Secretion of Elastinolytic Enzymes and Their Propeptides by Pseudomonas aeruginosa

Abstract

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions.

The opportunistic gram-negative pathogen Pseudomonas aeruginosa secretes many proteins into the extracellular medium via the type II or general secretory pathway (25). Translocation across the inner membrane is mediated by a classical N-terminal signal peptide, probably via the Sec system. The subsequent translocation of periplasmic intermediates across the outer membrane is mediated by machinery composed of at least 12 proteins encoded by xcp genes (1, 4, 12). Elastase is one of the exoproteins secreted via the Xcp machinery. This metalloprotease is produced as a preproenzyme, in which the “pre-” part is the signal sequence (6). The propeptide is an intramolecular chaperone that mediates the folding of elastase in the periplasm (9, 21). Proelastase is processed by autoproteolytic cleavage (20). Mutants defective in autoproteolytic processing of the proenzyme are also defective in elastase secretion, suggesting that autoprocessing occurs within the cell prior to secretion (20). The propeptide remains noncovalently associated with the mature elastase (16) and inhibits the proteolytic activity of the enzyme (17). Next, elastase is secreted via the Xcp machinery, after which the propeptide has been thought to be degraded in the periplasm (16). However, in this study we demonstrate that the propeptide is secreted and extracellularly degraded. Furthermore, while manipulating the extracellular proteolytic activity in these experiments we noticed drastic changes in the extracellular protein pattern, which we studied in further detail.

The propeptide is secreted by P. aeruginosa.

To determine the final localization of the propeptide, proteins from supernatant fractions of overnight cultures of P. aeruginosa PAO25 were isolated and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, essentially as described previously (9). The propeptide was not detected in the extracellular medium (Fig. 1B, lane 3). Because it was not detected intracellularly either (data not shown), the propeptide may have been degraded by proteases after its secretion. Since the proteolytic activity might vary during growth, culture supernatants from different growth stages were analyzed. Indeed, two forms of the propeptide, migrating with molecular masses of 21 (P₁) and 18 (P₂) kDa, respectively, were detected by immunoblotting in the supernatant of late-log-phase cells (Fig. 1B, lane 1) and not in that of the lasB mutant (data not shown), but they disappeared during further growth (Fig. 1B, lanes 2 and 3). P₂ is probably a proteolytic fragment of P₁, or, alternatively, P₁ and P₂ represent different conformations of the propeptide that are maintained during SDS-PAGE. In the same time period, elastase accumulated extracellularly (Fig. 1A, lanes 1 to 3), consistent with the notion that expression of the structural gene for elastase, lasB, is induced when cells enter stationary phase (22). Apparently, the propeptide can be detected in the extracellular medium as long as the amount of elastase remains low. Alkaline protease did not appear to be essential for degradation of the propeptide, since the propeptide secreted by the aprE mutant strain PAO25ME3 was degraded as well (Fig. 1B, lanes 4 to 6). Therefore, degradation of the extracellular propeptide might be mediated by the activity of elastase itself. This possibility was investigated by imposing conditions under which the secreted elastase is inactive but the autoproteolytic processing of the proenzyme is not prevented. Such conditions can be achieved by using a medium (2) which contains Zn²⁺, needed for autoproteolytic processing, but is depleted of Ca²⁺ ions, which are required for full elastase activity (23). The total proteolytic activity in the extracellular medium of overnight cultures, measured as described previously (13), appeared to be 10-fold reduced by the chelation of Ca²⁺ ions (data not shown). Growth of the wild-type strain PAO25 in this medium resulted in the secretion of mature elastase (Fig. 2A and B, lanes 2), and significant amounts of the 18-kDa form (P₂) of the propeptide were detected both on a stained gel and on an immunoblot (Fig. 2A and C, lanes 2). After blotting on a polyvinylidene difluoride membrane, the N-terminal amino acid sequence of the accumulated P₂ was determined by Edman degradation with a Protein Sequencer, model 476A (Perkin-Elmer Corp.). The N terminus (ADLID) was found to be identical to that of the propeptide (18), which proves the identity of the P₂ band. Furthermore, it shows that if P₂ is indeed generated from P₁ by a proteolytic event, then this proteolysis occurs near the C terminus of the propeptide. The complete absence of the propeptide in the supernatant fraction of xcpR mutant strain PAO7510 (Fig. 2A and C, lanes 3) demonstrates that the extracellular localization of the propeptide is Xcp dependent. Together, these results demonstrate that the propeptide of elastase is secreted by P. aeruginosa in an Xcp-dependent manner and is degraded by an extracellular protease, probably elastase.

