Legionella micdadei phosphatase catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate in human neutrophils.
The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself.
Saha
A K
AK
Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Dowling
J N
JN
Pasculle
A W
AW
Glew
R H
RH
D000135:Acid Phosphatase / Q000302:isolation & purification / Q000502:physiology*; D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D004075:Diglycerides / Q000096:biosynthesis; D006801:Humans; D006868:Hydrolysis; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000096:biosynthesis; D007875:Legionella / Q000201:enzymology*; D009504:Neutrophils / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D000099182:Phosphoinositide 5-Phosphatases
D000135:Acid Phosphatase; D000596:Amino Acids; D004075:Diglycerides; D007294:Inositol; D007295:Inositol Phosphates; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010744:Phosphoric Monoester Hydrolases; D000099182:Phosphoinositide 5-Phosphatases; C037178:phosphatidylinositol 4-phosphate