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  3. Medizin - Open Access LMU - Teil 17/22 Podcast
  4. Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling
Background: The human pathogen Listeria monocytogenes resides and
proliferates within the cytoplasm of epithelial cells. While the
virulence factors essentially contributing to this step of the
infection cycle are well characterized, the set of listerial genes
contributing to intracellular replication remains to be defined on
a genome-wide level. Results: A comprehensive library of L.
monocytogenes strain EGD knockout mutants was constructed upon
insertion-duplication mutagenesis, and 1491 mutants were tested for
their phenotypes in rich medium and in a Caco-2 cell culture assay.
Following sequencing of the plasmid insertion site, 141 different
genes required for invasion of and replication in Caco-2 cells were
identified. Ten in-frame deletion mutants were constructed that
confirmed the data. The genes with known functions are mainly
involved in cellular processes including transport, in the
intermediary metabolism of sugars, nucleotides and lipids, and in
information pathways such as regulatory functions. No function
could be ascribed to 18 genes, and a counterpart of eight genes is
missing in the apathogenic species L. innocua. Mice infection
studies revealed the in vivo requirement of IspE (Lmo0190) involved
in mevalonate synthesis, and of the novel ABC transporter
Lmo0135-0137 associated with cysteine transport. Based on the data
of this genome-scale screening, an extreme pathway and elementary
mode analysis was applied that demonstrates the critical role of
glycerol and purine metabolism, of fucose utilization, and of the
synthesis of glutathione, aspartate semialdehyde, serine and
branched chain amino acids during intracellular replication of L.
monocytogenes. Conclusion: The combination of a genetic screening
and a modelling approach revealed that a series of transporters
help L. monocytogenes to overcome a putative lack of nutrients
within cells, and that a high metabolic flexibility contributes to
the intracellular replication of this pathogen.

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„Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling“

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