PROJECT SUMMARY/ABSTRACT Multidrug resistant (MDR) infections caused by the bacterial pathogen Acinetobacter baumannii are increasing at alarming rates. Currently, over 60 % of global A. baumannii clinical isolates are MDR, leading the Centers for Disease Control and Prevention and the World Health Organization to categorize it as a top priority for the research and development of new antimicrobial therapies. In addition to accumulating resistance mechanisms, A. baumannii strains develop tolerance to antibiotics, which can frequently lead to poor therapeutic outcomes even with antibiotic susceptible strains. However, the mechanisms used by A. baumannii to adapt to and tolerate hostile conditions remain largely unknown. We found that A. baumannii employs a novel stress response pathway in which phenylacetic acid (PAA), a metabolite derived from phenylalanine catabolism, acts as a signaling molecule. We established that, in the presence of sub-inhibitory concentrations of different antibiotics, such as trimethoprim/sulfamethoxazole, A. baumannii dramatically increases the transcription of the paa operon which encodes enzymes required to degrade PAA. Conversely, other conditions, like hydrogen peroxide treatment, lead to a repression of the paa operon. The regulation of the paa operon triggers a physiological adaptive response that includes the modulation of pili biosynthesis and biofilm formation. Importantly, we found that artificial augmentation of PAA levels, through the addition of commercially available PAA-derivatives or mutations in PAA degradative genes, disrupts this response Furthermore, mutating initial steps of PAA degradation leads to increased sensitivity to antibiotics and oxidative stress in multiple strains. Here we propose to use our expertise in A. baumannii genetics and pathogenesis to investigate the PAA-mediated stress response in Acinetobacter and determine its importance in virulence. We will determine the breadth of PAA signaling using reporter assays, and we will explore PAA-mediated changes in cell physiology by profiling gene expression under different stress conditions. Further, we will characterize the PAA-dependent mechanisms of cell signaling under stress by measuring cellular levels of PAA and determining the role of important regulatory proteins in this cascade. Finally, we will test the virulence of strains unable to regulate PAA levels in the catheter-associated urinary tract infection and lung infection murine models. Our work will establish the role of PAA as a key regulatory molecule in A. baumannii, determine the biological processes regulated by PAA, and uncover the mechanisms by which PAA triggers adaptations to promote survival under stress. Understanding the fundamental aspects of the PAA stress response will provide a foundation to future clinical studies.