Listeria monocytogenes Requires Phosphotransferase Systems to Facilitate Intracellular Growth and Virulence

The metabolism of bacterial pathogens is exquisitely evolved to support growth and survival in the nutrient-limiting host. Many bacterial pathogens utilize bipartite metabolism to support intracellular growth by splitting carbon utilization between two carbon sources and dividing flux to distinct metabolic needs. For example, previous studies suggest that the professional cytosolic pathogen Listeria monocytogenes (L. monocytogenes) utilizes glycerol and hexose phosphates (e.g. Glucose-6-Phosphate) as catabolic and anabolic carbon sources in the host cytosol, respectively. However, the role of this putative bipartite glycerol and hexose phosphate metabolism in L. monocytogenes virulence has not been fully assessed. Here, we demonstrate that when L. monocytogenes is unable to consume either glycerol ({Delta}glpD/{Delta}golD), hexose phosphates ({Delta}uhpT), or both ({Delta}glpD/{Delta}golD/{Delta}uhpT), it is still able to grow in the host cytosol and is minimally attenuated in vivo suggesting that L. monocytogenes consumes alternative carbon source(s) in the host. An in vitro metabolic screen using BioLogs phenotypic microarrays demonstrated that both WT and PrfA* L. monocytogenes, a strain with constitutive virulence gene expression mimicking cytosolic replication, use phosphotransferase system (PTS) mediated carbon sources. These findings contrast with the existing metabolic model that cytosolic L. monocytogenes expressing PrfA does not use PTS mediated carbon sources. We next demonstrate that two independent and universal phosphocarrier proteins (PtsI [EI] and PtsH [HPr]), essential for the function of all PTS, are critical for intracellular growth and virulence in vivo. Finally, virulence phenotypes of these mutants were additive to mutants unable to consume glycerol and hexose phosphates ({Delta}glpD/{Delta}golD/{Delta}uhpT) in vivo, suggesting that hexose phosphates and glycerol are relevant metabolites in vivo in addition to those derived from PTS. Taken together, these studies indicate that PTS are critical virulence factors for the cytosolic growth and virulence of L. monocytogenes.