Environmental sensing and energy metabolism profiling of Pseudomonas aeruginosa PA14: a multidisciplinary approach to study multidrug-resistant biofilm

Bacteria are capable of establishing structured communities, known as biofilms, which enable them to adhere to surfaces and successfully withstand both antimicrobial therapies and host immune responses. Among the bacteria capable of forming biofilms, Pseudomonas aeruginosa has been designated by the WHO as one of the most dangerous to human health, necessitating the development of new antimicrobial therapies. The key regulator of the biofilm formation, dispersion and maintenance processes is the second messenger c-di-GMP, whose intracellular levels are regulated by the rate of its synthesis, carried out by diguanylate cyclases (DGC) carrying the GGDEF domain, and its degradation, carried out by phosphodiesterases carrying the EAL domain. Among those proteins, transmembrane multi-domain one-component system protein (OCSs) are capable of sensing of environmental/intracellular stimuli through sensory domains which in turn can finely modulate the catalytic activity of GGDEF/EAL downstream domains. These OCSs are deeply involved in c-di-GMP metabolism, which makes them target of choice for the development of new antimicrobial strategies. In this work, by integrating different biochemical and biophysical methods we were able to characterize two OCSs from the clinically relevant strain PA14 from P. aeruginosa, namely PA14_53310 and PA14_37690. We found that PA14_53310 is a heme-dependent DGC, capable of sensing heme through its CHASE4 periplasmic domain to finally decrease its DGC turnover. On the other hand, the CHASE4 periplasmic domain found in PA14_37690, binds to copper, suggesting a role in metal sensing. We also compared both PA14_53310 and PA14_37690 with their PAO1 orthologue, PA0847 and PA2072 respectively, both capable of sensing ferric iron through their CHASE4 periplasmic domain. Both these OCS represent promising targets for interfering with mature biofilm. Nevertheless, the identification and characterization of novel targets requires the development of a fast antimicrobial susceptibility test (AST). To identify the appropriate antimicrobial treatment for biofilm infection (and for testing in a medium-scale new antimicrobial agents under development), we set up a protocol based on Seahorse instrument. As a proof-of-concept we measured the effect of the AMP esc(1-21) on PA14 strain finding a dose-dependent effect on the oxygen consumption rate (OCR) measurement. This pilot study paves the way to evaluate the flexibility of the Seahorse instrument in this field.