Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression

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 muM/min and 234 muM, respectively, and the substrate DL-Val-Leu-Arg-pNA with V-max and K-m values of 0.3830 muM/min and 429 muM, 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 23degreesC. Proteolytic activity was reduced by 90% at 60degreesC for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease H. 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.