Protease IV production in Pseudomonas aeruginosa from the lungs of adults with cystic fibrosis

Smith, Lucas; Rose, Barbara; Tingpej, Pholawat; Zhu, Hua; Conibear, Tim; Manos, Jim; Bye, Peter; Elkins, Mark R.; Willcox, Mark; Bell, Scott C.; Wainwright, Claire; Harbour, C.

doi:10.1099/jmm.0.46845-0

Cited by 29 publications

(23 citation statements)

References 19 publications

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“…These results are consistent with those of our previous study of protease IV activity using the same isolates (40). The focus on proteases reflects previous reports that both the United Kingdom CF LES (34) and an epidemic ocular P. aeruginosa strain had increased protease activity (26).…”

Section: Discussionsupporting

confidence: 83%

“…The CF Liverpool epidemic strain (LES) has shown high levels of expression of virulence factors, including elastase and staphylolysin, associated with the premature activation of QS systems (34), and a virulent epidemic clone isolated from patients with microbial keratitis has been characterized by high activities of an elastase variant (26). Our previous study of AES-1, AES-2, and nonclonal P. aeruginosa isolates from subjects with CF showed that clonal isolates were more likely to have protease IV activity than nonclonal isolates (40). Enhanced protease IV activity may promote tissue destruction and inflammation and facilitate host-to-host spread.…”

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confidence: 99%

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Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

et al. 2007

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The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P ‫؍‬ 0.02), to show elastase and alkaline protease activity by zymography (P ‫؍‬ 0.04 and P ‫؍‬ 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P ‫؍‬ 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P ‫؍‬ 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.Pseudomonas aeruginosa lung infection is a major determinant of morbidity and mortality in cystic fibrosis (CF) patients (16). The pathogenesis of P. aeruginosa infection depends on multiple cell-associated and extracellular virulence factors including proteases (elastase [LasB], alkaline protease [AprA], and staphylolysin [LasA]), hemolysins (rhamnolipids), and toxins such as exoenzyme S (ExoS) and exotoxin A (13). Proteases contribute to pathogenesis in the lung through the induction of tissue necrosis and inflammation, destruction of surface receptors on neutrophils resulting in the inhibition of chemotaxis, phagocytosis, and the oxidative burst and degradation of surfactant proteins (27, 32).Many P. aeruginosa virulence factors, including the proteases, are regulated by cell-to-cell communication systems that rely on diffusible N-acylhomoserine lactones (AHLs) to monitor population size in a process known as "quorum sensing" (QS) (7, 31). P. aeruginosa has two AHL-regulated circuits: las, consisting of the transcriptional activator LasR and the AHL synthase LasI, which directs the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and rhl, consisting of RhlR and RhlI, which directs the synthesis of N-...

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Section: Discussionsupporting

confidence: 83%

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confidence: 99%

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

et al. 2007

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“…The metalloproteases Pseudomonas elastase B, alkaline protease (AprA), and protease LasA (staphylolysin) are released by P. aeruginosa (41). P. aeruginosa also produces serine protease IV, which can degrade surfactant (1).…”

Section: Discussionmentioning

confidence: 99%

Serine Proteases Degrade Airway Mucins in Cystic Fibrosis

et al. 2011

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Airway mucins are the major molecular constituents of mucus. Mucus forms the first barrier to invading organisms in the airways and is an important defense mechanism of the lung. We confirm that mucin concentrations are significantly decreased in airway secretions of subjects with cystic fibrosis (CF) who have chronic Pseudomonas aeruginosa infection. In sputum from CF subjects without a history of P. aeruginosa, we found no significant difference in the mucin concentration compared to mucus from normal controls. We demonstrate that mucins can be degraded by synthetic human neutrophil elastase (HNE) and P. aeruginosa elastase B (pseudolysin) and that degradation was inhibited by serine proteases inhibitors (diisopropyl fluorophosphates [DFP], phenylmethylsulfonyl fluoride [PMSF], and 1-chloro-3-tosylamido-7-amino-2-heptanone HCl [TLCK]). The mucin concentration in airway secretions from CF subjects is similar to that for normal subjects until there is infection by P. aeruginosa, and after that, the mucin concentration decreases dramatically. This is most likely due to degradation by serine proteases. The loss of this mucin barrier may contribute to chronic airway infection in the CF airway.

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“…protease, and hemolytic phospholipase (2)(3)(4)(5)(6). Secreted toxins and enzymes frequently assemble in the periplasm as proenzymes and are exported to the growth media as inactive proteins.…”

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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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Protease IV production in Pseudomonas aeruginosa from the lungs of adults with cystic fibrosis

Cited by 29 publications

References 19 publications

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

Serine Proteases Degrade Airway Mucins in Cystic Fibrosis

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

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