Pneumococcal transition from nasopharyngeal biofilm carriage to otitis media

描述（由申请人提供）：肺炎链球菌（肺炎球菌）是人类鼻咽部（NP）的有效定植菌，有时会引起疾病。然而，由于广泛的定植，疾病的发生频率足以使肺炎链球菌成为许多感染的主要原因，包括儿童中耳炎（OM），并伴有相关并发症，如听力损失、语言和认知发育障碍。尽管肺炎球菌的发病机制已经研究了很长时间，但我们对人类宿主与细菌之间复杂的相互作用，特别是与定植和向OM过渡有关的相互作用的了解尚不完整。从流行病学上讲，从定植到OM的转变与伴随的病毒感染或对宿主防御系统的其他攻击有关，但所涉及的具体机制尚不清楚。我们最近进行了新的观察，即肺炎球菌在体内携带NP与生物膜的形成有关，并表明这种表型在体外的重现需要NP环境的各个方面，包括较低的温度、与上皮细胞的相互作用和有限的营养可用性。此外，我们已经发现，这种环境的扰动特异性地从生物膜中释放细菌，这些细菌容易传播到中耳（ME），导致比生物膜细菌或在肉汤中生长的细菌更严重的疾病，并且具有独特的基因表达谱，这使我们能够确定细菌释放，传播和诱导OM所需的几个分子。这些研究的目的是通过我们新的基于上皮细胞的生物膜模型和动物定植和感染模型，从机制上探索肺炎球菌从NP生物膜（包括肺炎球菌生物膜和具有常见NP定植物种的多微生物生物膜）释放、传播和在ME中存活的信号，我们假设用温度升高（发烧）等分散剂扰动NP环境，营养物质和ATP（与细胞损伤相关）和病毒感染（常见的疾病诱因），这与肺炎球菌性肺泡炎高度相关，将诱导生物膜细菌的释放，其表型改变与引起肺泡炎感染的能力增加有关。在本文所述的研究中，我们将探索暴露于正常生物膜/NP环境的生理变化（目的1）或暴露于甲型流感病毒和呼吸道合胞病毒感染（目的2）后，细菌在体外和体内从生物膜中释放的情况。然后，我们将研究体外或体内释放的细菌诱导OM的能力。最后，我们将利用RNA-seq扩展我们对细菌群体的转录组分析，并鉴定和表征参与细菌生物膜释放、传播和诱导OM的分子（目的3）。这些研究将提供关于从体外和体内生长的生物膜中释放有毒细菌的机制信息，并提供负责体内毒性诱导的环境和细菌因素的具体信息，这些因素可用于抑制向中耳炎的过渡。

DESCRIPTION (provided by applicant): 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 n 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.