| Summary: | <i>Aspergillus fumigatus</i> is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of <i>A. fumigatus</i> colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of <i>A. fumigatus</i> supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o<sup>−</sup>) and HBE (16HBE14o<sup>−</sup>) trans-epithelial electrical resistance (TEER) was measured following exposure to <i>A. fumigatus</i> reference and clinical isolates, a gliotoxin-deficient mutant (Δ<i>gliG</i>) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. <i>A. fumigatus</i> conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while Δ<i>gliG</i> mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by <i>A. fumigatus</i> supernatants but not by Δ<i>gliG</i> supernatants, suggesting that gliotoxin is involved in this process. The fact that Δ<i>gliG</i> conidia were still capable of disrupting epithelial monolayers indicates that direct cell–cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.
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