Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix

Abstract

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell–cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications. Pseudomonas aeruginosa causes life-threatening, persistent infections in cystic fibrosis patients, despite highly aggressive antimicrobial therapy. Persistence is due, in part, to the ability of these bacteria to form surface-associated communities (biofilms) enmeshed in an extracellular matrix. This matrix is a poorly defined mixture of protein, polysaccharide, and DNA. An understanding of the organization and composition of the biofilm matrix will assist in the development of therapeutics aimed at disrupting biofilms. Using reagents that specifically recognize the P. aeruginosa Psl exopolysaccharide, we visualized matrix formation in real time during a biofilm development cycle. This revealed a highly organized and coordinated assembly of both polysaccharide and DNA components of the matrix. At late stages of biofilm morphogenesis, a Psl-free matrix cavity, occupied with numerous motile cells, developed. Mutants with reduced cell lysis were unable to form the Psl matrix cavity, whereas those with elevated cell death and lysis formed a larger matrix cavity, leading to accelerated dispersion. We propose that programmed cell death and autolysis are critical for the proper timing of biofilm development and dispersion. The data indicate that Psl is a key scaffolding component of the biofilm matrix, a property that likely plays a critical role in P. aeruginosa persistence.