Role of epigenetic regulation in the persistence of NTHI during colonization, ascension of the Eustachian tube and chronic otitis media

项目摘要 中耳感染或中耳炎(OM)是儿童最常见的细菌性疾病，导致 在美国和全球都有显著的发病率。多动症发作会严重影响儿童的听力 通常是这样的。由于对语言的长期影响，这种听力损失是非常令人担忧的 发展。多种不同的细菌和病毒会引起中耳感染。不可打印 流感嗜血杆菌是慢性OM、复发性OM和复发性OM的主要致病菌，其中 治疗失败了。包括NTHI在内的几种人类病原体已经进化出一种新的遗传系统， 称为相变子(相变调节子)，它介导了一种快速和可逆的变化。 许多基因在整个染色体上的表达。这种表观遗传调控通过时相变化发生。 编码DNA甲基转移酶的单个基因(Moda)，并导致两个不同的表型 亚群，断断续续。我们的初步数据清楚地表明，基因的差异 这些亚群之间的表达对已知的关键体外表型有显著影响 与OM的病程密切相关。我们进一步显示了Phasevarion的状态，以及从 在实验性OM的龙猫模型中，一种状态对另一种状态显著影响疾病严重程度。 然而，在提升过程中，modA2-从定植部位、鼻咽、 到了发病的地方，中耳是未知的。在特定目标1中，我们将评估特定部位的表达 MODA2相变调控基因在模拟上行实验模型中的作用 儿童的疾病病程。在实验性OM期间，确定宿主内基因表达的研究是 完全理解相变子对两者的作用机制是绝对必要的。 疾病的严重性和细菌在各种解剖环境中的持久性。此外，相变调节 通过表观遗传甲基化发生，在硅胶中无法识别。在具体目标2中，我们将定义 免疫细胞招募，定义细胞因子表达谱，并识别针对每种细胞因子开发的抗体 ModA2种群以及在疾病发展过程中modA2状态的变化。一个全面的 了解Phasevarion何时、何地和如何调控毒力因子组合的表达 随着随后的宿主对这些变化的免疫反应，NTHI基因的表达对于 制定最佳的疾病治疗和预防战略。我们希望为以下方面做出重大贡献 这提高了对相变子在细菌-宿主相互作用中作用的理解，通过成功地 完成本提案的具体目标。

Project Summary Middle ear infection or otitis media (OM) is the most predominant bacterial disease of childhood and results in significant morbidity in the U.S. and globally. Episodes of OM can substantially affect a child’s ability to hear normally. This loss of hearing is of great concern due to the long-term consequences on language development. Multiple different bacterial species and viruses cause middle ear infections. Nontypeable Haemophilus influenza (NTHI) is the primary bacterial pathogen of chronic OM, recurrent OM and OM wherein there is treatment failure. Several human pathogens, including NTHI, have evolved a novel genetic system, termed the phasevarion (for phase variable regulon), which mediates a rapid and reversible change in the expression of many genes throughout the chromosome. This epigenetic regulation occurs via phase variation of a single gene (modA) that encodes a DNA methyltransferase, and results in two phenotypically distinct subpopulations, ON and OFF. Our preliminary data clearly demonstrated that differences in the gene expression between these subpopulations have a significant affect on in vitro phenotypes known to be critical to the disease course of OM. We further showed that the status of the phasevarion, as well as switching from one status to the other, significantly affects disease severity in a chinchilla model of experimental OM. However, ModA2-regualted gene expression during ascension from the site of colonization, the nasopharynx, to the site of disease, the middle ear is unknown. In Specific Aim 1, we will assess site-specific expression of genes regulated by the ModA2 phasevarion in a model of ascending experimental model that mimics the disease course in children. Studies to determine gene expression within the host during experimental OM are absolutely necessary to fully understand the mechanisms by which the phasevarion contributes to both disease severity and bacterial persistence in various anatomical niches. Additionally, phasevarion regulation occurs through epigenetic methylation and cannot be discerned in silico. In Specific Aim 2, we will define immune cell recruitment, define cytokine expression profiles and identify antibodies developed against each modA2 population as well as a shift in ModA2 state over the course of disease development. A comprehensive understanding of when, where and how the phasevarion regulates expression of virulence factors combined with subsequent host immune responses to these changes in NTHI gene expression is required for the development of optimal disease treatment and prevention strategies. We expect to contribute significantly to this improved understanding of the role of the phasevarion in bacterial-host interactions via successful completion of Specific Aims of this proposal.