Journal of Periodontal and Implant Science

Korean Academy of Periodontology (대한치주과학회)
권호 표지

Co-expression of CdtA and CdtC subunits of cytolethal distending toxin from Aggregatibacter actinomycetemcomitans

  • Lee, Seung-Jae (Department of Periodontology, College of Dentistry, Chonbuk National University) ;
  • Lee, Kyung-Yeol (Department of Oral Microbiology and Institute of Oral Bioscience, Brain Korea 21 Project, Chonbuk National University) ;
  • Kim, Hyung-Seop (Department of Periodontology, College of Dentistry, Chonbuk National University)
  • Published : 2009.08.15
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

Keywords

References

  1. Henderson B, Nair SP, Ward JM, Wilson M. Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans. Annu Rev Microbiol 2003;57:29-55 https://doi.org/10.1146/annurev.micro.57.030502.090908
  2. Lara-Tejero M, Galan JE. Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions. Trends Microbiol 2002;10:147-152 https://doi.org/10.1016/S0966-842X(02)02316-8
  3. Leung WK, Ngai VKS, Yau JYY et al. Characterization of Actinobacillus actinomycetemcomitans isolated from young Chinese aggressive periodontitis patients. J Periodontal Res 2005;40:258-268 https://doi.org/10.1111/j.1600-0765.2005.00805.x
  4. Ahmed HJ, Svensson LA, Cope LD et al. Prevalence of cdtABC genes encoding cytolethal distending toxin among Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains. J Med Microbiol 2001;50: 860-864 https://doi.org/10.1099/0022-1317-50-10-860
  5. Fabris AS, DiRienzo JM, Wikstrom M, Mayer MP. Detection of cytolethal distending toxin activity and cdt genes in Actinobacillus actinomycetemcomitansisolates from geographically diverse populations. Oral Microbiol Immunol 2002;17:231-238 https://doi.org/10.1034/j.1399-302X.2002.170405.x
  6. Belibasakis G, Johansson A, Wang Y et al. Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin. Eur J Oral Sci 2002;110:366-373 https://doi.org/10.1034/j.1600-0722.2002.21350.x
  7. Kanno F, Korostoff J, Volgina A, DiRienzo JM. Resistance of human periodontal ligament fibroblasts to the cytolethal distending toxin of Actinobacillus actinomycetemcomitans. J Periodontol 2005;76:1189-1201 https://doi.org/10.1902/jop.2005.76.7.1189
  8. DiRienzo JM, Song M, Wan LSY, Ellen RP. Kinetics of KB and HEp-2 cell responses to an invasive, cytolethal distending toxin producing strain of Actinobacillus actinomycetemcomitans. Oral Microbiol Immunol 2002;17:245-251 https://doi.org/10.1034/j.1399-302X.2002.170407.x
  9. Belibasakis GN, Johansson A, Wang Y et al. The cytolethal distending toxin induces receptor activator of NF-kappaB ligand expression in human gingival fibroblasts and periodontal ligament cells. Infect Immun 2005;73:342-351 https://doi.org/10.1128/IAI.73.1.342-351.2005
  10. Belibasakis GN, Brage M, Lagergard T, Johansson A. Cytolethal distending toxin upregulates RANKL expression in Jurkat T-cells. APMIS 2008;116:499-506 https://doi.org/10.1111/j.1600-0463.2008.01017.x
  11. Mogi M, Otogoto J, Ota N, Togari A. Differential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with periodontitis. J Dent Res 2004;83:166-169 https://doi.org/10.1177/154405910408300216
  12. Pickett CL, Cottle DL, Pesci EC, Bikah G. Cloning, sequencing, and expression of the Escherichia coli cytolethal distending toxin genes. Infect Immun 1994;62:1046-1051
