Phage liquid crystalline droplets form occlusive sheaths that encapsulate and protect infectious rod-shaped bacteria

Abstract

The opportunistic pathogenPseudomonas aeruginosais a major cause of antibiotic-tolerant infections in humans.P. aeruginosaevades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens includingNeisseria meningitidis,Vibrio cholerae,andSalmonella entericaharbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.

The Impact of Pf Bacteriophages on the Fitness ofPseudomonas aeruginosa; A Mathematical Modeling Approach

“…We have previously reported that Pf phages promote the ordering of biofilm polymers into a liquid crystalline structure that prevents antibiotic diffusion and inhibits bacterial clearance (33)(34)(35). In addition, some antibiotics are bound to Pf phage structures and are prevented from killing embedded bacteria (29,36). This liquid crystalline organization has also been observed in other filamentous phages (37).…”

Protect thy host: Pf4 phages shield Pseudomonas aeruginosa from antibiotics

“…This allows the encapsulated cell to remain metabolically active and interact with its immediate environment while being protected from the antibiotic. Furthermore, the authors showed that also Pf4 phages devoid of DNA were able to form this protective crystalline shield and that such a shield could be formed around non-bacterial colloidal rods (Tarafder et al, 2020). As such, protective Pf4 phage shields represent a novel antibiotic resistance mechanism through liquid-liquid phase separation within prokaryotic systems.…”

“…The helical filaments have a diameter of 62 Å and an inner cavity of 22 Å, which is filled with linear singlestranded DNA. The authors also found that some of the filaments did not contain any DNA and hypothesised that the viral genome is not required for phage integrity (Tarafder et al, 2020).…”

“…A recent study now shed light on the phenomenon how Pf4 confers antibiotic resistance to P. aeruginosa (Tarafder et al, 2020). Using cryo-EM, optical microscopy and biochemical reconstitution experiments, Tarafder et al solved the structure of Pf4 and visualized Pf4 filaments together with bacterial cells.…”

“…These liquid crystalline structures confer adhesiveness to the biofilm, protect it from desiccation and promote tolerance to cationic antibiotics (Secor et al, 2015). In the study by Tarafder et al, researchers imaged this liquid crystalline structure together with a bacterial cell and proposed a mechanism for antibiotic resistance (Tarafder et al, 2020). The authors reason that the liquid crystalline structure wraps around or closely aligns with the bacterial cell acting as a physical barrier to shield the cell from the antibiotic.…”