Rôle des protéines SNARE au niveau de la vacuole bactérienne durant les phases précoces de linfection par yersinia pseudotuberculosis dans un contexte dautophagie

Rˆ ole des prot´ eines SNARE au niveau de la vacuole bact´ erienne durant les phases pr´ ecoces de l’infection par Yersinia pseudotuberculosis dans un contexte d’autophagie.. Summary of

Rˆ ole des prot´ eines SNARE au niveau de la vacuole

Yersinia pseudotuberculosis dans un contexte

d’autophagie Laure-Anne Ligeon

To cite this version:

Laure-Anne Ligeon Rˆ ole des prot´ eines SNARE au niveau de la vacuole bact´ erienne durant les phases pr´ ecoces de l’infection par Yersinia pseudotuberculosis dans un contexte d’autophagie M´ edecine humaine et pathologie Universit´ e du Droit et de la Sant´ e - Lille II, 2013 Fran¸cais.

<NNT : 2013LIL2S043> <tel-01252226v2>

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Hannibal lecteur, Le silence des agneaux

Remerciements

Professeur

Table des matières

Résumé

Summary

Table des illustrations et Abréviations Revue Bibliographique

Résumé

Summary

of Yersinia

pseudotuberculosis replicates mainly in vacuoles positive for LC3, a hallmark of autophagy

Surprisingly, this LC3-positive-vacuole presents only single limiting membrane Second, we showed

that SNARE proteins play a role in Y pseudotuberculosis intracellular traffic VAMP3 and VAMP7 are sequentially recruited to Yersinia-containing vacuoles (YCVs) VAMP7 is involved in the LC3

recruitment to YCVs with single- and double-membrane We proposed that VAMP3 is a component of the molecular checkpoint for bacterial commitment to either single- or double-membrane LC3-positive pathway Third, we characterized the traffic of endosomal proteins recruited to LC3-positive-YCV with single membrane in epithelial cells We showed that markers of early endosome and proteins involved in autophagosome formation, are recruited to YCVs during the early stage of infection Then, the vacuole acquire late endosomal and lysosomal proteins but acidification is not observed Finally,

we initiated a high-content screening approach for the identification of SNARE partners

Overall this work illustrates the importance of LC3-positive compartment ultrastructure analysis Our result demonstrate how bacterial subvert the molecular machinery of the host in order to create a replicative niche Finally, we present the importance of autophagy regulation by highlighting for the first times the existence of a molecular checkpoint between two LC3-positive vacuoles with different morphologies

Table des illustrations

Et Abréviations

Tableau des illustrations

Abréviations

Chapitre I : Les Yersiniae

1972).

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2+

Chapitre II : Les voies de dégradations : Autophagie et Endosome

Membrane

Plamsique

Destruction et Échappement de

la vacuole

UbUbUb

Salmonella

Gal3

Gal8Gal8

Gal3

Salmonella

S flexneriXÉNOPHAGIE

-­‐/-­‐

Plamsique

Destruction et Échappement de

Reconnaissance par l’autophagie

Acidification de l’autophagosome

Autophagosome acide

C burnetti Autophagosomenon-acide

A phagocytophilum

Y pseudotuberculosis(BMDM)

B pseudomallei Δ T3SSE.coli

Formation de l’autophagosome

indépendamment de Atg5/Atg7

L monocytogenes

LLO-S aureus

Chapitre III : Le trafic membranaire

Compartiment donneur

Initiation

Scission Bourgeonnement

Compartiment donneur

GEF

Rab-GDP Cytosol

Rab -GTP

Rab -GTP

Rab -GTP

Qa

Syntaxine-­‐17 ER

Golgi

l’autolysosomeVAMP8

Autolysosome/ vacuole

Amphisome

Lysosome Phagophore

Sec9p Sso2 Ykt6

Atg9

Stx5

VAMP7 VAMP8 VTI1B Stx17

VAMP3

Stx-7 Stx-8 STX-17 VTIB VAMP7

noyau

Réticulum

Endoplasmique

Appareil de Golgi

-­‐/-­‐ +/+

Article

Et Résultats

Qu’est ce qu’un auteur scientifique ? »

Chapitre I : Le rôle de deux protéines SNARE dans le trafic intracellulaire de Y pseudotuberculosis

•

•

Role of VAMP3 and VAMP7 in the commitment of Yersinia pseudotuberculosis to

LC3-associated pathways involving single- or double-membrane vacuoles

Delphine-Armelle Lacorre4,5, Elisabeth Werkmeister5,6, Véronique Proux-Gillardeaux7,8, Thierry Galli7,8, and Frank Lafont1,2,3,4,5

