Abstract
Background Legionella pneumophila, is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis.Methodology/Principal FindingsPhagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85α subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells.Conclusion/SignificanceEntry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.
Highlights
Legionnaire’s disease is a severe bacterial infection of the respiratory tract caused by Legionella pneumophila
Changes in the activation status of components in the signal transduction cascades that affect phagocytosis can modulate this response. Such an association exists between changes in phosphoinositidespecific phospholipase C (PI-PLC) [9], protein kinase C (PKC) [10], phosphatidylinositol 3-kinase (PI3K) [11] and Rho GTPases [12] can affect subsequent signal transduction events
The data presented in the current study indicate a role for host signal transduction processes involving tyrosine kinases PI3K and Akt during entry into murine macrophages
Summary
Legionnaire’s disease is a severe bacterial infection of the respiratory tract caused by Legionella pneumophila. The disease was named for the 29 Legionnaires killed by mysterious pneumonia at an American Legion Convention in Philadelphia in 1976 [1] The cause of this mysterious pneumonia was identified to be Legionella pneumophila, a bacteria that was proliferating in the hotel’s airconditioning system [2]. Macrophages are the primary cell type that L. pneumophila replicates within during infection [5], and has been shown to enter monocytes in an unusual mechanism called coiling phagocytosis [6]. Phagocytosis is a complex cellular function to both lower and higher organisms Lower organisms such as slime molds and protozoa utilize this response primarily for acquisition of nutrients; while in higher organisms it is a critical component of the immune system, primarily through ‘‘professional’’ phagocytes such as macrophages, dendritic cells, monocytes and neutrophils [7]. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis
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