Vection des bactéries par les insectes : la cuticule importe t’elle ?

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Vection des bactéries par les insectes : la cuticule importe t’elle ?

Yves Rahbé, Natalia Guschinskaya, Baptiste Monsion, Marilyne Uzest

To cite this version:

Yves Rahbé, Natalia Guschinskaya, Baptiste Monsion, Marilyne Uzest. Vection des bactéries par les insectes : la cuticule importe t’elle ?. 10. Réunion annuelle du Réseau de Biologie Adaptative des Pucerons et Organismes Associés : Bapoa, Nov 2017, Colmar, France. 35 p. �hal-01651301�

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Intro Methods Results Discussion & now

Vection des bactéries par les insectes : la cuticule importe t’elle ?

Rahbé, Yvan

Guschinskaya, Natalia

Monsion, Baptiste

Uzest, Marilyne

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MAP

• Part A: Panoramic

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Epicuticle: the outermost envelope (lipids + polyphenol + few proteins)

Exocuticle: (or inner epicuticle): lipids + proteins

Endocuticle: chitin + matrix of proteins +

lipids + pigments and N-acylcatecholamines à cross-linking

➔ The composition of the insect cuticle with

respect to protein content can be highly variable

Building Parts of insect Cuticle :

From Cuticle to cuticular proteins

Intro a: Glandular duct b: cement c: wax d: epicuticle e: exocuticle f: endocuticle g: glandular cell h: epidermal cell

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4 https://www.shmoop.com/animal-evolution-diversity/land-animals.html

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MPMI Vol. 29, No. 7, 2016, pp. 535–544. http:// dx.doi.org/10.1094/MPMI-02-16-0032-R

Blocking the

Transmission of a

Noncirculative

Vector-Borne Plant

Pathogenic Bacterium

Oui, des bactéries peuvent s’accrocher spécifiquement à des territoires

cuticulaires définis,

• par certains déterminants bactériens identifiés (eg PD1764, HxfB) et

• par des déterminants insectes ciblés (chitine)

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Intro Methods Results Discussion & now 14 Scanning electron micrograph of Yersinia pestis on proventricular spines of Xenopsylla cheopis

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Oui, des bactéries ont vraisemblablement le potentiel de se lier spécifiquement à des territoires cuticulaires, notamment internes des pièces buccales et de l’intestin antérieur (stylets &

foregut), dans d’autres modèles que   la maladie de Pierce

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Exploring the interaction between bacteria and insect

cuticular proteins by peptide Arrays using the pea aphid

(Acyrthosiphon pisum) model

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Encadrant: Yvan RAHBE, DR INRA

Natalia GUSCHINSKAYA, Post doct. Collaboration: Guy Condemine, DR CNRS

Laboratoire d'accueil: MAP UMR5240 CNRS UCBL INSA de Lyon

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Stagiaire: Hoang Thong KIEU, M2 biochimie-biologie moléculaire

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Screening the interactions, at the molecular level,

between the cuticular proteins of  

Acyrthosiphon pisum  

and the phythopathogenic bacteria Dickeya dadantii,

by peptide arrays

24 Main objective

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25 What is a peptide array ?

CelluSpotsTM_{peptide arrays - Intavis}

- Size: 76 × 26 mm (microscope slide)

- Peptides are covalently bound to cellulose via C_ter.

- 18 aa peptides immobilised on the spots.

- Spots: 2× 384 spots _{(technical repeat)}

- Distance between spots: 1,2 mm. - Spot diameter 0.8 mm.

- Cellulose mesh of : 8 – 10 μm.

- 2 versions: V1 and V2 (peptides / proteins)

Methods Methods

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Array design

26 Methods

à 143 Cuticular proteins in the genome of the pea aphid 

Belonging to 5 classes, among which the largest is the CPR family (PFAM PF00379), or « RR motif family »

à 3 Subclasses: RR1, RR2 and Unclassified CPRs à V1 contains a complete set of RR2 proteins  

(core set, full coverage)

à V2 contains:

à All RR1 (full set, full coverage)

à Remaining RR2  

(partial set, partial coverage)

à Remaining, = Unclassified CPR  

(partial set, partial coverage)

à 18 AA peptides with 3 AA overlap

à Peptides are immobilized as 2 duplicates in array à Example of chemiluminescence detection of hybridation 

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Intro Methods Results Discussion & now Detection method 27 Arrays V1 & V2 Fluorescence Chemiluminescence and - Bio-Rad imager. - Excitation wavelength: 530 nm - Emission wavelemgth: 605 nm - Acquisition time: 1s

- Analysis intensity by Image Lab - Vilber imager.

- Antibody anti-KdgM / Antibody coupled HRP / Luminata®

Crescendo•.

