Google Scholar]
Peyrani P, Allen M, Seligson D, Roberts C, Chen A, Haque N, Zervos M, Wiemken T, Harting J, Christensen D, et al. Clinical outcomes of osteomyelitis patients infected with methicillin-resistant Staphylococcus aureus USA-300 strains. The American journal of orthopedics (Belle Mead NJ) 2012;41:117–122. [PubMed] [Google Scholar]
Rogasch K, Ruhmling V, Pane-Farre J, Hoper D, Weinberg C, Fuchs S, Schmudde M, Broker BM, Wolz C, Hecker M, et al. Influence of the two-component system SaeRS on global gene expression in two different Staphylococcus aureus strains. Journal of bacteriology. 2006;188:7742–7758. doi: 10.1128/JB.00555-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
Somayaji SN, Ritchie S, Sahraei M, Marriott I, Hudson MC. Staphylococcus aureus induces expression of receptor activator of NF-kappaB ligand and prostaglandin E2 in infected murine osteoblasts. Infection and immunity. 2008;76:5120–5126. doi: 10.1128/IAI.00228-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
Voyich JM, Vuong C, DeWald M, Nygaard TK, Kocianova S, Griffith S, Jones J, Iverson C, Sturdevant DE, Braughton KR, et al. The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus. The Journal of infectious diseases. 2009;199:1698–1706. doi: 10.1086/598967. [DOI] [PMC free article] [PubMed] [Google Scholar]
Wang R, Braughton KR, Kretschmer D, Bach TH, Queck SY, Li M, Kennedy AD, Dorward DW, Klebanoff SJ, Peschel A, et al. Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. Nature medicine. 2007;13:1510–1514. doi: 10.1038/nm1656. [DOI] [PubMed] [Google Scholar]
Weichert S, Sharland M, Clarke NM, Faust SN. Acute haematogenous osteomyelitis in children: is there any evidence for how long we should treat? Current opinion in infectious diseases. 2008;21:258–262. doi: 10.1097/QCO.0b013e3283005441. [DOI] [PubMed] [Google Scholar]
Xiong YQ, Willard J, Yeaman MR, Cheung AL, Bayer AS. Regulation of Staphylococcus aureus alpha-toxin gene (hla) expression by agr, sarA, and sae in vitro and in experimental infective endocarditis. The Journal of infectious diseases. 2006;194:1267–1275. doi: 10.1086/508210. [DOI] [PubMed] [Google Scholar]
Yates JR, 3rd, Eng JK, McCormack AL, Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Analytical chemistry. 1995;67:1426–1436. doi: 10.1021/ac00104a020. [DOI] [PubMed] [Google Scholar]
Young AB, Cooley ID, Chauhan VS, Marriott I. Causative agents of osteomyelitis induce death domain-containing TNF-related apoptosis-inducing ligand receptor expression on osteoblasts. Bone. 2011;48:857–863. doi: 10.1016/j.bone.2010.11.015. [DOI] [PubMed] [Google Scholar]
Zielinska AK, Beenken KE, Joo HS, Mrak LN, Griffin LM, Luong TT, Lee CY, Otto M, Shaw LN, Smeltzer MS. Defining the strain-dependent impact of the Staphylococcal accessory regulator (sarA) on the alpha-toxin phenotype of Staphylococcus aureus. Journal of bacteriology. 2011;193:2948–2958. doi: 10.1128/JB.01517-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
Zielinska AK, Beenken KE, Mrak LN, Spencer HJ, Post GR, Skinner RA, Tackett AJ, Horswill AR, Smeltzer MS. sarA-mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates. Molecular Microbiology. 2012;86:1183–1196. doi: 10.1111/mmi.12048. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

