The role of O-specific antigen expression and its impact on virulence regulation and adaptation of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile bacterium found in various environments and is a well-known opportunistic human pathogen, particularly in hospital-acquired infections. It causes both acute and chronic biofilm-associated infections, posing a significant threat due to its ability to form biofilms, increasing rates of multi-drug resistance, and its large repertoire of virulence factors. Among these, bacterial motility is a key virulence factor, playing a crucial role in enhancing host-pathogen interactions and facilitating invasion. This study aimed to unravel contributors to bacterial motility and infection establishment, providing deeper insights into P. aeruginosa pathogenicity and adaptability. To achieve this, we employed a combinatorial approach, conducting a global motility screen with a collection of clinical P. aeruginosa PA14-like isolates, alongside transcriptional profiling of these isolates. Through this approach, we identified the O-specific antigen (OSA) biosynthesis gene cluster of P. aeruginosa PA14, a component of lipopolysaccharides (LPS), as potentially involved in swimming and twitching motility. We generated clean knockout mutants, to confirm its role in OSA biosynthesis and resistance to human serum, demonstrating that the absence of OSA increases cell surface hydrophobicity by exposing the hydrophobic lipid A molecule. Phenotypic characterization revealed an environment-dependent effect on bacterial motility and antimicrobial resistances. Further investigation highlighted the role of OSA in biofilm formation, altering biofilm structure and enhancing the biofilm maturation process. Additionally, we gained insights into the role of OSA during infection. We demonstrated that OSA expression is critical during the early stages of infection, providing protection against the host immune response, enabling P. aeruginosa to reach its full virulence potential, and promoting infection establishment. Transcriptional profiling and antibiotic treatment studies during ongoing infections further clarified the role of OSA in bacterial adaptability and infection progression. Overall, this study contributes to our understanding of the O-specific antigen as a key marker of adaption during infection. We shed light into the regulatory mechanism underlying its downregulation, which facilitates the transition from a motile, planktonic state to a sessile, biofilm-associated lifestyle, underlying its importance in infection progression.