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Chapter 71 : A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of

Authors: Stefan S. Weber1, Curdin Ragaz2, Katrin Reus3, Hubert Hilbi4
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Affiliations: 1: Institute of Microbiology, Swiss Federal Institute of Technology (ETH), 8093 Zürich, Switzerland; 2: Institute of Microbiology, Swiss Federal Institute of Technology (ETH), 8093 Zürich, Switzerland; 3: Institute of Microbiology, Swiss Federal Institute of Technology (ETH), 8093 Zürich, Switzerland; 4: Institute of Microbiology, Swiss Federal Institute of Technology (ETH), 8093 Zürich, Switzerland
Content Type: Monograph
Publication Year: 2006

Category: Bacterial Pathogenesis

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A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of , Page 1 of 2

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Abstract:

is a facultative intracellular bacterium which replicates within amoebae and macrophages by a similar mechanism. Intracellular replication within both protozoa and mammalian cells requires the bacterial genes, encoding a conjugation apparatus related to type IV secretion systems (T4SSs). The inositol carbohydrate moiety of phosphoinositides (PIs) is phosphorylated at positions 3, 4, and/or 5 by specific kinases or dephosphorylated by phosphatases, respectively. Phagocytosis of by was determined by a gentamicin protection assay and by flow cytometry. Wild-type (but not ) replicated more efficiently in or in wild-type treated with PI3K inhibitors. To investigate whether Icm/Dot secreted proteins bind in vitro to PIs immobilized on nitrocellulose membranes, the authors performed a lipid protein overlay assay using glutathione S-transferase fusion proteins and an anti-glutathione S-transferase antibody. Thus, SidC and its paralogue SdcA were found to specifically bind to PI phosphate but not to other PIs or lipids. The result was confirmed in a pull-down assay, where the authors employed phospholipid vesicles containing either PI(4)P or other PIs.

Citation: S. Weber S, Ragaz C, Reus K, Hilbi H. 2006. A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of , p 292-296. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch71
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A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of Legionella pneumophila
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Citation: S. Weber S, Ragaz C, Reus K, Hilbi H. 2006. A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of , p 292-296. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch71
/content/10.1128/9781555815660.ch71.ch71fig01
FIGURE 1
Icm/Dot-dependent phagocytosis of by as determined by a gentamicin protection assay. 5 × 10 wild type (Ax3, Ax2) and (PI3K; a A × 3 derivative lacking the phosphatidylinositol-3 kinases 1 and —2) were infected at a multiplicity of infection of 10 in a 96-well plate with wild-type or mutant strain grown for 3 days on charcoal-yeast extract agar plates. Where indicated, the strains were treated with the P13K inhibitor wortmannin (Wm; 0.1 μM, 1 h). The infection was synchronized by centrifugation (10 min, 880 × ), and after 10 min of incubation, extracellular bacteria were killed by the addition of medium containing gentamicin (0.1 mg/ml). After 1 h at 25°C, the medium was aspirated, and the infected amoebae were lysed in medium containing saponin (0.1%, 15 min). The number of protected from gentamicin within the amoebae was determined by plating 10 µl of the lysate.
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10.1128/9781555815660/f0293-01.gif
Image of FIGURE 1
FIGURE 1

Icm/Dot-dependent phagocytosis of by as determined by a gentamicin protection assay. 5 × 10 wild type (Ax3, Ax2) and (PI3K; a A × 3 derivative lacking the phosphatidylinositol-3 kinases 1 and —2) were infected at a multiplicity of infection of 10 in a 96-well plate with wild-type or mutant strain grown for 3 days on charcoal-yeast extract agar plates. Where indicated, the strains were treated with the P13K inhibitor wortmannin (Wm; 0.1 μM, 1 h). The infection was synchronized by centrifugation (10 min, 880 × ), and after 10 min of incubation, extracellular bacteria were killed by the addition of medium containing gentamicin (0.1 mg/ml). After 1 h at 25°C, the medium was aspirated, and the infected amoebae were lysed in medium containing saponin (0.1%, 15 min). The number of protected from gentamicin within the amoebae was determined by plating 10 µl of the lysate.

Citation: S. Weber S, Ragaz C, Reus K, Hilbi H. 2006. A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of , p 292-296. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch71
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/content/10.1128/9781555815660.ch71.ch71fig02
FIGURE 2
Phosphatidylinositol-3 kinases modulate trafficking of within and affect the amount of the Icm/Dot-secreted protein SidC on the vacuole. Confocal laser scanning micrographs are shown of LCVs in calnexin-green fluorescent proteinlabeled wild-type strain Ax3 (upper panel) or lacking the phosphatidylinositol-3 kinases-1 and -2 (lower panel). The amoebae were infected with DsRed-expressing wild-type (JR32) for 1 h and immuno-labeled for the Icm/Dot-secreted protein SidC with an affinity-purified antibody and Cy5-conjugated secondary antibody. In wild-type spacious LCVs were formed preferentially, which bound lower amounts of SidC. However, in a higher number of tight LCVs were observed, which accumulated on average 1.5 times more SidC. Fluorescence intensity was quantified by subtracting the averaged intensity within background areas from the intensity of a sample area. Bar, 2 μm.
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10.1128/9781555815660/f0294-01.gif
Image of FIGURE 2
FIGURE 2

Phosphatidylinositol-3 kinases modulate trafficking of within and affect the amount of the Icm/Dot-secreted protein SidC on the vacuole. Confocal laser scanning micrographs are shown of LCVs in calnexin-green fluorescent proteinlabeled wild-type strain Ax3 (upper panel) or lacking the phosphatidylinositol-3 kinases-1 and -2 (lower panel). The amoebae were infected with DsRed-expressing wild-type (JR32) for 1 h and immuno-labeled for the Icm/Dot-secreted protein SidC with an affinity-purified antibody and Cy5-conjugated secondary antibody. In wild-type spacious LCVs were formed preferentially, which bound lower amounts of SidC. However, in a higher number of tight LCVs were observed, which accumulated on average 1.5 times more SidC. Fluorescence intensity was quantified by subtracting the averaged intensity within background areas from the intensity of a sample area. Bar, 2 μm.

Citation: S. Weber S, Ragaz C, Reus K, Hilbi H. 2006. A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of , p 292-296. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch71
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References

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