Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant-associated biofilm infection and S. aureus bacteremia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease-dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared to the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison to the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease-mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.

Bone is a common site of cancer metastasis. Breast, prostate, and lung cancers show a predilection to metastasize to bone. Recently, we reported that the chemokine interleukin 8 (IL-8) stimulates both human osteoclast formation and bone resorption. IL-8 mRNA expression was surveyed in a panel of human breast cancer lines MDA-MET, MDA-MB-231, MDA-MB-435, MCF-7, T47D, and ZR-75, and the human lung adenocarcinoma cell line A549. IL-8 mRNA expression was higher in cell lines with higher osteolytic potential in vivo. Human osteoclast formation was increased by MDA-MET or A549 cell-conditioned medium, but not by MDA-MB-231. Pharmacologic doses of receptor activator of nuclear factor-KB (RANK)-Fc or osteoprotogerin had no effect on the pro-osteoclastogenic activity of the conditioned medium; however, osteoclast formation stimulated by conditioned medium was inhibited 60% by an IL-8-specific neutralizing antibody. The data support a model in which tumor cells cause osteolytic bone destruction independently of the RANK ligand (RANKL) pathway. Tumorproduced IL-8 is a major contributor to this process. The role of secreted IL-8 isoforms was examined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, which detected distinct IL-8 isoforms secreted by MDA-MET and MDA-231 cells, suggesting different pro-osteoclastogenic activities of the two IL-8-derived peptides. These data indicate that (a) osteoclast formation induced by MDA-MET breast cancer cells and A549 adenocarcinoma cells is primarily mediated by IL-8, (b) cell-specific isoforms of IL-8 with distinct osteoclastogenic activities are produced by tumor cells, and (c) tumor cells that support osteoclast formation independent of RANKL secrete other pro-osteoclastogenic factors in addition to IL-8. (Cancer Res 2005; 65(23): 11001-9)

The effects of type 1 diabetes on de novo bone formation during tibial distraction osteogenesis (DO) and on intact trabecular and cortical bone were studied using nonobese diabetic (NOD) mice and comparably aged nondiabetic NOD mice. Diabetic mice received treatment with insulin, vehicle, or no treatment during a 14-day DO procedure. Distracted tibiae were analyzed radiographically, histologically, and by microcomputed tomography (CT). Contralateral tibiae were analyzed using CT. Serum levels of insulin, osteocalcin, and cross-linked C-telopeptide of type I collagen were measured. Total new bone in the DO gap was reduced histologically (P < 0.001) and radiographically (P < 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by CT and mechanical testing demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals, which were prevented by insulin treatment. These studies demonstrate that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration. Diabetes 54:2875-2881, 2005 T ype 1 diabetes is associated with several disorders of skeletal health, including decreased bone density, an increased risk for osteoporosis (1-6), and fragility fracture (7-9), as well as poor bone healing and regeneration characteristics (10), conditions which all rely, in part, upon an intramembranous component to bone formation. Increasing evidence suggests that skeletal abnormalities in type 1 diabetes may, in part, result from the detrimental effects of type 1 diabetes on bone formation. For example, decreased expression of transcription factors that regulate osteoblast differentiation have been demonstrated in animal models of type 1 diabetes (11). Numerous reports of bone histology in diabetic animals demonstrate decreased osteoblast number, osteoid volume, and mineral apposition rates (rev. in 12). In diabetic rats, plasma osteocalcin concentrations, a marker of osteoblast activity, acutely decline beginning on the 1st day of glucosuria (13). Similarly, serum concentrations of osteocalcin in children with newly diagnosed type 1 diabetes are significantly lower at the onset of disease (14). Serum markers correlated with bone formation (IGF-I, alkaline phosphatase, and osteocalcin) also are significantly lower in diabetic patients with osteopenia compared with those without osteopenia (2), and studies have demonstrated that lower bone mineral density (BMD) in type 1 diabetes is correlated with decreased markers of bone formation and more exaggerated dysregulation of the IGF system (15).The present study was designed to test the hypothesis that type 1 diabetes specifically impedes intramembranous bone formation by using a model of tibial distraction osteogenesis uniquely modified for use ...

We previously described a rabbit osteomyelitis model that involved the direct introduction of Staphylococcus aureus into devascularized bone. To further evaluate the model, we performed experiments aimed at correlating the microbiological, radiographic, and histologic parameters involved in the development of experimental osteomyelitis. Using the strain UAMS-1, we achieved an infection rate of 75% with an inoculum as small as 2 x 10(3) colony-forming units. However, development of significant radiographic and histologic signs of disease required an inoculum of at least 2 x 10(4) colony-forming units. Radiographic signs were minimal 1 week after infection and progressed steadily to a maximum 3 weeks after infection. In contrast, histologic signs of disease were observed within 1 week and remained essentially unchanged throughout the 4-week evaluation period. Unlike the results obtained with UAMS-1, rabbits infected with the heavily encapsulated Staphylococcus aureus strain Smith diffuse exhibited little evidence of disease even when infected with 2 x 10(6) colony-forming units. The reduced virulence of strain Smith diffuse was surprising given its greatly enhanced virulence (relative to UAMS-1) in a murine peritonitis model of staphylococcal disease. These results suggest that UAMS-1 expresses virulence factors that are important in the pathogenesis of osteomyelitis and that some or all of these virulence factors are either absent or are not expressed in strain Smith diffuse. Most importantly, the results suggest that our model may be appropriate for the identification and characterization of these virulence factors.

To examine the role of the accessory gene regulator (agr) in staphylococcal osteomyelitis, we compared a Staphylococcus aureus osteomyelitis isolate (UAMS-1) with a derivative of the same strain (UAMS-4) carrying an inactivated agr locus. Virulence was assessed with a rabbit model of acute, exogenous osteomyelitis. Bacteria were delivered by microinjection into the midradial region of the forelimb. After 4 weeks, UAMS-1 was identified in the bone of 12 of 13 rabbits infected with >2 ؋ 10 6 CFU and 5 of 6 infected with <2 ؋ 10 5 CFU. In contrast, UAMS-4 was found in 6 of 13 infected with the higher dose and 1 of 6 infected with the lower dose. Additionally, on the basis of a five-point scale assessing radiographic evidence of disease, rabbits infected with UAMS-1 had average scores of 2.64 ؎ 0.30 (high dose) and 1.43 ؎ 0.39 (low dose) while rabbits infected with UAMS-4 had average scores of 0.95 ؎ 0.23 (high dose) and 0.63 ؎ 0.20 (low dose). Uninfected controls had an average score of 0.53 ؎ 0.08. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < 0.047). The results obtained with UAMS-4 were not significantly different from those obtained with the controls at either dose of UAMS-4 (P > 0.150). On the basis of a similar five-point scale assessing histopathological evidence of disease, rabbits infected with UAMS-1 had average scores of 2.31 ؎ 0.22 (high dose) and 1.96 ؎ 0.36 (low dose) while rabbits infected with UAMS-4 had average scores of 1.58 ؎ 0.29 (high dose) and 0.83 ؎ 0.32 (low dose). Controls had an average score of 0.33 ؎ 0.05. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < 0.040). However, the results obtained with UAMS-4 were significantly different from the controls only at the high dose of UAMS-4 (P ‫؍‬ 0.025). We conclude that mutation of agr reduces the incidence and severity of disease but does not eliminate the ability to colonize bone and cause histopathological evidence of osteomyelitis.