Virulence of Emerging Clostridium difficile

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1 Virulence of Emerging Clostridium difficile Jimmy D. Ballard, Ph.D. Professor and Chairman Department of Microbiology and Immunology University of Oklahoma HSC

2 TcdB 027/NAP1/BI A Toxin Better Than a Very Good Toxin

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4 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

5 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

6 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?

7 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%

8 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 E E GTD CPD TMD CROP

9 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 +

10 W D D GTD % Identity

11 D H C 698 CPD % Identity

12 E E TMD % Identity

13 CROP % Identity

14 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

15 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

16 Organ Pathologies in TcdB-Treated Mice Liver Spleen Bone Marrow Brain TcdB HIST TcdB HV

17 Survival of TcdB Treated Mice

18 Cysteine Protease Activity of Autoproteoytic Bacterial Toxins Egerer and Satchell, PLoS Path 2010

19 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

20 Enhanced processing of TcdB HV IP6 (μm) TcdB TcdB TcdB HIST TcdB TcdB TcdB TcdB HIST EC 50 IP6 = 9.20 ± 1.70 μm TcdB HV EC 50 IP6 = 4.97 ± μm TcdB HV TcdB Lanis et al. Mol. Micro. 2012

21 Activation of the Cysteine Protease Domain (CPD) Shen et al. Nat Struct Mol Biol 2011 Pruitt et al. (2009) JBC, 284, 33

22 TcdB HIST can exist in an activated unprocessed form Time (min) TcdB TcdB HIST Fluorescence TcdB Coomassie TcdB HV Fluorescence TcdB TcdB Coomassie TcdB HIST binds more probe in the unprocessed form than TcdB HV Lanis et al. Mol. Micro. 2012

23 TcdB HIST is predominantly labeled in the uncleaved form AWP19 (μm) TcdB TcdB HIST Fluorescence TcdB Coomassie TcdB HV Fluorescence TcdB TcdB Coomassie The intermediate form of TcdB HV is only evident with high concentrations of probe Lanis et al. Mol. Micro. 2012

24 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

25 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

26 % of Max TcdB CTERM Interactions (4 C) c-term 027 c-term HIST HV FL4-H TcdB CROP * + CROP + CROP

27 % of Max TcdB CTERM Interactions (37 C) c-term 027 c-term HV HIST FL4-H TcdB CROP * + CROP + CROP

28 TcdB Neutralization with TcdB HIST CTD antiserum * TcdB HIST +serum TcdB HV + serum

29 Solid Phase Peptide ELISA Solid Phase Peptide ELISA TcdB N- -C Enzymatic Domain CP D Translocation C-terminal antigenic region (Receptor Binding Domain) Peptides

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31 % Viability TcdB HIST CTD HIST CTD HV >CTD HIST

32 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 core genes 505 variant genes

33 Sequence Comparison TcdB 078,012,027 Ribotype GTD CPD TMD CROP AA differences Green, match to 012 Red and blue, different in all strains White 078, 027 identical Yellow, unique to % Identity % Identity % Identity

34 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

35 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