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How glucosylating clostridial toxins modify the actin cytoskeleton and intercellular junctions and are all cellular effects dependent of their enzymatic.

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Presentation on theme: "How glucosylating clostridial toxins modify the actin cytoskeleton and intercellular junctions and are all cellular effects dependent of their enzymatic."— Presentation transcript:

1 How glucosylating clostridial toxins modify the actin cytoskeleton and intercellular junctions and are all cellular effects dependent of their enzymatic activity on small GTPases?

2 glucosylating clostridial toxins C. difficile ToxA, ToxB (pseudomembranous colitis, postantibiotherapy enteritis) C. sordellii LT, HT (gangrenes, hémorragic enteritis, enterotoxemia, fatal shock in woman) C. novyi Tox  (gangrenes)

3 N 5462366 repeats hydrophobic 1 956 1128 1852 A domainT domainR domain glucosylating clostridial toxins : global organization N-ter ToxB ToxA 31 short repeats 7 long repeats  -hairpin + loop of 7-10 residues SR  -hairpin + loop of 18 residues LR Receptor : Gal-  1,3-Gal-  1,4-GlcNAc C 698 653 HD 587 Cysteine protease domain DxD

4 binding to receptor H+H+ Gal-  1-3Gal-  1-4GlcNac for ToxA autocatalytic cleavage early endosomes + inositol- hexaphosphate UDP-glucose UDP-N-acetyl-glucosamine Rho-GTPasesRho-Thr-Gluc Entry into cell

5 Rho-GTP Glucose-Thr35 tightly bound to the membrane Rho-GDP Thr35 Glucosylating clostridial toxins glucosylation Impairment of the recognition of the effectors GAP activity inhibited wt Rho-GTP UDP-glucose

6

7 LT82/HUVEC LT82 / 3T3 actine-GFP HUVEC Ctrl

8 Epithelial cell barrier MCCD Adherens junctions E-cadherin, catenins Tight junctions Occludin, ZO TER TER (% of control) LT basal LT apical 10 -7 10 -8 10 -9 1O -10 M Dextran-FITC paracellular way Flux (pmol/cm 2 per h) 4,4 kDa 77 kDa h

9 Ctrl Apical Median Basal LT82 ToxB LT depolymerizes first baso-lateral actin filaments, whereas ToxB depolymerizes both apical and baso-lateral actin filaments

10 LT82Ctrl E-cadherin  -catenin ZO-1 ToxB LT desorganizes first basolateral junctions, ToxB both tight and basolateral junctions

11 controlTcsL-82 (6h) thoracic fluid LT82 induces dehydration and thoracic fluid accumulation Geny et al. Am. J. Pathol. 2007, 170:1003-1017 volume of thoracic fluid (ml) 2468 16 0.0 0.1 0.2 0.3 0.4 0.5 Time (hours after LT injection (10 -7 M)] lung edema Ctrl LT82 6 h

12 LT glucosylates Rac in lung tissue and desorganizes E-cadherin distribution in lung vessels hypovolemia and anoxia (albumin EPO ) no inflammatory response

13         E-cadherin IQGAP Rac-GTP IQGAP Rac-GDP TPA Which signal pathway controlling AJs is altered by LTs? actin Rac-IQGAP pathway   Rac-actin depolymerization

14 E-cadherin is removed from the cell surface to cytosol in detergent-soluble fractions LT82 0246 LT9048 Time (h) LT82 LT9048 Cell surface biotinylated E-cadherin Time (h) E-cadherin in TX-100 insoluble fraction LT82 LT9048 00.511.5246Time (h) 00.511.5246Time (h) LT82 LT9048 E-cadherin in TX-100 soluble fraction 0 20 40 60 80 0O,511,5246

