A secreted aminopeptidase of Pseudomonas aeruginosa. Identification, primary structure, and relationship to other aminopeptidases.

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56). Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56) and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides.

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.

Inhibitors and specificity of Pseudomonas aeruginosa LasA.

LasA is an extracellular protease of Pseudomonas aeruginosa that enhances the elastolytic activity of Pseudomonas elastase and other proteases by cleaving elastin at unknown sites. LasA is also a staphylolytic protease, an enzyme that lyses Staphylococcus aureus cells by cleaving the peptidoglycan pentaglycine interpeptides. Here we showed that the staphylolytic activity of LasA is inhibited by tetraethylenepentamine and 1,10-phenanthroline (zinc chelators) as well as excess Zn2+ and dithiothreitol. However, LasA was not inhibited by several serine or cysteine proteinase inhibitors including diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, leupeptin, and N-ethylmaleimide. LasA staphylolytic activity was also insensitive to Nalpha-p-tosyl-L-lysine chloromethyl ketone or phosphoramidon. EDTA and EGTA were inhibitory only at concentrations greater than 20 mM. Without added inhibitors, LasA obtained by DEAE-cellulose fractionation was active toward beta-casein, but the same cleavage patterns were observed with column fractions containing little or no LasA. The beta-casein cleaving activity was fully blocked in the presence of inhibitors that did not affect staphylolytic activity. In the presence of such inhibitors, purified LasA was inactive toward acetyl-Ala4 and benzyloxycarbonyl-Gly-Pro-Gly-Gly-Pro-Ala, but it degraded soluble recombinant human elastin as well as insoluble elastin. N-terminal amino acid sequencing of two fragments derived from soluble elastin indicated that both resulted from cleavages of Gly-Ala peptide bonds located within similar sequences, Pro-Gly-Val-Gly-Gly-Ala-Xaa (where Xaa is Phe or Gly). In addition, Ala was identified as the predominant N-terminal residue in fragments released by LasA from insoluble elastin. A dose-dependence study of elastase stimulation by LasA indicated that a high molar ratio of LasA to elastase was required for significant enhancement of elastolysis. The present results suggest that LasA is a zinc metalloendopeptidase selective for Gly-Ala peptide bonds within Gly-Gly-Ala sequences in elastin. Substrates that contain no Gly-Gly peptide bonds such as beta-casein appear to be resistant to LasA.

The propeptide of Pseudomonas aeruginosa elastase acts an elastase inhibitor.

Elastase, an extracellular protease of Pseudomonas aeruginosa, is synthesized as a preproenzyme containing a large amino-terminal propeptide. The propeptide is cleaved within the periplasm to form a noncovalent complex with the elastase moiety. The propeptide-elastase complex was purified from the cell extract of P. aeruginosa by affinity chromatography on Gly3-D-Phe-Sepharose. The purified fraction was proteolytically inactive and contained the propeptide-elastase complex as the major protein component. Activation by limited proteolysis with trypsin was associated with the disappearance of the propeptide. To correlate individual proteins in the preparation with proteolytic activity, the purified fraction was subjected to polyacrylamide gel electrophoresis under nondenaturing conditions and subsequent incubation of the separation gel over a skim milk-agarose-indicator gel. Clearing zones due to proteolysis were produced either by mature elastase (control) or the free processed periplasmic enzyme, a low level of which was present in the purified propeptide-elastase complex preparation. No clearing was evident with the propeptide-elastase complex, indicating inhibition by the bound propeptide. Proteolytic activity of mature elastase was inhibited by various Pseudomonas cell fractions. This inhibition was abolished by antipropeptide antibodies, and, as evident from immunoblotting analysis, was consistent with propeptide presence in the effective fraction, whole cell extract, cytosol, and one of the two periplasmic fractions obtained upon conversion of P. aeruginosa cells to spheroplasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-blotting of the various cell fractions onto nitrocellulose membranes followed by incubation of the membranes with elastase and subsequent probing with antielastase antibodies revealed elastase propeptide binding. This binding of mature elastase to the propeptide was prevented by antibodies to the propeptide but not by inhibitors of elastase activity. Thermolysin, a neutral metalloprotease homologous to elastase, was not recognized by the elastase propeptide. In addition, propeptide containing P. aeruginosa fractions that were inhibitory to elastase had no effect on thermolysin activity. We conclude that elastase propeptide functions as an elastase inhibitor. Inhibition is specific, effective against both periplasmic and mature elastase, and depends on enzyme propeptide binding.

Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease.

