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Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase.

Tachado SD, Samrakandi MM, Cirillo JD - PLoS ONE (2008)

Bottom Line: 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.In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells.These results suggest an important role for PI3K and Akt in the L. pneumophila infection process.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, Texas, USA.

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 p85alpha 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 for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.

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L. pneumophila infection stimulates PI3K-dependent phosphorylation of Akt.Macrophages were treated or not treated with PI3K inhibitors and infected with L. pneumophila 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.
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pone-0003324-g003: L. pneumophila infection stimulates PI3K-dependent phosphorylation of Akt.Macrophages were treated or not treated with PI3K inhibitors and infected with L. pneumophila 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.

Mentions: To further demonstrate the involvement of PI3K in mediating phagocytosis of L. pneumophila by macrophages, we tested whether protein kinase B (Akt) is activated during phagocytosis. Akt mediates the effects of PI3K downstream in the PI3K signaling pathway. We utilized Western blot analysis with antibodies specific to the activated (phosphorylated) form of Akt to determine the levels of activated Akt in L. pneumophila infected macrophages. Macrophages were pre-treated in the presence or absence of PI3K inhibitors for 1 hr followed by L. pneumophila (MOI 100∶1) infection for 15 min and lysates prepared. Western blots were probed with an antibody recognizing Akt phosphorylated Ser473 (Cell Signaling, Beverly, MA). Infection of macrophages by L. pneumophila significantly elevated the level of Ser473 phosphorylated Akt as compared to the control, suggesting that Akt is activated during L. pneumophila infection (Fig. 3). Activation of Akt was blocked by pretreatment with Wortmannin. In addition, a second PI3K inhibitor LY294002 reduced the amount of Ser473 phosphorylated Akt to nearly basal levels. These data indicate that L. pneumophila infection of macrophages activates Akt which mediates the effects of PI3K distal in the signaling pathway.


Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase.

Tachado SD, Samrakandi MM, Cirillo JD - PLoS ONE (2008)

L. pneumophila infection stimulates PI3K-dependent phosphorylation of Akt.Macrophages were treated or not treated with PI3K inhibitors and infected with L. pneumophila 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553182&req=5

pone-0003324-g003: L. pneumophila infection stimulates PI3K-dependent phosphorylation of Akt.Macrophages were treated or not treated with PI3K inhibitors and infected with L. pneumophila 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.
Mentions: To further demonstrate the involvement of PI3K in mediating phagocytosis of L. pneumophila by macrophages, we tested whether protein kinase B (Akt) is activated during phagocytosis. Akt mediates the effects of PI3K downstream in the PI3K signaling pathway. We utilized Western blot analysis with antibodies specific to the activated (phosphorylated) form of Akt to determine the levels of activated Akt in L. pneumophila infected macrophages. Macrophages were pre-treated in the presence or absence of PI3K inhibitors for 1 hr followed by L. pneumophila (MOI 100∶1) infection for 15 min and lysates prepared. Western blots were probed with an antibody recognizing Akt phosphorylated Ser473 (Cell Signaling, Beverly, MA). Infection of macrophages by L. pneumophila significantly elevated the level of Ser473 phosphorylated Akt as compared to the control, suggesting that Akt is activated during L. pneumophila infection (Fig. 3). Activation of Akt was blocked by pretreatment with Wortmannin. In addition, a second PI3K inhibitor LY294002 reduced the amount of Ser473 phosphorylated Akt to nearly basal levels. These data indicate that L. pneumophila infection of macrophages activates Akt which mediates the effects of PI3K distal in the signaling pathway.

Bottom Line: 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.In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells.These results suggest an important role for PI3K and Akt in the L. pneumophila infection process.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, Texas, USA.

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 p85alpha 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 for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.

Show MeSH
Related in: MedlinePlus