An air-liquid interface system to study Bordetella pertussis interactions with respiratory epithelia

百日咳杆菌是一种引起百日咳的细菌。 据估计，每年有2400万例疫苗可预防的疾病，导致超过 每年有17万人死亡。重要的是，百日咳在高发国家的发病率 疫苗覆盖率正在上升，这归因于无症状传播是由 当前无细胞百日咳疫苗免疫力不完善和减弱。由于这些和其他原因 美国疾病控制与预防中心和美国国立卫生研究院都已将百日咳杆菌列为新出现的优先(重新)病原体。 担忧。B.百日咳有效地在人类呼吸道内定居和生长， 需要它接触并穿过粘液和溶胶层才能找到、紧密附着和生长 纤毛上皮细胞。这些能力对它的非凡成功至关重要，但也很困难。 在活体内进行详细研究。它们主要是在深层细胞培养中进行研究， 宿主细胞和百日咳杆菌的单层都浸没在丰富的哺乳动物细胞生长介质中。 这些条件不能复制柱状呼吸道上皮的结构或功能。 并且完全缺乏自然呼吸道的覆盖粘液、溶胶和空气界面， 百日咳杆菌自然生长的环境。我们的团队有几十年的经验， 波尔德氏菌，并从人的支气管组织中进行极化原代培养， 包含具有代表性的细胞类型，最重要的是包括纤毛在保护范围内跳动 粘液层和溶胶层。在这里，我们将第一次使用这种气液界面(ALI)系统， 结合百日咳杆菌遗传学和生物学技术，探讨Key在百日咳中的作用 宿主-病原体与真实的人类呼吸道上皮相互作用中的细菌因素。 具体地说，我们提出三个目标来(1)确定通过以下途径调节渗透的因素 (2)确定了B。 百日咳因子诱导和/或调节上皮细胞产生促炎/抗炎作用 信号，以及(3)确定百日咳杆菌如何破坏细胞和破坏上皮屏障。 这些研究产生的数据将揭示特定的百日咳杆菌因子在 它们与纤毛呼吸道上皮相互作用的各种可测量的方面 告知能够阻断呼吸道相互作用的新疫苗靶点的选择。

Bordetella pertussis, the bacterial pathogen responsible for “whooping cough” causes an estimated 24 million cases of vaccine-preventable illness per year, resulting in an excess of 170,000 deaths annually. Importantly, the incidence of whooping cough in nations with high vaccine coverage is on the rise, attributed to asymptomatic transmission that is enabled by the imperfect and waning immunity of current acellular pertussis vaccines. Due to these and other factors, both the CDC and NIH have listed B. pertussis as a priority (re)emerging pathogen of high concern. B. pertussis efficiently colonizes and grows within the human respiratory tract, requiring that it access and cross mucus and sol layers to find, tightly attach to and grow on ciliated epithelial cells. These abilities are critical to its remarkable success, but are difficult to study in detail in vivo. They have been studied primarily in submerged cell culture, with monolayers of host cells and B. pertussis all submerged in rich mammalian cell growth media. These conditions do not replicate the structure or function of the columnar airway epithelia and completely lack the overlying mucus, sol and air-interface of a natural airway, and the milieu in which B. pertussis naturally grows. Our team has decades of experience with Bordetella spp and with polarized primary culture from human broncho-tracheal tissues which contains representative cell types, most importantly including cilia beating within protective mucus and sol layers. Here, for the first time, we will use this air-liquid interface (ALI) system, combined with techniques in genetics and biology of B. pertussis, to probe the roles of key bacterial factors in host-pathogen interactions with realistic human respiratory epithelia. Specifically, we propose three aims to (1) identify factors that mediate infiltration through mucosal layers and enable bacterial growth on ciliated epithelia, (2) define the effect of B. pertussis factors on inducing and/or modulating epithelial cell produced pro/anti-inflammatory signals, and (3) determine how B. pertussis damages cells and disrupts the epithelial barrier. The data generated from these studies will reveal the roles of specific B. pertussis factors in various measurable aspects of their interactions with ciliated respiratory epithelia and should inform the choice of new vaccine targets capable of interrupting the airway interactions.