Full expression of the elastolytic phenotype of Pseudomonas aeruginosa depends on LasA, an extracellular protease with restricted specificity whose mode of action on elastin and biological role is not understood. LasA exhibits amino acid sequence homology to some bacteriolytic proteases and shares several physicochemical properties with the staphylolytic protease of P. aeruginosa. This led us to examine whether the two proteases are the same. Production of LasA and staphylolytic protease by prototrophic and lasA mutant strains of P. aeruginosa was investigated. The two prototrophic strains examined, PAO1 and FRD2, exhibited extracellular staphylolytic activity and secreted LasA. LasA mutants, PAO-E64 lasA1 (Ts), FRD2128 delta lasA, and FRD244 lasA::mTn10, did not exhibit staphylolytic activity. A low level of the LasA protein was detected in the culture filtrate of the temperature-sensitive lasA mutant PAO-E64, but none was detectable in those of the deletion and insertion mutants, FRD2128, and FRD244, respectively. The staphylolytic protease was purified from the culture filtrate of P. aeruginosa strain FRD2 by DEAE-cellulose chromatography. The purified enzyme hydrolyzed pentaglycine into the respective di- and tripeptides and reacted specifically with antibodies against a synthetic peptide identical in sequence to positions 77-98 in LasA. The amino-terminal sequence of the first 15 amino acid residues of the staphylolytic protease was found to be identical with that of the secreted LasA. These results clearly indicate that LasA is a staphylolytic protease. In addition to lysing staphylococci, it may enhance elastolysis by cleaving Gly-Gly bonds, which are abundant in elastin.

Identification of cleavage sites involved in proteolytic processing of Pseudomonas aeruginosa preproelastase.

The extracellular elastase (33 kDa) of Pseudomonas aeruginosa is synthesized as a 53.6 kDa preproenzyme containing a long, N-terminal propeptide. The free propeptide and the elastase precursor generated upon propeptide removal were isolated from P. aeruginosa cells and subjected to N-terminal amino acid sequence analysis. The results identified Ala-174 and Ala+1 as the amino terminal residues of the propeptide and the elastase precursor, respectively, indicating that: (1) the signal peptide consists of 23 amino acid residues and its molecular weight is 2.4 kDa, (2) the propeptide contains 174 amino acid residues and is of 18.1 kDa molecular weight, and (3) no additional N-terminal proteolytic cleavage is required for elastase maturation.

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.

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

An inactive precursor of the extracellular elastase of Pseudomonas aeruginosa was extensively purified by immunoadsorption chromatography of the soluble bacterial cell fraction on a column of Sepharose coupled to antielastase antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified precursor fraction revealed two major protein bands with molecular weights of about 36,000 (P36) and 20,000 (P20) that in the absence of sodium dodecyl sulfate were associated with each other. The following findings identify P36 as the elastase precursor and indicate that proteolytic processing of this molecule is required for activation: (i) P36 is larger than the elastase, and it binds antielastase antibodies; (ii) trypsin activation is associated with the disappearance of P36 and the appearance of a new protein band migrating identically with the elastase and reacting with antibodies against the elastase; (iii) peptide maps generated from P36 and the elastase are similar although not identical. P20 by itself was not recognized by antielastase antibodies. Its association with P36 accounts for its adsorption to the immunoaffinity column and suggests that it may serve in elastase secretion.

Growth of Pseudomonas aeruginosa and secretion of protease in the presence of the protease inhibitors.

The compounds benzyloxycarbonyl-L-leucyl-hydroxamate (Z-Leu-NHOH), benzyloxycarbonyl-glycyl-hydroxamate (Z-Gly-NHOH) and 2-mercaptoacetyl-L-phenylalanyl-L-leucine (HSAc-Phe-Leu) are potent inhibitors of Pseudomonas aeruginosa elastase. The effect of these inhibitors on growth and protease secretion by the bacteria was studied under conditions where the organisms secrete elastase as the major proteolytic constituent. Z-Gly-NHOH and Z-Leu-NHOH inhibited bacterial growth and enzyme secretion by 40 and 30%, respectively, although the inhibition by Z-Leu-NHOH was expressed only when growth was in dialysed medium. Inhibition of growth by both compounds was first observed at the end of the logarithmic phase of growth, suggesting that these compounds are not toxic to the bacteria. HSAc-Phe-Leu did not inhibit growth or enzyme secretion. The level of HSAc-Phe-Leu in the medium decreased constantly during incubation, probably because of gradual oxidation of the -SH group. The rate of this decrease was markedly enhanced in presence of the bacteria, suggesting that HSAc-Phe-Leu is either consumed or destroyed by the organisms. We propose that the partial reduction of growth exerted by the hydroxamate derivatives is the result of inhibition of the extracellular proteases by these compounds. HSAc-Phe-Leu failed to inhibit growth because of its rapid loss during incubation with the bacteria. Since the three inhibitors have no antibacterial effects, their therapeutic potential should be examined in combination with antibiotics. Experimental treatment with HSAc-Phe-Leu should be frequent in order to overcome its loss in presence of the organisms.