First Principles and Molecular Dynamics Modelling of a Mucoid Pseudomonas aeruginosa Biofilm Extracellular Matrix

Bacterial biofilms consist of bacterial populations enveloped and buried deep within an extracellular matrix (ECM). The ECM is a dense, tightly aggregated, scaffold of organic and inorganic material which behaves as a highly effective diffusion barrier, able to sequester incoming antimicrobials and functioning as a mode of adaptive antimicrobial resistance. The Cystic Fibrosis (CF) lung is a prime infection site for mucoid Pseudomonas aeruginosa which, upon colonisation of the CF lung epithelia, establishes a biofilm matrix rich in acetylated, anionic, alginate exopolysaccharide (EPS). The EPS affords the enveloped bacteria resistance to host defenses and antimicrobial attack, manifesting a chronic infection state in the CF lung, which significantly shortens an already truncated life expectancy. As such, there is an urgent need to identify, at the atomic-scale, critical links between EPS biomolecular structure and pathogenic virulence. In particular, this includes elucidating the atomistic mechanisms behind mucoid biofilm stabilisation, penetration and eradication. The aim of this research was to develop the first atomistic model of the mucoid P. aeruginosa biofilm EPS that was structurally representative of the EPS observed in the lungs of CF patients in vivo. Particular attention is given to the thermodynamically stable accommodation of key structural motifs unique to mucoid P. aeruginosa, as well as the EPS conformational change and intermolecular associations that drive cation-induced stabilisation, govern cell-to-cell signal distributions and facilitate mature matrix disruption. The atomistic model critically implicated calcium ions in biofilm matrix chronicity, identified the molecular functionality pivotal for the interactions that govern molecular motion through the EPS, elucidated the atomistic mechanism underpinning mature EPS matrix disruption and extended the mechanistic understanding of exogenous gallium therapy. This information will prime pharmacologists with the understanding required to develop novel small molecule antimicrobials capable of treating chronic mucoid P. aeruginosa biofilm infections in the lungs of CF patients.