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.     

Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions.

The ability of Pseudomonas aeruginosa to degrade elastin, a major component of connective tissue, likely contributes to its pathogenicity and multiplication in human tissues. Two extracellular enzymes are required for P. aeruginosa elastolytic activity: elastase and LasA. Elastase is a zinc metalloprotease, but little is known about the structure of LasA. When grown under metal ion-deficient conditions, P. aeruginosa culture supernatants were found to exhibit a low level of elastolytic activity, which coincided with production of low levels of the 51-kDa proelastase and no detectable LasA. By using this fact to identify factors that promote elastolytic activity, P. aeruginosa PAO1, FRD2, and DG1 were grown in metal ion-deficient medium supplemented with zinc (10(-4) M ZnCl2), calcium (2.5 x 10(-3) M CaCl2), or iron (10(-4) M FeCl3). High levels of proteolytic and elastolytic activity were exhibited by all strains when cultured in the presence of both zinc and calcium, and this was associated with the production of mature 33-kDa elastase and 21-kDa LasA. Supplementing DG1 and PAO1 cultures with zinc alone stimulated the production of 33-kDa elastase, which, because of the calcium-deficient conditions, exhibited low proteolytic and elastolytic activities. Zinc also stimulated the production of a 41-kDa form of LasA in DG1 and PAO1 culture supernatants. Elastase production by FRD2 cultured in the presence of zinc alone differed from that by the other two strains in that supernatants contained 33-kDa elastase, a 21-kDa form of LasA, and exhibited high proteolytic and elastolytic activities. Such strain-associated differences in LasA processing and elastase activity can be explained by differences in metal ion-scavenging mechanisms adapted by the strains. Supplementing cultures with calcium stimulated the production of elastase but had no effect on LasA production. The elastase produced exhibited variable sizes, possibly resulting from aberrant processing reactions, and showed little proteolytic activity. Proteolytic activity could be recovered from 33-kDa elastase produced in the presence of calcium by inclusion of zinc in the enzymatic assay. Although iron was previously found to exert a repressive effect on P. aeruginosa elastolytic activity, iron exerted little effect on elastolytic activity when added to cultures containing both zinc and calcium. These studies support the conclusion that elastase production and processing are promoted by both zinc and calcium. LasA production, in comparison, is stimulated by zinc, with both zinc and calcium facilitating its processing. The association of 41-kDa LasA with a low level of elastolytic activity and of 21-kDa LasA with a high level of activity supports the conclusion that lasA encodes a larger, precursor protein which is processed to an active 21-kDa form during 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.     

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.     

Enzymatic depilation of animal hide: identification of elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a depilating protease

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.     

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.     

Synthesis, processing, and transport of Pseudomonas aeruginosa elastase.

Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were identified in Pseudomonas aeruginosa cells by pulse-labeling with [35S]methionine and immunoprecipitation. In the absence of inhibitors, cells of a wild-type strain as well as those of the secretion-defective mutant PAKS 18 accumulated Pro II as the only elastase-related radioactive protein. EDTA but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] inhibited the formation of Pro II, and this inhibition was accompanied by the accumulation of Pro I. P accumulated in cells labeled in the presence of ethanol (with or without EDTA), dinitrophenol plus EDTA, or carbonyl cyanide m-chlorophenyl hydrazone plus EDTA. Pro I and Pro II were localized to the periplasm, and as evident from pulse-chase experiments, Pro I was converted to the mature extracellular enzyme with Pro II as an intermediate of the reaction. P was located to the membrane fraction. Pro I but not Pro II was immunoprecipitated by antibodies specific to a protein of about 20,000 molecular weight (P20), which, as we showed before (Kessler and 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 required both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.     

Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene.

Pseudomonas aeruginosa PAO-E64 is a mutant which produces parental levels of elastase antigen but has no elastolytic activity at 37 degrees C. The lesion (lasA1) in PAO-E64 is not a mutation in the structural gene for P. aeruginosa elastase (P.A. Schad, R.A. Bever, T.I. Nicas, F. Leduce, L.F. Hanne, and B.H. Iglewski, J. Bacteriol. 169: 2691-2696, 1987). A 1.7-kilobase segment of DNA that complements the lasA1 lesion was sequenced. Computer analysis of the DNA sequence showed that it contained an open reading frame which encoded a 41,111-dalton protein. The lasA gene was expressed under an inducible PT-7 promoter, and a 40,000-dalton protein was detected in Escherichia coli lysates. The lasA protein was localized in the outer membrane fraction of E. coli. This lasA protein produced in E. coli activated the extracellular elastase produced by the P. aeruginosa mutant, PAO-E64.     

