Cellular and Molecular Biology of Cholesteatoma

描述(申请人提供)：中耳和乳突的胆脂瘤发展为中耳炎的并发症。这些表皮结构通常会受到需氧细菌和厌氧细菌的混合感染，其中最常见的是Ps。铜绿假单胞菌。细菌在胆脂瘤内形成生物膜，导致慢性感染。这些感染的胆脂瘤具有侵袭性，并导致骨溶解增加。我们已经鉴定出耳源性肺炎链球菌的菌株。铜绿假单胞菌(Oppa)增加了对角质形成细胞的黏附，增加了生物膜的产生。我们将通过检测群体感应基因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。在致病机制方面，我们将利用粘附性OPPA分离株的微型Tn5转座子的随机定向突变来研究这些细菌与角质形成细胞的粘附性。突变筛选将丰富非粘附性突变细菌，随后将对其进行测序，以确定新的粘附素基因。关于毒力，初步研究表明，我们的OPPA菌株通过内毒素依赖和非内毒素依赖的机制产生破骨细胞。脂多糖依赖的研究将用脂多糖敏感的小鼠破骨细胞前体进行。与破骨细胞形成相关的信号(如RANKL、TNFa)的表达将被确定以了解Ps的机制。铜绿假单胞菌脂多糖介导的破骨细胞生成。为了研究内毒素非依赖性破骨细胞的形成，我们将使用来自Toll样受体4缺陷小鼠的内毒素不敏感的破骨细胞前体细胞来确定诱导破骨细胞发育途径的哪些部分。

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.