title: Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression.
abstract: Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V(max) and K(m) values of 0.0551 microM/min and 234 microM, respectively, and the substrate DL-Val-Leu-Arg-pNA with V(max) and K(m) values of 0.3830 microM/min and 429 microM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23C. Proteolytic activity was reduced by 90% at 60 degrees C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.
authors.affiliation: Centro de Investigação de Recursos Naturais and Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Açores, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Caldas
authors.forename: A
authors.initials: A
authors.lastname: Cherqui
authors.forename: A
authors.initials: A
authors.lastname: Pereira
authors.forename: N
authors.initials: N
authors.lastname: Simões
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006458:Hemolymph / Q000378:metabolism; D007915:Lepidoptera / Q000276:immunology*; D008969:Molecular Sequence Data; D009348:Nematoda / Q000382:microbiology*; D013559:Symbiosis; D014774:Virulence; D020593:Xenorhabdus / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity
chemical_list: D023181:Antimicrobial Cationic Peptides; C031161:cecropin A; D010450:Endopeptidases