<b>Barrier formation by primary human airway epithelial cells sets limits to </b><b><i>Staphylococcus aureus</i></b><b> adhesion, invasion and cytotoxicity </b>-<b> </b>Supplemental Materials

<p dir="ltr"><i>Staphylococcus aureus</i> is an opportunistic pathogen that can cause severe infections of the human respiratory tract. The airway epithelium forms the first line of defense against this pathogen, but how exactly this layer of multiple differentiated cell types fends off an <i>S. aureus</i> challenge was so far poorly understood. Therefore, our present study aimed to assess how primary airway epithelial cells exert their protective effect against <i>S. aureus</i> infection. To this end we cultured primary bronchial and tracheobronchial epithelial cells at an air-liquid interface over a 28-day period, and we related the increase in transepithelial electrical resistance (TEER) to staphylococcal cell invasion. The results show that formation of a tight primary epithelial cell barrier sets a major limit to <i>S. aureus</i> invasion. The involvement of epithelial cell-cell junctions was corroborated with E-cadherin-deficient bronchial epithelial cells, which are significantly more susceptible to bacterial adhesion, invasion and cytotoxicity than their wild-type counterparts. As shown by RNA sequencing, prior to barrier formation, primary airway epithelial cells respond to an <i>S. aureus</i> challenge by increased expression of cadherin genes, implying a compensatory mechanism to reinforce epithelial junctions. Conversely, airway epithelial cells in a tight and differentiated mucociliary barrier respond to <i>S. aureus</i> by inducing cell death- and apoptosis-related pathways, potentially to eliminate infected cells. We conclude that formation of a tight epithelial barrier, combined with an appropriate temporal shift in the cellular responses to <i>S. aureus</i>-imposed ‘infection stress’, are critical aspects of the airway epithelial strategy to manage and resolve staphylococcal infections.</p>