NIHMS484604-supplement-01.pdf^{(3.7MB, pdf)}

NIHMS484604-supplement-02.xlsx^{(82.3KB, xlsx)}

NIHMS484604-supplement-03.xlsx^{(70.6KB, xlsx)}

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[R11] Gerber JS, Coffin SE, Smathers SA, Zaoutis TE. Trends in the incidence of methicillin-resistant Staphylococcus aureus infection in children’s hospitals in the United States. Clinical infectious diseases. 2009;49:65–71. doi: 10.1086/599348. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] Goerke C, Fluckiger U, Steinhuber A, Bisanzio V, Ulrich M, Bischoff M, Patti JM, Wolz C. Role of Staphylococcus aureus global regulators sae and sigmaB in virulence gene expression during device-related infection. Infection and immunity. 2005;73:3415–3421. doi: 10.1128/IAI.73.6.3415-3421.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] Gonzalez BE, Teruya J, Mahoney DH, Jr, Hulten KG, Edwards R, Lamberth LB, Hammerman WA, Mason EO, Jr, Kaplan SL. Venous thrombosis associated with staphylococcal osteomyelitis in children. Pediatrics. 2006;117:1673–1679. doi: 10.1542/peds.2005-2009. [DOI] [PubMed] [Google Scholar]

[R14] Gonzalez DJ, Okumura CY, Hollands A, Kersten R, Akong-Moore K, Pence MA, Malone CL, Derieux J, Moore BS, Horswill AR, et al. Novel phenol-soluble modulin derivatives in community-associated methicillin-resistant Staphylococcus aureus identified through imaging mass spectrometry. The Journal of biological chemistry. 2012;287:13889–13898. doi: 10.1074/jbc.M112.349860. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] Hudson MC, Ramp WK, Nicholson NC, Williams AS, Nousiainen MT. Internalization of Staphylococcus aureus by cultured osteoblasts. Microbial pathogenesis. 1995;19:409–419. doi: 10.1006/mpat.1995.0075. [DOI] [PubMed] [Google Scholar]

[R16] Kaito C, Saito Y, Nagano G, Ikuo M, Omae Y, Hanada Y, Han X, Kuwahara-Arai K, Hishinuma T, Baba T, et al. Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence. PLoS pathogens. 2011;7:e1001267. doi: 10.1371/journal.ppat.1001267. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] Lew DP, Waldvogel FA. Osteomyelitis. The New England journal of medicine. 1997;336:999–1007. doi: 10.1056/NEJM199704033361406. [DOI] [PubMed] [Google Scholar]

[R18] Lew DP, Waldvogel FA. Osteomyelitis. Lancet. 2004;364:369–379. doi: 10.1016/S0140-6736(04)16727-5. [DOI] [PubMed] [Google Scholar]

[R19] Li M, Diep BA, Villaruz AE, Braughton KR, Jiang X, DeLeo FR, Chambers HF, Lu Y, Otto M. Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:5883–5888. doi: 10.1073/pnas.0900743106. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] Li M, Gray W, Zhang H, Chung CH, Billheimer D, Yarbrough WG, Liebler DC, Shyr Y, Slebos RJ. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling. Journal of proteome research. 2010;9:4295–4305. doi: 10.1021/pr100527g. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] Liang X, Yu C, Sun J, Liu H, Landwehr C, Holmes D, Ji Y. Inactivation of a two-component signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus. Infection and immunity. 2006;74:4655–4665. doi: 10.1128/IAI.00322-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] Ma ZQ, Dasari S, Chambers MC, Litton MD, Sobecki SM, Zimmerman LJ, Halvey PJ, Schilling B, Drake PM, Gibson BW, et al. IDPicker 2s: Improved protein assembly with high discrimination peptide identification filtering. Journal of proteome research. 2009;8:3872–3881. doi: 10.1021/pr900360j. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] MacCoss MJ, McDonald WH, Saraf A, Sadygov R, Clark JM, Tasto JJ, Gould KL, Wolters D, Washburn M, Weiss A, et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:7900–7905. doi: 10.1073/pnas.122231399. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] Marriott I, Gray DL, Tranguch SL, Fowler VG, Jr, Stryjewski M, Scott Levin L, Hudson MC, Bost KL. Osteoblasts express the inflammatory cytokine interleukin-6 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue. The American journal of pathology. 2004;164:1399–1406. doi: 10.1016/S0002-9440(10)63226-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] Martinez MN, Emfinger CH, Overton M, Hill S, Ramaswamy TS, Cappel DA, Wu K, Fazio S, McDonald WH, Hachey DL, et al. Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones. Journal of lipid research. 2012;53:379–389. doi: 10.1194/jlr.M019752. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] Mazmanian SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P, Jelenska J, Joachmiak A, Missiakas DM, Schneewind O. Passage of heme-iron across the envelope of Staphylococcus aureus. Science. 2003;299:906–909. doi: 10.1126/science.1081147. [DOI] [PubMed] [Google Scholar]

