Identification and characterization of a phospholipase A1 activity type three secreted protein, PP _ExoU from Pseudomonas plecoglossicida NB 2011, the causative agent of visceral granulomas disease in large yellow croaker ( Larimichthys crocea )

Pseudomonas plecoglossicida NB 2011 , the causative agent of visceral granulomas disease in farmed Larimichthys crocea in China, encodes a predicted type three effector PP _ExoU, a homolog of the cytotoxin ExoU of Pseudomonas aeruginosa . In this study, secretion of PP _ExoU was tested in various broth, the protein was expressed with the pET 30a prokaryotic system, the phospholipase A ( PLA ) activity of the recombinant protein was determined with fluorogenic phospholipid substrates, fusion expression with green fluorescent protein in transfected HeLa cells was investigated, and the lactate dehydrogenase ( LDH ) level was measured. The results showed the protein was type three secreted in several media; the recombinant protein displayed significant PLA 1 activity with ubiquitin. Fluorescence was observed on the cell membrane and scattered in the cytoplasm of HeLa cells expressing catalytic wild‐type PP _ExoU, blebbing and stretching developed in the cell membranes indicating of membrane damage. Fluorescence scattered in the cytoplasm of cells expressing the catalytic inactive protein. A significant LDH level was detected in HeLa cells expressing wild‐type PP _exoU, but not in the Ser/Asp‐mutated protein, suggestion mutation of predicted catalytic residues abolished the PLA activity. This is the first report on the function of a secreted type three protein from P. plecoglossicida .