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.     

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.     

Biosynthesis of the pyoverdine siderophore of Pseudomonas aeruginosa involves precursors with a myristic or a myristoleic acid chain

Pyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa to import iron. Biosynthesis of this chelator involves non-ribosomal peptide synthetases and other enzymes. PvdQ is a periplasmic enzyme from the NTN hydrolase family and is involved in the final steps of PVDI biosynthesis. A pvdQ mutant produces two non-fluorescent PVDI precursors with a higher molecular mass than PVDI. In the present study, we describe the use of mass spectrometry to determine the structure of these PVDI precursors and show that they both contain a unformed chromophore like ferribactin, and either a myristic or myristoleic chain that must be removed before PVDI is secreted into the extracellular medium.     

Comparative molecular docking analysis of essential oil constituents as elastase inhibitors

Elastase is a protease or proteolytic enzyme, responsible for the breakdown of protein. There are eight human genes encoding for elastase, of which Elastase-1 (CELA-1) and Elastase-2 (ELANE) has significant implications on human diseases. Elastase-1 is primarily expressed in skin keratinocytes and is regarded as the major cause for the blistering in bullous pemphigoid, which affects the skin. On the other hand, Elastase-2 (ELANE), is expressed in the azurophil granules of neutrophils, is responsible for pulmonary emphysema and cyclic hematopoiesis a rare genetic disorder. Elastase is also produced by bacteria such as Pseudomonas aeruginosa, and forms the virulent factor in human. The ingredients from essential natural oils were found to have wound healing effects on non-healing wounds that is interfered by elastase due to microbial infection. Essential oils such as citral, citronellal, geranial, geraniol, and thymol were screened for their inhibitory activity on elastase produced by neutrophil, skin, and Pseudomonas aeruginosa by docking and were analyzed for their subcutaneous ADMET properties by ADME - TOX - Web server.     

Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.     

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.     

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.     

蛋壳内膜分解菌所产蛋白酶的纯化及其N-末端氨基酸序列测定

从土壤中分离得到的1株产蛋壳内膜分解酶(ESM protease)的铜绿假单胞菌(Pseudomonasaeruginosa).通过对其发酵液进行饱和硫酸铵盐析,二次离子交换层析得到蛋壳内膜分解活性达到304.5 U/mg的目标蛋白,SDS-PAGE电泳显示该酶分子量约为32 kD,通过测定其N-末端15个氨基酸残基为:Ala、Glu、Ala、Gly、Gly、Val、Ala、Gly、Lys、Glu、Asp、Ala、Ala、Glu和Leu.     

Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression

Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V(max) and K(m) values of 0.0551 microM/min and 234 microM, respectively, and the substrate DL-Val-Leu-Arg-pNA with V(max) and K(m) values of 0.3830 microM/min and 429 microM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23C. Proteolytic activity was reduced by 90% at 60 degrees C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.     

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.     

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.     

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.     

Identification of a member of the serralysin family isolated from a psychrotrophic bacterium, Pseudomonas fluorescens 114.

An extracellular metalloprotease named No. 114 protease is one of the major secretions of a psychrotrophic bacterium, Pseudomonas fluorescens 114, the cold-adaptation mechanism of which has not been identified. In this study, we purified and cloned No. 114 protease, which is a single polypeptide having a molecular mass of 47 kDa. This protease contains a zinc-binding motif (HEXXHXUGUXH: X, arbitrary amino acid; U, bulky hydrophobic amino acid), glycine-rich repeats (GGXGXD) and no cysteine residue, which are the features specifically found in serralysin subfamily. No. 114 protease has its maximum activity at the temperature of 35-40 degrees C, which is about 20 degrees C lower than that of a serralysin from a mesophilic bacterium, Pseudomonas aeruginosa. All these results imply that No. 114 protease from this psychrophilic bacterium is a unique member of the serralysin group characterized by a low optimal temperature.     

Annexin II is a novel receptor for Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa (P. aeruginosa) are critical in ventilated and poly-traumatized patients. Most important, these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis. Therefore, identification of molecular mechanisms that mediate the infection of mammalian cells with P. aeruginosa is urgently required. Here, we aimed to identify novel receptors that are involved in internalization of P. aeruginosa into mammalian epithelial cells. Employing SDS-PAGE purification and mass spectrometry we demonstrate that annexin II specifically binds to P. aeruginosa. The significance of the interaction of annexin II with P. aeruginosa for the infection of mammalian cells is indicated by the finding that neutralization of the ligands on P. aeruginosa by incubation of the bacteria with recombinant, soluble annexin II prevents internalization of P. aeruginosa into human epithelial cells.     

Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene, pvdY(II) (for the pvdY gene present in type II P. aeruginosa strains), that is only present in strains that make type II pyoverdine. A mutation in pvdY(II) prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of pvdY(II) is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of pvdY(II) provides insights into the molecular basis for production of different pyoverdines by different strains of P. aeruginosa.     

Protease-deficient mutants of Pseudomonas aeruginosa: pleiotropic changes in activity of other extracellular enzymes.

Mutants of Pseudomonas aeruginosa strain PAKS-I which are defective in the formation of extracellular protease activity have been characterized. The mutants produced between approximately 1 and 25% of the protease activity of the wild type and no strains completely lacking extracellular protease were found, even after repeated mutagen treatment. Most mutants also had changed activities of extracellular staphylolytic enzyme, lipase and lecithinase. Four of 13 mutants were unable to release alkaline phosphatase and staphylolytic enzyme into the medium in contrast to the wild type. Serotype, phage type and biochemical reactions were essentially unchanged. The results indicate that some of the mutations affected the cell envelope structure of function leading to decreased ability to release extracellular proteins, and that other mutations possibly affected a common regulatory mechanism for extracellular enzymes.     

群体感应淬灭酶克隆表达及其对铜绿假单胞菌的影响

【背景】由于抗生素的大量使用,导致细菌耐药性越来越强,寻找新的抗细菌感染药物成为研究热点。【目的】克隆表达群体感应淬灭酶,探究其对铜绿假单胞菌毒力及致病性的影响。【方法】利用PCR技术从产群体感应淬灭酶的芽孢杆菌QSI-1基因组DNA中克隆出aiiA基因,将其克隆到表达载体pET30a并导入大肠杆菌E.coliBL21(DE3)中进行诱导表达,通过镍柱亲和层析获得纯化的N-酰基高丝氨酸内酯酶。用不同浓度的淬灭酶作用于铜绿假单胞菌,检测其对铜绿假单胞菌毒力因子产生以及生物膜形成能力的影响;以秀丽隐杆线虫为模型,考察其对线虫感染铜绿假单胞菌存活率的影响。【结果】克隆表达出群体感应淬灭酶,该酶能显著抑制铜绿假单胞菌毒力因子产生和生物膜的形成,并能降低铜绿假单胞菌对感染线虫的致死率。【结论】群体感应淬灭酶可作为一种能高效抑制细菌致病性的物质,为临床治疗细菌性感染提供新的策略。     

A copper-containing protein that inhibits nitrite reductase from Pseudomonas aeruginosa

A non-blue copper-containing glycoprotein was isolated from Pseudomonas aeruginosa. The protein has a molecular mass of 10 kDa and contains 1 atom of EPR-detectable type II copper. The protein inhibits oxidation of both azurin and cytochrome c-551 catalyzed by nitrite reductase from Ps. aeruginosa. Thus, it may be considered as an endogenous inhibitor of nitrite reductase.     

Flagellin/TLR5 responses induce mucus hypersecretion by activating EGFR via an epithelial cell signaling cascades

Mucus hypersecretion is an important manifestation in patients with chronic inflammatory airway diseases. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium Pseudomonas aeruginosa (P. aeruginosa), an important human respiratory pathogen, induced MUC5AC mucin expression via an epithelial cell signaling cascade in human airway epithelial cells. The flagellin purified from P. aeruginosa up-regulated MUC5AC expression by activating its receptor Toll-like receptor 5 (TLR5) in 16HBE cells. This effect was inhibited by NADPH oxidase inhibitor (DPI), small interfering RNA of dual oxidase 2 (Duox2) and reactive oxygen species (ROS) scavengers (nPG and DMSO). Flagellin induced TGF-α release, and stimulated phosphorylated epidermal growth factor receptor (EGFR) and MUC5AC overproduction. These effects were prevented by EGFR and TGF-α neutralizing antibodies, metalloprotease inhibitors (GM6001 and TNF-α protease inhibitor-1) and specific knockdown of TNF-α-converting enzyme (TACE) with TACE siRNA. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for mucin overproduction in patients with P. aeruginosa infections.