Elastase and the LasA Protease of Pseudomonas aeruginosa Are Secreted with Their Propeptides

Kessler, Efrat; Safrin, Mary; Gustin, Jean K.; Ohman, Dennis E.

doi:10.1074/jbc.273.46.30225

Cited by 116 publications

(88 citation statements)

References 34 publications

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“…3A) demonstrated that much less elastase protein accumulated in an 18-h culture supernatant of the dsbA mutant than in the wild type. As a test for the ability to secrete elastase protein, which may be unstable over time, washed cells were tested for the ability to release the protein after just 30 and 60 min into fresh medium as previously de- (18). Under these conditions (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The examination of elastase in stationary-phase culture supernatants using a Western blot analysis showed reduced levels of elastase, suggesting that the dsbA mutation compromised elastase accumulation. However, examination of 30-min washed-cell cultures, which can detect unstable secreted proteins (18), showed that elastase was secreted. Thus, the lack of elastase accumulation in the supernatant over time probably reflects its instability in the absence of proper folding by DsbA.…”

Section: Discussionmentioning

confidence: 99%

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Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in Pseudomonas aeruginosa Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA

et al. 2000

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Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation. Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little difference in levels of expression in the two strains. However, the dsbA-lacZ fusion showed two-to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase. Although efficient secretion of elastase in the dsbA mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of dsbA in the mucoid PDO300 background did not affect alginate production. Thus, even though dsbA is coregulated with mucoid conversion, it was not required for alginate production. This suggests that mucA mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in Pseudomonas aeruginosa Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA

et al. 2000

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“…Additional studies have shown that HA/protease has bifunctional properties; the 45-kDa form is responsible for the enterotoxic response and the fully processed 35-kDa protease causes the HA activity responsible for the cytotoxic effects caused by this organism (10). We note that the processing of the pro-LasA to mature LasA protease in P. aeruginosa has been shown to involve the action of other secreted proteases, including elas-tase, lysine-specific protease (protease IV or PrpL), and alkaline proteinase (14). Kessler et al (14) showed that purified preparations of each protease were able to convert the secreted 42-kDa pro-LasA into the mature 20-kDa LasA, with the transient accumulation of a 28-kDa intermediate.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

et al. 2008

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The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ϳ10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.Vibrio anguillarum, a marine bacterium, is the causative agent of vibriosis, a systemic disease of both wild and cultured marine fish characterized by hemorrhagic septicemia (2). Outbreaks of vibriosis result in high mortalities among infected fish, and this disease continues to be a major obstacle for the aquaculture industry (2). V. anguillarum enters its fish host through the gastrointestinal (GI) tract and quickly colonizes this nutrient-rich environment (21,22). Garcia et al. (9) have shown that V. anguillarum grows extremely well in salmon intestinal mucus and that mucus-grown cells specifically express a number of different proteins, including several outer membrane proteins (9) and the extracellular metalloprotease EmpA (7).The zinc metalloprotease EmpA has been identified as a virulence factor for V. anguillarum and is important for virulence during infection of the GI tract of Atlantic salmon (Salmo salar) (7,16,19). In V. anguillarum wild-type strain M93Sm, EmpA is expressed during stationary phase when cells are incubated in Atlantic salmon GI mucus (6, 7). The creation of an empA null mutant (M99) by insertional mutagenesis showed that EmpA is responsible for the protease activity observed for wild-type strain M93Sm (7). However, EmpA activity is dependent on successful secretion and processing of the nascent protein to a...

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“…The pro-sequences of P. aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin act as molecular chaperones that mediate folding of the mature enzymes within the periplasm (Braun et al, 1996;Marie-Claire et al, 1999;McIver et al, 1995;O'Donohue & Beaumont, 1996). The propeptides remain non-covalently associated with the mature enzymes to inhibit their proteolytic activity until liberation from the cell (Braun et al, 1998;Kessler & Safrin, 1994;Kessler et al, 1998;O'Donohue & Beaumont, 1996).…”

Section: Discussionmentioning

confidence: 99%

An extracellular zinc metalloprotease gene of Burkholderia cepacia

et al. 2003

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Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence. A B. cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe. The predicted amino acid sequences of these B. cepacia and a B. pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway. zmpA isogenic mutants were constructed in B. cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes. The zmpA mutants produced less protease than the parent strains. The B. cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B. cepacia strain Pc715j and a Pc715j zmpA mutant. The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B. cepacia.

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Elastase and the LasA Protease of Pseudomonas aeruginosa Are Secreted with Their Propeptides

Cited by 116 publications

References 34 publications

Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in Pseudomonas aeruginosa Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA

Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in Pseudomonas aeruginosa Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

An extracellular zinc metalloprotease gene of Burkholderia cepacia

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