Ellis, Holly R. PhDAdindu, Chukwuemeka Steve2024-08-292024-07July 2024July 2024http://hdl.handle.net/10342/13666Pseudomonas aeruginosa PAO1 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 PAO1 pathogenicity including hydrogen sulfide. Sulfide (HS-) at sub-micromolar concentrations protects P. aeruginosa PAO1 from antibiotic-induced oxidative damage and host-produced reactive oxygen species. However, elevated sulfide levels result in cellular toxicity and affect 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 levels of HS- results in the formation of low molecular weight persulfides which are the substrates for the sulfide oxidation enzymes. In P. aeruginosa PAO1, 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 (3MPA) as a substrate; however, P. aeruginosa PAO1 could not utilize 3MPA in microbial growth studies to ascertain the physiological relevance of the thiol substrate. MDO was able to oxidize 3-mercaptopyruvate (3MPR), 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 that are ubiquitous in cells. The ST expressed on the mdo operon has four rhodanese domains with two of the domains containing putative catalytic Cys residues (Cys191 and Cys435) with the potential to mediate sulfur transfer through an enzyme cysteine persulfide intermediate. Cys435 was the only accessible thiol in thiol assays. Results from HDX-MS investigations supported a more solvent-accessible region surrounding Cys435. The accessible thiol was identified as Cys435 in HDX-MS investigations, which corresponded to the role of this residue as the sulfide mediator. Only Cys435 formed a persulfide intermediate with either thiosulfate or 3MPR as the sulfur donor in cysteine persulfidation assays. These studies support Cys435 as the catalytic cysteine with thiosulfate and 3MPR serving as the sulfur donor. ST showed a similar affinity for 3MPR and 3MPA suggesting that each substrate could serve as a putative sulfur acceptor for the enzyme to form a persulfide. MDO was able to oxidize the persulfides of 3MPA and 3MPR, suggesting a metabolic link between MDO and ST. Although 3MPA could bind ST and serve as a substrate for MDO, it was not a viable sulfur source in growth studies. Proteomic studies were performed to identify the changes in protein expression when the organism was grown in sulfur-free media supplemented with sulfide. When P. aeruginosa PAO1 is under sulfide stress, the genes that were high in abundance were those involved in biological processes including amino acid breakdown, arginine deaminase pathway, and tRNA metabolism amongst other pathways. The data from these investigations point to the potential of the enzymes of the mdo operon to mobilize and assimilate sulfide in P. aeruginosa PAO1 thereby enhancing its viability and pathogenicity. Understanding the strategies for the acquisition of sulfur from less preferred sources and their regulation provides insights into the metabolic versatility and pathogenicity of P. aeruginosa PAO1, highlighting potential targets for therapeutic interventions.application/pdfEnglishChemistry, BiochemistryDoctoral Dissertation2024-08-27