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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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Virulent but not avirulent L. pneumophila induce association of PI3K with tyrosine-phosphorylated proteins.Macrophages were infected with the virulent (AA100) and avirulent (Lp-14) strains of L. pneumophila 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.
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pone-0003324-g004: Virulent but not avirulent L. pneumophila induce association of PI3K with tyrosine-phosphorylated proteins.Macrophages were infected with the virulent (AA100) and avirulent (Lp-14) strains of L. pneumophila 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.

Mentions: Recruitment of the p85/p110-type PI3K to the plasma membrane and its association with tyrosine phosphorylated proteins occurs following stimulation is a key event in promoting its activation [11]. Tyrosine phosphorylation and activation of PI3K in Trypanosoma cruzi [9], Listeria monocytogenes [30] and in Cryptosporidium parvum [15], have been shown to play a key role in host cell invasion. A role for tyrosine phosphorylation in L. pneumophila entry into host cells has been previously observed [21]–[23]. We hypothesized that virulence depends on these tyrosine phosphorylation events mediating activation of p85/p110-type PI3K. It was tested by infecting macrophages with virulent (AA100) and avirulent Lp-14 L. pneumophila and analyzing for PI3K activity. We tested whether tyrosine phosphorylated proteins are induced by L. pneumophila, and whether PI3K activity is associated with this response. Infection of macrophages by L. pneumophila consistently increased phosphorylation of several major components migrating with apparent molecular weights (Mr) of 27, 30, and 35 kDa (Fig. 4A). Importantly proteins migrating with apparent molecular weights of 20, 22, and 37 KDa are de novo proteins and inhibited by wortmannin suggesting that phosphorylation is mediated by PI3K.


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 L. pneumophila induce association of PI3K with tyrosine-phosphorylated proteins.Macrophages were infected with the virulent (AA100) and avirulent (Lp-14) strains of L. pneumophila 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553182&req=5

pone-0003324-g004: Virulent but not avirulent L. pneumophila induce association of PI3K with tyrosine-phosphorylated proteins.Macrophages were infected with the virulent (AA100) and avirulent (Lp-14) strains of L. pneumophila 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.
Mentions: Recruitment of the p85/p110-type PI3K to the plasma membrane and its association with tyrosine phosphorylated proteins occurs following stimulation is a key event in promoting its activation [11]. Tyrosine phosphorylation and activation of PI3K in Trypanosoma cruzi [9], Listeria monocytogenes [30] and in Cryptosporidium parvum [15], have been shown to play a key role in host cell invasion. A role for tyrosine phosphorylation in L. pneumophila entry into host cells has been previously observed [21]–[23]. We hypothesized that virulence depends on these tyrosine phosphorylation events mediating activation of p85/p110-type PI3K. It was tested by infecting macrophages with virulent (AA100) and avirulent Lp-14 L. pneumophila and analyzing for PI3K activity. We tested whether tyrosine phosphorylated proteins are induced by L. pneumophila, and whether PI3K activity is associated with this response. Infection of macrophages by L. pneumophila consistently increased phosphorylation of several major components migrating with apparent molecular weights (Mr) of 27, 30, and 35 kDa (Fig. 4A). Importantly proteins migrating with apparent molecular weights of 20, 22, and 37 KDa are de novo proteins and inhibited by wortmannin suggesting that phosphorylation is mediated by PI3K.

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