FIG. 1.

Secretion of the propeptide of elastase. Proteins in the supernatant fractions of cultures at different growth stages, grown in LB medium with agitation at 37°C, were precipitated with 5% trichloroacetic acid, and samples corresponding to 360 μl of supernatant were analyzed by SDS-PAGE and Western blotting. Gels of 12% polyacrylamide were stained with Coomassie brilliant blue (A), and blots were probed with an antipropeptide antiserum which was preadsorbed with a cell lysate of the elastase-negative mutant strain AP103-II (15) (B). The optical densities at 600 nm of the cultures analyzed were 2.0 (lanes 1 and 4), 3.5 (lanes 2 and 5) and 5.5 (overnight growth) (lanes 3 and 6 through 10). Strains are all derivatives of PAO25. Lanes 1 through 3, PAO25 (wild type) (Holloway collection); lanes 4 through 6, PAO25ME3 (aprE::ΩHg [11]); lane 7, PAN8 (lasB::Km aprEΩHg [7]); lane 8, PAN9 (lasB::Km^r aprEΩHg xcpQ::Gm^r [7]); lane 9, PAN10 (lasB::Km^r [8]); lane 10, PAN11 (lasB::Km^r xcpR-54 [8]). Elastase (LasB), propeptide form 1 (P₁), propeptide form 2 (P₂), pro-LasD, pro-LasA, 58-kDa protein, 23-kDa protein, and 21-kDa protein are indicated on the right, and molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 2.

Inhibition of extracellular degradation of the propeptide. Secreted proteins of cultures grown overnight in LB medium (lane 1) or in brain heart infusion broth supplemented with 20 mM sodium oxalate as a chelator of Ca²⁺, 20 mM MgCl₂, and 1.75% glycerol (lanes 2 and 3) were precipitated with 5% trichloroacetic acid, and samples corresponding to 360 μl of supernatant were analyzed by SDS-PAGE and Western blotting. Gels of 12% polyacrylamide were stained with Coomassie brilliant blue (A), and blots were probed with antielastase (B) or antipropeptide (C) antisera. Lanes 1 and 2, PAO25 (wild-type); lane 3, PAO7510 (xcpR-54 [mutant of PAO25]) (13). Elastase (LasB), propeptide form 2 (P₂), pro-LasA, 23-kDa protein, and 21-kDa protein are indicated on the right, and molecular mass markers (in kilodaltons) are indicated on the left.

Additional alterations in the extracellular protein pattern upon inhibition of proteolytic activity.

