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Understanding the Cellular Effects of Glucosylating Clostridial Toxins on Actin Cytoskeleton and Intercellular Junctions

Explore how clostridial toxins modify actin cytoskeleton and cell junctions, and the impact on cellular function. Investigate enzymatic activities of glucosylating toxins and their effects on small GTPases.

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Understanding the Cellular Effects of Glucosylating Clostridial Toxins on Actin Cytoskeleton and Intercellular Junctions

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  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 Toxa (gangrenes)

  3. 653 587 698 D H C glucosylating clostridial toxins : global organization A domain T domain R domain 546 2366 1 hydrophobic N 956 1852 1128 repeats Cysteine protease domain ToxA 31 short repeats 7 long repeats DxD b-hairpin + loop of 7-10 residues SR b-hairpin + loop of 18 residues LR N-ter ToxB Receptor : Gal-a1,3-Gal-b1,4-GlcNAc

  4. Entry into cell early endosomes binding to receptor Gal-a1-3Gal-b1-4GlcNac for ToxA H+ + inositol- hexaphosphate autocatalytic cleavage Rho-Thr-Gluc Rho-GTPases UDP-glucose UDP-N-acetyl-glucosamine

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

  6. LT82 / 3T3 actine-GFP HUVEC Ctrl LT82/HUVEC

  7. Epithelial cell barrier MCCD Tight junctions Occludin, ZO Flux (pmol/cm2 per h) TER (% of control) Adherens junctions E-cadherin, catenins TER LT apical LT basal 1O-10M 10-9 10-8 10-7 Dextran-FITC 4,4 kDa 77 kDa paracellular way h

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

  9. E-cadherin b-catenin ZO-1 Ctrl LT82 ToxB LT desorganizes first basolateral junctions, ToxB both tight and basolateral junctions

  10. control TcsL-82 (6h) 0.5 0.4 0.3 0.2 2 4 6 8 16 0.1 0.0 LT82 induces dehydration and thoracic fluid accumulation thoracic fluid volume of thoracic fluid (ml) Time (hours after LT injection (10-7M)] LT82 6 h lung edema Ctrl Geny et al. Am. J. Pathol. 2007, 170:1003-1017

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

  12. b b b a a a IQGAP Rac-GTP Which signal pathway controlling AJs is altered by LTs? Rac-IQGAP pathway E-cadherin IQGAP IQGAP b b b a Rac-GDP actin a a TPA Rac-actin depolymerization

  13. Time (h) 0 2 4 6 LT82 Cell surface biotinylated E-cadherin LT9048 Time (h) E-cadherin in TX-100 insoluble fraction Time (h) Time (h) 0 0 0.5 0.5 1 1 1.5 1.5 2 2 4 4 6 6 LT82 LT9048 LT82 E-cadherin in TX-100 soluble fraction LT9048 LT82 LT9048 0 O,5 1 1,5 2 4 6 80 60 40 20 0 E-cadherin is removed from the cell surface to cytosol in detergent-soluble fractions

  14. immuno-precipitation E-cadherin LT82 LT9048 Time (h) 0 2 4 6 2 4 6 h E-cadherin a-catenin b-catenin IQGAP1 LTs 250 % of protein bound to E-cadherin beads Control 200 TPA Control TPA 150 E- cadherin 100 Time (h) b-catenin 50 a-catenin 0 E-cadherin a-catenin IQGAP1 b-catenin IQGAP1 LTs remove the whole E-cadherin-catenin complex from the cell surface LTs do not impair the Rac-IGAP pathway IP: E- cadherin a-catenin E-cadherin IQGAP1 b-catenin

  15. 0 30 60 90 120 160 min 250 200 Glucosylation of GTPases 150 100 50 P-paxillin Y118 0 HeLa LT induces paxillin dephosphorylation in a Rac-dependent manner 0 30 60 90 120 min HeLa Rac1G12V P-paxillin in FAs (TIRF) P-paxillin Rac wt RacG12V-c-myc GFP GFP RacG12V RacG12V LT82 2 h Ctrl LT82

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

  17. 120 100 80 60 40 20 0 0 60 120 180 240 120 100 80 120 % of control 60 100 40 80 20 60 0 40 20 120 0 0 60 120 180 240 100 80 60 40 20 0 0 60 120 180 240 LT decreases PIP, PIP2, PIP3 % of control PI4P PI4,5P2 PI3,4,5P3 Time (min) LT decreases PIP2 in lipid microdomains PIP2 in DRM Fraction number 1 2 3 4 5 6 7 8 9 10 Ctrl LT82

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

  19. 0 30 60 90 120 180 min LT incubation P-SAPK/JNK LT, ToxB stimulate MAPK Phospho- p42/44MAPK total ATF2 p38MAPK p-ATF2 total SAPK/JNK C-JUN pSer63 glucosylation of Rho/Ras (non-glucosylated forms) pSer73 0 30 60 90 120 180 min ToxB incubation

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

  21. 0 30 60 90 120 180 min 0 60 90 120 min Don cells phospho-SAPK-JNK DonQ deficient in UDP-glucose the same stimulation level of SAPK-JNK activation JNK pathway facilitates LT-dependent glucosylation of small G-proteins ToxB LT82 0 30 60 90 120 HeLa cells pretreated with JNK inhibitor retardation of glucosylation JNK stimulation is independent of LT glucosylation activity

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

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

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

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