An adequate multiplication rate in vivo is crucial for an infectious agent to cause clinical
disease and achieve its transmission to new hosts. Despite their key importance, the
nutritional and metabolic determinants of pathogen proliferation within the host remain a neglected area of research in infection biology. In this MSc thesis I have investigated the
carbon metabolites used by the bacterial intracellular pathogen Listeria monocytogenes to
grow within host cells.
Previous work in the Vazquez-Boland laboratory demonstrated that, via a specific permease
named Hpt, L. monocytogenes steals hexose phosphates from the host cell to fuel its rapid
intracellular growth. Albeit more slowly, mutants lacking this permease are still able to
replicate intracellularly, indicating that Listeria uses other carbon substrates from the host
cell. Evidence suggested that free glucose could be this additional carbon substrate. To test
this hypothesis, I sought to obtain a glucose utilisation- deficient mutant by disabling the main
transport systems involved in glucose uptake by L. monocytogenes. A double mutant was
constructed which lacked the central component of the phosphoenolpyruvate:sugar
phosphotransferase system (PTS) Hpr (PtsH) necessary for PTS- dependent sugar transport,
plus the main non -PTS specific glucose transporter GIcU1. The double ptsHIglucU1 knockout mutant was virtually unable to grow on glucose in vitro and to proliferate within HeLa
epithelial cells in conditions in which free glucose is in excess, indicating that glucose is a main intracellular carbon source for L. monocytogenes.
also sought to provide evidence for this conclusion from the host cell side. Using siRNA
knockdown assays, HeLa cells were depleted of hexokinase, the enzyme that converts all
the incoming free glucose into glucose -6- phosphate. A Ahpt mutant unable to use hexose
phosphates showed wild -type growth in the hexokinase -depleted host cells, further indicating
that free glucose, before conversion into glucose -6- phosphate and entering glycolysis, is a major carbon substrate for intracellular Listeria.