[R27] Mrak LN, Zielinska AK, Beenken KE, Mrak IN, Atwood DN, Griffin LM, Lee CY, Smeltzer MS. saeRS and sarA act synergistically to repress protease production and promote biofilm formation in Staphylococcus aureus. PloS one. 2012;7:e38453. doi: 10.1371/journal.pone.0038453. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] Nickerson NN, Prasad L, Jacob L, Delbaere LT, McGavin MJ. Activation of the SspA serine protease zymogen of Staphylococcus aureus proceeds through unique variations of a trypsinogen-like mechanism and is dependent on both autocatalytic and metalloprotease-specific processing. The journal of biologic chemistry. 2007;282:34129–34138. doi: 10.1074/jbc.M705672200. [DOI] [PubMed] [Google Scholar]

[R29] Nygaard TK, Pallister KB, Ruzevich P, Griffith S, Vuong C, Voyich JM. SaeR binds a consensus sequence within virulence gene promoters to advance USA300 pathogenesis. The Journal of infectious diseases. 2010;201:241–254. doi: 10.1086/649570. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] Peyrani P, Allen M, Seligson D, Roberts C, Chen A, Haque N, Zervos M, Wiemken T, Harting J, Christensen D, et al. Clinical outcomes of osteomyelitis patients infected with methicillin-resistant Staphylococcus aureus USA-300 strains. The American journal of orthopedics (Belle Mead NJ) 2012;41:117–122. [PubMed] [Google Scholar]

[R31] Rogasch K, Ruhmling V, Pane-Farre J, Hoper D, Weinberg C, Fuchs S, Schmudde M, Broker BM, Wolz C, Hecker M, et al. Influence of the two-component system SaeRS on global gene expression in two different Staphylococcus aureus strains. Journal of bacteriology. 2006;188:7742–7758. doi: 10.1128/JB.00555-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] Somayaji SN, Ritchie S, Sahraei M, Marriott I, Hudson MC. Staphylococcus aureus induces expression of receptor activator of NF-kappaB ligand and prostaglandin E2 in infected murine osteoblasts. Infection and immunity. 2008;76:5120–5126. doi: 10.1128/IAI.00228-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] Voyich JM, Vuong C, DeWald M, Nygaard TK, Kocianova S, Griffith S, Jones J, Iverson C, Sturdevant DE, Braughton KR, et al. The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus. The Journal of infectious diseases. 2009;199:1698–1706. doi: 10.1086/598967. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] Wang R, Braughton KR, Kretschmer D, Bach TH, Queck SY, Li M, Kennedy AD, Dorward DW, Klebanoff SJ, Peschel A, et al. Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. Nature medicine. 2007;13:1510–1514. doi: 10.1038/nm1656. [DOI] [PubMed] [Google Scholar]

[R35] Weichert S, Sharland M, Clarke NM, Faust SN. Acute haematogenous osteomyelitis in children: is there any evidence for how long we should treat? Current opinion in infectious diseases. 2008;21:258–262. doi: 10.1097/QCO.0b013e3283005441. [DOI] [PubMed] [Google Scholar]

[R36] Xiong YQ, Willard J, Yeaman MR, Cheung AL, Bayer AS. Regulation of Staphylococcus aureus alpha-toxin gene (hla) expression by agr, sarA, and sae in vitro and in experimental infective endocarditis. The Journal of infectious diseases. 2006;194:1267–1275. doi: 10.1086/508210. [DOI] [PubMed] [Google Scholar]

