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Elastinolytic and Proteolytic Enzymes

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Pseudomonas Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1149))

Abstract

Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion.

Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth.

Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of staphylolytic activity in which the rate of S. aureus cell lysis is determined spectrophotometrically, and methods for determination of peptidase activity of pseudolysin, staphylolysin, lysyl endopeptidase, and the aminopeptidase. The latter methods employ chromogenic or fluorogenic peptide derivatives comprising a short amino acid sequence matching the preferred cleavage site of the protease as substrates. As only one peptide bond is cleaved in each substrate, these assays permit kinetic studies.

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Abbreviations

Abz:

2-Aminobenzoyl

AMC:

7-Amino-4-methylcoumarin (or 7-amido-4-methylcoumarin)

APS:

Ammonium persulfate

Dabsyl:

4-(Dimethylamino) azobenzene-4 sulfonyl chloride

DDW:

Double distilled water (de-ionized water)

DFP:

Diisopropyl fluorophosphate

DMF:

Dimethylformamide

EDANS:

5-(2-Aminoethylamino)-1-naphthalene sulfonic acid

FA:

Furylacryloyl

FITC:

Fluorescein isothiocyanate

LB:

Luria broth

Nba:

4-Nitrobenzylamide

PBS:

Phosphate buffered saline

PMSF:

Phenylmethylsulfonyl fluoride

pNA:

para-Nitroanilide

RFU:

Relative fluorescence units

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SGAP:

Streptomyces griseus aminopeptidase

Su:

Succinyl

TCA:

Trichloro acetic acid

TEMED:

N,N,N′,N′-Tetramethylethylenediamine

TLCK:

N α-p-Tosyl-l-lysine chloromethyl ketone

Tris:

2-Amino-2-(hydroxymethyl)-1,3-propanediol

TSB:

Tryptic soy broth

Z:

N-Carbobenzyloxy-

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Authors and Affiliations

  1. Sackler Faculty of Medicine, Maurice and Gabriela Goldschleger Eye Research Institute, Tel Aviv University, Sheba Medical Center, Tel-Hashomer, 52621, Israel

    Efrat Kessler & Mary Safrin

Authors
  1. Efrat Kessler
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  2. Mary Safrin
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Correspondence to Efrat Kessler .

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Editors and Affiliations

  1. MRC Centre for Molecular Bacteriology and Infection (CMBI) Department of Life Sciences, Imperial College, London, UK

    Alain Filloux

  2. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain

    Juan-Luis Ramos

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Kessler, E., Safrin, M. (2014). Elastinolytic and Proteolytic Enzymes. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_13

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  • DOI: https://doi.org/10.1007/978-1-4939-0473-0_13

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