Biofilm assembly becomes crystal clear - filamentous bacteriophage organize the Pseudomonas aeruginosa biofilm matrix into a liquid crystal

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with many types of chronic infection. At sites of chronic infection, such as the airways of people with cystic fibrosis (CF), P. aeruginosa forms biofilm-like aggregates. These are clusters of bacterial cells encased in a polymer-rich matrix that shields bacteria from environmental stresses and antibiotic treatment. When P. aeruginosa forms a biofilm, large amounts of filamentous Pf bacteriophage (phage) are produced. Unlike most phage that typically lyse and kill their bacterial hosts, filamentous phage of the genus Inovirus, which includes Pf phage, often do not, and instead are continuously extruded from the bacteria. Here, we discuss the implications of the accumulation of filamentous Pf phage in the biofilm matrix, where they interact with matrix polymers to organize the biofilm into a highly ordered liquid crystal. This structural configuration promotes bacterial adhesion, desiccation survival, and antibiotic tolerance - all features typically associated with biofilms. We propose that Pf phage make structural contributions to P. aeruginosa biofilms and that this constitutes a novel form of symbiosis between bacteria and bacteriophage.

Keywords: Pseudomonas aeruginosa; biofilm; chronic infection; cystic fibrosis; filamentous bacteriophage; liquid crystal; soft matter physics.

Figure 1. FIGURE 1: Schematic of how depletion attraction drives liquid crystal assembly from Pf phage within polymer-rich environments.

(A) Dashed lines around phage particles represent an exclusion volume not accessible to the polymer coil’s center of mass. When phages are dispersed, the volume inaccessible to the polymers is maximized. (B) When phages come into close proximity to each other, their exclusion volumes overlap, increasing the total volume accessible to the polymers. This maximizes the entropy of the system. Arrows represent an osmotic imbalance that polymers exert on the liquid crystalline phage bundle. This osmotic pressure is what physically holds the liquid crystal together. (C) Fluorescently labeled Pf phages (green) are initially dispersed when mixed with the host polymer hyaluronan. (D) As time progresses, the phages form liquid crystalline structures.

Figure 2. FIGURE 2: Pf phages organize the P. aeruginosa biofilm matrix into a liquid crystal.

The birefringence of the liquid crystalline biofilm matrix can be visualized by placing colony biofilms on an agar pad between crossed polarizing lenses. The liquid crystalline matrix can change the polarization of light allowing it to pass through both polarizing lenses. Thus, non-liquid crystalline biofilms appear opaque (red arrow) while liquid crystalline biofilms appear bright (blue arrow). The organization of the biofilm matrix into a liquid crystal enhances adhesion, antibiotic tolerance, and desiccation survival. Further, Pf phage can increase the viscosity of host polymers such as mucin and DNA.