Exocellular oxidases: tools to assess the exposure of soil fungi to xenobiotics?

(1)

HAL Id: hal-01600787

https://hal.archives-ouvertes.fr/hal-01600787

Submitted on 4 Jun 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Exocellular oxidases: tools to assess the exposure of soil fungi to xenobiotics?

Claude Jolivalt, Nathalie Demont-Caulet, Isabelle Touton, Agathe Brault, Albert Kollmann, Christian Mougin

To cite this version:

Claude Jolivalt, Nathalie Demont-Caulet, Isabelle Touton, Agathe Brault, Albert Kollmann, et al.. Exocellular oxidases: tools to assess the exposure of soil fungi to xenobiotics?. 4th International Symposium on Environmental Aspects of Pesticide Microbiology, SETAC, Sep 2004, Thessaloniki, Greece. 2004. �hal-01600787�

(2)

Exocellular oxidases

Tools to assess the exposure of soil fungi

to xenobiotics

?

Claude Jolivalt,

Biochemistry, UMR CNRS 7573, ENSCP Paris

Nathalie Demont-Caulet

Cellular Biology, INRA Versailles / Univ.-Paris VII

Isabelle Touton, Agathe Brault, Albert Kollmann, Christian Mougin

Xenobiotics and Environment, INRA Versailles

(3)

Research areas of the team Xenobiotics and Environment

Structural and metabolic biochemistry

structural data, regulation of expression, enzyme evolution

Biocatalysis,

bioconversion, biotransformation

Fate and impacts of xenobiotics in soils

Fungi

(4)

The aims of the present project

1 - Improvement of the knowledge on fungal laccases

2 - Development of tools to assess the adverse effects of

pollutants on soil ecosystems

● Effect of xenobiotics on laccase expression and activity

in liquid cultures (identification of biomarkers)

● Effect of xenobiotics on laccase expression and activity

in soils inoculated with fungi (validation of biomarkers)

(5)

Ecological relevance

● Provide one of the highest biomass in the soil ● Initiate numerous trophic chains

Functional relevance

● Responsible of the breakdown and mineralization of organic matter ● Catalyze the biotransformation of numerous xenobiotics

Practical relevance

● Secrete exocellular oxidases: peroxidases, laccases

(6)

The main characteristics of laccases

oxidases mainly produced by fungi (Trametes versicolor)

and also by higher plants (Rhus vernicifera) insects (Bombyx)

bacteria (Azospirillum lipoferum) extracellular glycoproteins containing copper

520 to 550 aa, 60 to 80 kD

high redox potential: 0.7 V, very robust

Cu1 Cu3a Cu3b Cu2 (T1) (T2/T3) O₂ 2 H₂O reducing substrate

e

-4 RH 4 R• radical

(7)

Functions and uses of fungal laccases

1 - Natural

● biodegradation of ligno-cellulosic materials

● morphogenesis, pathogenesis….

2 - Industrial

● textile and food industry

● pulp paper bleaching

3 - Environmental

● biotransformation of xenobiotics, bioremediation

● biomarkers for the assessment of adverse

(8)

The laccases of T. versicolor

2 families of isozymes

● type A: N-terminale sequence GIGP

constitutive and induced

gene AF 414109

►

lac A

● type B: N-terminale sequence AIGP

constitutive, could be induced ???

gene U44430

(9)

Effect of xenobiotics on laccase activity

● Treatment of 4-day liquid cultures of T. versicolor by xenobiotics ● Measurement of laccase activity after 2 and/or 3 days (ABTS)

Screening for chemicals efficient in increasing laccase activity Dose/response relationships

(10)

0 1 2 3 4 5 6 water xylid ine e-vin iferi n atrazi ne 2HF nony lphe nol 4OH5C lF 9-fluor enon e Laccase activity (unit / mL)

Laccase activity after 2 ( ) and 3 ( ) days of treatment

(11)

acet one xylid ine fenu ron chlor oto luron isonor uron nebu ron isopr otuo n linu ron diur on meto xuron fluom eturo n Laccase activity (unit / mL) 0 2 4 6 8 10 12 14

