Virulence of Emerging Clostridium difficile Jimmy D. Ballard, Ph.D. Professor and Chairman Department of Microbiology and Immunology University of Oklahoma HSC
TcdB 027/NAP1/BI A Toxin Better Than a Very Good Toxin
Epidemic CDAD North American PFGE Type 1 (NAP1) Restriction Endonuclease Assay type BI Ribotype 027 NAP1/BI/027 Strain Nomenclature TcdB HV = TcdB produced by NAP1/BI/027 Strains TcdB HIST = TcdB produced strains commonly encountered before 2003
Epidemic/Hypervirulent C. difficile? Epidemic/Emerging ( Mortality, Recurrence) Wilcox et al. CID 55(8) Miller et al. CID 50 McDonald et al. NEJ 353 (23) Marsh et al. JCM 50 (12) Loo et al. NEJ 353 (23) Goorhuis et al. CID 45 (6) Petrella et al. CID 55 (3) Non-Hypervirulent Wilson et al. CID 50 (12) Morgan et al. PloS One 3 (3) Cloud et al. CG&H 7 Walk et al. CID Oct. 2012
Mot (+) Mot (-) Aggregation (-) HV/Ep Aggregation (+) Hist HV/Ep Hist Flagella CwpV PaLoc CDT: Binary Toxin Gyrase Hist tcdr tcdb tcde tcda tcdc cdtr gyra Fq S HV/Ep tcdr tcdb tcde tcda cdtr cdta cdtb gyra Fq R 3247 core genes 505 variant genes Sporulation? Toxin Levels?
C. difficile PaLoc Comparison of C. difficile Pathogenicity Locus between Historical and Hypervirulent Strain DNA Homology 100% 93% 99% 98% 95% Protein tcdd tcdb tcde tcda tcdc Similarity 100% 96% 100% 99% Identity 100% 92% 99% 98%
MLD Large Clostridial Toxin 270 kda Glucosyltransferase TcdB Major virulence factor Found in all clinically relevant isolates Immunity to the TcdB provides protection from CDI TcdB(-) strains are attenuated in virulence W D D D H C 102 286 288 653 587 698 E E 976 970 GTD CPD TMD CROP 1 543 767 1852 2366
TcdB TcdB Cellular Intoxication by TcdB/TcdA RME Glc UDP Rho Rac Cdc42 UDP-Glc IP 6 H + UDP H + IP 6 H + H + H + H +
W D D 102 286 288 GTD 1 543 96 % Identity
D H 653 587 C 698 CPD 544 767 96 % Identity
E E 976 970 TMD 768 1852 91 % Identity
CROP 1853 2366 88 % Identity
Impact of TcdB on Zebrafish TcdB HIST TcdB HV 10 nm 1 nm Untreated Hamm et al. (2006) PNAS Lanis et al. (2010) PLoS Pathogens
No difference in enzymatic activity of TcdB HIST and TcdB HV No correlative difference in cell binding between TcdB HIST and TcdB HV TcdB HV gains access to the cytoplasm (translocates) more rapidly than TcdB HIST
Organ Pathologies in TcdB-Treated Mice Liver Spleen Bone Marrow Brain TcdB HIST TcdB HV
Survival of TcdB Treated Mice
Cysteine Protease Activity of Autoproteoytic Bacterial Toxins Egerer and Satchell, PLoS Path 2010
Differences in autoproteolytic activity between various CPDs C. sordellii Lethal Toxin requires low ph for processing TcdA is much less efficient at processing despite equal IP6 affinity MARTX toxins reset to process multiple substrates
Enhanced processing of TcdB HV IP6 (μm) TcdB 1-2366 TcdB 544-2366 TcdB HIST TcdB 1-543 TcdB 1-2366 TcdB 544-2366 TcdB HIST EC 50 IP6 = 9.20 ± 1.70 μm TcdB HV EC 50 IP6 = 4.97 ± 0.918 μm TcdB HV TcdB 1-543 Lanis et al. Mol. Micro. 2012
