Characterization of phospholipase activity of the Pseudomonas aeruginosa type III cytotoxin, ExoU

Abstract

Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.

FIG. 1.

Optimization of an in vitro assay. (A) One microgram of rExoU and a range of concentrations of yeast extract from 0 to 200 μg were incubated with radiolabeled liposomes (3.3 mM). After 3 h of incubation at 30°C, phospholipase activity was measured using radiolabeled POPC in POPC-POPS (50:50) liposomes and thin-layer chromatography. Fifty micrograms of yeast extract (open circle) in the absence of rExoU was used as a negative control. (Inset) Assay in the presence of 0 to 10 μg of yeast extract proteins. (B) rExoU was titrated using 4 μg of yeast extract. A noncatalytic mutant, rExoUS142A (open circle), did not hydrolyze the substrate, even in the presence of yeast extract. (C) rExoU (1 μg) was incubated with yeast extract (5 μg) and radiolabeled substrates at various pHs at 30°C for 3 h, and phospholipase activity was measured. (D) The incubation temperature of in vitro assays was varied, and rExoU activity was quantified using 1 μg of rExoU and 5 μg of yeast extract. (E) One microgram of rExoU and 30 μg of yeast extract were incubated in a buffer containing various concentrations of NaCl at 30°C for 3 h.

FIG. 2.

The activity of the rExoU cofactor is susceptible to heat and protease treatment. (A) Yeast extract (10 μg) was incubated at 30 or 56°C for 30 min or boiled for 5 min, followed by the addition of 1 μg of rExoU and radiolabeled liposomes. (B) Twenty micrograms of yeast extract was digested with chymotrypsin at 30°C for 15 min. After the proteolysis reaction was stopped by the addition of protease inhibitors, the treated extract was added to 1 μg of rExoU and substrate liposomes.

FIG. 3.

Incubation conditions affecting the stability of rExoU and phospholipase activity. (A) The degradation of rExoU (1 μg) during incubation at pH 7.4 or 6.5 at 30°C in the presence (U ext) or absence (U) of yeast extract (5 μg) was monitored by Western blotting. Aprotinin (2.3 μg) was added to the incubation mixture at pH 6.5 to minimize rExoU degradation. The samples were used after 1 or 3 h of incubation for the in vitro assay. For positive controls, 1 μg of rExoU or 5 μg of extract was incubated individually, and fresh extract or rExoU was added immediately prior to the assay. Percentages indicate levels of rExoU activity relative to those exhibited by positive controls. (B) The incubation temperature of in vitro assays was varied, and rExoU activity was quantified in the presence of a protease inhibitor. Samples containing 5 μg of rExoU, 5 μg of yeast extract proteins, and 6 μg of aprotinin were incubated with liposomes for 15 min.