Synthesis, processing, and transport of Pseudomonas aeruginosa elastase

Abstract

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...

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

“…We excised the bands and identified the proteins by N-terminal sequence analysis. The 49-kDa band was identified as alkaline metalloprotease AprA (24), the 33-kDa band was identified as elastase LasB (4,31), and the 27-kDa band is the PrpL protease (65). To ensure that the increased amounts of proteases were due to Ca 2ϩ addition and not due to PO 4 3Ϫ limitation (by possible PO 4 3Ϫ precipitation as calcium phosphate), experiments were performed with various concentrations of Ca 2ϩ and PO 4 3Ϫ as described in Materials and Methods.…”

An extracellular zinc metalloprotease gene of Burkholderia cepacia

“…The deduced amino acid sequences of the B. cepacia and B. pseudomallei extracellular zinc metalloproteases have a preproenzyme structure similar to members of the thermolysin-like metalloprotease family (Hase & Finkelstein, 1991;Kessler & Safrin, 1988;McIver et al, 1991). The preproenzyme structure dictates secretion through the general secretory pathway (Pugsley, 1993).…”

Cloning and Overexpression of Extracellular Elastase from Pseudomonas Aeruginosa

“…Elastase is initially synthesized as a precursor with a pre-pro-mature domain structure consisting of a signal peptide (23 residues), a propeptide (174 residues) and a carboxy-terminal catalytic domain (301 residues) (25). Elastase requires these properties for both proper folding and secretion in P. aeruginosa (26).…”