Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI

Proteome Science

Andreas Pich¹  Ingo Just¹  Ralf Gerhard¹  Nelli Jochim¹ 

[1] Hannover Medical School, Institute of Toxicology, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

关键词: Toxin A;    relative quantification;    ICPL™;    colonic cells;    C. difficile-associated diarrhea;   

Others  :  821160
DOI  :  10.1186/1477-5956-9-48

 received in 2011-05-31, accepted in 2011-08-17,  发布年份 2011

【 摘 要 】

Background

The anaerobe Clostridium difficile produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induce reorganization of the cytoskeleton and cell death in colonic cells. Whether all toxin effects on target cells depend on catalytic glucosyltransferase activity is unclear at present. Thus, we conducted a proteome approach to compare the protein profile of target cells treated either with wild type toxin A (rTcdA wt) or with a catalytically inactive mutant toxin A (mutant rTcdA). Relative protein quantification was feasible using isotope-coded protein labeling techniques (ICPL) and mass spectrometry (LC-MALDI).

Results

Altogether we found a significant differential expression of thirty proteins after treatment with rTcdA wt or mutant rTcdA. Mutant rTcdA caused up-regulation of seven proteins and sixteen proteins were responsive to rTcdA wt after 5 h. Long-term effect of rTcdA wt on protein expression was the down-regulation of eleven proteins. Up- or down-regulation of several proteins was verified by western blot analysis confirming the MS results.

Conclusion

Our results indicate incubation time-dependent effects of the clostridial glucosylating toxin A on colonic cells. The rTcdA wt impact more cellular functions than actin cytoskeleton reorganization and apoptosis. Furthermore, these data give insight into glucosyltransferase independent effects of clostridial glucosylating toxins on target cells after short incubation time. Additionally, our data reveal pro-inflammatory and proliferative effects of mutant rTcdA after short-term incubation.

