PURIFICATION AND CHARACTERIZATION OF A PROTEASE FROM PSEUDOMONAS-PSEUDOMALLEI

Pseudomonas pseudomallei is the causative agent of melioidosis, a glan ders-like disease of humans and animals. The pathogenesis of melioidos is is not well understood, and the role of various extracellular enzym es produced by P. pseudomallei in the development of this disease is n ot 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 36000 that was optimally active at 60 degrees C a nd at pH 8.0. The P. pseudomallei protease was shown to be a metalloen zyme requiring iron for maximal activity, and activity was inhibited i n the presence of ethylenediaminetetraacetic acid (150 mM). Antibodies directed against an alkaline protease produced by Pseudomonas aerugin osa cross-reacted with the P. pseudomallei protease. These data indica te that the P. pseudomallei protease belongs to the family of alkaline proteases sensitive to metal chelators. Purified P. pseudomallei prot ease was capable of digesting a variety of eucaryotic protein substrat es including immunoglobulins. A P. pseudomallei strain deficient in pr otease production was shown to be less virulent than the parental stra in in an animal model of lung infection. These data suggest that this protease may be a significant pathogenic determinant in infections cau sed by P. pseudomallei.