Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a wide ecological niche and causes a number of diseases in human and animals. It invades mammalian host cells and escapes from the vacuoles prior to replication in the host cell cytoplasm and infecting adjacent cells via actin-based mobility. Phosphoinositide (PIP) metabolism is essential to mammalian cells in signal transduction, actin remodelling, endosome dynamics and membrane trafficking. Modulation of host PIP metabolism by bacteria PIP phosphatases is important for pathogenicity and virulence of many human pathogens.In this study the function of two L. monocytogenes tyrosine and inositol phosphatases LipA and LipB were studied in vitro. The lipA and lipB deletion mutants generated in EGDe and InlA strains were not affected in invasion but were attenuated in intracellular growth in Caco-2 and Hela M cell lines but not in mouse macrophages. Deletion of lipA or lipB did not affect the actin polymerisation but caused reduced plaque number in the plaque assay. The turnover of five PIPs in Hela M cells during L. monocytogenes infection were studied by expression of fluorescent protein tagged domains that specifically recognizes individual PIPs. L. monocytognenes did not affect the metabolism of PI4P, PI(4,5)P2, PI(3,4,5)P3 but co-localised with PI3P at 1.5 hr post-infection and with PI(3,4)P2 at 6 hr to 24 hr post-infection. The PI(3,4)P2 effector protein lamellipodin was discovered to be recruited to actin-associated L. monocytogenes at 4 hr to 24 hr post-infection in Hela M cells. This discovery leads to the hypothesis of a novel mechanism of lamellipodin-dependant cell-to-cell spread. The lipA mutant was found to be attenuated in PI(3,4)P2 recruitment and therefore hypothesized to participate in the proposed lamellipodin pathway by converting PI(3,5)P2 into PI5P, leading to the activation of PI3K and subsequent production of PI(3,4)P2. LipB showed partial localisation at the Golgi complex when over-expressed in Hela M cells, and it was assumed to act mainly as a protein-tyrosine phosphatase.In summary, this study provides some evidence on L. monocytogenes modulating host PIP metabolism by the production of inositol phosphatases. It gives us a better understanding on the intracellular growth of this pathogenic bacterium, and on the interaction between host and parasite.
|Date of Award||1 Aug 2012|
- The University of Manchester
|Supervisor||Ian Roberts (Supervisor)|
- Listeria monocytogenes