In Vivo Virulence Gene Expression in Acute Otitis Media

描述（由申请人提供）：肺炎链球菌是急性中耳炎（OM）的主要原因，每年导致超过100万婴儿死于肺炎、脑膜炎和菌血症。随着抗生素耐药性肺炎球菌菌株的出现和传播，加上毒力模式的变化和可用疫苗的不足，这些“肺炎球菌疾病”的医疗管理变得越来越复杂和昂贵。S.肺炎链球菌高度适应并无症状地定殖于鼻咽的粘膜表面。S.肺炎球菌与宿主中耳粘膜（MEM）结合是启动OM的关键步骤。控制S.肺炎是否定植于鼻咽部或侵入MEM引起OM尚不清楚。S.肺炎克雷伯氏菌在菌落形态上经历自发的菌株内变异，其影响多个细胞表面结构、病原体-宿主相互作用和毒力。透明（T）变体更能够定殖于鼻咽，而不透明（O）变体在全身感染期间显示出增加的毒力。环境因素（例如，氧和压力），并鉴定了与表型变异相关的几个基因。中耳是一个无菌的充满空气的腔体，其所含气体的体积决定了其压力，而压力受粘膜-血液扩散和咽鼓管功能的调节。ET是从鼻咽部进入ME的重要通道。肺炎和宿主ET功能-ME环境，其触发病原体突破宿主防御，易位和侵入ME导致感染尚未建立。我们推测ET功能障碍和相关的ME气体/压力失调诱导了毒性表型的优势选择，并增强了肺炎球菌的毒力，从而导致急性OM。我们有三个具体目标：目的1是确定肺炎球菌的表型变化在ME气体/压力失调在体内（急性OM的大鼠模型构建与ET阻塞，然后ME接种的T或O变种，然后通过通气管插入）和体外（人ME上皮细胞系暴露于选择性压力和T或O变种）。目的2是评估编码先前在体内或体外测试的与肺炎球菌相变异和毒力相关的毒力因子的基因表达。目的3是连接主机响应表型变异和毒力的评价选定的促炎介质中的MEM。识别肺炎球菌的变异体和相关的毒力基因，优先转录在改变ME环境下的表型开关是中央我们了解肺炎球菌如何安装一个成功的ME感染。从这项研究中获得的信息，为临床分子诊断和治疗的毒力靶向基因中断的承诺。

DESCRIPTION (provided by applicant): Streptococcus pneumoniae is a leading cause of acute otitis media (OM) and responsible for over 1 million infant deaths each year from pneumonia, meningitis, and bacteremia. With the emergence and dissemination of antibiotic resistant pneumococcal strains, coupled to changing patterns of virulence and the inadequacy of available vaccines, medical management of these "pneumococcal diseases" has become increasingly complex and costly. S. pneumoniae is highly adapted to and colonizes asymptomatically the mucosal surface of the nasopharynx. The ability of S. pneumoniae to bind to the host middle ear mucosa (MEM) is a critical step to initiate OM. The mechanisms that control S. pneumoniae either to colonize the nasopharynx or to invade to the MEM causing OM are unclear. S. pneumoniae undergoes spontaneous intrastrain variations in colony morphology that affect multiple cell-surface structures, pathogen-host interaction and virulence. Transparent (T) variants are more capable of colonizing the nasopharynx, whereas the opaque (O) variants show increased virulence during systemic infections. Environmental factors (e.g., oxygen and pressure) and several genes associated with the phenotypic variations were identified. Middle ear (ME) is a sterile and air-filled cavity and its volume of the contained gas determines its pressure, which is regulated by mucosal-blood diffuse and Eustachian tube (ET) function. ET is a critical passageway from the nasopharynx into the ME. The dynamic interactions between S. pneumoniae and host ET function-ME environment, which trigger pathogen to breach host defense, translocate and invade to the ME leading to infection have yet to be established. We hypothesize that ET dysfunction and associated ME gas/pressure dysregulation induce the predominant selection of the virulent phenotypes and enhance pneumococcal virulence, which cause acute OM. We have three specific aims: Aim 1 is to identify pneumococcal phenotypic variations during ME gas/pressure dysregulations in vivo (a rat model of acute OM constructed with ET obstruction followed by ME inoculation of either T or O variants, then by ventilation tube insertion) and in vitro (human ME epithelial cell line exposed to selective pressures and T or O variants). Aim 2 is to assess gene expression coding for virulence factors that were previously tested either in vivo or in vitro to be associated with pneumococcal phase variations and virulence. Aim 3 is to link host response to phenotypic variations and virulence by evaluation of the selected proinflammatory mediators in the MEM. Identification of pneumococcal variants and associated virulence genes that are preferentially transcribed during the phenotypic switch under the altered ME environment is central to our understanding of how pneumococcus mounts a successful ME infection. The information gained from this study holds a promise for clinical molecular diagnostics and therapy by targeted gene interruptions of virulence.