Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.
LasA is an extracellular protease of Pseudomonas aeruginosa that enhances the elastolytic activity of Pseudomonas elastase and other proteases by cleaving elastin at unknown sites. LasA is also a staphylolytic protease, an enzyme that lyses Staphylococcus aureus cells by cleaving the peptidoglycan pentaglycine interpeptides. Here we showed that the staphylolytic activity of LasA is inhibited by tetraethylenepentamine and 1,10-phenanthroline (zinc chelators) as well as excess Zn 2؉ and dithiothreitol. However, LasA was not inhibited by several serine or cysteine proteinase inhibitors including diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, leupeptin, and N-ethylmaleimide. LasA staphylolytic activity was also insensitive to N ␣ -p-tosyl-L-lysine chloromethyl ketone or phosphoramidon. EDTA and EGTA were inhibitory only at concentrations greater than 20 mM. Without added inhibitors, LasA obtained by DEAE-cellulose fractionation was active toward -casein, but the same cleavage patterns were observed with column fractions containing little or no LasA. The -casein cleaving activity was fully blocked in the presence of inhibitors that did not affect staphylolytic activity. In the presence of such inhibitors, purified LasA was inactive toward acetyl-Ala 4 and benzyloxycarbonyl-Gly-Pro-Gly-Gly-Pro-Ala, but it degraded soluble recombinant human elastin as well as insoluble elastin. N-terminal amino acid sequencing of two fragments derived from soluble elastin indicated that both resulted from cleavages of Gly-Ala peptide bonds located within similar sequences, Pro-Gly-Val-Gly-Gly-Ala-Xaa (where Xaa is Phe or Gly). In addition, Ala was identified as the predominant N-terminal residue in fragments released by LasA from insoluble elastin. A dose-dependence study of elastase stimulation by LasA indicated that a high molar ratio of LasA to elastase was required for significant enhancement of elastolysis. The present results suggest that LasA is a zinc metalloendopeptidase selective for Gly-Ala peptide bonds within Gly-Gly-Ala sequences in elastin. Substrates that contain no Gly-Gly peptide bonds such as -casein appear to be resistant to LasA.
Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were Safrin, J. Bacteriol. 170:1215-1219, 1988, forms a complex with an inactive periplasmic elastase precursor of about 36,000 molecular weight. Our results suggest that the elastase is made by the cells as a preproenzyme (P), containing a signal sequence of about 4,000 molecular weight and a "pro" sequence of about 20,000 molecular weight. Processing and export of the preproenzyme involve the formation of two periplasmic proenzyme species: proelastase I (56 kilodaltons [kDa]) and proelastase II (36 kDa). The former is short-lived, whereas proelastase II accumulates temporarily in the periplasm, most likely as a complex with the 20-kDa propeptide released from proelastase I upon conversion to proelastase II. The final step in elastase secretion seems to require both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.Unlike most gram-negative bacteria, Pseudomonas aeruginosa secretes several proteins into the medium. Many of these proteins, including exotoxins A and S, the proteases elastase and alkaline proteinase, and phospholipase C, are toxic to humans and animals and are thought to enhance the virulence of the organism (5, 13,25,29,42). Although the properties of most Pseudomonas exoenzymes and the role that each of them may play in the pathogenesis of Pseudomonas infections have been studied in detail, relatively little is known about their synthesis and secretion. DNA sequencing of the structural genes of exotoxin A (10) and phospholipase C (34) indicated that like most exported proteins of other bacteria or higher organisms (for reviews, see references 2 and 35), both the exotoxin and the phospholipase are synthesized by the cells as larger precursors, each containing a typical amino-terminal leader sequence which is removed during the secretion process. Lory et al. (27) demonstrated that processing and secretion of the exotoxin precursor are both inhibited in the presence of ethanol, leading to accumulation of the precursor in the outer membrane. No exotoxin was found in the periplasm. These authors proposed that the exotoxin precursor is secreted cotranslationally and directly to the outer membrane via zones of adhesion between the inner and outer membranes. According to their model, the exotoxin precursor is proteolytically processed to the mature toxin upon release into the medium. Secretion of phospholipase C and of the elastase seems to follow a different route, since * Corresponding author. phospholipase activity (32) and an inactive elastase precursor (19,22) were demonstrated in the periplasmic fraction of P. aeruginosa cells. The inactive periplasmic elastase precursor is activated by controlled proteolysis (19,22), yet this precursor was reported to have the same molecular weight and the same N-terminal amino acid (alanine) as the extracellular elastase (7,8,19,24). Fecycz and Campbell (7) suggested that activati...
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