Investigator: R. Lane Smith, PhD Project Staff: David J. Schurman, MD Project Category: Arthritis - 2000 Background: Staphylococcal septic arthritis continues to be recognized as a serious clinical problem and has been the focus of a number of clinical approaches to treat bacterial infection and prevent residual cartilage damage. One major difficulty with Staphylococcal joint infections is that the onset of cartilage destruction begins within hours of infection. Our studies using a rabbit model of Staphylococcal arthritis demonstrates that 40% of total proteoglycan is loss within 48 hours of intra-articular inoculation with 105 colony forming units of S. aureus. Early treatment with antibiotic slows but does not prevent the onset of cartilage destruction. Recent studies have analyzed the effects of combining steroid treatment in an effort to control the contribution of the inflammatory reaction to the bacteria on the cartilage destruction. Generally, the extent of cartilage breakdown was lessened with approaches to decrease inflammatory processes. However, in all cases the onset of cartilage breakdown had occurred in spite of the treatment methods in the models studied. Our studies demonstrate that direct effects of Staphylococcus aureus on the articular cartilage that are evident in vitro may contribute to the rapid onset of cartilage destruction in the septic joint. The direct effects of S. aureus on cartilage destruction result from the release of a soluble protein factor (Staph Factor) that activates chondrocyte-mediated autocatabolism. Staph Factor requires living chondrocytes and induces the upregulation of the matrix metalloproteinases while selectively inhibiting the synthesis of cartilage matrix macromolecules. Staph Factor has been partially purified and the range of its biological activity has been confirmed using in vitro cartilage assays and in vivo in a rabbit knee model. However, the precise molecular structure of Staph Factor is as yet unknown. The purpose of this proposal is to address the fundamental question regarding the molecular nature of this potent destructive mediator. Preliminary data suggest that the molecule does not fall into the peptidoglycan or endotoxin superantigen paradigms. Hypothesis: The hypothesis being addressed is that the unique biological properties of Staph Factor derive from a molecular structure that activates cartilage destruction and imparts resistance to proteolytic and glycolytic attack. The increased number of antibiotic-resistant strains of Staphylococcal organisms makes the characterization of Staph Factor critical to the fields of pediatric orthopaedics, rheumatology and adult orthopaedics. Objectives: The specific aim of this proposal is to purify the Staph Factor to homogeneity so that the molecular mechanisms by which Staphylococcus aureus induces cartilage destruction may be further understood. The work will involve determination of the primary protein sequence of the Staph Factor and include characterization of the carbohydrate modifications of the protein. Procedures: The laboratory techniques involved will include ultrafiltration, lectin affinity chromatography and FPLC protein chromatography, two-dimensional SDS-PAGE, microsequencing and carbohydrate analysis using mass-spectrometry. Funding Source: NIH |
