Project Summary Listeria monocytogenes (LM) is a very dangerous foodborne pathogen with a high mortality rate (20 - 30%). A major reason why LM is dangerous is because it can survive in a variety of environmental stresses encountered during its infection cycle, including low pH, high salt, low temperature, and the intracellular environments. The ability of LM to survive in different environmental stresses is directly related to its ability to adjust its metabolism depending on the type of stress and nutrient availability. There is a knowledge gap in our understanding of the regulatory mechanisms controlling LM metabolic adaptations to stressful environmental conditions. We have evidence that DeoR-family regulators serve this critical function by regulating LM metabolism. Members of the DeoR-family regulators often serve as transcriptional repressors or activators in sugar metabolism. By analyzing the genome of LM strain F2365, we found seven members of the DeoR-family regulators. We constructed a LM deletion strain by targeting FruR-encoding DeoR-family regulator (LMOf2365_2307). We found that F2365?fruR is attenuated and has decreased growth in macrophages relative to the virulent parent strain F2365. Furthermore, deletion of fruR causes strong upregulation of phosphofructokinase (fruK) and fructose-specific PTS system transporter subunit IIABC (fruA). Our central hypothesis is that the DeoR-family transcriptional regulators contribute to the ability of LM to adapt to environmental changes and replicate in host cells by acting as global regulators for many metabolic genes, including phosphotransferase transport system (PTS) and ABC transporters. The specific goal of this proposal is to determine the contribution of DeoR-family regulators to LM adaptation to harsh environmental conditions. This goal will be achieved by performing the following two specific aims: Aim 1 elucidate the role of FruR in L. monocytogenes intracellular replication. In this aim, we will analyze the glucose incorporation and metabolism in F2365?fruR replicating in macrophage by 13C-perturbation techniques. We will compare the transcriptome of parent and F2365?fruR strain during growth in macrophage cell line. We will determine whether the intracellular growth defect of F2365?fruR is due to delayed vacuolar escape or due to a defect in the hemolysin production.; Aim 2 define the role of other DeoR-family regulators in Listeria's response to stressful conditions. This aim will determine the effect of DeoR-family genes in virulence and in growth defect in macrophages cell lines. We will also identify the role of DeoR-family regulators in LM adaptation to food related stress conditions, including exposure to acid and high salt. The rationale for this project is to determine mechanisms that allow LM to tolerate and grow within stressful food-related environmental conditions and survive in intracellular host cells, which will assist in the development of intervention strategies to control LM and will lead to advancements in therapies to combat foodborne infection.