CARTILAGE AUTOCATABOLISM AND INFECTIOUS ARTHRITIS

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Staphylococcal septic arthritis remains a serious clinical problem in very young children, in individuals with chronic rheumatoid arthritis and in immunocompromised patients. The consequence of Staphylococcal joint infection is residual joint destruction often in spite of appropriate antibiotic therapy. The etiology of Staphylococcal joint destruction involves multiple parameters that include the inflammatory response to the bacteria, bacterial expression of virulence factors, the state of the host immune system and bacterial adherence factors. Our studies demonstrate that direct effects of Staphylococcus aureus on cartilage metabolism accelerate extracellular matrix destruction in vitro. The direct effects of S. aureus on cartilage occur through the release of a soluble protein factor (Staph Factor) that triggers chondrocyte-mediated autocatabolism. Staph Factor requires living chondrocytes and induces the expression of matrix metalloproteinases, interleukin-6 and prostaglandin E2 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 in vitro and in vivo using cartilage degradation assays and proteoglycan loss in a rabbit knee model. However, the precise moleculer structure of Staph Factor remains unknown. The purpose of this proposal is to address the fundamental question regarding the nature of this potent destructive mediator. Preliminary data suggest that the molecule does not fall into the peptidoglycan or endotoxin superantigen paradigms. 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. Specific Aim 1 will consist of a Part A that will focus on the characterization of the primary protein sequence of the Staph Factor and a Part B that will focus on the characterization of the carbohydrate modifications of the protein structure. The hypothesis being addressed is that the unique biological properties of Staph Factor derive from the unique molecular structure. The structure of the molecule renders its capacity to induce cartilage destruction resistant to heat, proteolytic attack and glycolytic cleavage. With the advent of greater numbers of antibiotic resistant strains of Staphylococcal organisms, the importance of this work is critical to the fields of pediatric orthopaedics, rheumatology and adult orthopaedics. The activity of Staph Factor on dermal endothelial cells makes it important to dermatology. The laboratory techniques involved will include ultrafiltration, lectin affinity chromatography, FPLC protein chromatography, two-dimensional SDS-PAGE, microsequencing and carbohydrate analysis using mass- spectrophotometry.