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.