Biophysical Society Thematic Meeting | Ascona 2026

Mechanobiology of Infection

Poster Abstracts

10-POS Board 10 FROM MONOMER TO FILAMENT: MULTISCALE REAL-TIME IMAGING REVEALS TYPE IV PILI ASSEMBLY DYNAMICS IN NEISSERIA MENINGITIDIS Foad Ghasemi 1 ; Daiki Nishiguchi 2 ; Sylvie Goussard 1 ; Benoit Lelandais 3,4 ; Jean-Yves Tinevez 3 ; Guillaume Duménil 1 ; 1 Institut Pasteur, Université Paris Cité, INSERM UMR1225, Pathogenesis of Vascular Infections, Paris, France 2 Department of Physics, School of Science, The University of Tokyo, Tokyo, Japan 3 Institut Pasteur, Université Paris Cité, Image Analysis Hub, Paris, France 4 Institut Pasteur, Université Paris Cité, Imaging and Modeling Unit, Paris, France Type IV pili (T4P) are dynamic filaments that serve as key virulence factors in diverse bacteria. Pilin monomers are maintained as a pool in the inner membrane (IM). They dynamically assemble into and disassemble from pili at rates up to ~1000 subunits/s, driven by distinct extension and retraction motors. Yet how bacteria balance massive monomer flux between the membrane reservoir and growing pili, and how extension-retraction switching is regulated, remain unknown. We addressed these questions in the human pathogen Neisseria meningitidis. We combined maleimide labeling of cysteine-mutant pilin, high-speed fluorescence microscopy, and microfluidics to visualize pili and IM pilin pools in single cells. Using an in-house automated single-pilus tracking pipeline developed in Python, we captured complete extension-retraction cycles across hundreds of bacteria. We then extracted key dynamical parameters, including pilus length and distribution, piliation frequency, extension and retraction speeds, and IM pilin monomer concentration. Moreover, fluorescence recovery after photobleaching (FRAP) yielded diffusion coefficients for pilin in the IM. Finally, by tuning the wild-type to cysteine-mutant pilin ratio, we achieved single-molecule resolution of pilus dynamics in live bacteria and, for the first time, during active infection of host cells. These measurements reveal diverse aspects of T4P dynamics, and our analysis continues to uncover key mechanistic insights. Among the emerging findings, we observed that pilus length inversely correlates with IM pilin signal in the vicinity of the pilus base, consistent with local monomer depletion during extension and replenishment during retraction. Within individual pili, extension and retraction speeds are positively correlated, suggesting a mechanistic coupling between extension and retraction motors. Furthermore, piliation rate decreases with cell size, linking T4P dynamics to the bacterial cell cycle. Our work establishes a quantitative framework linking IM pilin availability, motor activity, T4P dynamics, and coordination among multiple pili, and provides a mechanistic foundation for targeting T4P-mediated pathogenesis.

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