[R37] Yates JR, 3rd, Eng JK, McCormack AL, Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Analytical chemistry. 1995;67:1426–1436. doi: 10.1021/ac00104a020. [DOI] [PubMed] [Google Scholar]

[R38] Young AB, Cooley ID, Chauhan VS, Marriott I. Causative agents of osteomyelitis induce death domain-containing TNF-related apoptosis-inducing ligand receptor expression on osteoblasts. Bone. 2011;48:857–863. doi: 10.1016/j.bone.2010.11.015. [DOI] [PubMed] [Google Scholar]

[R39] Zielinska AK, Beenken KE, Joo HS, Mrak LN, Griffin LM, Luong TT, Lee CY, Otto M, Shaw LN, Smeltzer MS. Defining the strain-dependent impact of the Staphylococcal accessory regulator (sarA) on the alpha-toxin phenotype of Staphylococcus aureus. Journal of bacteriology. 2011;193:2948–2958. doi: 10.1128/JB.01517-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] Zielinska AK, Beenken KE, Mrak LN, Spencer HJ, Post GR, Skinner RA, Tackett AJ, Horswill AR, Smeltzer MS. sarA-mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates. Molecular Microbiology. 2012;86:1183–1196. doi: 10.1111/mmi.12048. [DOI] [PMC free article] [PubMed] [Google Scholar]

