Abstract 1435 Role of the Pseudomonas aeruginosa mdo Operon in Maintaining Hydrogen Sulfide Levels

Pseudomonas aeruginosa is an opportunistic human pathogen that is especially problematic for individuals with cystic fibrosis and immunocompromised patients. It is a leading cause of nosocomial infections and is responsible for 10% of all hospital-acquired infections. The CDC classifies the organism as a serious threat mainly due to its emerging development of multidrug resistance. Several virulence factors contribute to P. aeruginosa pathogenicity including hydrogen sulfide. Sulfide (HS-) at sub-micromolar concentrations protects P aeruginosa from antibiotic-induced oxidative damage and host-produced reactive oxygen species. However, elevated HS- level results in cellular toxicity and affects the organism's ability to form a biofilm. Therefore, HS- concentrations must be tightly regulated to balance the potential toxicity with bacterial virulence. In several organisms, toxic levels of HS- are converted to usable sulfur forms by the combined actions of dioxygenases and sulfurtransferases. Increased level of HS- results in the formation of low molecular weight persulfides which are the substrates for the sulfide oxidation enzymes. In P. aeruginosa, enzymes expressed on the mdo operon have been identified but their mechanism of action and metabolic roles have not been elucidated. In P. aeruginosa PAO1, the mdo operon expresses 3-mercaptopropionate dioxygenase (MDO) and a sulfurtransferase (ST). MDO was initially characterized as utilizing 3-mercaptopropionate (MP) as a substrate; however, P. aeruginosa PAO1 could not utilize MP in microbial growth studies to ascertain the physiological relevance of the thiol substrate. MDO was able to oxidize 3-mercaptopyruvate, which is a physiologically relevant substrate and can also be linked with ST activity. Even though MDO is expressed from the same operon as an annotated ST, the functional role of the ST enzyme has not been recognized. ST enzymes have conserved cysteine residues that mediate sulfur transfer from a sulfur donor to a sulfur acceptor. These sulfur donors and acceptors are usually low molecular weight thiols which are ubiquitous in cells. The ST expressed on the mdo operon has two conserved cysteine residues (Cys191 and Cys435) with the potential to mediate sulfur transfer through an enzyme Cys-persulfide intermediate. Protein cysteine persulfidation assays were performed to identify the sulfur donor and catalytic Cys. Only Cys435 was able to form a persulfide intermediate. There was only one accessible Cys in thiol assays. The accessible thiol was identified as Cys435 in HDX-MS investigations, which corresponded to the role of this residue as the sulfide mediator. These studies support Cys435 as the catalytic cysteine. Proteomic studies were performed to identify the changes in protein expression when the organism was grown in sulfur free media supplemented with sulfide. The data from these investigations point to the potential of the enzymes of the mdo operon to mobilize and assimilate sulfide in P. aeruginosa thereby enhancing its viability and pathogenicity.