Mechanisms of activation and secretion of a cell‐associated precursor of an exocellular protease of Pseudomonas aeruginosa 34362A

Abstract

An inactive precursor to the active exocellular protease 1 of Pseudomonas aeruginosa is cell-associated and located primarily in the periplasmic space. We have studied factors that bring about activation of the precursor in vitro in order to shed some light on the process of its activation and secretion in vivo. A variety of diverse procedures were shown to effect irreversible activation. Several mild non-enzymatic procedures were effective, such as dialysis of an ammonium sulfate precipitate against neutral buffers, gel filtration (Sephadex G-loo), and ion-exchange chromatography (DEAE-cellulose). Activation also resulted following treatment with anionic detergents (sodium dodecyl sulfate, N-lauroyl sarcosine) and deoxycholate. Limited exposure to any of-several proteases with different specificities also resulted in activation. The kinetics of detergent-catalyzed activation reveals a long lag followed by rapid activation, suggesting at least a two-stage process. The precursor and the mature protease 1 have indistinguishable molecular masses (33 kDa), as measured by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis of these proteins purified by immunoabsorbance chromatography under denaturing conditions. Further, both precursor and protease have identical N-terminal alanine. Our results suggest that it is improbable that activation is the result of proteolytic processing of the precursor itself, but rather that it may involve the removal of a non-covalently associated inhibitor molecule. Hydrophobic interaction chromatography on octyl-Sepharose revealed that activation was accompanied by a significant change in the hydrophobicity, pointing to a significant change in the conformation of the precursor and the mature protease.A mutant has been studied which accumulates activatable precursor in the periplasm but releases no active enzyme into the culture medium, supporting the hypothesis that secretion through the inner and outer membranes proceed by different mechanisms. Comparison of outer membranes of protease-secreting strains (34 362A and PAKS 1) and a protease-negative mutant (PAKS 18) which accumulates precursor has shown that there is a change in the outer membrane protein profile in the latter.

Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion

“…Our failure to detect enzymatically active elastase within MVs can be explained by previous work (12,28). It has been shown that the enzyme is first made as an inactive preproelastase in the cytoplasm and a proelastase in the periplasm (12,28,29). This proenzyme becomes modified as it crosses the outer membrane and becomes active.…”

Partial purification and characterization of an inactive precursor of Pseudomonas aeruginosa elastase

“…Previous attempts to purify this precursor resulted in its activation. Thus, the purified precursor and the elastase were indistinguishable, and the possibility was raised that activation did not result from proteolytic processing of the precursor itself, but rather that it involved the removal of a non-covalently associated elastase inhibitor (5). In the present study, we describe the purification of an inactive periplasmic elastase precursor that is structurally distinct from the elastase and demonstrate that activation involves proteolytic processing of the precursor itself.…”

Synthesis, processing, and transport of Pseudomonas aeruginosa elastase

“…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 activation did not result from proteolytic processing of the precursor itself, but rather involved dissociation of the elastase from a non-covalently bound elastase inhibitor.…”