Abstract
Within-host environments are complex and multidimensional, making it challenging to link the evolutionary responses of colonizing pathogens to causal selective drivers. Loss of quorum sensing (QS) via mutation of the master regulator, lasR, is a common adaptation of Pseudomonas aeruginosa during chronic infections. Here, we use experimental evolution in host-mimicking media to show that loss of QS is constrained by environmental factors associated with host inflammation. Specifically, environments combining oxidative stress and abundant free amino acids limited loss of QS, whereas QS loss was rapid in the absence of oxidative stress, regardless of amino acids. Under oxidative stress, lasR mutations were contingent upon first decoupling regulation of oxidative stress responses from QS via mutations in the promoter region of the primary catalase, katA, or in the oxidative stress regulator, oxyR, enabling maintenance of oxidative stress tolerance. Together, our findings suggest that host inflammatory responses likely delay the loss of QS while colonizers undergo stepwise evolution, first adapting to survive lethal stressors before responding to other nutritional selective drivers that favor loss of QS. IMPORTANCE: Pseudomonas aeruginosa is a common cause of chronic infections characterized by persistent inflammation. Host inflammatory responses alter within-host environments, including by increasing levels of antimicrobial stressors and releasing free amino acids through proteolysis. Here, we show stepwise adaptation of experimental P. aeruginosa populations to inflammation-like environments, first adapting to survive lethal stress by decoupling oxidative stress responses from quorum sensing (QS), before then adapting to the nutritional conditions, delaying the loss of quorum sensing. These results highlight the power of using laboratory evolution experiments to disentangle the multidimensional selective forces driving pathogen adaptation in complex within-host environments.