【 授权许可】

   
2011 Jochim et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Just I, Gerhard R: Large clostridial cytotoxins. Rev Physiol Biochem Pharmacol 2004, 152:23-47.
• [2]Voth DE, Ballard JD: Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 2005, 18(2):247-263.
• [3]Bartlett JG: Narrative review: the new epidemic of Clostridium difficile-associated enteric disease. Ann Intern Med 2006, 145(10):758-764.
• [4]Genth H, Dreger SC, Huelsenbeck J, Just I: Clostridium difficile toxins: more than mere inhibitors of Rho proteins. Int J Biochem Cell Biol 2008, 40(4):592-597.
• [5]Teichert M, Tatge H, Schoentaube J, Just I, Gerhard R: Application of mutated Clostridium difficile toxin A for determination of glucosyltransferase-dependent effects. Infect Immun 2006, 74(10):6006-6010.
• [6]Aktories K, Just I: Clostridial Rho-inhibiting protein toxins. Curr Top Microbiol Immunol 2005, 291:113-145.
• [7]Jaffe AB, Hall A: Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 2005, 21:247-269.
• [8]Warny M, Keates AC, Keates S, Castagliuolo I, Zacks JK, Aboudola S, Qamar A, Pothoulakis C, LaMont JT, Kelly CP: p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis. J Clin Invest 2000, 105(8):1147-1156.
• [9]He D, Sougioultzis S, Hagen S, Liu J, Keates S, Keates AC, Pothoulakis C, Lamont JT: Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation. Gastroenterology 2002, 122(4):1048-1057.
• [10]Chen ML, Pothoulakis C, LaMont JT: Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A. J Biol Chem 2002, 277(6):4247-4254.
• [11]Nottrott S, Schoentaube J, Genth H, Just I, Gerhard R: Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases. Apoptosis 2007, 12(8):1443-1453.
• [12]Sutton PA, Li S, Webb J, Solomon K, Brazier J, Mahida YR: Essential role of toxin A in C difficile 027 and reference strain supernatant-mediated disruption of Caco-2 intestinal epithelial barrier function. Clin Exp Immunol 2008, 153(3):439-447.
• [13]Lottspeich F, Kellermann J: ICPL labeling strategies for proteome research. Methods Mol Biol 2011, 753:55-64.
• [14]Paradela A, Marcilla M, Navajas R, Ferreira L, Ramos-Fernandez A, Fernandez M, Mariscotti JF, Garcia-del Portillo F, Albar JP: Evaluation of isotope-coded protein labeling (ICPL) in the quantitative analysis of complex proteomes. Talanta 2010, 80(4):1496-1502.
• [15]Lee YY, Yu CP, Lin CK, Nieh S, Hsu KF, Chiang H, Jin JS: Expression of survivin and cortactin in colorectal adenocarcinoma: association with clinicopathological parameters. Dis Markers 2009, 26(1):9-18.
• [16]Matarrese P, Falzano L, Fabbri A, Gambardella L, Frank C, Geny B, Popoff MR, Malorni W, Fiorentini C: Clostridium difficile toxin B causes apoptosis in epithelial cells by thrilling mitochondria. Involvement of ATP-sensitive mitochondrial potassium channels. J Biol Chem 2007, 282(12):9029-9041.
• [17]Muetzelburg MV, Hofmann F, Just I, Pich A: Identification of biomarkers indicating cellular changes after treatment of neuronal cells with the C3 exoenzyme from Clostridium botulinum using the iTRAQ protocol and LC-MS/MS analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2009, 877(13):1344-1351.
• [18]Larsson M, Majeed M, Ernst JD, Magnusson KE, Stendahl O, Forsum U: Role of annexins in endocytosis of antigens in immature human dendritic cells. Immunology 1997, 92(4):501-511.
• [19]Oddo M, Calandra T, Bucala R, Meylan PR: Macrophage migration inhibitory factor reduces the growth of virulent Mycobacterium tuberculosis in human macrophages. Infect Immun 2005, 73(6):3783-3786.
• [20]Nguyen MT, Lue H, Kleemann R, Thiele M, Tolle G, Finkelmeier D, Wagner E, Braun A, Bernhagen J: The cytokine macrophage migration inhibitory factor reduces pro-oxidative stress-induced apoptosis. J Immunol 2003, 170(6):3337-3347.
• [21]Gerhard R, Tatge H, Genth H, Thum T, Borlak J, Fritz G, Just I: Clostridium difficile toxin A induces expression of the stress-induced early gene product RhoB. J Biol Chem 2005, 280(2):1499-1505.
• [22]Fatica A, Tollervey D: Making ribosomes. Curr Opin Cell Biol 2002, 14(3):313-318.
• [23]Warner JR, McIntosh KB: How common are extraribosomal functions of ribosomal proteins? Mol Cell 2009, 34(1):3-11.
• [24]Schafer T, Maco B, Petfalski E, Tollervey D, Bottcher B, Aebi U, Hurt E: Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 2006, 441(7093):651-655.
• [25]Naora H, Takai I, Adachi M, Naora H: Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis. J Cell Biol 1998, 141(3):741-753.