  13. Elwell CA, Dreyfus LA.DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Mol Microbiol 2000;37:952-963 https://doi.org/10.1046/j.1365-2958.2000.02070.x
  14. Nesic D, Hsu Y, Stebbins CE. Assembly and function of a bacterial genotoxin. Nature 2004;429:429-433 https://doi.org/10.1038/nature02532
  15. Peres SY, Marches O, Daigle F et al. A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase. Mol Microbiol 1997;24:1095-1107 https://doi.org/10.1046/j.1365-2958.1997.4181785.x
  16. Lee RB, Hassane DC, Cottle DL, Pickett CL. Interactions of Campylobacter jejuni cytolethal distending toxin subunits CdtA and CdtC with HeLa cells. Infect Immun 2003;71:4883-4890 https://doi.org/10.1128/IAI.71.9.4883-4890.2003
  17. McSweeney LA, Dreyfus LA. Carbohydrate-binding specif icity of the Escherichia coli cytolethal distending toxin CdtA-II and CdtC-II subunits. Infect Immun 2005;73:2051-2060 https://doi.org/10.1128/IAI.73.4.2051-2060.2005
  18. Mise K, Akifusa S, Watarai S et al. Involvement of ganglioside GM3 in G2/M cell cycle arrest of human monocytic cells induced by Actinobacillus actinomycetemcomitans cytolethal distending toxin. Infect Immun 2005;73:4846-4852 https://doi.org/10.1128/IAI.73.8.4846-4852.2005
  19. Yamada T, Komoto J, Saiki K, Konishi K, Takusagawa F. Variation of loop sequence alters stability of cytolethal distending toxin (CDT): crystal structure of CDT from Actinobacillus actinomycetemcomitans. Protein Sci 2006;15:362-372 https://doi.org/10.1110/ps.051790506
  20. Boesze-Battaglia K, Besack D, McKay T et al. Cholesterolrich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal-distending toxin. Cell Microbiol 2006;8:823-836 https://doi.org/10.1111/j.1462-5822.2005.00669.x
  21. Saiki K, Konishi K, Gomi T, Nishihara T, Yoshikawa M. Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans. Microbiol Immunol 2001;45:497-506 https://doi.org/10.1111/j.1348-0421.2001.tb02650.x
  22. Ko SY, Jeong DK, Ryu SH, Kim HS. Cloning and protein expression of Actinobacillus actinomycetemcomitans cytolethal distending toxin subunit CdtA. J Korean Acad Periodontol 2007;37:339-351 https://doi.org/10.5051/jkape.2007.37.Suppl.339
  23. Lee ES, Park SY, Lee EU, Kim HS. Cloning and protein expression of Aggregatibacter actinomycetemcomitans cytolethal distending toxin C. J Korean Acad Periodontol 2008;38:317-324 https://doi.org/10.5051/jkape.2008.38.Suppl.317
  24. Mao X, DiRienzo JM. Functional studies of the recombinant subunits of a cytolethal distending holotoxin. Cell Microbiol 2002;4:245-255 https://doi.org/10.1046/j.1462-5822.2002.00186.x
  25. Nesic D, Stebbins CE. Mechanisms of assembly and cellular interactions for the bacterial genotoxin CDT. PLoS Pathog 2005;1:214-224
  26. Ueno Y, Ohara M, Kawamoto T et al. Biogenesis of the Actinobacillus actinomycetemcomitans cytolethal distending toxin holotoxin. Infect Immun 2006;74:3480-3487 https://doi.org/10.1128/IAI.00739-05
  27. Lagergard T, Lundqvist A, Wising C, Gabrielsson V, Ahlman K. Formaldehyde treatment increases the immunogenicity and decreases the toxicity of Haemophilus ducreyi cytolethal distending toxin. Vaccine 2007;25:3606-3614 https://doi.org/10.1016/j.vaccine.2007.01.061
  28. Mbwana J, Ahmed HJ, Ahlman K et al. Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid. Microb Pathog 2003;35:133-137 https://doi.org/10.1016/S0882-4010(03)00111-6