title: Identification and characterization of a phospholipase A1 activity type three secreted protein, PP_ExoU from Pseudomonas plecoglossicida NB2011, the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea).
abstract: Pseudomonas plecoglossicida NB2011, 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 pET30a 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 PLA1 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.
authors.affiliation: Biological and Environmental College, Zhejiang Wanli University, Ningbo, China.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Biological and Environmental College, Zhejiang Wanli University, Ningbo, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China.
authors.forename: H
authors.initials: H
authors.lastname: Guo
authors.affiliation: Biological and Environmental College, Zhejiang Wanli University, Ningbo, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Mao
authors.affiliation: Biological and Environmental College, Zhejiang Wanli University, Ningbo, China.
authors.forename: C
authors.initials: C
authors.lastname: Ge
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002681:China; D005393:Fish Diseases / Q000382:microbiology*; D006099:Granuloma / Q000382:microbiology / Q000662:veterinary*; D006367:HeLa Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010473:Perciformes; D054466:Phospholipases A1 / Q000737:chemistry* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011552:Pseudomonas Infections / Q000382:microbiology / Q000662:veterinary*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D054466:Phospholipases A1
keywords: 
  Pseudomonas plecoglossicida
  ; PP_ExoU; phospholipase A11; type three secreted protein