Skip to main content

Advertisement

SpringerLink
Log in

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

  • Published:
Journal of Biosciences Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

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

T m :

midpoint temperature

References

  • Andrews AT 1986 Electrophoresis, theory techniques and biochemical and clinical applications (Oxford: Clarendon Press)

    Google Scholar 

  • Aneas MA, Portaro FC, Lebrun I, Juliano L, Palma MS and Fernandes BL 2001 ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity. Braz. J. Med. Biol. Res 34 1397–1403

    Article  CAS  Google Scholar 

  • Bayoudh A, Gharsallah N, Chamkha M, Dhouib A, Ammar S and Nasri M 2000 Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MNI. J. Ind. Microbiol. Biotechnol. 24 291–295

    Article  CAS  Google Scholar 

  • Cheng M, Takenaka S, Aoki S, Murakami S and Aoki K 2009 Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4. J. Biosci. Bioeng. 107 373–378

    Article  CAS  Google Scholar 

  • Divakar K, Priya J and Gautam P 2010 Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01. J. Mol. Catal. B Enzym. 66 311–318

    Article  CAS  Google Scholar 

  • Frees D, Brøndsted L, and Ingmer H 2013 Bacterial proteases and virulence; in Regulated Proteolysis in Microorganisms, Subcellular Biochemistry (eds) DA Dougan (Springer Science+Business Media Dordrecht)

    Google Scholar 

  • Fukuchi S and Nishikawa K 2001 Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria. J. Mol. Biol. 309 835–843

    Article  CAS  Google Scholar 

  • Ghorbel B, Sellami-Kamoun A and Nasri M 2003 Stability studies of protease from Bacillus cereus BG1. Enzyme Microb. Technol. 32 513–518

    Article  CAS  Google Scholar 

  • Ghorbel-Frikha B, Sellami-Kamoun A, Fakhfakh N, Haddar A, Manni L and Nasri M 2005 Production and purification of a calcium-dependent protease from Bacillus cereus BG1. J. Ind. Microbiol. Biotechnol. 32 186–194

    Article  CAS  Google Scholar 

  • Kagawa TF, Conney JC, Baker HM, Mc Swenney S, Liu M, Gubba S, Musser JM and Baker EN 2000 Crystal structure of the zymogen form of the group A Streptococcus virulence factor Sep B: An integrin-binding cysteine protease. Microbiology 97 2235–2240

    CAS  Google Scholar 

  • Kantardjieff KA and Rupp B 2004 Protein isoelectric point as a predictor for increased crystallization screening efficiency. Bioinformatics 20 2162–2168

    Article  CAS  Google Scholar 

  • Karadžić I, Masui A and Fujiwara N 2004 Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil. J. Biosci. Bioeng. 98 145–152

    Article  Google Scholar 

  • Khan AB, Naim A, Orth D, Grif K, Mohsin M, Prager R, Dierich MP and Würzner R 2009 Serine protease espP subtype alpha, but not beta or gamma, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups. Int. J. Med. Microbiol. 299 247–254

    Article  CAS  Google Scholar 

  • Kotb E, Abouel-Hawa MI, Tohamy EY and El-Msalamy K 2013 Purification of toxic protease from Brevibacterium otitidis KB76 with both metal and hydrosulfuryl at the active site. Biologia 68 797–802

    Article  CAS  Google Scholar 

  • Lee CY, Cheng MF, Yu MS and Pan MJ 2002 Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus. FEMS Microbiol. Lett. 209 31–37

    Article  CAS  Google Scholar 

  • Martínez-García S, Rodríguez-Martínez S, Cancino-Diaz ME and Cancino-Diaz JC 2018 Extracellular proteases of Staphylococcus epidermidis: roles as virulence factors and their participation in biofilm. APMIS 126 177–185

    Article  Google Scholar 

  • Palpperumal S, Sankaralingam S, Kathiresan D, Harinathan B, Shankar T and Prabhu D 2016 Partial purification and characterization of a haloalkaline protease from Pseudomonas aeruginosa. BMRJ 15 1–7

    Article  Google Scholar 

  • Patil U and Chaudhari A 2011 Optimal production of alkaline protease from solvent-tolerant alkalophilic Pseudomonas aeruginosa MTCC 7926. Indian J. Biotechnol. 10 329–339

    CAS  Google Scholar 

  • Puri S, Beg QK and Gupta R 2002 Optimization of alkaline protease production from Bacillus sp. using response surface methodology. Curr. Microbiol. 44 286–290

    Article  CAS  Google Scholar 

  • Ridgway ID, Small HJ, Atkinson RJA, Birkbeck HT, Taylor AC and Neil DM 2008 Extracellular proteases and possible disease related virulence mechanisms of two marine bacteria implicated in an opportunistic bacterial infection of Nephrops norvegicus. J. Invertebr. Pathol. 99 14–19

    Article  CAS  Google Scholar 

  • Secades P and Guijarro JA 1999 Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production. App. Environ. Microbiol. 65 3969–3975

    CAS  Google Scholar 

  • Shastry S and Prasad MS 2002 Extracellular protease from Pseudomonas sp, CL 1457 active against Xanthomonas campestris. Process Biotechnol. 37 611–621

    CAS  Google Scholar 

  • Stehling EG, Da Silveira WD and Leite D 2008 study of biological characteristics of Pseudomonas areuginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections. Braz. J. Infect. Dis. 12 86–88

    Article  Google Scholar 

  • Yadav JS, Chowghury S and Chaudhuri SR 2010 purification and characterization of an extracellular protease from pseudomonas aeruginosa isolated from East Calcutta Wetland. J. Biol. Sci. 10 424–431

    Article  CAS  Google Scholar 

  • Young JA and Collier RJ 2007 Anthrax Toxin: Receptor Binding, Internalization, Pore Formation, and Translocation. Annu. Rev. Biochem. 76 243–265

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Author information

Authors and Affiliations

  1. Department of Microbiology and Botany, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt

    Essam Kotb, Yehia A El-Zawahry & Ghadeer E Saleh

Authors
  1. Essam Kotb
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yehia A El-Zawahry
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ghadeer E Saleh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Essam Kotb.

Additional information

Communicated by Sudha Bhattacharya.

Corresponding editor: Sudha Bhattacharya

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 526 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kotb, E., El-Zawahry, Y.A. & Saleh, G.E. Isolation of a putative virulence agent, cytotoxic serine-elastase, from a newly isolated Pseudomonas aeruginosa ZuhP13. J Biosci 44, 7 (2019). https://doi.org/10.1007/s12038-018-9829-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12038-018-9829-3

Keywords

Search

Navigation