Our overarching goal is to develop efficacious strategies to combat multidrug-resistant (MDR) pathogens, a growing healthcare threat. Quorum sensing (QS) inhibition is an attractive anti-microbial approach, as it represents an anti-virulence strategy fundamentally different from current traditional therapies. QS a highly conserved bacterial communication signaling regulatory mechanism controlling numerous virulence functions and inter- and intraspecies interactions in mono- and polymicrobial communities. The recalcitrant ESKAPE pathogen Pseudomonas aeruginosa (PA) exemplifies a highly problematic nosocomial pathogen with astounding complex interconnected QS systems controlling multiple virulence functions and regulating antagonism or synergism with other microbes. QS relevance in pathogenesis has been recognized; however, the regulatory interrelationships of these systems during infection and in polymicrobial settings have been studied primarily in vitro, limiting relevance to the human environment during infection and leaving answered fundamental questions that will aid in combatting MDR infections by utilizing the anti-virulence/anti-QS approach. We hypothesize that the interrelationships between the three QS systems are significant drivers of infection severity, interspecies interactions, and microbial community composition. In turn, we anticipate that our anti-QS lead compound will restrict infection exacerbations and promote microbial homeostasis. We propose to assess this hypothesis paradigmatically by leveraging an extensive collection of PA QS mutants and a novel anti-QS compound in dynamic and human-relevant host environments to identify specific infection-relevant host-microbe and microbe-microbe interactions and dissect the mechanisms underlying these interactions. Specifically, in the proposed three Aims, we will address three fundamental questions that will fill existing gaps: 1) What are the regulatory relationships and functions of the three major PA QS systems? 2) How do anti-QS therapies impact infection severity and QS regulatory interactions in PA mono- and polymicrobial settings? and 3) How do the QS systems and inhibitors impact the development of resistance and limit perturbations of host-associated microbial communities? Answering these questions will not only provide critical insights into long-standing questions about the anti-virulence approach and the role of QS mutations in PA virulence but will also uncover how the in vivo environment impacts the regulatory interrelationships of the three QS systems and QS-dependent inter- and intraspecies interactions. By tapping into bacterial QS pathways with novel potent and highly specific anti-QS compounds, we aim to treat infections while preserving the composition and protective function of the microbiota. Results from these dynamic settings may aid in the fight against antibiotic resistance and provide fundamental insights into the molecular mechanisms underlying the virul...