A) Proteins significantly decreased in abundance upon inactivation of sae
Protein¹	Mean LAC spectra	Mean LACΔsae spectra	LAC/LACΔsae²	Description	Quasi-p value³
Toxins, proteases, and exoenzymes
Q2FHS2\|Q2FHS2_STAA3	242.3333	0.6667	363.5000	Alpha-hemolysin; SAUSA300_1058	0.0014
Q2FFA2\|LUKL2_STAA3	157.6667	1.0000	157.6667	LukB; SAUSA300_1975	0.0270
Q2FFA3\|LUKL1_STAA3	152.6667	0.0000	152.6667	LukA; SAUSA300_1974	0.0117
Q2FE78\|Q2FE78_STAA3	53.6667	0.6667	80.5000	Gamma-hemolysin component A; HlgA	0.0100
Q2FFT1\|SPLC_STAA3	24.3333	0.6667	36.5000	Serine protease SplC	0.0242
Q2FJP4\|Q2FJP4_STAA3	58.6667	1.6667	35.2000	Putative staphylococcal enterotoxin; SAUSA300_0370	0.0080
Q2FJV7\|Q2FJV7_STAA3	67.0000	2.0000	33.5000	5′-nucleotidase, lipoprotein e(P4) family; SAUSA300_0307	0.0024
Q2FFS9\|SPLA_STAA3	57.6667	2.6667	21.6250	Serine protease SplA	0.0272
Q2FFT2\|SPLD_STAA3	71.0000	5.3333	13.3125	Serine protease SplD	0.0203
Q2FE77\|Q2FE77_STAA3	19.0000	1.6667	11.4000	Gamma-hemolysin component C; HlgC	0.0150
Q2FFT0\|SPLB_STAA3	261.6667	24.0000	10.9028	Serine protease SplB	0.0150
Q2FGV0\|Q2FGV0_STAA3	600.0000	58.0000	10.3448	Panton-Valentine leukocidin, LukF-PV	0.0150
Q2FI16\|Q2FI16_STAA3	6.6667	0.6667	10.0000	Chitinase-related protein; SAUSA300_0964	0.0427
Q2FFT4\|SPLF_STAA3	75.3333	8.0000	9.4167	Serine protease SplF	0.0217
Q2FJL6\|Q2FJL6_STAA3	6.0000	0.6667	9.0000	Exotoxin; SAUSA300_0398	0.0399
Q2FFR8\|Q2FFR8_STAA3	26.6667	3.3333	8.0000	Leukotoxin LukE	0.0178
Q2FFT3\|SPLE_STAA3	93.6667	12.6667	7.3947	Serine protease SplE	0.0279
Q2FIK2\|Q2FIK2_STAA3	216.6667	30.0000	7.2222	Thermonuclease; Nuc	0.0148
Q2FGU9\|Q2FGU9_STAA3	191.0000	61.0000	3.1311	Panton-Valentine leukocidin, LukS-PV	0.0446
Immunomodulatory
Q2FE79\|SBI_STAA3	85.6667	0.3333	257.0000	Immunoglobulin-binding protein Sbi	0.0153
Q2FFF7\|CHIPS_STAA3	856.0000	7.6667	111.6522	Chemotaxis inhibitory protein; Chp	0.0034
Q2FHS7\|FLIPR_STAA3	10.0000	0.0000	10.0000	FPRL1 inhibitory protein; Flr	0.0150
Metabolic, replication, or cell division-associated
Q2FJ94\|RS7_STAA3	22.0000	0.3333	66.0000	30S ribosomal protein S7; RpsG	0.0242
Q2FHI2\|RS2_STAA3	22.0000	0.3333	66.0000	30S ribosomal protein S2; RpsB	0.0272
Q2FJ08\|Q2FJ08_STAA3	16.6667	0.3333	50.0000	Na+/H+ antiporter; SAUSA300_0617	0.0399
Q2FEQ4\|RL6_STAA3	7.3333	0.3333	22.0000	50S ribosomal protein L6; RplF	0.0426
Q2FG78\|Q2FG78_STAA3	13.3333	0.6667	20.0000	Cell shape-determining protein MreC	0.0272
Q2FF50\|ATKC_STAA3	9.3333	0.0000	9.3333	Potassium-transporting ATPase C chain; KdpC	0.0178
Q2FG80\|RL21_STAA3	6.3333	0.0000	6.3333	50S ribosomal protein L21; RplU	0.0081
Q2FEQ7\|RL30_STAA3	5.6667	0.0000	5.6667	50S ribosomal protein L30; RpmD	0.0341
Q2FEQ1\|RL5_STAA3	11.3333	2.6667	4.2500	50S ribosomal protein L5; RplE	0.0085
Q2FJ90\|Q2FJ90_STAA3	3.3333	0.0000	3.3333	Putative pyridoxal phosphate-dependent acyltransferase; SAUSA300_0535	0.0080
Q2FEQ5\|RL18_STAA3	3.3333	0.0000	3.3333	50S ribosomal protein L18; RplR	0.0133
Q2FFQ4\|Q2FFQ4_STAA3	3.3333	0.0000	3.3333	cbf1; 3′-5′ exoribonuclease YhaM; SAUSA300_1791	0.0150
Q2FG82\|RL27_STAA3	3.0000	0.0000	3.0000	50S ribosomal protein L27; RpmA	0.0153
Q2FKN2\|RLMH_STAA3	3.0000	0.0000	3.0000	rRNA large subunit methyltransferase; SAUSA300_0026	0.0372
Q2FEP4\|RL22_STAA3	2.0000	0.0000	2.0000	50S ribosomal protein L22; RplV	0.0024
Regulatory
Q2FIT2\|Q2FIT2_STAA3	235.3333	0.0000	235.3333	SaeP; SAUSA300_0693	0.0016
Surface/cell-wall associated
Q2FE03\|FNBA_STAA3	19.3333	0.3333	58.0000	Fibronectin-binding protein A; FnbA	0.0272
Q2FJK6\|Q2FJK6_STAA3	30.3333	0.6667	45.5000	Putative surface protein; SAUSA300_0408	0.0063
Q2FHS5\|Q2FHS5_STAA3	31.3333	0.0000	31.3333	Fibrinogen-binding protein; Efb; SAUSA300_1055	0.0014
Q2FKM6\|Q2FKM6_STAA3	49.6667	2.6667	18.6250	Penicillin-binding protein 2′; MecA	0.0150
