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好氧和厌氧细菌的混合物感染，其中最常见的是铜绿假单胞菌。细菌在胆脂瘤内形成生物膜，导致慢性感染。这些受感染的胆脂瘤具有侵袭性，可导致骨质溶解增加。我们已经确定了耳致病性铜绿假单胞菌（OPPA）的菌株，增加了对角质形成细胞的粘附性，增加了生物膜的产生。我们将通过检测群体感应基因的表达和海藻酸盐基因的表达和产生来进一步表征这些分离株。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，我们将使用随机定向的mini-Tn5转座子诱变粘附OPPA分离物来研究这些生物对角质形成细胞的粘附性。突变筛选将丰富非粘附突变细菌，随后将测序以鉴定新的粘附素基因。在毒力方面，初步研究表明，我们的OPPA菌株通过lps依赖性和不依赖性机制产生破骨细胞。脂多糖依赖性研究将用脂多糖敏感的小鼠破骨细胞前体进行。通过测定破骨细胞生成相关信号（如RANKL、TNFa）的表达，了解铜绿假单胞菌LPS介导破骨细胞生成的机制。为了研究不依赖于脂多糖的破骨细胞发生，我们将使用来自toll样受体4缺失小鼠的脂多糖不敏感破骨细胞前体来确定诱导破骨细胞发育途径的哪一部分。

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.