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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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PI3K inhibitors block L. pneumophila entry into host cells.Murine J774A.1 (A, B) that were treated or not treated with different concentrations of LY294002 or (A) Wortmannin (B) for 30 min and subsequently infected with L.pneumophila for 1 hr. Entry in the absence of the inhibitor was arbitrarily set to 100%. Data presented are means+/−SEM for assays done in triplicate. Similar results were obtained in at least two independent experiments.
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pone-0003324-g001: PI3K inhibitors block L. pneumophila entry into host cells.Murine J774A.1 (A, B) that were treated or not treated with different concentrations of LY294002 or (A) Wortmannin (B) for 30 min and subsequently infected with L.pneumophila for 1 hr. Entry in the absence of the inhibitor was arbitrarily set to 100%. Data presented are means+/−SEM for assays done in triplicate. Similar results were obtained in at least two independent experiments.

Mentions: To determine whether PI3K pathway is involved in non-opsonic entry of L. pneumophila by macrophages, we tested the effects of two structurally unrelated compounds, wortmannin and LY294002, both of which specifically inhibit PI3K activity. Cultures of J774A.1 macrophages were treated with different concentrations of wortmannin and LY294002 for 30 min, followed by co-incubation with L. pneumophila (MOI 100∶1). As shown in Fig. 1A, entry of L. pneumophila into macrophages was inhibited by LY294002 in a dose-dependent fashion. Significant inhibition started at 10 µM and became maximal at 50 µM (p<0.01). Similar results were observed when macrophages were treated with different concentrations of wortmannin, a structurally unrelated PI3K inhibitor. Significant inhibition started at 0.5 nM (p<0.05) and reached a maximum value at 100 nM. Moreover, wortmannin was more effective in inhibiting L. pneumophila entry into macrophages compared to LY294002. The former inhibited entry with an IC50 of ∼2.5 nM, while the latter inhibited with an IC50 of 8.0 µM. These results are similar to those reported elsewhere [27], [28]. Furthermore, IC50 values of this study were similar to those reported for PI3K inhibition but not PI 4-kinase [27], and MLCK [29]. Similar results were obtained using primary human macrophages differentiated from peripheral blood monocytes (data not shown). These studies suggest that non-opsonic entry of L. pneumophila by murine macrophages is PI3K-dependent, as has been shown in several other microorganisms [9], [15], [30].


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

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

PI3K inhibitors block L. pneumophila entry into host cells.Murine J774A.1 (A, B) that were treated or not treated with different concentrations of LY294002 or (A) Wortmannin (B) for 30 min and subsequently infected with L.pneumophila for 1 hr. Entry in the absence of the inhibitor was arbitrarily set to 100%. Data presented are means+/−SEM for assays done in triplicate. 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-g001: PI3K inhibitors block L. pneumophila entry into host cells.Murine J774A.1 (A, B) that were treated or not treated with different concentrations of LY294002 or (A) Wortmannin (B) for 30 min and subsequently infected with L.pneumophila for 1 hr. Entry in the absence of the inhibitor was arbitrarily set to 100%. Data presented are means+/−SEM for assays done in triplicate. Similar results were obtained in at least two independent experiments.
Mentions: To determine whether PI3K pathway is involved in non-opsonic entry of L. pneumophila by macrophages, we tested the effects of two structurally unrelated compounds, wortmannin and LY294002, both of which specifically inhibit PI3K activity. Cultures of J774A.1 macrophages were treated with different concentrations of wortmannin and LY294002 for 30 min, followed by co-incubation with L. pneumophila (MOI 100∶1). As shown in Fig. 1A, entry of L. pneumophila into macrophages was inhibited by LY294002 in a dose-dependent fashion. Significant inhibition started at 10 µM and became maximal at 50 µM (p<0.01). Similar results were observed when macrophages were treated with different concentrations of wortmannin, a structurally unrelated PI3K inhibitor. Significant inhibition started at 0.5 nM (p<0.05) and reached a maximum value at 100 nM. Moreover, wortmannin was more effective in inhibiting L. pneumophila entry into macrophages compared to LY294002. The former inhibited entry with an IC50 of ∼2.5 nM, while the latter inhibited with an IC50 of 8.0 µM. These results are similar to those reported elsewhere [27], [28]. Furthermore, IC50 values of this study were similar to those reported for PI3K inhibition but not PI 4-kinase [27], and MLCK [29]. Similar results were obtained using primary human macrophages differentiated from peripheral blood monocytes (data not shown). These studies suggest that non-opsonic entry of L. pneumophila by murine macrophages is PI3K-dependent, as has been shown in several other microorganisms [9], [15], [30].

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