Aural cholesteatomas are epithelial cyst-like structures that expand into adjacent bone, resulting in chronic infection and hearing loss. The overall goal of this proposal is to investigate the mechanisms of bacterial-host interactions in the pathogenesis and virulence of cholesteatomas infected with Pseudomonas aeruginosa (PA). These bacteria form biofilms within the cholesteatoma resulting in chronic infection which is recalcitrant to antibiotics and host defenses. The pathophysiology of infected cholesteatomas is complex involving interactions among bacterial factors, the underlying epithelium and adjacent bone. In Specific Aim I, we will use a gerbil model of experimental cholesteatoma to evaluate the pathogenesis of otopathogenic PA isolates isolated from human cholesteatomas. We have chosen a representative strain, OPPA8 for sequencing and analysis. Evaluation of site directed deletion mutants will provide insight into the genes necessary for cholesteatoma development and enhanced aggressiveness. We also plan to evaluate a number of known virulence factors in the gerbil model by using targeted deletions of key virulence genes (galU, algC, algD, lasI, lasR, fliC, exsA). In Specific Aim II, our goal is to elucidate the mechanisms of PA LPS in host injury and response, specifically deregulated keratinocyte proliferation and bone resorption. We will examine the effect of lipopolysaccharide in the proliferation and differentiation of keratinocytes in vitro using an immortalized cell line, primary human and primary mouse keratinocytes. We will further examine the mechanism of PA LPS induced proliferation in vitro by evaluating downstream effectors involved in PA LPS mediated proliferation and differentiation. By comparing cytokine profiles from PA LPS stimulated keratinocytes derived from TLR2-/-, TLR4-/- and MyD88-/- knockout mice we hope to delineate the mechanism by which this process occurs. Additionally, we will evaluate the mechanisms of PA LPS induced bone resorption in vivo using a murine model of localized bone resorption. Previous studies in our laboratory demonstrated PA LPS induces bone resorption in vitro through a TLR4, MyD88 dependent pathway but preliminary in vivo data also suggests a role for TLR2. An understanding of the pathogenicity and virulence of PA in cholesteatoma may lead to new targets of drug therapy or treatment modalities that will prevent or ameliorate the hearing loss associated with this disease.