Activation of the Cysteine Protease Domain (CPD) Shen et al. Nat Struct Mol Biol 2011 Pruitt et al. (2009) JBC, 284, 33
TcdB HIST can exist in an activated unprocessed form Time (min) TcdB 1-2366 TcdB HIST Fluorescence TcdB 544-2366 Coomassie TcdB HV Fluorescence TcdB 1-2366 TcdB 544-2366 Coomassie TcdB HIST binds more probe in the unprocessed form than TcdB HV Lanis et al. Mol. Micro. 2012
TcdB HIST is predominantly labeled in the uncleaved form AWP19 (μm) TcdB 1-2366 TcdB HIST Fluorescence TcdB 544-2366 Coomassie TcdB HV Fluorescence TcdB 1-2366 TcdB 544-2366 Coomassie The intermediate form of TcdB HV is only evident with high concentrations of probe Lanis et al. Mol. Micro. 2012
TcdB HV shows enhanced affinity for intramolecular substrate TcdB HV IP6 TcdB HIST Key TcdB Substrate (Enzymatic Domain) AWP19 probe / Inhibitor Lanis et al. Mol. Micro. 2012
Differences in Cell Entry TcdB HV TcdB HIST H + H + H + H + H + H + H + H + H + H + H + H+ H+ H+ H + IP 6 IP 6 H+ IP 6 IP 6
% of Max TcdB CTERM Interactions (4 C) 100 80 012 c-term 027 c-term HIST 60 40 HV 20 0 10 0 10 1 10 2 10 3 10 4 FL4-H TcdB 1645 + CROP 2366 2366 1645 * + CROP + CROP 1851 1851
% of Max TcdB CTERM Interactions (37 C) 100 80 60 40 012 c-term 027 c-term HV HIST 20 0 10 0 10 1 10 2 10 3 10 4 FL4-H TcdB 1645 + CROP 2366 2366 1645 * + CROP + CROP 1851 1851
TcdB Neutralization with TcdB HIST CTD antiserum * TcdB HIST +serum TcdB HV + serum
Solid Phase Peptide ELISA Solid Phase Peptide ELISA TcdB N- -C Enzymatic Domain CP D Translocation C-terminal antigenic region (Receptor Binding Domain) 1 2 3 4 5 6 7 8 9 10 11 12 358 Peptides
% Viability 80 60 40 20 0 TcdB HIST + + + + + CTD HIST - + - + - CTD HV - - - - + >CTD HIST - - + + +
078 078 Mot (+) Mot (-) Aggregation (-) HV/Ep Aggregation (+) Hist HV/Ep Hist Flagella CwpV Hist PaLoc tcdr tcdb tcde tcda tcdc CDT: Binary Toxin cdtr Gyrase gyra Fq S 078 HV/Ep tcdr tcdb tcde tcda cdtr cdta cdtb gyra Fq R 078 078 3247 core genes 505 variant genes
Sequence Comparison TcdB 078,012,027 Ribotype GTD CPD TMD CROP 012 027 078 237 AA differences Green, match to 012 Red and blue, different in all strains White 078, 027 identical Yellow, unique to 078 012 027 92% Identity 012 078 96% Identity 078 027 91% Identity
Conclusion TcdB HV is a more potent toxin than TcdB HIST TcdB HV undergoes more efficient autoprocessing than TcdB HIST TcdB HV and TcdB HIST vary in putative neutralizing epitopes within the CTD The CTD of TcdB HV may contribute to more efficient cell entry TcdB 078 sequence varies from TcdB HV and TcdB HIST
Acknowledgements Ballard Research Group Soumitra Barua, Ph.D. Jason Larabee, Ph.D. Jonathan Hunt, Ph.D. Logan Hightower Jordi Lanis Latisha Heinlen, M.D., Ph.D. Collaborators Judith James, M.D., Ph.D. (OMRF) Aimee Shen, Ph.D. University of Vermont