The contribution of exoproducts to virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa produces a large number of extracellular products which may contribute to its virulence. We have employed a genetic approach to determine the contribution of toxin A, exoenzyme S, elastase and alkaline protease to the pathogenesis of P. aeruginosa. Mutations have been introduced with chemicals or transposons. Mutants have been identified using immunological, chemical, or toxicity assays. Mutants were extensively characterized in vitro to ascertain that they were identical to their parent strain except for the production of the desired product. Appropriate mutants were compared with their parent strains in several animal models: the burned mouse model, the mouse corneal infection model, and a rat model of chronic lung infection. The data indicate that virulence of P. aeruginosa is multifactorial. Further, the relative contribution of a given P. aeruginosa product may vary with the type of infection.     

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.     

Elastase and alkaline protease production by Pseudomonas aeruginosa strains: comparison of two procedures

Pseudomonas aeruginosa is an opportunistic pathogen that can cause fatal infections in immunocompromised hosts. The virulence of P. aeruginosa is associated with the presence of various extracellular factors like elastase and alkaline protease. These enzymes are suggested to contribute to tissue destruction and assist bacterial invasion during infection. Therefore it seems likely that determination of these virulence factors will be an important prognostic marker in the near future especially for follow up of cystic fibrosis patients, to start antimicrobial agents that are directly or indirectly inhibit microbial growth or virulence factor production. Herein, we suggest a simple test procedure to be used in routine laboratories for estimation of elastase and alkaline protease levels and compare them with quantitative methods in the literature. We detected the amount of elastase and alkaline protease in 49 clinical P. aeruginosa isolates by comparing agar plate method and colorimetric assay. The resulting values were in the range reported in the literature and differed from one strain to another(elastase: 0-1390 mg/ml, alkaline protease: 0- 770 mg/ml). Linear relationships were found when assays compared in pairs and significant correlation coefficients were obtained(r>0.788 for alkaline protease, p<0.0001- r>0.926 for elastase, p<0.0001). Our method can be applied in laboratories regardless of the availability of technical equipment.     

Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease.     

The role of Pseudomonas aeruginosa elastase as a potent inflammatory factor in a rat air pouch inflammation model

Pseudomonas aeruginosa, an opportunistic pathogen, can cause life threatening infections in patients compromised by underlying respiratory disease like bronchiectasis, cystic fibrosis and diffuse panbronchiolitis. Most strains of P. aeruginosa produce some kind of protease with broad substrate specificities during the infectious state in the host. P. aeruginosa elastase, one of the strongest exotoxins, has a tissue-damaging proteolytic activity and is capable of degrading such plasma proteins as immunoglobulins, complement factor and cytokines. The present study focused on the effect of P. aeruginosa elastase and was designed to evaluate the neutrophil accumulation at the inflammation site mediated by P. aeruginosa elastase in the inflammatory response in the host. An air pouch model in rats, considered as a useful model of inflammation, was used to analyze the number of leukocytes, the volume of exudate and the concentration of interleukin-8 after the injection of P. aeruginosa elastase into the pouch cavity. The number of neutrophils and the volume of exudate in the pouch cavity increased significantly at 4 h, peaked at 8 h in a dose-dependent manner and then decreased at 24 h. The concentration of interleukin-8 in pouch fluid peaked 4 h earlier than the peak of the neutrophil number. The enzymatic activity of P. aeruginosa elastase seemed to reinforce the inflammation process. The influence of lipopolysaccharide contamination was negligible. Although these observations were made in the subcutaneous cavity, they indicate that P. aeruginosa elastase plays a role as an immunoprovocative factor in the inflammatory response in cases of infection with P. aeruginosa.     

Pseudomonas aeruginosa mutants altered in their sensitivity to the effect of iron on toxin A or elastase yields

Iron affects yields of toxin A, alkaline protease, elastase, pyochelin, and pyoverdin in Pseudomonas aeruginosa. Mutants of P. aeruginosa PAO1 resistant to the effect of iron on toxin (toxC) or elastase (elaC) yields were isolated. Two types of mutants were isolated: iron transport and iron regulatory mutants. The toxC regulatory mutants produced toxin A in medium containing iron; however, yields of elastase and alkaline protease remained sensitive to regulation by iron. The elaC regulatory mutants were resistant to the effect of iron on elastase yields, but toxin A and alkaline protease yields were decreased by iron, analogous to the parent strain. These data suggest that toxin A, elastase, and alkaline protease yields can be independently regulated by iron.     