The extracellular protein pattern of cells grown in Ca²⁺-depleted brain heart infusion medium was quite different from that of cells grown in Luria-Bertani (LB) medium (Fig. 2A [compare lanes 1 and 2]). Although culture conditions may affect the expression of various genes encoding secreted proteins, the activity of extracellular proteases could be the main cause for the differences observed, since mutations in lasB, encoding elastase (Fig. 1A, lane 9), or in aprE and lasB (Fig. 1A, lane 7) had a similar effect on the extracellular protein pattern. For example, an additional protein migrating with an M_r of 42,000 was abundantly present in the spent culture medium of the lasB aprE mutant strain PAN8 (Fig. 1A, lane 7) but not in that of the wild-type strain (Fig. 1A, lane 3) unless this strain was grown under protease-inhibiting conditions (Fig. 2A, lane 2). Determination of the N-terminal amino acid sequence (HDDGLPAFRY) revealed that this protein corresponds to the nonprocessed form of LasA, pro-LasA. The lasA gene encodes a staphylolytic protease which is, like elastase, produced as a preproenzyme (10). Processing of pro-LasA has been reported to require another extracellular protease, which is not elastase (14). Consistent with this notion, pro-LasA did not accumulate in the supernatant fraction of the lasB mutant strain PAN10 (Fig. 1A, lane 9). However, the mutant impaired in alkaline protease production also showed no pro-LasA accumulation (Fig. 1A, lane 6). Apparently, pro-LasA accumulates only when both elastase and alkaline protease are absent. Since Ca²⁺ depletion inhibits both elastase and alkaline protease (5) activities, the presence of pro-LasA in the culture medium of Ca²⁺-depleted wild-type cells (Fig. 2A, lane 2) (23) can be explained. The accumulation of pro-LasA was always attended by the disappearance of a protein migrating with a molecular mass of 21 kDa, probably mature LasA (e.g., compare lanes 3 and 7 in Fig. 1A). The absence of pro-LasA in the supernatant fraction of the xcpQ mutant derivative of strain PAN8 (Fig. 1A, lane 8) shows that LasA is secreted via the Xcp pathway.

The N-terminal sequence of another secreted protein, migrating with an M_r of 43,000 and prominently present in the supernatant fraction of the aprE lasB mutant strain (Fig. 1A, lane 7), was determined. This sequence (HGSMETPPSR) perfectly matches that of the N terminus of mature LasD, which has a reported molecular mass of 23 kDa (24). Therefore, we conclude that LasD is apparently also produced and secreted as a precursor, although the additional domain of LasD has to be located at the C terminus. We therefore cannot explain the report of a 30-kDa LasD precursor with an N-terminal amino acid sequence different from that of the mature protein (24). pro-LasD also accumulated in the supernatant fraction of the lasB mutant strain PAN10 (Fig. 1A, lane 9), which shows that elastase is required for the processing of pro-LasD. The accumulation of pro-LasD was attended by the absence of a protein migrating with a molecular mass of 23 kDa, probably mature LasD (24) (e.g., compare lanes 3 and 9 in Fig. 1A). LasD is secreted via the Xcp pathway, since it was not detectable in the supernatant of an xcpR mutant derivative of PAN10 (Fig. 1A [compare lanes 9 and 10]). Furthermore, culture conditions affected the expression of LasD, since neither the precursor nor the mature LasD were detected in the supernatant of cultures grown in the Ca²⁺-depleted medium (Fig. 2A, lane 2).

The N-terminal amino acid sequence of a third protein abundantly present in the extracellular medium of the lasB aprE mutant strain PAN8 and having an M_r of 58,000 (Fig. 1A, lane 7) was determined. The sequence obtained (APSEAQQFTE) showed a perfect match (3) with an unidentified protein in the P. aeruginosa database that shows 43% identity with the C-terminal half of an aminopeptidase of Streptomyces griseus (19). Secretion of this 58-kDa protein appears to be Xcp dependent, since it was not detected in the supernatant of the xcpQ mutant strain (Fig. 1A [compare lanes 7 and 8]).

Concluding remarks.

Previously it was suggested that the propeptide of elastase is degraded in the periplasm after the secretion of mature elastase. Here we report the secretion of the propeptide of elastase. The propeptide is probably secreted in complex with the folded elastase, and dissociation of the complex occurs in the extracellular medium. Consequently, whereas the roles of the propeptide as a chaperone required for the folding of elastase and as an inhibitor of the proteolytic activity have been documented previously, an additional role in the targeting of the enzyme to the secretion apparatus can be envisaged. Furthermore, like LasA, LasD appears to be secreted as a proenzyme. Therefore, the secretion of propeptide-enzyme complexes may represent a common theme in the secretion of proteases across the outer membrane via the type II pathway. However, in contrast to elastase, both pro-LasA and pro-LasD are processed only after translocation across the outer membrane. For the processing of pro-LasA, either elastase or alkaline protease is required, whereas elastase is essential for the processing of pro-LasD.