Understanding the Role of Pyruvate Dehydrogenase in Listeria monocytogenes Virulence

To survive within restrictive host niches, bacterial pathogens must possess finely tuned physiological adaptations. One such niche inhabited by Listeria monocytogenes (L. monocytogenes) is the host cell cytosol--a compartment characterized by significant barriers to entry, metabolic limitation, and immune surveillance. Previously, we identified L. monocytogenes transposon mutants defective for intracellular survival due to disruptions in key metabolic pathways, including cell wall biosynthesis, menaquinone production, and pyruvate metabolism. One of these mutants mapped to a central component of the pyruvate dehydrogenase (PDH) complex, pdhC::Tn. Notably, this mutant exhibits pronounced survival defects during infection, despite retaining robust growth and survival in nutrient-rich media. We go on to show that disruption of pdhA::Tn and pdhD::Tn similarly led to virulence attenuation during intra-macrophage growth, plaquing assays, and murine infections. Respiro-fermentative metabolic profiling revealed that pdhC::Tn mutants have an altered respiro-fermentative metabolism with more prominent secretion of lactate. Further, unbiased metabolomic profiling revealed a global starvation phenotype with lower levels of upper glycolytic intermediates and TCA cycle intermediates coupled with elevated intra-bacterial levels of pyruvate and lactate. We then demonstrate that PDH mutants are unable to efficiently utilize phosphotransferase (PTS)-dependent carbon sources and that their growth can be rescued using non-PTS-mediated carbon sources such as hexose phosphates. To identify genetic suppressors of PDH deficiency, we performed an EMS mutagenesis screen using fructose--a PTS-transported carbon source--as the sole carbon source. Five suppressors each contained a single independent mutation in the redox sensing regulator rex. Subsequently, we show that loss of rex restores pdhC::Tns ability to consume PTS-mediated carbon sources through the alleviation of fermentative repression. Further, pdhC::Tn suppressor mutants show restored intracellular growth, but not virulence in vivo. Together, these findings indicate that a key defect in PDH mutants is the inability to import and metabolize PTS-dependent carbon sources in the host cytosol. We posit this impairment leads to disruptions in redox balance and a shift in respiro-fermentative metabolism, ultimately contributing to the loss of intracellular fitness and virulence.