Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity.

A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P.A. Schad and B.H. Iglewski, J. Bacteriol. 170:2784-2789, 1988). However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.     

Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveais elastolytic activity due to alkaline proteinase and the LasA fragment

The extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to investigate the role of LasB in virulence and LasA processing, a LasB-negative mutant, PAO1E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201-1. The resulting plasmid, pBRMOB-LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB-positive P. aeruginosa strain, PAO1. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PAO1E supernatant fluids yielded no detectable LasB (less than 1 ng ml-1 LasB). The absence of LasB in PAO1E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunoglobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PAO1E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PAO1 and PAO1E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.     

Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion.

Pseudomonas aeruginosa secretes elastase in a multistep process which begins with the synthesis of a preproelastase (53.6 kDa) encoded by lasB, is followed by processing to proelastase (51 kDa), and concludes with the rapid accumulation of mature elastase (33 kDa) in the extracellular environment. In this study, mutants of P. aeruginosa were constructed by gene replacement which expressed lasB1, an allele altered in vitro at an active-site His-223-encoding codon. The lasB1 allele was exchanged for chromosomal lasB sequences in two strain backgrounds, FRD2 and PAO1, through a selectable-cassette strategy which placed a downstream Tn501 marker next to lasB1 and provided the selection for homologous recombination with the chromosome. Two lasB1 mutants, FRD720 and PDO220, were characterized, and their culture supernatants contained greatly reduced proteolytic (9-fold) and elastolytic (14- to 20-fold) activities compared with their respective parental lasB+ strains. This was primarily due to the effect of His-223 substitution on substrate binding by elastase and thus its proteolytic activity. However, the concentration of supernatant elastase antigen was also reduced (five- to sevenfold) in the mutant strains compared with the parental strains. An immunoblot analysis of cell extracts showed a large accumulation of 51-kDa proelastase within lasB1 mutant cells which was not seen in wild-type cell extracts. A time course study showed that production of extracellular elastase was inefficient in the lasB1 mutants compared with that of parental strains. This showed that expression of an enzymatically defective elastase inhibits proper processing of proelastase and provides further evidence for autoproteolytic processing of proelastase in P. aeruginosa. Unlike the parental strains, culture supernatants of the lasB1 mutants contained two prominent elastase species that were 33 and 36 kDa in size. Extracellular 51-kDa proelastase was barely detectable, even though it accumulated to high concentrations within the lasB1 mutant cells. These data suggest that production of an enzymatically defective elastase affects proper secretion because autoproteolytic processing of proelastase is necessary for efficient localization to the extracellular milieu. The appearance of reduced amounts of extracellular elastase and their sizes of 33 and 36 kDa suggest that lasB1-encoded elastase was processed by alternate, less-efficient processing mechanisms. Thus, proelastase must be processed by removal of nearly all of the 18-kDa propeptide before elastase is a protein competent for extracellular secretion.     

A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.     

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.     

Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments

With the determination of the three-dimensional structure of elastase and the probable identification of the active site and key residues involved in proteolytic activity, our knowledge of the molecular details of this interesting protease is rapidly increasing. Pseudomonas elastase appears to be remarkably similar to the Bacillus metalloproteinase thermolysin. A further significant development has been the discovery of the lasA gene and the fact that Pseudomonas elastase and alkaline proteinase appear to act in concert with the LasA protein to display the notable elastolytic activity exhibited by isolates of this organism. Biochemical and genetic studies indicate that LasA is a second elastase which may be an important virulence factor that has been overlooked in previous studies.     

Pseudomonas aeruginosa proteases and corneal virulence

Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses. Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P. aeruginosa corneal infections. Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes. Allelic exchange was used to construct mutants of P. aeruginosa PAO1-V deficient in AP (PAO1-V AP[ - ]), LasB and LasA protease (PDO801 LasB[ - ]), or all three proteases (PDO801 TM). These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease. Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection. Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100). The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110). Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection. Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.     

Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase.

The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasB1) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species. These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.     

Physico-chemical and biological characteristics of Pseudomonas aeruginosa elastase

Elastase isolated from P. aeruginosa clinical strain hydrolyzes elastin, casein, hemoglobin, ovalbumin, gelatin, fibrin, collagen. The optimum pH ensuring the activity of the enzyme is 7.8-8.0. Elastase shows maximum stability at pH 6.6-9.0. Heating at 80 degrees C for 10 minutes results in its practically complete inactivation. Elastase is a highly radiosensitive enzyme. Chelating agents and zinc, cobalt, mercury ions suppress its activity. Sodium and ammonium chlorides selectively inhibit the elastolytic, but not proteolytic activity of the enzyme. Elastase shows pronounced dermonecrotic and keratolytic action.     

Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.     

Activation of an elastase precursor by the lasA gene product of Pseudomonas aeruginosa.

To study the role of the lasA gene product in the secretion of enzymatically active elastase by Pseudomonas aeruginosa, we constructed mutants by gene replacement with in vitro-derived insertion and deletion mutations in the cloned lasA gene. lasA mutants were deficient in the production of elastolytic activity. A membrane-associated, higher-molecular-weight (approximately 47,000) precursor of elastase was observed in both the wild-type and the lasA mutants. Unlike the wild-type strain, the lasA mutant accumulated the 47,000-molecular weight elastase species in the soluble fraction of the cell, suggesting that the lasA gene product has a role in elastase secretion. Although lasA mutants were deficient in elastolytic activity, they produced a proelastase with a mature molecular weight (approximately 37,000) that still retained general proteolytic activity. Final yields of elastase-related material were approximately the same in both the wild-type strain and lasA mutant supernatants. The lasA gene was expressed in Escherichia coli, and the approximate molecular weight of the lasA gene product was 31,000. Extracts of E. coli containing the lasA gene product were shown in vitro to activate the proelastase produced by P. aeruginosa lasA mutants to an enzyme with elastolytic activity. Thus the lasA gene product has a direct effect on broadening the substrate specificity of secreted proelastase, as well as a second role (direct or indirect) in the secretion of elastase.     

Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes

A highly sensitive assay based on new internally quenched fluorogenic peptide substrates has been developed for monitoring protease activities. These novel substrates comprise an Edans (5-(2-aminoethylamino)-1-naphthalenesulfonic acid) group at the C terminus and a Dabsyl (4-(dimethylamino)azobenzene-4'-sulfonyl chloride) fluorophore at the N terminus of the peptide chains. The Edans fluorescence increases upon peptide hydrolysis by Pseudomonas aeruginosa proteases, and this increase is directly proportional to the amount of substrate cleaved, i.e., protease activity. The substrates Dabsyl-Ala-Ala-Phe-Ala-Edans and Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans were used for testing the peptidasic activities of P. aeruginosa elastase and LasA protease, respectively. Elastase and LasA kinetic parameters were calculated and a sensitive assay was designed for the detection of P. aeruginosa proteases in bacterial supernatants. The sensitivity and the small sample requirements make the assay suitable for high-throughput screening of biological samples. Furthermore, this P. aeruginosa protease assay improves upon existing assays because it is simple, it requires only one step, and even more significantly it is enzyme specific.     

Cloning and characterization of elastase genes from Pseudomonas aeruginosa.

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.     

Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa

The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.     

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.     

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Secretion of Elastinolytic Enzymes and Their Propeptides by Pseudomonas aeruginosa

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.