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.

Cell migration is critical to a wide range of physiological and pathological events and is central to disease progression of B lymphocyte (B cell)-derived leukemia and lymphoma as well as many other types of cancer. It is extensively controlled by phosphoinositide 3-kinase (PI3K), which generates PI(3,4,5)P3 (PIP3) and PI(3,4)P2, lipid messengers that recruit pleckstrin homology (PH)-domain-containing signaling proteins. While PIP3 is known to regulate cell migration, it remains a major unanswered question in the field whether PI(3,4)P2 is also implicated in this cellular function. A series of investigations here on PI(3,4)P2-specific lipid phosphatases and binding proteins in the context of chemotaxing malignant B cells provide the first insights into a previously unappreciated role of PI(3,4)P2 signaling in cell migration. First, I used physiological regulators of PI(3,4)P2, the inositol polyphosphate 4-phosphatase (INPP4) enzymes, as tool to manipulate PI(3,4)P2 levels to determine the function of this lipid second messenger. PI(3,4)P2 depletion by INPP4A or INPP4B relative to phosphatase-dead mutants indicated an essential role of PI(3,4)P2 in mediating both the speed and directionality of chemotaxis. Gene silencing of the authenticated PI(3,4)P2-specific binding protein TAPP2 leads to reduced migration speed and directionality, similar to PI(3,4)P2 depletion. The impaired migration is underlain by alterations in chemokine-induced rearrangement of the actin cytoskeleton, loss of migratory polarity and dysregulation of the leading edge activator Rac. A putative PI(3,4)P2-binding protein, lamellipodin (Lpd), is found to strongly colocalize with PI(3,4)P2 depending on the Lpd PH domain. Lpd knock-down rescue experiments indicated that PI(3,4)P2 controls directionality through Lpd, while Lpd also promotes motility independently of PH domain binding to PI(3,4)P2. The PI(3,4)P2-binding protein kinase Akt/PKB (also binds to PIP3) is found to play a positive role in the B cell context. Here, PI(3,4)P2 depletion does not inhibit phosphorylation of Akt but seemingly reduces its activity. It is likely that PI(3,4)P2 mediates malignant B cell migration in part through promoting Akt activity. Taken together, the thesis work establishes the PI(3,4)P2 pathway as a novel branch of the PI3K signaling network controlling cell migration and suggests that PI(3,4)P2 may integrate diverse downstream migratory pathways to impact on cell migration.