Uncovering mechanisms of nitric oxide detoxification and reactivity in Pseudomonas aeruginosa

PROJECT SUMMARY Pseudomonas aeruginosa is a Gram-negative bacterial pathogen and a major source of nosocomial infections globally. This bacterium infects numerous anatomical sites, which results in wound infections, bloodstream infections, and lung infections, among others. Further, P. aeruginosa infections are often antibiotic resistant, which limits treatment options. Therefore, understanding how P. aeruginosa interfaces with the host environment may provide additional insight into therapeutic development. As part of the host immune response to P. aeruginosa, infiltrating immune cells like macrophages produce nitric oxide (NO) to control and eliminate the pathogen. NO is a gaseous free radical that has been harnessed throughout life as a crucial signaling molecule, but within the infection context, NO and its reactivity is weaponized against invading microbes to control and eliminate them. Despite robust immune cell influx and NO production, opportunistic pathogens like P. aeruginosa often establish chronic infections in immunocompromised hosts, posing a conundrum: how do bacteria overcome NO exposure to persist within this hostile environment? We hypothesized that microbial resistance to NO relies on its metabolism within the bacterial cell as well as components of the extracellular environment modulating NO reactivity. To interrogate NO intracellular metabolism, P. aeruginosa genetic mutants were screened for their ability to survive NO exposure, which identified the highly conserved FtsH protease as being important for the response of P. aeruginosa to NO toxicity. To investigate the extracellular reactivity of NO, we wondered whether the chemical landscape NO encounters might change its reactivity. One component of this landscape is the bacterial secreted metabolite pyocyanin, which serves as an important molecule for the survival of P. aeruginosa. We found that when pyocyanin and NO are present simultaneously, a modified version of pyocyanin with NO stably bound forms and has unique chemical properties, which changes the toxicity and function of NO and pyocyanin. Collectively, these preliminary results demonstrated that accounting for microbe-intrinsic regulatory pathways and external small molecule interactions provide a context-dependent framework to consider NO toxicity and reactivity. These findings will be bolstered by experiments outlined in this proposal. Specifically, this application seeks to expand on this concept by 1) further elucidating cell-intrinsic NO resistance pathways in P. aeruginosa and 2) discovering cell-extrinsic NO- metabolite interactions and reaction mechanisms. These results will provide proof-of-principle that NO’s ability to shape microbial survival is influenced by both microbial regulatory circuits and the small molecule landscape that NO encounters. Defining these contributions is critical for understanding P. aeruginosa survival within the host.