Biophysical Society Thematic Meeting | Ascona 2026

Mechanobiology of Infection

Poster Abstracts

29-POS Board 29 STRUCTURAL BASIS OF TYPE IV PILI MECHANICAL FUNCTION IN PSEUDOMONAS AERUGINOSA Gani Chia-Ni Tsai 1 ; Laure Le Blanc 1 ; Michael Nilges 2 ; Olivera Francetic 3 ; Yasaman Karami 4 ; Alexandre Persat 1 ; 1 EPFL, Institute of Bioengineering and Global Health Institute, School of Life Sciences, Lausanne, Switzerland 2 Institut Pasteur, CNRS UMR3528, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, Paris, France 3 Institut Pasteur, CNRS UMR3528, Biochemistry of Macromolecular Interactions Unit, Pathogens have developed strategies to sense and overcome mechanical challenges in order to navigate dynamic host environments and establish infection. Pseudomonas aeruginosa uses type IV pili (T4P), dynamic bacterial appendages essential for twitching motility, mechanosensing, virulence, and biofilm formation. T4P extend and retract through the cell envelope via polymerization and depolymerization of the major pilin PilA. The retraction generates ~100 pN of tension in the T4P filament, placing an extreme mechanical load on the protein assembly. The intrinsic rigidity of the filament is thus critical for driving cell movement and transmitting retraction forces to trigger downstream intracellular responses. However, the molecular mechanisms governing this mechanical stability remain unclear. Here, we combined cryo-EM reconstruction, classical molecular dynamics (MD) simulations and steered MD (SMD) simulations, T4P functionality assays, and optical tweezer measurements to investigate the structural and mechanical determinants of PAO1 T4P filament rigidity and its functional consequences. We extended the simulations to the P. aeruginosa PAK strain and four other Gram-negative species where pili fulfill distinct biological roles, including surface adhesion and autoaggregation in Neisseria meningitidis, Neisseria gonorrhoeae, and E. coli, and collective behavior in Myxococcus xanthus. By measuring atomic fluctuations and structural extension under applied force, we found that the PAO1 pilus possesses the most rigid and least extensible structure among the species compared. To pinpoint the basis of this rigidity, we computed intersubunit salt bridge and hydrogen bond strengths, guiding targeted point mutations in PAO1 PilA. A phage infection assay dependent on functional T4P and active retraction revealed that several mutations conferred phage resistance, indicating lost functionality. In contrast, R30E and G75L mutants remained susceptible and maintained twitching motility. Optical tweezer measurements, pulling purified pili between two hydrophobic beads, confirmed that these mutations reduced pilus flexibility and increased the filament spring constant. These findings demonstrate that the intrinsic mechanical rigidity of the T4P filament is critical for force transmission and biological responses. Department of Structural Biology and Chemistry, Paris, France 4 Université de Lorraine, CNRS, Inria, Loria, Nancy, France

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