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。随着耐抗生素肺炎球菌菌株的出现和传播，再加上毒力模式的变化和现有疫苗的不足，这些“肺炎球菌疾病”的医疗管理变得越来越复杂和昂贵。肺炎链球菌高度适应并无症状地定植于鼻咽部粘膜表面。肺炎链球菌与宿主中耳黏膜（MEM）结合的能力是引发OM的关键步骤。控制肺炎链球菌定殖鼻咽部或侵入MEM引起OM的机制尚不清楚。肺炎链球菌在菌落形态上经历自发的菌株内变异，影响多种细胞表面结构、病原体-宿主相互作用和毒力。透明(T)变异体更能在鼻咽部定植，而不透明(O)变异体在全身感染时表现出更高的毒力。环境因素（如氧气和压力）和几个与表型变异相关的基因被确定。中耳（ME）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，其压力受粘膜-血液扩散和耳咽管（ET）功能的调节。鼻咽部是鼻咽部进入胃内窥镜的重要通道。肺炎链球菌与宿主ET功能-ME环境之间的动态相互作用，触发病原体突破宿主防御，易位并侵入ME导致感染的机制尚未建立。我们假设ET功能障碍和相关的ME气体/压力失调诱导了毒性表型的主要选择，并增强了肺炎球菌的毒力，从而导致急性OM。我们有三个具体目的：目的1是在体内（一种由ET阻塞构建的急性OM大鼠模型，随后接种T或O变体的ME，然后插入通气管）和体外（暴露于选择性压力和T或O变体的人ME上皮细胞系）中确定肺炎球菌在ME气体/压力失调期间的表型变化。目的2是评估先前在体内或体外测试中与肺炎球菌期变异和毒力相关的毒力因子的基因表达编码。目的3是通过评价MEM中选定的促炎介质，将宿主反应与表型变异和毒力联系起来。在改变的ME环境下，在表型转换过程中优先转录的肺炎球菌变异和相关毒力基因的鉴定对于我们理解肺炎球菌如何成功感染ME至关重要。从这项研究中获得的信息为临床分子诊断和靶向毒性基因中断治疗提供了希望。

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.