15 E-cadherin  -catenin  -catenin IQGAP1 0246 h246 LT82LT9048 Time (h) immuno-precipitation E-cadherin LTs remove the whole E-cadherin-catenin complex from the cell surface IP: E-cadherin E-cadherin ControlTPA  catenin  catenin IQGAP1 E-cadherin  -catenin  -catenin IQGAP1 0 50 100 150 200 250 Control TPA LTs do not impair the Rac-IGAP pathway Time (h) LTs E-cadherin  -catenin  -catenin IQGAP1 % of protein bound to E-cadherin beads

16 HeLa Rac1 G12V 0 30 60 90 120 min Glucosylation of GTPases P-paxillin Y118 0 30 60 90 120 160 min HeLa 0 50 100 150 200 250 GFP Rac G12V CtrlLT82 P-paxillin in FAs (TIRF) LT induces paxillin dephosphorylation in a Rac-dependent manner P-paxillin Rac G12V -c-mycRac wt LT82 2 h

17 LT induces a dissociation and redistribution of focal adhesion and adherens junction proteins p120-catenin Control GM1 Flotillin E-cadherin  -catenin Talin 0 20 40 60 80 100 120 % of control raftsAJsFAs * ** immuno precipitation -Paxillin - + redistribution from detergent- insoluble to soluble fractions dissociation of  -catenin and talin from paxillin  -catenin talin P-paxillin Total paxillin AJ protein FA proteins LT treatment

18 240180120600 0 20 40 60 80 100 120 240180120600 0 20 40 60 80 100 120 PI3,4,5P3 240180120600 0 20 40 60 80 100 120 PI4PPI4,5P2 LT decreases PIP, PIP2, PIP3 Time (min) % of control 0 20 40 60 80 100 120 PIP2 in DRM 12435678910 Fraction number Ctrl LT82 LT decreases PIP2 in lipid microdomains

19 Rac LT glucosylation (inactivation) FA and adherens junction disorganization increase in cell barrier permeability actin filament depolymerization IQGAP PAK LIMK cofilin - paxillin dephosphorylation - redistribution of AJ complexes from membrane to cytosol - dissociation of FA from AJ proteins phosphatases ? - decrease in PIP PI4,5P kinase ?

20 LT, ToxB stimulate MAPK p42/44MAPK Phospho- p38MAPK SAPK/JNK 0 30 60 90 120 180 min LT incubation glucosylation of Rho/Ras (non-glucosylated forms) total p-ATF2 pSer63 pSer73 C-JUN ATF2 0 30 60 90 120 180 min P-SAPK/JNK ToxB incubation

21 LT82 [M] Polymerized actin Percent of control (%) 10 -7 10 -8 10 -9 0 0 20 40 60 80 100 120 140 Ctrl JNK inhibitor JNK pathway is involved in LT effect on actin cytoskeleton + JNK inhibitor II (25 mM) 50  m 10  m LT82CtrlToxB Control PD98059 p44 inhibitor SB203580 p38 inhibitor SP600125 JNK inhibitor ToxB [M]

22 JNK pathway facilitates LT-dependent glucosylation of small G-proteins 0306090120180 min LT82 HeLa cells pretreated with JNK inhibitor retardation of glucosylation JNK stimulation is independent of LT glucosylation activity 06090120 min Don cells phospho-SAPK-JNK DonQ deficient in UDP-glucose the same stimulation level of SAPK-JNK activation 0306090120 ToxB

23 Rac LT glucosylation (inactivation) stimulation of p42/44MAPK p38MAPK SAPK/JNK facilitation Transcriptional factors

24 LT mitochondria Ras APOPTOSIS caspase 9 caspase 3 PLD1 cleavage Rac glucosylation (inactivation) - paxillin dephosphorylation - dissociation of FA from AJ proteins - decrease in PIP actin cytoskeleton and intercellular junction alterations facilitation stimulation of p42/44MAPK p38MAPK SAPK/JNK PI3K/ Akt RhoB

25 Geny B. Boehm C. Gibert M. Grassard A. Sauvonnet N. Payrastre B. Biologie des Interactions cellulaires CNRS Toulouse Bactéries anaérobies et Toxines Huerre M. Khun H. Histotechnologie et Pathologie


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