Exploration of H2S-producing enzymes as a potential antimicrobial strategy targeting Pseudomonas aeruginosa

The growing resistance of microorganisms to multiple drugs highlights the need to introduce novel therapeutic strategies. A recently explored defense system in resistant pathogens relies on the biogenesis of hydrogen sulfide (H2S), an important signal molecule proven to induce a drug-tolerant state and confer protection against antibiotic-induced oxidative stress. Clearly, gaining a comprehensive understanding of the fundamental mechanisms employed by bacteria to counteract antibiotics defines a crucial starting point to develop efficacious antimicrobial therapies. In the search for new druggable candidates, we focused our attention on two enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) from the human pathogen Pseudomonas aeruginosa (Pa). Pa is one of the most threatening bacteria (ESKAPE pathogens), and it is one of the leading causes of hospital infections. The rise in diversity of antimicrobial resistance phenotypes seen in Pa is becoming a serious antibiotic management problem and Pa is recognized as a prototype of Gram-negative multidrug-resistant (MDR) “superbug” for which the identification of new pharmacological interventions is urgently needed. CBS and CGL represent appealing drug targets because they belong to the reverse transsulfuration pathway, which is the key route for the generation of H2S in several opportunistic pathogens. In this study, we overexpressed the recombinant forms of CGL and CBS from this pathogen and pursued a biophysical and biochemical study to explore their structural and kinetic properties. We characterized these enzymes in term of kinetics and regulation, and successfully obtained the crystal structure of CGL in collaboration with Professor Martinez Cruz from CIC-bioGUNE (Spain). Additionally, we performed a side-by-side comparison of CBS from Pa and its orthologues from higher and lower eukaryotes to shed light on the complexity of domain organization, allosteric modulation, and catalytic specificity of CBS enzyme. Overall, this research may help support the potential draggability of CGL and CBS enzymes as part of the H2S biogenetic repertoire, paving the way for the design of new inhibitors.