title: Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions.
abstract: The ability of Pseudomonas aeruginosa to degrade elastin, a major component of connective tissue, likely contributes to its pathogenicity and multiplication in human tissues. Two extracellular enzymes are required for P. aeruginosa elastolytic activity: elastase and LasA. Elastase is a zinc metalloprotease, but little is known about the structure of LasA. When grown under metal ion-deficient conditions, P. aeruginosa culture supernatants were found to exhibit a low level of elastolytic activity, which coincided with production of low levels of the 51-kDa proelastase and no detectable LasA. By using this fact to identify factors that promote elastolytic activity, P. aeruginosa PAO1, FRD2, and DG1 were grown in metal ion-deficient medium supplemented with zinc (10(-4) M ZnCl2), calcium (2.5 x 10(-3) M CaCl2), or iron (10(-4) M FeCl3). High levels of proteolytic and elastolytic activity were exhibited by all strains when cultured in the presence of both zinc and calcium, and this was associated with the production of mature 33-kDa elastase and 21-kDa LasA. Supplementing DG1 and PAO1 cultures with zinc alone stimulated the production of 33-kDa elastase, which, because of the calcium-deficient conditions, exhibited low proteolytic and elastolytic activities. Zinc also stimulated the production of a 41-kDa form of LasA in DG1 and PAO1 culture supernatants. Elastase production by FRD2 cultured in the presence of zinc alone differed from that by the other two strains in that supernatants contained 33-kDa elastase, a 21-kDa form of LasA, and exhibited high proteolytic and elastolytic activities. Such strain-associated differences in LasA processing and elastase activity can be explained by differences in metal ion-scavenging mechanisms adapted by the strains. Supplementing cultures with calcium stimulated the production of elastase but had no effect on LasA production. The elastase produced exhibited variable sizes, possibly resulting from aberrant processing reactions, and showed little proteolytic activity. Proteolytic activity could be recovered from 33-kDa elastase produced in the presence of calcium by inclusion of zinc in the enzymatic assay. Although iron was previously found to exert a repressive effect on P. aeruginosa elastolytic activity, iron exerted little effect on elastolytic activity when added to cultures containing both zinc and calcium. These studies support the conclusion that elastase production and processing are promoted by both zinc and calcium. LasA production, in comparison, is stimulated by zinc, with both zinc and calcium facilitating its processing. The association of 41-kDa LasA with a low level of elastolytic activity and of 21-kDa LasA with a high level of activity supports the conclusion that lasA encodes a larger, precursor protein which is processed to an active 21-kDa form during secretion.
authors.affiliation: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston 29425.
authors.forename: J C
authors.initials: JC
authors.lastname: Olson
authors.forename: D E
authors.initials: DE
authors.lastname: Ohman
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D007501:Iron / Q000494:pharmacology; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D007501:Iron; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C066371:staphylolytic protease; D015032:Zinc; D002118:Calcium