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A Role for Phosphoinositide Metabolism in Phagocytosis and Intracellular Replication of Legionella pneumophila, Page 1 of 2
< Previous page Next page > /docserver/preview/fulltext/10.1128/9781555815660/9781555813901_Chap71-1.gif /docserver/preview/fulltext/10.1128/9781555815660/9781555813901_Chap71-2.gifAbstract:
Legionella pneumophila 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 icm/dot 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 L. pneumophila by Dictyostelium was determined by a gentamicin protection assay and by flow cytometry. Wild-type L. pneumophila (but not ΔicmT) replicated more efficiently in ΔPI3K1/2 or in wild-type Dictyostelium 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.