Identification of the active site residues of Pseudomonas aeruginosa protease IV. Importance of enzyme activity in autoprocessing and activation

Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence. Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity. To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad. Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV. In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV. These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.     

Cloning, expression and characterization of recombinant elastase from Pseudomonas aeruginosa in Picha pastoris

The gene lasB from Pseudomonas aeruginosa, which encoded elastase, was cloned and firstly successfully expressed in Pichia pastoris stain KM71 under the control of AOX promoter. The effects on the recombinant elastase activities of different pH, different temperatures and different metal ions were assayed. The full-length gene (1497 bp) encodes a preproenzyme including an N-terminal signal peptide (23 aa), a propeptide (197 aa) and mature elastase (301 aa). The recombinant elastase was secreted into culture supernatants using signal sequence from lasB and showed a single band at about 34 kDa by SDS-PAGE. The recombinant elastase expression hit the highest level of approximately 450 mg/L and the specific elastolytic activity of the recombinant elastase was 130 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The optimal temperature and pH of the recombinant elastase was 28 degrees C and 7.4, respectively. The enzyme possessed high resistance to heat, and can be activated by Ca(2+). These enzyme properties suggested that it could be produced in an industrial scale and has the potential to be a commercial enzyme.     

Antibacterial activity in vivo and in vitro in the hemolymph of Galleria mellonella infected with Pseudomonas aeruginosa

The antibacterial activity of hemolymph from Galleria mellonella infected with entomopathogenic strain of Pseudomonas aeruginosa and non-pathogenic bacterium Escherichia coli was studied. In vivo, the antimicrobial activity appeared shortly after P. aeruginosa infection, reached the maximum level 18 h postinjection, while 30 h later only trace activity was noted. The activity induced by E. coli sustained on the high level until 48 h after infection. We also noted that the antimicrobial activity level induced by the non-pathogenic bacterium was higher in comparison to that measured in insects infected with the pathogenic strain of P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides of G. mellonella larvae were digested by P. aeruginosa elastase B. After 1 h incubation of cell-free hemolymph of immune-challenged larvae with elastase B, no antibacterial activity was observed. It was also shown that elastase B degraded synthetic cecropin B while in the presence of 6 mM EDTA antibacterial activity of cell-free hemolymph as well as cecropin B, was not changed which confirmed that the activity was abolished by the metalloprotease.     

Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia

Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [³⁵S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A₁, a specific inhibitor of vacuolar-type H⁺-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.     

Homologous and heterologous expression and maturation processing of extracellular glutamyl endopeptidase of Staphylococcus epidermidis

The extracellular serine endopeptidase GluSE (EC 3.4.21.19) is considered to be one of the virulence factors of Staphylococcus epidermidis. The present study investigated maturation processing of native GluSE and that heterologously expressed in Escherichia coli. In addition to the 28-kDa mature protease, small amounts of proenzymes with molecular masses of 32, 30, and 29 kDa were identified in the extracellular and cell wall-associated fractions. We defined the pre (M1-A27)- and pro (K28-S66)-segments, and found that processing at the E32-S33 and D48-I49 bonds was responsible for production of the 30- and 29-kDa intermediates, respectively. The full-length form of C-terminally His-tagged GluSE was purified as three proenzymes equivalent to the native ones. These molecules possessing an entire or a part of the pro-segment were proteolytically latent and converted to a mature 28-kDa form by thermolysin cleavage at the S66-V67 bond. Mutation of the essential amino acid S235 suggested auto-proteolytic production of the 30- and 29-kDa intermediates. Furthermore, an undecapeptide (I56-S66) of the truncated pro-segment not only functions as an inhibitor of the protease but also facilitates thermolysin processing. These findings could offer clues to the molecular mechanism involved in the regulation of proteolytic activity of pathogenic proteases secreted from S. epidermidis.