Synergistic effects of Candida albicans and Porphyromonas gingivalis biofilms on epithelial barrier function in a 3D aspiration pneumonia model

Ewelina Wronowska Ibeth Guevara-Lora Aleksandra Brankiewicz Marcin Zawrotniak Dorota Satala Justyna Karkowska-Kuleta Joanna Budziaszek Joanna Koziel Maria Rapala-Kozik ^*

• Jagiellonian University, Kraków, Poland

Polymicrobial infections involving Candida albicans and Porphyromonas gingivalis represent a significant challenge in epithelial barrier integrity. This study explores their synergistic effects on epithelial cells using an air-liquid interface (ALI) model. Mixed-species biofilms significantly increased epithelial permeability and disrupted tight junction proteins, as evidenced by the reduction of ZO-1 and E-cadherin levels. These alterations were accompanied by oxidative stress, characterized by decreased HO-1 expression and enhanced Bax/Bcl-xL ratios, indicating increased pro-apoptotic activity. Supernatants from dual-species biofilms demonstrated a pronounced effect on epithelial cells, modulating IL-8 secretion and exacerbating oxidative damage. While C. albicans was identified as the dominant driver of pro-inflammatory responses, P. gingivalis contributed through immune modulation and enzymatic activity, primarily via gingipains. The role of P. gingivalis proteases was assessed using a gingipain-null mutant (∆K∆RAB). Compared to the wild-type strain (W83), ∆K∆RAB biofilms caused less epithelial disruption and oxidative stress, highlighting the critical role of gingipains in pathogenesis. The survival experiments using Galleria mellonella larvae supported these findings, highlighting the reduced survival associated with dual-species biofilms and the potential for high-dose antimicrobial therapies to mitigate this effect. These results emphasize the cooperative mechanisms of these pathogens in compromising epithelial barriers and underline the importance of combination therapies targeting fungal and bacterial components in polymicrobial infections.