Pseudomonas aeruginosa elastase: affinity chromatography and some properties as a metallo-neutral proteinase.

Morihara, Kazuyuki; Tsuzuki, Hiroshige

doi:10.1271/bbb1961.39.1123

Cited by 41 publications

(24 citation statements)

References 2 publications

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“…Zinc (Zn) and calcium (Ca) ions have previously been shown to be required for enzyme activity and stability of elastase, respectively (21,22). Iron (Fe) (2) and, more recently, Zn (3) have also been implicated in the regulation of elastase production.…”

Section: Resultsmentioning

confidence: 99%

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

Olson

Ohman

1992

J Bacteriol

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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 (l0-4 M ZnCl2), calcium (2.5 x i0-3 M CaCl2), or iron (1O-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.Pseudomonas aeruginosa is an opportunistic pathogen known to cause severe and lethal infections in compromised hosts. Elastase is one of several extracellular enzymes produced by P. aeruginosa that is beli...

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Section: Resultsmentioning

confidence: 99%

“…Proelastase is then further processed to its mature 33-kDa form via autoproteolytic processing mechanisms. In this regard, McIver et al (20) have recently shown that substitutions at the active site residue His-223 resulted in both loss of proteolytic activity and accumulation of the unprocessed 51-kDa form of elastase when expressed in Escherichia coli (22).…”

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confidence: 99%

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

Olson

Ohman

1992

J Bacteriol

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“…Many studies have been carried out on the mechanisms of the bacterial colonization and the pathogenesis of chronic infections in the lower respiratory tracts, and some cilio-inhibitory substances, produced by P. aeruginosa, St. pneumoniae, and H. influenzae have already been isolated [14][15][16][17][18]26]. It is well-known that P. aeruginosa, in particular, releases several cilio-inhibitory substances, such as phenazine pigments (pyocyanin, 1-hydroxyphenazine) [14,26], haemolysin (rhamonolipid) [15], and at least two kinds of proteinases (elastase, alkaline protease) [16,[27][28][29]. It is also known that human neutrophil elastase causes damage to the host tissues, including respiratory ciliated epithelium, as well as to pathogenic micro-organisms [16,23].…”

Section: Discussionmentioning

confidence: 99%

Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro

Amitani¹,

Murayama²,

Nawada³

et al. 1995

Eur Respir J

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A As sp pe er rg gi il ll lu us s c cu ul lt tu ur re e f fi il lt tr ra at te es s a an nd d s sp pu ut tu um m s so ol ls s f fr ro om m p pa at ti ie en nt ts s w wi it th h p pu ul lm mo on na ar ry y a as sp pe er rg gi il ll lo os si is s c ca au us se e d da am ma ag ge e t to o h hu um ma an n r re es sp pi ir ra at to or ry y c ci il li ia at te ed d e ep pi it th he el li iu um m i in n v vi it tr ro o We investigated the in vitro effects of culture filtrates of Aspergillus species and sputum sols from patients with pulmonary aspergillosis on ciliary beat frequency (CBF) and epithelial integrity of human respiratory ciliated epithelium. Culture filtrates of 25 clinically isolated fungi (16 Aspergillus fumigatus, three Aspergillus niger, one Aspergillus flavus, three Candida albicans, and two Cryptococcus neoformans) were obtained by culturing the fungi in Medium-199 at 37˚C for 7 days, and five sputum sols were obtained from patients with pulmonary aspergillosis infected by A. fumigatus.During 6 h experiments using a photometric technique, 14 out of 16 A. fumigatus culture filtrates caused progressive and significant reduction in CBF associated with marked epithelial disruption, whilst the culture filtrates of A. niger and A. flavus caused minor epithelial damage without slowing of CBF, and Medium-199 alone (Control) showed neither epithelial damage nor slowing of CBF. All of the sputum sols also caused significant slowing of CBF as well as epithelial disruption. Culture filtrates of C. albicans and Cr. neoformans had no effects on human respiratory epithelium.We conclude that Aspergillus species, especially A. fumigatus release a factor (or factors) which causes damage to respiratory epithelium and slows CBF, and that these factors may contribute to the colonization of the lower respiratory tracts by the Aspergillus species and may possibly contribute to the further proliferation and spread of the lesions in pulmonary aspergillosis.

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“…Heat denaturation, or formaldehyde inactivation of enzyme activity with preservation of immunogenicity, completely abolish mucin release activity of P. aeruginosa elastase. In addition, removal of Zn+2 from the assay system with phenanthroline under conditions of calcium sufficiency significantly diminished the activity of this metalloproteinase (38). This approach was required because the simple addition of EDTA or other chelators of divalent cations alters the secretory response of tracheal epithelium (39).…”

Section: Discussionmentioning

confidence: 99%

Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

et al. 1984

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Abstract. We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells in response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other proteinases play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections.

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Pseudomonas aeruginosa elastase: affinity chromatography and some properties as a metallo-neutral proteinase.

Abstract: Pseudomonas aeruginosa elastase was found to be a typical metallo-neutral proteinase.

Cited by 41 publications

References 2 publications

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

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

Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro

Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

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