Pseudomonas aeruginosa A2 elastase: purification, characterization and biotechnological applications

An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34 kDa. Temperature and pH with highest activity were 60 °C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 40 °C with an enzyme/substrate (E/S) ratio of 5 U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes.     

Cloning and sequencing of a gene of organic solvent-stable protease secreted from Pseudomonas aeruginosa PST-01 and its expression in Escherichia coli

A gene of organic solvent-stable protease (PST-01 protease) secreted by Pseudomonas aeruginosa PST-01 was cloned and its nucleotide was sequenced. The nucleotide sequence analysis revealed that the PST-01 protease was a pseudolysin, which was an elastase produced by P. aeruginosa and was well characterized by the previous investigators. The PST-01 protease produced in recombinant Escherichia coli was not secreted into the extracellular medium, but its proenzyme was released by the lysis of the cells and became a 33.1kDa mature enzyme autoproteolytically. Its characteristics including organic solvent stability were as same as those of the PST-01 protease secreted by P. aeruginosa PST-01.     

Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.

The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.     

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.     

Degradation of IgA proteins by Pseudomonas aeruginosa elastase

Human colostral IgA and myeloma proteins of both IgA1 and IgA2 subclasses were susceptible to cleavage by Pseudomonas aeruginosa elastase. Detailed analysis of the cleavage products of IgA myeloma proteins revealed complete degradation of Fab with no evidence of intact Fab fragments as intermediate cleavage products. In contrast, both IgA1 and IgA2 proteins were resistant to cleavage by alkaline protease from P. aeruginosa. The susceptibility of human IgA proteins to elastase suggests a mechanism by which P. aeruginosa might evade the potentially protective function of IgA by producing this enzyme.     

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.     

Interaction of pseudomonas exoproducts with phagocytic cells

Polymorphonuclear leukocytes play the major role in host defense against infections with Pseudomonas aeruginosa; however, mononuclear cells also may contribute to defense against pulmonary infections with P. aeruginosa. Therefore, we examined the effects of three extracellular products of P. aeruginosa, exotoxin A, alkaline protease, and elastase, on the function of phagocytic cells. Phagocytosis or killing, protein synthesis, and membrane integrity were used as assays of cellular function. Pseudomonas toxin readily inhibited protein synthesis in mouse peritoneal macrophages; in contrast, proteolytic enzymes did not alter protein synthesis, but transiently decreased the sensitivity of macrophages to toxin. High levels of toxin reduced protein synthesis in human peripheral polymorphonuclear leukocytes but did not alter the ability of these cells to kill P. aeruginosa. Elastase and alkaline protease did not cause release of marker enzymes and did not directly inhibit the bactericidal activity of polymorphonuclear leukocytes; killing was reduced due to inactivation of complement components. In conclusion, these potential virulence products do not modify phagocyte function directly and thus do not directly interfere with host response in pseudomonas infections.     

Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.     

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.     

Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa.

The isoelectric points of three proteases (I, II and III), separated from culture supernatants of Pseudomonas aeruginosa strain PAKS-I by isoelectric focusing, were 8.5, 6.6 and 4.5 respectively. Collagenase activity was not detected. More than 75% of the extracellular protease activity of this strain was due to protease II. This enzyme also possessed elastase activity. When purified by ammonium sulphate precipitation, isoelectric focusing and gel chromatography, protease II showed one band on disc electrophoresis and one band on conventional immunoelectrophoresis. The pH optimum, stability and effect of inhibitors and substrate concentration were examined. The molecular weight was 23000 +/- 5000. Protease II was lethal for mice when injected intraperitoneally at a high dose (minimum lethal dose 0.1 mg). Dermonecrosis and subcutaneous haemorrhages were produced in new-born mice upon subcutaneous injection of 10 microgram protease II. A sensitive test for cytotoxicity showed no evidence of cytoplasmic membrane damage to HeLa cells or human diploid embryonic lung fibroblasts by protease II. Morphological changes similar to those produced by trypsin were found.     

Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.     

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.     

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.     

Genetic mapping and characterization of Pseudomonas aeruginosa mutants that hyperproduce exoproteins

We isolated two mutants of Pseudomonas aeruginosa PAO with defective iron uptake. In contrast to the wild-type strain, the mutants produced extracellular protease activity in media containing high concentrations of salts or iron and hyperproduced elastase, staphylolytic enzyme, and exotoxin A in ordinary media (Xch mutants). The mutations were located in the 55' region of the chromosome, between the markers met-9011 and pyrD.