Multi-faceted exploration of the novel active γ-carbonic anhydrase PaCAγ1 in the human pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium, is an opportunistic pathogen that causes severe health issues in both immunocompetent and immunocompromised individuals. It is a member of the ESKAPE group of pathogens, and a leading cause of nosocomial infections worldwide. With the aim of identifying novel tools to combat P. aeruginosa infections, and considering that carbonic anhydrases are emerging as promising targets to overcome antibiotic resistance, we present here the identification and detailed characterization of a γ-class carbonic anhydrase, PaCAγ1, from P. aeruginosa using a multidisciplinary approach. Specifically, the identified enzyme was recombinantly produced and subjected to an extensive biochemical and biophysical characterization, proving its enzymatic function and revealing its overall stability at high temperatures. We determined that PaCAγ1 plays a role in pathogen fitness under carbon-limiting conditions and contributes to its survival within the macrophages. Various sulfonamide, sulfamide, and sulfamate derivatives were evaluated for their inhibitory properties against PaCAγ1 following a drug repurposing approach. This study reports on the discovery and characterization of a previously unrecognized γ-class CA, which enhances our understanding of the biochemical capacity and physiological importance of these enzymes in human pathogens.