Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1994 May;62(5):1768–1775. doi: 10.1128/iai.62.5.1768-1775.1994

Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity.

P R Austin 1, P E Jablonski 1, G A Bohach 1, A K Dunker 1, C J Hovde 1
PMCID: PMC186405  PMID: 8168939

Abstract

Escherichia coli Shiga-like toxin type I (SLT-I) is a potent cytotoxin consisting of an enzymatically active A subunit and a pentameric B subunit that mediates toxin binding to susceptible eukaryotic cells. Evidence that the carboxy-terminal 38 amino acids of the A subunit are involved in holotoxin 1A:5B association is presented. We compared the ability of purified recombinant SLT-I B subunit (Slt-IB) to combine in vitro with purified recombinant SLT-I A subunit (Slt-IA; full-length subunit A includes amino acids 1 to 293) and its ability to combine with purified recombinant SLT-I A1 subunit (Slt-IA1; truncated subunit A includes amino acids 1 to 255). Each mixture was analyzed for biological and physical evidence of toxin assembly. Although Slt-IA successfully combined with Slt-IB to form a molecular species similar to holotoxin that was detectable by nondenaturing polyacrylamide gel electrophoresis and immunoblotting and yielded a molecule which was cytotoxic to cultured Vero cells, Slt-IA1 did not have this ability. Slt-IA1 was 36-fold more active than Slt-IA in an in vitro protein synthesis inhibition assay. These findings suggest that the Slt-IA2 fragment is crucial for formation of SLT holotoxin and stabilizes the interaction between the A and B subunits.