• [26]Lee SB, Kwon IS, Park J, Lee KH, Ahn Y, Lee C, Kim J, Choi SY, Cho SW, Ahn JY: Ribosomal protein S3, a new substrate of Akt, serves as a signal mediator between neuronal apoptosis and DNA repair. J Biol Chem 2010.
• [27]Dreyfuss G, Kim VN, Kataoka N: Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol 2002, 3(3):195-205.
• [28]Dreyfuss G, Matunis MJ, Pinol-Roma S, Burd CG: hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem 1993, 62:289-321.
• [29]Kim JH, Hahm B, Kim YK, Choi M, Jang SK: Protein-protein interaction among hnRNPs shuttling between nucleus and cytoplasm. J Mol Biol 2000, 298(3):395-405.
• [30]Wagner EJ, Garcia-Blanco MA: Polypyrimidine tract binding protein antagonizes exon definition. Mol Cell Biol 2001, 21(10):3281-3288.
• [31]Iervolino A, Santilli G, Trotta R, Guerzoni C, Cesi V, Bergamaschi A, Gambacorti-Passerini C, Calabretta B, Perrotti D: hnRNP A1 nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL leukemogenesis. Mol Cell Biol 2002, 22(7):2255-2266.
• [32]Perrotti D, Neviani P: From mRNA metabolism to cancer therapy: chronic myelogenous leukemia shows the way. Clin Cancer Res 2007, 13(6):1638-1642.
• [33]Kamath RV, Leary DJ, Huang S: Nucleocytoplasmic shuttling of polypyrimidine tract-binding protein is uncoupled from RNA export. Mol Biol Cell 2001, 12(12):3808-3820.
• [34]Koch G, Smith M, Macer D, Webster P, Mortara R: Endoplasmic reticulum contains a common, abundant calcium-binding glycoprotein, endoplasmin. J Cell Sci 1986, 86:217-232.
• [35]Hornef MW, Normark BH, Vandewalle A, Normark S: Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells. J Exp Med 2003, 198(8):1225-1235.
• [36]Paris S, Denis H, Delaive E, Dieu M, Dumont V, Ninane N, Raes M, Michiels C: Up-regulation of 94-kDa glucose-regulated protein by hypoxia-inducible factor-1 in human endothelial cells in response to hypoxia. FEBS Lett 2005, 579(1):105-114.
• [37]Randow F, Seed B: Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability. Nat Cell Biol 2001, 3(10):891-896.
• [38]Haug G, Leemhuis J, Tiemann D, Meyer DK, Aktories K, Barth H: The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol. J Biol Chem 2003, 278(34):32266-32274.
• [39]Haug G, Aktories K, Barth H: The host cell chaperone Hsp90 is necessary for cytotoxic action of the binary iota-like toxins. Infect Immun 2004, 72(5):3066-3068.
• [40]Na X, Kim H, Moyer MP, Pothoulakis C, LaMont JT: gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A. Infect Immun 2008, 76(7):2862-2871.
• [41]Treml K, Meimaroglou D, Hentges A, Bause E: The alpha-and beta-subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver. Glycobiology 2000, 10(5):493-502.
• [42]Breuer W, Bause E: Oligosaccharyl transferase is a constitutive component of an oligomeric protein complex from pig liver endoplasmic reticulum. Eur J Biochem 1995, 228(3):689-696.
• [43]Edwards EH, Sprague EA, Kelley JL, Kerbacher JJ, Schwartz CJ, Elbein AD: Castanospermine inhibits the function of the low-density lipoprotein receptor. Biochemistry 1989, 28(19):7679-7687.
• [44]Moore SE, Spiro RG: Inhibition of glucose trimming by castanospermine results in rapid degradation of unassembled major histocompatibility complex class I molecules. J Biol Chem 1993, 268(6):3809-3812.
• [45]Kim H, Rhee SH, Kokkotou E, Na X, Savidge T, Moyer MP, Pothoulakis C, LaMont JT: Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J Biol Chem 2005, 280(22):21237-21245.
• [46]Molitor JA, Walker WH, Doerre S, Ballard DW, Greene WC: NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells. Proc Natl Acad Sci USA 1990, 87(24):10028-10032.
• [47]Burger S, Tatge H, Hofmann F, Genth H, Just I, Gerhard R: Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system. Biochem Biophys Res Commun 2003, 307(3):584-588.
• [48]Gerhard R, Nottrott S, Schoentaube J, Tatge H, Olling A, Just I: Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells. J Med Microbiol 2008, 57(Pt 6):765-770.
• [49]Schmidt A, Kellermann J, Lottspeich F: A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 2005, 5(1):4-15.
• [50]Luecke N, Templin C, Muetzelburg MV, Neumann D, Just I, Pich A: Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix. Rapid Commun Mass Spectrom 2010, 24(5):561-570.
• [51]Vizcaino JA, Cote R, Reisinger F, Barsnes H, Foster JM, Rameseder J, Hermjakob H, Martens L: The Proteomics Identifications database: 2010 update. Nucleic Acids Res 2010, (38 Database):D736-42.
• [52]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227(5259):680-685.