- Excitation wavelength: UV - Acquisition time: 30s

- Analysis intensity by Spotfinder Methods

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Intro Methods Results Discussion & now 28 Arrays V1& V2 Fluorescence - Bio-Rad. - Excitation wavelength: V605. - Acquisition time: 1s Agrobacterium tumefaciens Dickeya dadantii

Detection by chemiluminescence (PepArray V1)

Erwinia aphidicola Pantoea agglomerans Chimiluminescence - Vilber imager. - Antibody coupled HRP/ luminata crescendo. - Exitation wavelength: UV. - Acquisition time: 30s - Analysis intensity by spotfinder Results

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Intro Methods Results Discussion & now 28 Arrays V1& V2 Fluorescence - Bio-Rad. - Excitation wavelength: V605. - Acquisition time: 1s Agrobacterium tumefaciens Dickeya dadantii

Erwinia aphidicola Pantoea agglomerans Chimiluminescence - Vilber imager. - Antibody coupled HRP/ luminata crescendo. - Exitation wavelength: UV. - Acquisition time: 30s - Analysis intensity by spotfinder à Reproducibility issues à Erratic spot issues à No real fixation of the

bacteria on V1 arrays ?

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Intro Methods Results Discussion & now 29 Arrays V1 &V2 Fluorescence - Bio-Rad. - Excitation wavelength: V605. - Acquisition time: 1s Dickeya dadantii Agrobacterium tumefaciens Erwinia aphidicola Pantoea agglomerans Chimiluminescence - Viber imager. - Antibody coupled HRP/ luminata crescendo. - Exitation wavelength: UV. - Acquisition time: 30s - Analysis intensity by spotfinder

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Intro Methods Results Discussion & now 29 Arrays V1 &V2 Fluorescence - Bio-Rad. - Excitation wavelength: V605. - Acquisition time: 1s Dickeya dadantii Agrobacterium tumefaciens Erwinia aphidicola Pantoea agglomerans Chimiluminescence - Viber imager. - Antibody coupled HRP/ luminata crescendo. - Exitation wavelength: UV. - Acquisition time: 30s - Analysis intensity by spotfinder

à Spots detected on the arrays à Fixation of the bacteria on the peptides of the V2 arrrays à Probable true positive

signal, visible especially with D. dadantii

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Intro Methods Results Discussion & now Analysis arrays (1)

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Quantification by TIGR

spotfinder software

Computer (excel, jmp) analysis and classification of signal intensities

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Computer (excel, jmp) analysis and classification of signal intensities Normalisation and substraction  

by the negative controls

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Computer (excel, jmp) analysis and classification of signal intensities Normalisation and substraction  

by the negative controls

corresponding peptides (and protein / families) bind bacteria, and are manually grouped by homology

groups are analysed (WebLogo)

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31 Analysis arrays (D.dadantii) (2)

B C

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B C

R

L

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che m ilum ine sc e nc e inte nsity 0 36250 72500 108750 145000 spots rang 0 96 193 289 385 B C

Chemiluminescence intensity spots of array incubed with D.dadantii (RFP) bacterias.

A

A: positive spots B: negative spots C: no signal detected

~40000 ~20000

L

à 50 - 60 spots considered positive (intensity > 40 000 ).

à 180 – 200 spots considered negative (intensity ~40 000 – 20 000). à 80 – 100 spots no dectected (intensity < 20 000).

R che m ilum ine sc e nc e inte nsity 0 31250 62500 93750 125000 spots rang 0 96 193 289 385 A B C ~40000 ~20000 R L Results

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Intro Methods Results Discussion & now Analysis (D.dadantii) (2)

32 à 50 peptide sequences correspond to 50 positive spots

à belong to 23 CuPs

à For example: 3 peptides sequences (326, 324, 325) from CuP ACYPI010088 Results

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For example

3 peptide sequences (pep-324, pep-325 and pep-326)   of CuP ACYPI010088 (CPR unclassified)

Results

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Intro Methods Results Discussion & now Conclusion

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- Interaction of D.dadantii with some CuPs proteins from CRP family was detected.

- No interaction of D.dadantii bacterias with the major CuP RR2 subfamily.

- 23 CuPs (RR1 and Unclassified) are able to take part in the interaction with D.dadantii in vitro.

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Intro Methods Results Discussion & now Perspectives

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- Test a colorimetric method - More reproducibile ? - Less sensitive ?

- Non re-usable

- SEM of hybridized arrays

- Non Dickeya : change to other primary antibody

- Test purified major bacterial envelope components: - Extract pilus and flagellum and hybridize (V1 ?, V2) - Incubation with mutant bacteria to test outer membrane

components (kdgM, ompA, ompF …) - Impact of growth phase on binding

- Go to in vivo situation in aphid anterior digestive tract   (SEM of dissected stylets - oesophagum). Entry OK, Binding ?