(12)

nonylphenol (µM) laccase activity (unit / mL) 0,0 0,5 1,0 1,5 2,0 4-n-nonylphenol t-nonylphenol 0 0.1 0.5 1 5 10 50 100 500

(13)

Effect of xenobiotics on the expression of laccase genes

● Treatment of 4-day liquid cultures of T. versicolor with xenobiotics ● Measurement of laccase activity (ABTS)

● Extraction of mRNAs (RNeasy Mini Kit Qiagen)

● Synthesis of cDNAs by RT-PCR (Advantage RT Clontech) ● relative quantification of genes by real time PCR

(Thermocycler Roche, ref: TEF1, gene X94913) ● detection by fluorescence (SYBR Green I)

(14)

High level of activity: xylidine, lac A

Time of induction (days)

0 Ctl-6 2 3 6 8 10 Relative expression 0,0 0,2 0,4 0,6 0,8 1,0 Activity (unit / mL) 0 2 4 6 8 mRNA Act Chemical concentration: 100 µM

(15)

Xylidine: dose-response relationship, lac A

Chemical concentration (µM) Relative expression 0,0 0,2 0,4 0,6 0,8 1,0 Activity (unit / mL) 0 1 2 3 4 5 6 7 mRNA

Act after 2 days

(16)

Moderate level of activity: diuron, lac A

Chemical concentration (µM) Relative expression 0,0 0,3 0,6 0,9 1,2 Activity (u nit / mL) 0 1 2 3 4 5 mRNA

Act after 2 days Act after 3 days

(17)

Low level of activity: 4-n-nonylphenol, lac A

Chemical concentration (µM) Relative expression 0,00 0,04 0,08 0,12 0,16 Activity (unit/mL) 0,0 0,2 0,4 0,6 0,8 1,0 mRNA

Act after 2 days Act after 3 days

(18)

Can lac B be induced by xenobiotics?

Chemical concentration: 100 µM

Time of induction (days)

Relative expression 0,0 0,2 0,4 0,6 0,8 1,0 lac A lac B 0 Ctl-6 2 3 6 8 10 0,003

(19)

-Effect of xenobiotics on the production of laccase proteins

● Treatment of 4-day liquid cultures of T. versicolor with xenobiotics ● Separation of the extracellular fluid

● Precipitation of polysaccharidic compounds with 10% acetone ● Diafiltration of the protein solution (cut off 10kD, Amicon)

● Purification on Concanavalin A-Sepharose 4B column (Sigma-Aldrich)

(elution with 0.2M Tris Hcl in the presence 0.5M NaCl and 0.2M o-methyl glucose)

● separation by SDS-Page (12%)

● Identification of proteins by Maldi-TOF (Bruker) profiles of proteins

(20)

Separation and identification of lac A isoforms

kDa 170 130 100 72 55 40 30 23

1

st

_{migration 2}

nd

_{migration Maldi-TOF}

mass pI

guaïacol blue nitrate pH 5 2.5 markers 5 isozymes pI 3.23 3.11 3 2.88 2.75 MW Da 58463 59776 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1400 1900 2400 A 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1 4 0 0 1 9 0 0 2 4 0 0 m / z 1400 1900 2400 A

(21)

Conclusions

● Laccase activity can be increased following fungal exposure to environmental pollutants

● Lac A (but not lac B) are induced by chemicals

● mRNA measurement is highly specific and well related to

enzymatic activities, but the response is not sensitive enough to be useful (induction by 15-20)

●

the proteomic approach should be developed

Could fungal laccases be used for point contamination (heavy pollution) assessment?

(22)

Perspectives

● Understand the effects of xenobiotics at the transcriptional, traductional and post-traductional levels

● Assess the effects of metals

● Develop tools to inoculate fungi into soils and perform assays in soil

(23)

Acknowledgements

Bertrand Dubreucq, Cellular Biology, INRA Versailles Christian Malosse, Phytopharmacy, INRA Versailles Agence De l’Environnement et de la Maitrise de L’Energie

(grant n° 0275048)