Integrative Genomic Analysis Identifies Isoleucine and CodY as Regulators of <em>Listeria monocytogenes</em> Virulence

<div><p>Intracellular bacterial pathogens are metabolically adapted to grow within mammalian cells. While these adaptations are fundamental to the ability to cause disease, we know little about the relationship between the pathogen's metabolism and virulence. Here we used an integrative Metabolic Analysis Tool that combines transcriptome data with genome-scale metabolic models to define the metabolic requirements of <em>Listeria monocytogenes</em> during infection. Twelve metabolic pathways were identified as differentially active during <em>L. monocytogenes</em> growth in macrophage cells. Intracellular replication requires <em>de novo</em> synthesis of histidine, arginine, purine, and branch chain amino acids (BCAAs), as well as catabolism of L-rhamnose and glycerol. The importance of each metabolic pathway during infection was confirmed by generation of gene knockout mutants in the respective pathways. Next, we investigated the association of these metabolic requirements in the regulation of <em>L. monocytogenes</em> virulence. Here we show that limiting BCAA concentrations, primarily isoleucine, results in robust induction of the master virulence activator gene, <em>prfA</em>, and the PrfA-regulated genes. This response was specific and required the nutrient responsive regulator CodY, which is known to bind isoleucine. Further analysis demonstrated that CodY is involved in <em>prfA</em> regulation, playing a role in <em>prfA</em> activation under limiting conditions of BCAAs. This study evidences an additional regulatory mechanism underlying <em>L. monocytogenes</em> virulence, placing CodY at the crossroads of metabolism and virulence.</p> </div>