Mechanisms associated with Acanthamoeba castellanii (T4) phagocytosis.
Using fluorescein isothiocyanate (FITC)-labelled Escherichia coli, phagocytosis in Acanthamoeba is studied. This assay is based on the quenching effect of trypan blue on FITC-labelled E. coli. Only intracellular E. coli retain their fluorescence, which are easily discriminated from non-fluorescent adherent bacteria. Acanthamoeba uptake of E. coli is significantly reduced in the presence of genistein, a protein tyrosine kinase inhibitor. In contrast, sodium orthovanadate (protein tyrosine phosphatase inhibitor) increases bacterial uptake by Acanthamoeba. Treatment of Acanthamoeba with cytochalasin D (actin polymerization inhibitor) abolished the ability of Acanthamoeba to phagocytose E. coli suggesting that tyrosine kinase-mediated signaling may play a role in Acanthamoeba phagocytosis. In addition, we showed that phosphatidylinositol 3-kinase (PI3K) plays an important role in Acanthamoeba uptake of E. coli. Role of mannose-binding protein in Acanthamoeba phagocytosis is discussed further.
Alsam
Selwa
S
School of Biological and Chemical Sciences, Birkbeck, University of London, London WC1E 7HX, UK.
Sissons
James
J
Dudley
Ricky
R
Khan
Naveed Ahmed
NA
D048248:Acanthamoeba castellanii / Q000187:drug effects / Q000201:enzymology* / Q000382:microbiology; D000818:Animals; D015638:Cytochalasin D / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004798:Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D015638:Cytochalasin D; D004791:Enzyme Inhibitors; D004798:Enzymes; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D019833:Genistein; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D014343:Trypan Blue; D014638:Vanadates; D020741:rho GTP-Binding Proteins