ETD

• Bacteroides thetaiotaomicron
• biofilm
• type V pili

The human gut microbiome is essential for host health and immunity, with biofilm formation promoting microbial persistance (J. Xu & Gordon, 2003; Sonnenburg et al., 2004). Bacteroides thetaiotaomicron, a prominent gut commensal, uses surface structures like Type V pili to mediate adhesion. While the reference strain VPI5482 is a poor biofilm former in vitro (Mihajlovic et al., 2019, acapsulated mutants (Δcps 1-8) show increased aggregative properties, suggesting that the capsule masks the surface adhesins (Béchon et al., 2020).
Type V pili are proteinaceous filaments consisting of anchor, stalk and tip pilins (Shibata et al., 2020), and their role in biofilm formation remains unclear.

Since these pili operons are repressed in standard laboratory conditions, previous studies engineered the constitutive plasmid- based expression of the BT2316-2319 operon. This expression reduced aggregation and epithelial adhesion in the Δcps 1-8 mutant, leading to the hypothesis that Type V pili increase interbacterial distance.

This thesis project aimed to further characterise these phenotypes. Using a phage-engineered promoter (Whitaker et al., 2017) inserted chromosomally upstream of Type V pili operons, successfully reproducing previous data and extending the method to another repressed operon, BT2655-2660. The results showed that constitutive expression of BT2655-2660 reduced biofilm formation and aggregation in the Δcps 1-8 background, further supporting the hypothesis that Type V pili increase interbacterial distance.