Atomistic modelling and NMR studies reveal that gallium can target the ferric PQS uptake system in P. aeruginosa biofilms.

Intravenous gallium nitrate therapy is a novel therapeutic strategy deployed to combat chronic Pseudomonas aeruginosa biofilm infections in the lungs of cystic fibrosis (CF) patients by interfering with iron (Fe3+) uptake. The therapy is a source of Ga3+, which competes with Fe3+ for siderophore binding, subsequently disrupting iron metabolism and inhibiting biofilm proliferation in vivo. It was recently demonstrated that the Pseudomonas quinolone signal (PQS) can chelate Fe3+ to assist in bacterial iron uptake. However, it is unknown whether exogenous gallium also targets [Fe(PQS)3] uptake, which, in turn, would extend the mechanism of gallium therapy beyond siderophore competition, potentially supporting use of the therapy against P. aeruginosa mutants deficient in siderophore uptake proteins. To that end, the thermodynamic feasibility of iron-for-gallium cation exchange into [Fe(PQS)3] was evaluated using quantum chemical density functional theory (DFT) modelling and verified experimentally using 1H nuclear magnetic resonance (NMR). We demonstrate here that Ga3+ can strongly bind to three PQS molecules and, furthermore, displace and substitute Fe3+ from the native chelate pocket within PQS complexes, through a Trojan horse mechanism, retaining the key structural features present within the native ferric complex. As such, [Fe(PQS)3] complexes, in addition to ferric-siderophore complexes, represent another target for gallium therapy.

A DFT study of the gallium ion-binding capacity of mature Pseudomonas aeruginosa biofilm extracellular polysaccharide.

Intravenous gallium therapy is a non-antibiotic approach to limit Pseudomonas aeruginosa biofilm proliferation, by outcompeting iron for siderophore binding. Gallium therapy represents a viable therapeutic strategy for cystic fibrosis (CF) patients harbouring mucoid P. aeruginosa biofilm lung infections. Siderophore deficient P. aeruginosa isolates still demonstrate a hindered biofilm proliferation when exposed to gallium but it is currently unknown whether exogenous gallium has any disruptive influence on the exopolysaccharide (EPS), the major mucoid P. aeruginosa CF lung biofilm matrix component. To that end, Density-Functional Theory (DFT) was deployed to assess whether gallium (Ga3+) could be substituted into the mature mucoid EPS scaffold in preference of calcium (Ca2+)-the native EPS cross-linking ion. Removal of the stable, bound native calcium ions offers a large enthalpic barrier to the substitution and the mature EPS fails to accommodate exogenous gallium. This suggests that gallium, perhaps, is utilising a novel, possibly unknown, ferric uptake system to gain entry to siderophore deficient cells.

Polyguluronate simulations shed light onto the therapeutic action of OligoG CF-5/20.

Chronic mucoid P. aeruginosa cystic fibrosis (CF) lung infections are associated with the development of a biofilm composed of anionic acetylated exopolysaccharide (EPS) alginate, electrostatically stabilised by extracellular Ca2+ ions. OligoG CF-5/20, a low molecular weight guluronate rich oligomer, is emerging as a novel therapeutic capable of disrupting mature P. aeruginosa biofilms. However, its method of therapeutic action on the mucoid biofilm EPS is not definitively known at a molecular level. This work, utilising molecular dynamics (MD) and Density-Functional Theory (DFT), has revealed that OligoG CF-5/20 interaction with the EPS is facilitated solely through bridging Ca2+ ions, which are not liberated from their native EPS binding sites upon OligoG CF-5/20 dispersal, suggesting that OligoG CF-5/20 does not cause disruptions to mature P. aeruginosa biofilms through breaking EPS-Ca2+-EPS ionic cross-links. Rather it is likely that the therapeutic activity arises from sequestering free Ca2+ ions and preventing further Ca2+ induced EPS aggregation.

Atomic-scale interactions between quorum sensing autoinducer molecules and the mucoid P. aeruginosa exopolysaccharide matrix.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung coloniser whose chronicity is associated with the formation of cation cross-linked exopolysaccharide (EPS) matrices, which form a biofilm that acts as a diffusion barrier, sequestering cationic and neutral antimicrobials, and making it extremely resistant to pharmacological challenge. Biofilm chronicity and virulence of the colony is regulated by quorum sensing autoinducers (QSAIs), small signalling metabolites that pass between bacteria, through the biofilm matrix, regulating genetic responses on a population-wide scale. The nature of how these molecules interact with the EPS is poorly understood, despite the fact that they must pass through EPS matrix to reach neighbouring bacteria. Interactions at the atomic-scale between two QSAI molecules, C4-HSL and PQS-both utilised by mucoid P. aeruginosa in the CF lung-and the EPS, have been studied for the first time using a combined molecular dynamics (MD) and density functional theory (DFT) approach. A large-scale, calcium cross-linked, multi-chain EPS molecular model was developed and MD used to sample modes of interaction between QSAI molecules and the EPS that occur at physiological equilibrium. The thermodynamic stability of the QSAI-EPS adducts were calculated using DFT. These simulations provide a thermodynamic rationale for the apparent free movement of C4-HSL, highlight key molecular functionality responsible for EPS binding and, based on its significantly reduced mobility, suggest PQS as a viable target for quorum quenching.

Cation complexation by mucoid Pseudomonas aeruginosa extracellular polysaccharide.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung colonizer, producing an extracellular matrix (ECM) composed predominantly of the extracellular polysaccharide (EPS) alginate. The ECM limits antimicrobial penetration and, consequently, CF sufferers are prone to chronic mucoid P. aeruginosa lung infections. Interactions between cations with elevated concentrations in the CF lung and the anionic EPS, enhance the structural rigidity of the biofilm and exacerbates virulence. In this work, two large mucoid P. aeruginosa EPS models, based on ß-D-mannuronate (M) and ß-D-mannuronate-α-L-guluronate systems (M-G), and encompassing thermodynamically stable acetylation configurations-a structural motif unique to mucoid P. aeruginosa-were created. Using highly accurate first principles calculations, stable coordination environments adopted by the cations have been identified and thermodynamic stability quantified. These models show the weak cross-linking capability of Na+ and Mg2+ ions relative to Ca2+ ions and indicate a preference for cation binding within M-G blocks due to the smaller torsional rearrangements needed to reveal stable binding sites. The geometry of the chelation site influences the stability of the resulting complexes more than electrostatic interactions, and the results show nuanced chemical insight into previous experimental observations.