Non-Opsonic Phagocytosis of Legionella pneumophila by Macrophages Is Mediated by Phosphatidylinositol 3-Kinase

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 Findings: Phagocytosis 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 32 P 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 p85a 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/Significance: Entry 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 fo

Virulent but not avirulent strains of <i>L. pneumophila</i> stimulate PI3K activity.

<p>Thin layer chromatography of lipids from <i>in vitro</i> kinase assays on infected macrophage lysates (A). Macrophages were stimulated with different ligands for 15 min. Lysates were immunoprecipitated with specific antiserum to p85^α, and immune complexes were subjected to <i>in vitro</i> kinase assays using exogenous ATP-γ³²P and PI as substrates followed by fluorography. The line (PIP) denotes the position of PI3P standard. Spots aligning at the same position as the PI3P standard were scraped and counted using scintillation counter (B). Similar results were obtained in at least two independent experiments.</p

Overexpression of PI3K mutant protein ablates <i>L. pneumophila</i> entry.

<p>Entry by <i>L. pneumophila</i> into J774A.1 macrophages expressing the p85α mutant PI3K (pSR1NeoΔp85) and containing vector alone (pSR1Neo) after 1 hour co-incubation (A). Western analysis using antibody against the PI3K p85 α subunit, demonstrating Δp85 expression in transfected macrophages (B). Macrophage lysates were immunoprecipitated with anti-p85α and run on SDS/PAGE followed by Western blot analysis. Entry into macrophages carrying the vector alone was arbitrarily set to 100%. Data are the means+/−SEM for assays done in duplicate. Similar results were obtained in at least two independent experiments.</p

L. pneumophila infection stimulates PI3K-dependent phosphorylation of Akt.

<p>Macrophages were treated or not treated with PI3K inhibitors and infected with <i>L. pneumophila</i> for 15 min. Lysates were prepared and Ser⁴⁷³ phospho Akt (activated) detected by Western blot analysis (ppAkt). The membrane was stripped and re-probed with antibody against total cellular Akt, which is normally phosphorylated at a single position (pAkt). Similar results were obtained in at least two independent experiments.</p

PI3K inhibitors block <i>L. pneumophila</i> entry into host cells.

<p>Murine J774A.1 (A, B) that were treated or not treated with different concentrations of LY294002 or (A) Wortmannin (B) for 30 min and subsequently infected with <i>L.pneumophila</i> for 1 hr. Entry in the absence of the inhibitor was arbitrarily set to 100%. Data presented are means+/−SEM for assays done in triplicate. Similar results were obtained in at least two independent experiments.</p

Virulent but not avirulent <i>L. pneumophila</i> induce association of PI3K with tyrosine-phosphorylated proteins.

<p>Macrophages were infected with the virulent (AA100) and avirulent (Lp-14) strains of <i>L. pneumophila</i> for 15 min. Lysates were then immunoprecipitated with anti-p85 antibody and probed with anti-phosphotyrosine antibody (A) or co-immunoprecipitated with anti-phosphotyrosine antibody and probed with anti-p85 (B). The levels of total protein present in each immunoprecipitate were similar as judged by Coomassie staining of identical gels run in parallel (lower panel). Similar results were obtained in at least two independent experiments.</p