Molecular Analysis of<i>Pseudomonas aeruginosa</i>Protease IV Expressed in<i>Pseudomonas putida</i>

Traidej, Mullika; Caballero, Armando R.; Marquart, Mary E.; Thibodeaux, Brett A.; O’Callaghan, Richard J.

doi:10.1167/iovs.02-0458

Cited by 33 publications

(61 citation statements)

References 20 publications

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“…A pH gradient, generated by mixing 10 mM ammonium acetate buffer at pH 9.0 with 10 mM ammonium acetate buffer at pH 6.4, was used to elute the protein. The eluted fractions containing protease IV were assayed by Western blotting with rabbit polyclonal antiserum prepared against a recombinant form of mature protease IV (33). The reactive fractions were pooled and then concentrated to 1.0 ml using a stirred ultrafiltration cell with a 10-kDa cutoff filter membrane (YM10; Amicon Inc., Beverly, MA) and loaded onto a Sephacryl S-300 molecular sieve matrix (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…Also detected was a 45-kDa protein (proenzyme) that contained the propeptide and mature protease domains but not the signal peptide. In an experimental model of keratitis, expression of protease IV by P. putida caused a 3-fold increase in the clinical score of infected eyes relative to eyes infected with P. putida lacking the protease IV gene (33).…”

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confidence: 98%

“…Similar to elastase B, this protein consists of three domains: signal sequence, propeptide domain, and mature protease. Protease IV is synthesized as a pre-proenzyme that is processed intracellularly and secreted into the extracellular milieu as the mature protease (33).…”

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confidence: 99%

“…The protease IV gene has been cloned and expressed in Pseudomonas putida providing a more abundant supply of protease than that obtained from the naturally expressed chromosomal P. aeruginosa gene (33). Expression of protease IV in P. putida revealed a protein of 48 kDa containing the three domains of protease IV: a signal sequence, a propeptide, and a mature protease.…”

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confidence: 99%

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Identification of the Active Site Residues of Pseudomonas aeruginosa Protease IV

Traidej¹,

Marquart²,

Caballero³

et al. 2003

Journal of Biological Chemistry

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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, sitedirected 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 lysinespecific 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.

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Section: Methodsmentioning

confidence: 99%

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confidence: 98%

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confidence: 99%

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confidence: 99%

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Identification of the Active Site Residues of Pseudomonas aeruginosa Protease IV

Traidej¹,

Marquart²,

Caballero³

et al. 2003

Journal of Biological Chemistry

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“…It was shown that P. aeruginosa strains possessing AP activity are more virulent than AP-deficient strains in an animal model of corneal infection (9,25,35). AP is also postulated to play a major role in the pathogenesis of acute and chronic lung infections caused by P. aeruginosa (18,32,39).…”

Section: Discussionmentioning

confidence: 99%

Human Pre-Elafin Inhibits a Pseudomonas aeruginosa -Secreted Peptidase and Prevents Its Proliferation in Complex Media

Bellemare

Vernoux

Morisset

et al. 2008

Antimicrob Agents Chemother

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Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gramnegative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated.Pseudomonas aeruginosa is an opportunistic pathogen that is life-threatening for immunocompromised individuals and for patients suffering from chronic respiratory diseases such as cystic fibrosis (CF) (10, 27). Chronic lung P. aeruginosa infection is the primary cause of morbidity and mortality in CF patients, and its prevalence increases with age. P. aeruginosa is also the predominant bacteria associated with nosocomial infections, and acute P. aeruginosa infection may result in sepsis and death (26). P. aeruginosa is characterized by the expression of numerous virulence factors, reflected by the unusually large genome of this bacteria and has developed sophisticated mechanisms to escape the host immune defenses (10,15,27,33). In addition, due to low membrane permeability, active efflux of drugs, and the presence of ␤-lactamase activity, P. aeruginosa infections are becoming increasingly difficult to treat (17). Hence, the development of novel antimicrobial agents, preferably acting on targets exposed at the bacterial cell surface, is urgently needed.Among the P. aeruginosa virulence factors, the secreted peptidases are thought to play a critical role in tissue invasion, nutrient accessibility, and degradation of the innate host defense molecules and therefore constitute potential therapeutic targets (18)(19)(20). Because opportunistic pathogens are taking advantage of a weakened host protective shield, augmentation therapy with lung natural defense molecules also appears to be a ...

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Assays for Studying Pseudomonas aeruginosa Secreted Proteases

Fortuna,

Collalto,

Rampioni

et al. 2023

Methods in Molecular Biology

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Molecular Analysis ofPseudomonas aeruginosaProtease IV Expressed inPseudomonas putida

Cited by 33 publications

References 20 publications

Identification of the Active Site Residues of Pseudomonas aeruginosa Protease IV

Identification of the Active Site Residues of Pseudomonas aeruginosa Protease IV

Human Pre-Elafin Inhibits a Pseudomonas aeruginosa -Secreted Peptidase and Prevents Its Proliferation in Complex Media

Assays for Studying Pseudomonas aeruginosa Secreted Proteases

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