title: Purification and characterization of a protease from Pseudomonas pseudomallei.
abstract: Pseudomonas pseudomallei is the causative agent of melioidosis, a glanders-like disease of humans and animals. The pathogenesis of melioidosis is not well understood, and the role of various extracellular enzymes produced by P. pseudomallei in the development of this disease is not known. The present studies were designed to purify and characterize an extracellular protease produced by P. pseudomallei isolates and to test the hypothesis that this protease may play a role in melioidosis. The protease was present in culture supernatants as an enzyme with a molecular weight of 36,000 that was optimally active at 60 degrees C and at pH 8.0. The P. pseudomallei protease was shown to be a metalloenzyme requiring iron for maximal activity, and activity was inhibited in the presence of ethylenediaminetetraacetic acid (150 mM). Antibodies directed against an alkaline protease produced by Pseudomonas aeruginosa cross-reacted with the P. pseudomallei protease. These data indicate that the P. pseudomallei protease belongs to the family of alkaline proteases sensitive to metal chelators. Purified P. pseudomallei protease was capable of digesting a variety of eucaryotic protein substrates including immunoglobulins. A P. pseudomallei strain deficient in protease production was shown to be less virulent than the parental strain in an animal model of lung infection. These data suggest that this protease may be a significant pathogenic determinant in infections caused by P. pseudomallei.
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Canada.
authors.forename: M M
authors.initials: MM
authors.lastname: Sexton
authors.forename: A L
authors.initials: AL
authors.lastname: Jones
authors.forename: W
authors.initials: W
authors.lastname: Chaowagul
authors.forename: D E
authors.initials: DE
authors.lastname: Woods
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016957:Burkholderia pseudomallei / Q000201:enzymology* / Q000472:pathogenicity; D002614:Chelating Agents; D003921:Diabetes Mellitus, Experimental; D006801:Humans; D007501:Iron / Q000378:metabolism; D008168:Lung / Q000382:microbiology / Q000473:pathology; D008171:Lung Diseases / Q000382:microbiology / Q000473:pathology; D008297:Male; D008554:Melioidosis / Q000382:microbiology / Q000473:pathology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D011480:Protease Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D014774:Virulence
chemical_list: D002614:Chelating Agents; D011480:Protease Inhibitors; D007501:Iron; D008666:Metalloendopeptidases