A Secreted Aminopeptidase of Pseudomonas aeruginosa

Cahan, Rivka; Axelrad, Itschak; Safrin, Mary; Ohman, Dennis E.; Kessler, Efrat

doi:10.1074/jbc.m106950200

Cited by 77 publications

(78 citation statements)

References 51 publications

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“…PA2939 encodes a recently characterized, secreted, zinc-dependent metalloprotease (Cahan et al, 2001). Culture supernatants derived from mutants of the las QS system (DlasR, DlasI, DlasRI) and Dvfr did not contain any of the major isoforms of this protein.…”

Section: Discussionmentioning

confidence: 99%

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

et al. 2003

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The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surfaceassociated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Dlas mutants, although they were unaffected in Drhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

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Section: Discussionmentioning

confidence: 99%

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

et al. 2003

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“…Deletions at the C Terminus Resulting in Active rLAP-Cahan et al (17) reported the existence of a processed form of PA-LAP beginning at Gly-39. We, therefore, attempted to recapitulate this protein via expression of a construct we termed rLAP53 (all wild type amino acids from 40 to 536 were included, with an initiating methionine in place of Gly-39; see Fig.…”

Section: N-terminal Sequence and Mass Spectrometry Analysis Of Rlap55-mentioning

confidence: 99%

“…ing protease, we generated active site mutants and re-assessed processing at the N terminus. PA-LAP is a zinc-dependent enzyme and exhibits high sequence homology in its activity domain to LAP from Streptomyces griseus (17). Sequence alignments of PA-LAP with the Streptomyces enzyme and other LAPs suggested that zinc ions coordinated at the active site likely involve the side chains of the amino acids His-296, Asp-308, Glu-341, Asp-369, and His-467 (17).…”

Section: Table 2 Specific Activity Of Protease-treated Rlap Proteinsmentioning

confidence: 99%

“…Cahan et al (17) purified PA-LAP from high density culture supernatants of P. aeruginosa and characterized some of its biochemical features including its preference for leucine and methionine over other substrates and its requirement for Zn 2ϩ . From their data 17 and that of Braun et al (9), at least two N-terminal sequences have been reported for PA-LAP isolated from culture supernatants; 1 beginning at amino acid 25, indicating the removal of the signal sequence, and 1 at amino acid 39, suggesting the removal an N-terminal prosequence (9,17). Here we investigate the activation of PA-LAP by expressing a full-length version of the enzyme (without the signal sequence) and various recombinant variants of the protein (recombinant LAP (rLAP) in Escherichia coli and measuring enzyme activity against a small molecular weight reporter substrate.…”

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confidence: 99%

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Proteolytic Cleavage of a C-terminal Prosequence, Leading to Autoprocessing at the N Terminus, Activates Leucine Aminopeptidase from Pseudomonas aeruginosa

Sarnovsky

Rea

Makowski

et al. 2009

Journal of Biological Chemistry

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At high bacterial cell density the gene expression program of Pseudomonas aeruginosa is regulated by quorum sensing. Among the gene products highly up-regulated by this system is an exoprotease, leucine aminopeptidase (PA-LAP), which is coexpressed with several known virulence factors and secreted as a proenzyme. We undertook a study of its activation by expressing the full-length proform of PA-LAP recombinantly in Escherichia coli (here termed, rLAP55) and characterizing individual steps in its conversion to an active enzyme. Activation is initiated with the proteolytic removal of a C-terminal prosequence. Removal of ϳ20 amino acids is accomplished by Pseudomonas elastase, which is also positively regulated by quorum sensing. Activation is also mediated by other proteases that cleave rLAP55 near its C terminus. The importance of the C terminus was confirmed by showing that C-terminal deletions of 1-24 amino acids produce a fully active enzyme. The removal of C-terminal prosequences either by proteolysis or deletion leads to an unusual autoprocessing event at the N terminus. Autoprocessing is apparently an intramolecular event, requires the active site of LAP, and results in the removal of 12 N-terminal amino acids. Furthermore, a detailed analysis of the C-terminal prosequence suggests that the proenzyme state is dependent on the presence of a basic side chain contributed by the last amino acid, lysine 536. Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus.

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“…The highly abundant band in untreated fraction 1, near 50 kDa, was identified as the aminopeptidase PepB (PaAP) (40), which is greatly diminished in the same fraction of the vesicles from EBHtreated cells. OprF, the next protein identified, at ϳ37 kDa, is much more apparent in both of the EBH fractions than in the untreated fractions, verifying the Western blot data described above.…”

Section: Epoxides Affect Cif Packaging In a Cifr-independent Fashionmentioning

confidence: 99%

Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

Ballok

Filkins

Bomberger

et al. 2014

Journal of Bacteriology

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Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or hostrelevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.

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A Secreted Aminopeptidase of Pseudomonas aeruginosa

Cited by 77 publications

References 51 publications

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

Proteolytic Cleavage of a C-terminal Prosequence, Leading to Autoprocessing at the N Terminus, Activates Leucine Aminopeptidase from Pseudomonas aeruginosa

Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

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