| Herein we reported on the 1500 fold purification, biochemical characterization, and cell biology of the hemolytic phospholipase C of Pseudomonas aeruginosa , the paradigm for a novel class of PC-PLC/phosphatases that includes homologs identified in a number of microbial pathogens including Mycobacterium tuberculosis, Francisella tularensis, and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography, and native preparative polyacrylamide gel electrophoresis. The enzyme purifies as a heterodimeric complex, PlcHR2, which is active only on choline containing phospholipids (e.g., phosphatidylcholine, sphingomyelin). Remarkably, PlcH is the first protein demonstrated to synthesize sphingomyelin. D609, a widely used, competitive inhibiter of the zinc-metallophospholipases C does not inhibit PlcHR2. PlcH, PlcR2, and the PlcHR 2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits both the cytotoxic and hemolytic activities of PlcHR 2. In addition to being required for the secretion of PlcH, PlcR 2 affects both the enzymatic and hemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.;Subsequent to purification and biochemical characterization, the effects of PlcHR2 on eukaryotic cells were examined. Treatment of various eukaryotic cell lines with PlcHR2 resulted in varying levels of cytotoxicity and release of calcium from internal stores. Primary human umbilical vein endothelial cells (HUVEC) and CHO cells are extremely sensitive to PlcHR 2, whereas HeLa, L929 fibroblasts, and primary human lung epithelial cells are 100–2000 fold less sensitive. Pretreatment of CHO cells with trypsin reduced the level of cytotoxicity ∼50% consistent with receptor mediated entry. In this regard, PlcH contains an integrin binding, arginine-glycine-aspartate (RGD) motif. Therefore, we hypothesize that integrin binding is required for the high susceptibility of eukaryotic cells to PlcH. In support of this hypothesis it was demonstrated that RGD peptides are able to inhibit both PlcHR 2 induced cytotoxicity and release of calcium from internal stores. Mutations in the RGD motif affect both cytotoxicity and binding of PlcH to cells. Finally, specific integrin antibodies are able to inhibit PlcH cytotoxicity in both CHO cells and HUVECs. |
