Limits...
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.

Show MeSH

Related in: MedlinePlus

Virulent but not avirulent strains of L. pneumophila stimulate PI3K activity.Thin layer chromatography of lipids from in vitro kinase assays on infected macrophage lysates (A). Macrophages were stimulated with different ligands for 15 min. Lysates were immunoprecipitated with specific antiserum to p85α, and immune complexes were subjected to in vitro 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2553182&req=5

pone-0003324-g005: Virulent but not avirulent strains of L. pneumophila stimulate PI3K activity.Thin layer chromatography of lipids from in vitro kinase assays on infected macrophage lysates (A). Macrophages were stimulated with different ligands for 15 min. Lysates were immunoprecipitated with specific antiserum to p85α, and immune complexes were subjected to in vitro 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.

Mentions: We examined the ability of avirulent strain Lp-14 to stimulate PI3K as compared to wild-type virulent L. pneumophila. Strain Lp-14 is an AA100 mutant that is sodium resistant and unable to replicate in monocytic cells [34]. In addition, we tested other ligands in these assays, including non-pathogenic E. coli (HB101) and the yeast cell wall extract zymosan. The virulent strain L. pneumophila activated PI3K 10-fold, as measured by the levels of phosphoinositide 3-phosphates (PIP3) obtained from extracts using phosphatidylinositol (PI) as a substrate (Fig. 5). In contrast, Lp-14 activated PI3K four-fold compared to that of the control, suggesting that there is a correlation between virulence and the degree of activation of PI3K. PI3K activity is barely detectable in uninfected macrophages and macrophages infected with non-pathogenic E. coli. Zymosan activates PI3K at levels comparable to that of Lp-14. These data suggest that induction of PI3K during L. pneumophila entry into macrophages correlates with virulence.


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

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

Virulent but not avirulent strains of L. pneumophila stimulate PI3K activity.Thin layer chromatography of lipids from in vitro kinase assays on infected macrophage lysates (A). Macrophages were stimulated with different ligands for 15 min. Lysates were immunoprecipitated with specific antiserum to p85α, and immune complexes were subjected to in vitro 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553182&req=5

pone-0003324-g005: Virulent but not avirulent strains of L. pneumophila stimulate PI3K activity.Thin layer chromatography of lipids from in vitro kinase assays on infected macrophage lysates (A). Macrophages were stimulated with different ligands for 15 min. Lysates were immunoprecipitated with specific antiserum to p85α, and immune complexes were subjected to in vitro 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.
Mentions: We examined the ability of avirulent strain Lp-14 to stimulate PI3K as compared to wild-type virulent L. pneumophila. Strain Lp-14 is an AA100 mutant that is sodium resistant and unable to replicate in monocytic cells [34]. In addition, we tested other ligands in these assays, including non-pathogenic E. coli (HB101) and the yeast cell wall extract zymosan. The virulent strain L. pneumophila activated PI3K 10-fold, as measured by the levels of phosphoinositide 3-phosphates (PIP3) obtained from extracts using phosphatidylinositol (PI) as a substrate (Fig. 5). In contrast, Lp-14 activated PI3K four-fold compared to that of the control, suggesting that there is a correlation between virulence and the degree of activation of PI3K. PI3K activity is barely detectable in uninfected macrophages and macrophages infected with non-pathogenic E. coli. Zymosan activates PI3K at levels comparable to that of Lp-14. These data suggest that induction of PI3K during L. pneumophila entry into macrophages correlates with virulence.

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