LONGIN SNARE TM GFP TIVAMP/ VAMP7

128

5 VAMP7

0VAMP7-GFP

255

128

3.6 VAMP7

0

I

Abstract

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking

autophagosome acidification In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy Here, we address the

question of the membrane traffic during internalization of Yersinia investigating the role of vesicle associated membrane proteins (VAMPs) First, we show that as well in epithelial cells, Yersinia

pseudotuberculosis replicates mainly in non-acidic LC3-positive vacuoles Second, in these cells, we

unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single-membranes using correlative light-electron microscopy Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3 Fourth, we show that VAMP7 knockdown alters LC3’s association with single-and multi-membrane-YCVs Finally, in non-infected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher numbers of double-membrane, LC3-positive vesicles Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion

proteins involved in Yersinia pseudotuberculosis’ intravesicular life cycle

33

(GFP) Merge

1µm

500nm

-LC3 VAMP3

infected]/[(LC3II/tubulin)non-­‐infected

25)

siCTRL Non Infected siCTRL Infected

Bacteria GFP-LC3 VAMP7-555

Position (µm)

Pixel intensity (grey scale) 0 4

Bacteria siCTRL

Bacteria siVAMP7

GFP-LC3

GFP-LC3 VAMP7-555

VAMP7-555

Merge

Merge

Bafilomycin Rapamycin Infected

- - - + + - - - - + + -

+ + + + + + + + + +

- - - - - - - -

LC3II Tubulin LC3I

Position (µm)

Bacteria GFP-LC3 VAMP7-555

Figure 5 Ligeon et al.

Bacteria mRFP-VAMP7

- -

+ + +

-

- - -

+

+ - +

+

+

+ +

siCTRL siVAMP7

infected non-­‐ infected

33

®

C

-± 1.6%

LC3-

59.9%

VAMP3 LC3

-± 10.1%

G F

HeLa-EGFP HeLa-EGFP-VAMP3

Rapamycin

500nm

HeLa EGFP

Rapamycin

Figure 7 Ligeon et al.

YCV distribution with VAMP3 or LC3

double single

-14.0%

x7

100 80

60 40

BMDM EGFP BMDM EGFP-VAMP3

11.4%

11.4%

double single

BMDM EGFP-VAMP3

500nm

HeLa HeLa siVAMP3

13-­‐15

36 12

25

Figure S1 Ligeon et al.

15 25 35 40 50 70 100

-

-15 25 35 40 50

15 25 35 40 50

3 2.5 2

1 1.5

0.5

3.5

4.5 4

Bafilomycin Rapamycin

- - +

-+

- + + + + +

EGFP-VAMP3 EGFP

VAMP3

Tubulin

D

B

Rapamycin

HeLa GFP-VAMP3 Cell 1 HeLa GFP-VAMP3 Cell 2 HeLa GFP-VAMP3 Cell 3 HeLa GFP-VAMP3 Cell 4 HeLa GFP-VAMP3 Cell 5

17 26 34 43 55 72 95

No drug Rapamycin

HeLa EGFP HeLa EGFP-VAMP3

N.S

N.S N.S

2

41 42

TM TM

600

®

®

®

TM

00:03 Bacteria GFP-VAMP3 Merge

-­‐/-­‐

Chapitre II : Interaction de la vacuole de Y pseudotuberculosis avec l’autophagie et la voie de l’endocytose

•

Laure-Anne Ligeon, Yann Ciczora, Delphine-Armelle Lacorre and Frank Lafont

H F

0

B

20 40 60 80

100 Atg16L1+ Atg16L1-

20 40 60 80

0

G

20 40 60 80 100

0.5 2 4

Rab7+ Rab7-

30min p.i.

Recepteur de la transeferine ULK1

Voie Lysosomale Voie Endosomale

Endosome précoce tardif Voie Autophagique

« La recherche fondamentale ne peut être pratiquée que

dans un climat de liberté intellectuelle »

Discussion Générale

Et Perspectives

« On fait la Science avec des faits comme une maison avec

des pierres ; mais une accumulation de faits n'est pas plus une science qu'un tas de pierres n'est une maison

Discussion Générale et Perspectives

Recepteur de la transeferine ULK1

Voie Lysosomale Voie Endosomale

Endosome précoce tardif

VAMP3 VAMP7

SNARE Voie Autophagique

Ubiquitine

Recrutement ?

Matériels

Et Méthodes

Matériels et Méthodes

2

L-­‐Glutamine Provenance

L-­‐Glutamine Provenance

TM

Nom Description Provenance

®

2

Annexes

Annexe II : Légendes des films

Annexe III : Figure crible (i)

Annexe IV : Figure crible (ii)

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Bibliographie

103(32):11821-7