Cellular and Molecular Biology of Cholesteatoma

描述（由申请人提供）：中耳和乳突胆脂瘤是中耳炎的并发症。这些表皮结构经常被需氧菌和厌氧菌的混合物感染，其中最常见的是Ps。铜绿。细菌在中耳瘤内形成生物膜，导致慢性感染。这些感染的中耳瘤是侵略性的，并导致骨质溶解增加。我们已经确定了耳致病性Ps菌株。铜绿假单胞菌（OPPA），其具有增加的对角质形成细胞的粘附和增加的生物膜产生。我们将通过检测群体感应基因的表达和藻酸盐基因的表达和生产来进一步表征这些菌株。我们建议研究这些慢性感染的发病机制和毒力。关于发病机制，我们将研究这些生物体的粘附角质形成细胞使用随机定向的小Tn5转座子诱变粘附OPPA分离株。诱变筛选将富集非粘附突变细菌，随后将测序以鉴定新的粘附素基因。关于毒力，初步研究表明，我们的OPPA菌株通过LPS依赖性和LPS非依赖性机制产生破骨细胞生成。将使用LPS敏感的鼠破骨细胞前体进行LPS依赖性研究。将测定与破骨细胞生成相关的信号（例如RANKL、TNFa）的表达以理解Ps的机制。铜绿假单胞菌LPS介导的破骨细胞生成。为了研究不依赖于LPS的破骨细胞生成，我们将使用来自Toll样受体4缺陷小鼠的LPS不敏感的破骨细胞前体来确定破骨细胞发育途径的哪些部分被诱导。

DESCRIPTION (provided by applicant): Cholesteatomas of the middle ear and mastoid develop as a complication of otitis media. These epidermal structures often become infected with a mixture of aerobic and anaerobic bacteria, the most common of which is Ps. aeruginosa. Bacteria form biofilms within cholesteatomas resulting in chronic infection. These infected cholesteatomas are aggressive and cause increased osteolysis. We have identified strains of otopathogenic Ps. aeruginosa (OPPA) that have increased adherence to keratinocytes and increased biofilm production. We will further characterize these isolates by examining the expression of quorum sensing genes and alginate gene expression and production. We propose to study the pathogenesis and virulence of these chronic infections. With regard to pathogenesis, we will study the adherence of these organisms to keratinocytes using randomly directed mini-Tn5 transposon mutagenesis of adherent OPPA isolates. The mutagenesis screen will be enriched for non-adherent mutant bacteria and will subsequently be sequenced to identify novel adhesin genes. With regard to virulence, preliminary studies show that our OPPA strains produce osteoclastogenesis by both an LPS-dependent and LPS-independent mechanism. LPS-dependent studies will be done with LPS sensitive murine osteoclast precursors. Expression of signals associated with osteoclastogenesis (e.g. RANKL, TNFa) will be determined to understand the mechanism of Ps. aeruginosa LPS mediated osteoclastogenesis. To study LPS-independent osteoclastogenesis, we will use LPS-insensitive osteoclast precursors derived from mice deficient in toll-like receptor 4 to determine which portions of the osteoclast development pathway are induced.