Physiological concentrations of calcium interact with alginate and extracellular DNA in the matrices of Pseudomonas aeruginosa biofilms to impede phagocytosis by neutrophils

Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or incorporated from the host environment. P. aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca²⁺). We investigated the structural and mechanical impacts of Ca²⁺ supplementation in alginate-dominated biofilms grown in vitro and evaluated the impact of targeted enzyme treatments on clearance by immune cells. We used multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. Our results suggest that the presence of Ca²⁺ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observed that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.