@phdthesis{Kessie2021,
  author    = {Kessie, David Komla},
  title     = {Characterisation of Bordetella pertussis virulence mechanisms using engineered human airway tissue models},
  doi       = {10.25972/OPUS-23571},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235717},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Pertussis is a highly contagious acute respiratory disease of humans which is mainly caused by the gram-negative obligate human pathogen Bordetella pertussis. Despite the availability and extensive use of vaccines, the disease persists and has shown periodic re-emergence resulting in an estimated 640,000 deaths worldwide in 2014. The pathogen expresses various virulence factors that enable it to modulate the host immune response, allowing it to colonise the ciliated airway mucosa. Many of these factors also directly interfere with host signal transduction systems, causing damage to the ciliated airway mucosa and increase mucous production. Of the many virulence factors of B. pertussis, only the tracheal cytotoxin (TCT) is able to recapitulate the pathophysiology of ciliated cell extrusion and blebbing in animal models and in human nasal biopsies. Furthermore, due to the lack of appropriate human models and donor materials, the role of bacterial virulence factors has been extrapolated from studies using animal models infected with either B. pertussis or with the closely related species B. bronchiseptica which naturally causes respiratory infections in these animals and produces many similar virulence factors. Thus, in the present work, in vitro airway mucosa models developed by co-culturing human airway epithelia cells and fibroblasts from the conduction zone of the respiratory tract on a decellularized porcine small intestine submucosa scaffold (SISser®) were used, since these models have a high correlation to native human conducting zone respiratory epithelia. The major aim was to use the engineered airway mucosa models to elucidate the contribution of B. pertussis TCT in the pathophysiology of the disease as well as the virulence mechanism of B. pertussis in general. TCT and lipopolysaccharide (LPS) either alone or in combination were observed to induce epithelial cell blebbing and necrosis in the in vitro airway mucosa model. Additionally, the toxins induced viscous hyper-mucous secretion and significantly disrupted barrier properties of the in vitro airway mucosa models. This work also sought to assess the invasion and intracellular survival of B. pertussis in the polarised epithelia, which has been critically discussed for many years in the literature. Infection of the models with B. pertussis showed that the bacteria can adhere to the models and invade the epithelial cells as early as 6 hours post inoculation. Invasion and intracellular survival assays indicated the bacteria could invade and persist intracellularly in the epithelial cells for up to 3 days. Due to the novelty of the in vitro airway mucosa models, this work also intended to establish a method for isolating individual cells for scRNA-seq after infection with B. pertussis. Cold dissociation with Bacillus licheniformis subtilisin A was found to be capable of dissociating the cells without inducing a strong fragmentation, a problem which occurs when collagenase and trypsin/EDTA are used. In summary, the present work showed that TCT acts possibly in conjunction with LPS to disrupt the human airway mucosa much like previously shown in the hamster tracheal ring models and thus appears to play an important role during the natural B. pertussis infection. Furthermore, we established a method for infecting and isolating infected cells from the airway mucosa models in order to further investigate the effect of B. pertussis infection on the different cell populations in the airway by single cell analytics in the future.},
  subject      = {Tissue engineering},
  language  = {en}
}