Listeria monocytogenes TcyKLMN cystine/cysteine transporter facilitates glutathione synthesis and virulence gene expression

Listeria monocytogenes ( Lm ) is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of Lm ’s master virulence regulator, PrfA. While glutathione synthesis by Lm was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized under changing environments, especially in light of the observation that Lm is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators: CodY, CymR and CysK, which sense and respond to changing concentrations of branched chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is up-regulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of Lm metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis, as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence. Importance Bacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into a pathogenic state to accomplish successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.