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Isolation of a putative virulence agent, cytotoxic serine-elastase, from a newly isolated Pseudomonas aeruginosa ZuhP13

  • Essam KotbEmail author
  • Yehia A El-Zawahry
  • Ghadeer E Saleh
Article
  • 70 Downloads

Abstract

A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39% recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and 40°C. It showed stability at pH 6–9 for 1 h and up to 60°C for 30 min with midpoint temperature (Tm) at 61.3°C and isoelectric value (pI) at 5.6±0.2. Its Km and catalytic efficiency (Kcat/Km) for the substrate azocasein were 1.3 mg/mL and 4.62×107 M−1s−1, respectively. On contrary to most P. aeruginosa proteases, Zn2+, EDTA, 2,2′-bipyridine and o-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitor II) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Taken together, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic and hemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformed hepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.

Keywords

Elastase Pseudomonas aeruginosa purification virulence factor 

Abbreviations

BoNT

botulinum neurotoxin

EDTA

ethylene diamine tetraacetic acid

i.p

intraperitoneal

LB broth

Luria-Bertani broth

LB plot

Linweaver-Bulk plot

pI

isoelectric point

PMSF

phenylmethyl sulfonyl fluoride

SBTI

soybean trypsin inhibitor

TEM

transmission electron microscope

TLCK

tosyl lysine chloromethyl ketone

Tm

midpoint temperature

Notes

Acknowledgements

We appreciate consultants of Zagazig University Hospitals for facilities provided in samples collection.

Supplementary material

12038_2018_9829_MOESM1_ESM.docx (527 kb)
Supplementary material 1 (DOCX 526 kb)

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Copyright information

© Indian Academy of Sciences 2019

Authors and Affiliations

  • Essam Kotb
    • 1
    Email author
  • Yehia A El-Zawahry
    • 1
  • Ghadeer E Saleh
    • 1
  1. 1.Department of Microbiology and Botany, Faculty of ScienceZagazig UniversityZagazigEgypt

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