Q2FHS4\|Q2FHS4_STAA3	6.0000	0.0000	6.0000	Fibrinogen-binding protein; SAUSA300_1056	0.0150
Q2FHU9\|Q2FHU9_STAA3	5.3333	1.6667	3.2000	IsdD; SAUSA300_1031	0.0469
Q2FF92\|Q2FF92_STAA3	2.0000	0.0000	2.0000	Serine-aspartate repeat family protein, SdrH	0.0024
Uncharacterized/Other
Q2FIG3\|Q2FIG3_STAA3	42.3333	4.0000	10.5833	Ear protein	0.0110
Q2FG28\|Y1656_STAA3	6.6667	0.0000	6.6667	Putative universal stress protein SAUSA300_1656	0.0178
Q2FDE2\|Q2FDE2_STAA3	420.0000	120.3333	3.4903	Putative uncharacterized protein; GN=SAUSA300_pUSA010004	0.0150
Q2FJ23\|Q2FJ23_STAA3	442.0000	133.3333	3.3150	Putative uncharacterized protein; GN=SAUSA300_0602	0.0174
B) Proteins significantly increased in abundance upon inactivation of sae
Protein	Mean LAC spectra	*Mean LACΔsae* spectra**	LAC/LACΔsae⁴	Description	Quasi-p value
Toxins, proteases, and exoenzymes
Q2FDM2\|Q2FDM2_STAA3	16.3333	134.0000	8.2041	Zinc metalloproteinase aureolysin; Aur	0.0080
Q2FKG2\|Q2FKG2_STAA3	114.0000	399.3333	3.5029	1-phosphatidylinositol phosphodiesterase; Plc	0.0080
Q2FI30\|Q2FI30_STAA3	50.3333	117.3333	2.3311	Cysteine protease; SspB	0.0379
Q2FFI6\|Q2FFI6_STAA3	439.0000	885.6667	2.0175	Staphopain A; SAUSA300_1890	0.0178
Q2FIH7\|Q2FIH7_STAA3	44.3333	79.0000	1.7820	Staphylococcal enterotoxin Q; Seq	0.0384
Q2FIH8\|Q2FIH8_STAA3	27.6667	39.6667	1.4337	Staphylococcal enterotoxin K; Sek	0.0069
Metabolism, replication, or cell division-associated
Q2FIL7\|ENO_STAA3	12.6667	58.0000	4.5789	Enolase; Eno	0.0252
Q2FHW6\|Q2FHW6_STAA3	3.6667	16.3333	4.4545	Pyruvate carboxylase; Pyc	0.0178
Q2FHY7\|Q2FHY7_STAA3	3.0000	11.0000	3.6667	Pyruvate dehydrogenase E1 component, alpha subunit; PdhA	0.0203
Q2FHY6\|Q2FHY6_STAA3	5.6667	20.6667	3.6471	Pyruvate dehydrogenase E1 component, beta subunit; PdhB	0.0117
Q2FJY7\|Q2FJY7_STAA3	2.3333	7.3333	3.1429	Putative staphyloxanthin biosynthesis protein; SAUSA300_0277	0.0178
Q2FDV3\|ROCA_STAA3	1.3333	3.3333	2.5000	1-pyrroline-5-carboxylate dehydrogenase; RocA	0.0272
Q2FFY7\|PEPVL_STAA3	10.3333	25.3333	2.4516	Putative dipeptidase; SAUSA300_1697	0.0372
Q2FDW0\|Q2FDW0_STAA3	4.6667	10.3333	2.2143	Hydroxymethylglutaryl-CoA synthase; SAUSA300_2484	0.0178
Q2FHY4\|Q2FHY4_STAA3	31.6667	69.0000	2.1789	Dihydrolipoyl dehydrogenase; LpdA	0.0178
Q2FF15\|GLYA_STAA3	6.0000	11.6667	1.9444	Serine hydroxymethyltransferase; GlyA	0.0385
Q2FIB6\|Q2FIB6_STAA3	74.3333	135.3333	1.8206	Glycerophosphoryl diester phosphodiesterase; GlpQ	0.0069
Q2FI76\|Q2FI76_STAA3	17.3333	26.6667	1.5385	Oligoendopeptidase F; PepF	0.0489
Q2FJ07\|Q2FJ07_STAA3	27.0000	41.0000	1.5185	ABC transporter, substrate-binding protein; SAUSA300_0618	0.0200
Q2FIM1\|Q2FIM1_STAA3	91.6667	138.6667	1.5127	Glyceraldehyde-3-phosphate dehydrogenase, type I; Gap	0.0279
Q2FG58\|RL20_STAA3	2.0000	3.0000	1.5000	50S ribosomal protein L20; RplT	0.0150
Regulatory
Q2FFR1\|TRAP_STAA3	5.6667	18.3333	3.2353	Signal transduction protein TRAP	0.0251
Q2FHI3\|CODY_STAA3	15.0000	35.3333	2.3556	GTP-sensing transcriptional pleiotropic repressor CodY	0.0279
Surface/cell wall-associated
Q2FHV2\|ISDB_STAA3	36.6667	46.0000	1.2545	Iron-regulated surface determinant protein B; IsdB	0.0255
Q2FJ78\|SDRD_STAA3	4.3333	19.0000	4.3846	Serine-aspartate repeat-containing protein D; SdrD	0.0081
Q2FDM9\|Q2FDM9_STAA3	2.0000	6.6667	3.3333	Clumping factor B; ClfB	0.0272
Q2FJ77\|SDRE_STAA3	4.0000	10.3333	2.5833	Serine-aspartate repeat-containing protein E; SdrE	0.0247
Q2FE08\|Q2FE08_STAA3	57.0000	88.3333	1.5497	Putative cell wall surface anchor family protein; SAUSA300_2436	0.0489
Uncharacterized/Other
Q2FIM3\|Q2FIM3_STAA3	3.0000	12.3333	4.1111	Putative uncharacterized protein; SAUSA300_0754	0.0161
Q2FGU0\|Q2FGU0_STAA3	0.0000	3.0000	3.0000	PhiSLT ORF527-like protein; SAUSA300_1391	0.0099
Q2FEC8\|Y2315_STAA3	5.6667	12.3333	2.1765	Uncharacterized lipoprotein; SAUSA300_2315	0.0490

