non-opsonic phagocytosis of legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinasenon-opsonic嗜肺性军团菌的吞噬巨噬细胞是由磷脂酰肌醇3-kinase.pdfVIP

Non-Opsonic Phagocytosis of Legionella pneumophila by Macrophages Is Mediated by Phosphatidylinositol 3-Kinase Souvenir D. Tachado¤a, Mustapha M. Samrakandi¤b, Jeffrey D. Cirillo* Department of Microbial and Molecular Pathogenesis, Texas AM Health Science Center, College Station, Texas, United States of America 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 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 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 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 signa