Pneumococcal transition from nasopharyngeal biofilm carriage to otitis media

项目摘要/摘要 肺炎链球菌(简称肺炎球菌)是人类鼻咽(NP)的一种有效的定殖物。 有时会导致疾病。然而，由于广泛的殖民统治，疾病经常发生，以至于S。 肺炎是包括儿童中耳炎(OM)在内的许多感染的主要原因，与 并发症，如听力损失、语言和认知发展障碍。虽然 肺炎球菌的发病机制研究由来已久，我们对其复杂的相互作用有一定的认识 人类宿主和细菌之间的关系，特别是当它们与定植和向OM的过渡有关时 不完整。从流行病学上讲，从定居到OM的转变与伴随的病毒感染有关 或其他对宿主防御系统的攻击，但涉及的具体机制还不是很清楚。 我们最近进行了一项新的观察，发现肺炎链球菌体内携带NP与生物被膜有关。 形成并已表明，在体外重复这种表型需要NP的某些方面 环境，包括较低的温度、与上皮细胞的相互作用和有限的养分供应。 此外，我们还发现，这种环境的扰动特别会将细菌从 容易扩散到中耳(ME)的生物膜，导致比这两种生物膜更严重的疾病 细菌或在肉汤中生长的细菌，具有独特的基因表达谱，这使我们能够识别 细菌释放、传播和诱导OM所需的几种分子。 这些研究的目标是机械地探索肺炎球菌释放所涉及的信号。 来自NP生物膜(肺炎球菌生物膜和具有常见NP定植的多微生物生物膜 物种)，扩散到ME，并在ME中存活使用我们新的基于上皮细胞的生物膜模型和 殖民和感染的动物模型，我们假设用来干扰NP环境 分散剂，如温度升高(发烧)、营养素和三磷酸腺苷(与细胞损伤有关)和病毒 感染(常见疾病触发)，与肺炎球菌肺炎高度相关，会导致释放 表型改变的生物被膜细菌与引起ME感染的能力增加有关。 在这里描述的研究中，我们将探索细菌在体外和体内生物膜中的释放。 暴露于正常生物膜/NP环境的生理变化(目标1)或接触甲型流感病毒和 呼吸道合胞病毒感染(目标2)。然后我们将研究细菌在体外或体内释放的能力 诱发多发性骨髓瘤。最后，我们将使用rna-seq扩展我们对细菌种群的转录组分析。 并鉴定和表征参与细菌生物被膜释放、传播和诱导的分子 OM(目标3)。这些研究将提供有关有毒细菌从 在体外和体内生长的生物膜，并提供环境和细菌因素的特定信息 负责体内毒力诱导，可用于抑制向中耳炎的转变。

Project Summary / Abstract Streptococcus pneumoniae (the pneumococcus) is an effective colonizer of the human nasopharynx (NP) that on occasion causes disease. However, due to widespread colonization, disease occurs often enough that S. pneumoniae is a leading cause of numerous infections including otitis media (OM) in children, with associated complications, such as hearing loss and impairment of language and cognitive development. Although pneumococcal pathogenesis has been studied for a long time, our understanding of the complex interactions between the human host and bacteria, especially as they relate to colonization and the transition to OM is incomplete. Epidemiologically, transition from colonization to OM is associated with concomitant virus infection or other assaults on the host defense system, but the specific mechanisms involved are not very clear. We recently made the novel observation that pneumococcal NP carriage in vivo is associated with biofilm formation and have shown that recapitulation of this phenotype in vitro requires aspects of the NP environment, including lower temperature, interaction with epithelial cells and limited nutrient availability. Moreover, we have discovered that perturbation of this environment specifically releases bacteria from the biofilm that readily disseminate to the middle ear (ME), causing more severe disease than either biofilm bacteria or bacteria grown in broth and have a unique gene expression profile, that has allowed us to identify several molecules required for bacterial release, dissemination, and induction of OM. The goals of these studies are to mechanistically explore the signals involved in pneumococcal release from NP biofilms (both pneumococcal biofilms and poly-microbial biofilms with common NP colonizing species), dissemination to, and survival in the ME using our novel epithelial cell-based biofilm models and animal models of colonization and infection, and we hypothesize that perturbing the NP environment with dispersants such as increased temperature (fever), nutrients and ATP (associated with cell damage) and virus infection (common disease trigger), that is highly associated with pneumococcal OM will induce release of biofilm bacteria with altered phenotypes associated with increased ability to cause infection of the ME. In the studies delineated herein, we will explore release of bacteria from biofilms in vitro and in vivo after exposure to physiological changes of the normal biofilm/NP environment (Aim 1) or to influenza A virus and respiratory syncytial virus infection (Aim 2). We will then study the ability of bacteria released in vitro or in vivo to induce OM. Finally, we will expand our transcriptome analyses of the bacterial populations using RNA-seq and identify and characterize molecules involved in bacterial biofilm release, dissemination to, and induction of OM (Aim 3). The studies will provide mechanistic information regarding the release of virulent bacteria from biofilms growing in vitro and in vivo and provide specific information of the environmental and bacterial factors responsible for virulence induction in vivo that can be used to inhibit the transition to otitis media.