PERMALINK

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

James E Cassat, M.D., Ph.D.

Neal D Hammer, Ph.D.

J Preston Campbell, Ph.D.

Meredith A Benson

Daniel S Perrien, Ph.D.

Lara N Mrak

Mark S Smeltzer, Ph.D.

Victor J Torres, Ph.D.

Eric P Skaar, Ph.D., M.P.H.

Summary

Introduction

Results

Establishment of a murine model of osteomyelitis for the study of pathologic bone remodeling and intraosseous bacterial survival

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

S. aureus secreted virulence factors contribute to the pathogenesis of osteomyelitis

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

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

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

Sae-regulated secreted virulence factors are cytotoxic to osteoblasts

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

Analysis of the Sae exoproteome identifies aureolysin as a significant determinant of the S. aureus secreted virulence repertoire

Table 1. Proteins altered in abundance upon inactivation of sae.

Table 2. Proteins altered in abundance upon concomitant inactivation of sae and aur.

Inactivation of aureolysin in the Δsae background restores osteoblast cytotoxicity and increases cortical bone destruction

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.

Alpha-type phenol soluble modulins are cytotoxic to murine and human osteoblasticcells and contribute to pathogen-induced bone destruction during osteomyelitis

Discussion

Experimental procedures

Ethics statement

Bacterial Strains and Growth Conditions

Murine Model of Osteomyelitis

Micro-computed tomography

Histopathology

Osteoblast cytotoxicity assays

Preparation of concentrated S. aureus culture supernatant

Proteomic analysis of S. aureus exoproteins

Supplementary Material

Highlights.

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

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