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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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Overexpression of PI3K mutant protein ablates L. pneumophila entry.Entry by L. pneumophila 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.
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pone-0003324-g002: Overexpression of PI3K mutant protein ablates L. pneumophila entry.Entry by L. pneumophila 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.

Mentions: To confirm our pharmacological data regarding wortmannin and LY294002 phagocytosis inhibition, J774A.1 cells were transiently transfected with a dominant negative PI3K mutant gene (J774A.1∶Δp85) or empty vector. Western blot analysis of the cells transfected with the dominant negative p85 mutant and the empty vector confirm Δp85 expression in these macrophages (Fig. 2A). The protein encoded by this gene lacks amino acids 479–513 located between the two src-homology 2 (SH2) domains. This region is necessary and sufficient for interaction of the p85 subunit with the N terminus of p110 catalytic subunit, required for PI3K activity [31]. The ability of L. pneumophila to enter J774A.1∶Δp85 cells were reduced 50% as compared to normal J774A.1 cells carrying the empty vector (Fig. 2B). Even though this inhibitory effect resulting from dominant negative p85 expression is less than that obtained following drug exposure, it supports the notion that the PI3K pathway mediates L. pneumophila host uptake. The smaller decline could be due to the partial suppression of PI3K activity owing to lack of complete replacement of wild type functional p85 protein with its mutant counterpart. In addition, these differences could be due to inhibition of other cellular signaling pathways by Wortmannin such as PKC-ζ [32], DNA-dependent protein kinase (DNA-PK) [33] also involved in phagocytosis. These studies confirm the pharmacological data and support the involvement of PI3K in the ability of L. pneumophila to infect macrophages.


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

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

Overexpression of PI3K mutant protein ablates L. pneumophila entry.Entry by L. pneumophila 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553182&req=5

pone-0003324-g002: Overexpression of PI3K mutant protein ablates L. pneumophila entry.Entry by L. pneumophila 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.
Mentions: To confirm our pharmacological data regarding wortmannin and LY294002 phagocytosis inhibition, J774A.1 cells were transiently transfected with a dominant negative PI3K mutant gene (J774A.1∶Δp85) or empty vector. Western blot analysis of the cells transfected with the dominant negative p85 mutant and the empty vector confirm Δp85 expression in these macrophages (Fig. 2A). The protein encoded by this gene lacks amino acids 479–513 located between the two src-homology 2 (SH2) domains. This region is necessary and sufficient for interaction of the p85 subunit with the N terminus of p110 catalytic subunit, required for PI3K activity [31]. The ability of L. pneumophila to enter J774A.1∶Δp85 cells were reduced 50% as compared to normal J774A.1 cells carrying the empty vector (Fig. 2B). Even though this inhibitory effect resulting from dominant negative p85 expression is less than that obtained following drug exposure, it supports the notion that the PI3K pathway mediates L. pneumophila host uptake. The smaller decline could be due to the partial suppression of PI3K activity owing to lack of complete replacement of wild type functional p85 protein with its mutant counterpart. In addition, these differences could be due to inhibition of other cellular signaling pathways by Wortmannin such as PKC-ζ [32], DNA-dependent protein kinase (DNA-PK) [33] also involved in phagocytosis. These studies confirm the pharmacological data and support the involvement of PI3K in the ability of L. pneumophila to infect macrophages.

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