Biophysical basis of phage liquid crystalline droplet-mediated antibiotic tolerance in pathogenic bacteria

Inoviruses are abundant filamentous phages infecting numerous prokaryotic phyla, where they can symbiotically promote host fitness and increase bacterial virulence. Due to their unique properties, inoviruses have also been utilised in biotechnology for phage display and as models for studying phase behaviour of colloidal rods. Inoviral phages secreted by bacteria can self-assemble into liquid crystalline droplets that protect bacterial cells in biofilms from antibiotics, however, factors governing the formation of such droplets and the mechanism of antibiotic protection are poorly understood. Here, we investigate the structural, biophysical, and protective properties of liquid crystalline droplets formed by Pseudomonas aeruginosa and Escherichia coli inoviral phages. We report a cryo-EM structure of the capsid from the highly studied E. coli fd phage, revealing distinct biochemical properties of fd compared to Pf4 phage from P. aeruginosa. We show that fd and Pf4 form liquid crystalline droplets with diverse morphologies governed by the underlying phage particle geometry and biophysics, rather than their surface biochemical properties. Finally, we show that these morphologically diverse droplets made of either phage can protect rod-shaped bacteria from antibiotic treatment, despite differing modes of association with cells. This study advances our understanding of phage assembly into liquid crystalline droplets, and provides insights into how filamentous molecules protect bacteria from extraneous molecules under crowding conditions, which are found in biofilms or on infected host tissues.