Full text

PDF
1771

Images in this article

1770Image
on p.1770
1771Image
on p.1771
1772Image
on p.1772

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brown J. E., Ussery M. A., Leppla S. H., Rothman S. W. Inhibition of protein synthesis by Shiga toxin: activation of the toxin and inhibition of peptide elongation. FEBS Lett. 1980 Aug 11;117(1):84–88. doi: 10.1016/0014-5793(80)80918-5. [DOI] [PubMed] [Google Scholar]
  2. Calderwood S. B., Acheson D. W., Goldberg M. B., Boyko S. A., Donohue-Rolfe A. A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit. Infect Immun. 1990 Sep;58(9):2977–2982. doi: 10.1128/iai.58.9.2977-2982.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Calderwood S. B., Auclair F., Donohue-Rolfe A., Keusch G. T., Mekalanos J. J. Nucleotide sequence of the Shiga-like toxin genes of Escherichia coli. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4364–4368. doi: 10.1073/pnas.84.13.4364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chanter N., Hall G. A., Bland A. P., Hayle A. J., Parsons K. R. Dysentery in calves caused by an atypical strain of Escherichia coli (S102-9). Vet Microbiol. 1986 Sep;12(3):241–253. doi: 10.1016/0378-1135(86)90053-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Grandis S., Ginsberg J., Toone M., Climie S., Friesen J., Brunton J. Nucleotide sequence and promoter mapping of the Escherichia coli Shiga-like toxin operon of bacteriophage H-19B. J Bacteriol. 1987 Sep;169(9):4313–4319. doi: 10.1128/jb.169.9.4313-4319.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeGrandis S., Law H., Brunton J., Gyles C., Lingwood C. A. Globotetraosylceramide is recognized by the pig edema disease toxin. J Biol Chem. 1989 Jul 25;264(21):12520–12525. [PubMed] [Google Scholar]
  7. Deresiewicz R. L., Austin P. R., Hovde C. J. The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain. Mol Gen Genet. 1993 Nov;241(3-4):467–473. doi: 10.1007/BF00284701. [DOI] [PubMed] [Google Scholar]
  8. Donohue-Rolfe A., Keusch G. T. Shigella dysenteriae 1 cytotoxin: periplasmic protein releasable by polymyxin B and osmotic shock. Infect Immun. 1983 Jan;39(1):270–274. doi: 10.1128/iai.39.1.270-274.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gannon V. P., Teerling C., Masri S. A., Gyles C. L. Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family. J Gen Microbiol. 1990 Jun;136(6):1125–1135. doi: 10.1099/00221287-136-6-1125. [DOI] [PubMed] [Google Scholar]
  10. Gill D. M. The arrangement of subunits in cholera toxin. Biochemistry. 1976 Mar 23;15(6):1242–1248. doi: 10.1021/bi00651a011. [DOI] [PubMed] [Google Scholar]
  11. Goldberg I., Mekalanos J. J. Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant. J Bacteriol. 1986 Mar;165(3):715–722. doi: 10.1128/jb.165.3.715-722.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Griffin D. E., Gemski P. Release of Shiga toxin from Shigella dysenteriae 1 by polymyxin B. Infect Immun. 1983 Apr;40(1):425–428. doi: 10.1128/iai.40.1.425-428.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haddad J. E., al-Jaufy A. Y., Jackson M. P. Minimum domain of the Shiga toxin A subunit required for enzymatic activity. J Bacteriol. 1993 Aug;175(16):4970–4978. doi: 10.1128/jb.175.16.4970-4978.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ito H., Yutsudo T., Hirayama T., Takeda Y. Isolation and some properties of A and B subunits of Vero toxin 2 and in vitro formation of hybrid toxins between subunits of Vero toxin 1 and Vero toxin 2 from Escherichia coli O157:H7. Microb Pathog. 1988 Sep;5(3):189–195. doi: 10.1016/0882-4010(88)90021-6. [DOI] [PubMed] [Google Scholar]
  15. Jacewicz M., Clausen H., Nudelman E., Donohue-Rolfe A., Keusch G. T. Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide. J Exp Med. 1986 Jun 1;163(6):1391–1404. doi: 10.1084/jem.163.6.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jackson M. P. Structure-function analyses of Shiga toxin and the Shiga-like toxins. Microb Pathog. 1990 Apr;8(4):235–242. doi: 10.1016/0882-4010(90)90050-z. [DOI] [PubMed] [Google Scholar]
  17. Keusch G. T., Donohue-Rolfe A., Jacewicz M., Kane A. V. Shiga toxin: production and purification. Methods Enzymol. 1988;165:152-62, 399-401. doi: 10.1016/s0076-6879(88)65025-7. [DOI] [PubMed] [Google Scholar]
  18. Konowalchuk J., Speirs J. I., Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun. 1977 Dec;18(3):775–779. doi: 10.1128/iai.18.3.775-779.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lindberg A. A., Brown J. E., Strömberg N., Westling-Ryd M., Schultz J. E., Karlsson K. A. Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1. J Biol Chem. 1987 Feb 5;262(4):1779–1785. [PubMed] [Google Scholar]
  21. Locht C., Keith J. M. Pertussis toxin gene: nucleotide sequence and genetic organization. Science. 1986 Jun 6;232(4755):1258–1264. doi: 10.1126/science.3704651. [DOI] [PubMed] [Google Scholar]
  22. Murzin A. G. OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J. 1993 Mar;12(3):861–867. doi: 10.1002/j.1460-2075.1993.tb05726.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nicosia A., Perugini M., Franzini C., Casagli M. C., Borri M. G., Antoni G., Almoni M., Neri P., Ratti G., Rappuoli R. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4631–4635. doi: 10.1073/pnas.83.13.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Olsnes S., Reisbig R., Eiklid K. Subunit structure of Shigella cytotoxin. J Biol Chem. 1981 Aug 25;256(16):8732–8738. [PubMed] [Google Scholar]
  26. Paton A. W., Paton J. C., Goldwater P. N., Heuzenroeder M. W., Manning P. A. Sequence of a variant Shiga-like toxin type-I operon of Escherichia coli O111:H-. Gene. 1993 Jul 15;129(1):87–92. doi: 10.1016/0378-1119(93)90700-d. [DOI] [PubMed] [Google Scholar]
  27. Reisbig R., Olsnes S., Eiklid K. The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit. J Biol Chem. 1981 Aug 25;256(16):8739–8744. [PubMed] [Google Scholar]
  28. Ribi H. O., Ludwig D. S., Mercer K. L., Schoolnik G. K., Kornberg R. D. Three-dimensional structure of cholera toxin penetrating a lipid membrane. Science. 1988 Mar 11;239(4845):1272–1276. doi: 10.1126/science.3344432. [DOI] [PubMed] [Google Scholar]
  29. Sandvig K., Garred O., Holm P. K., van Deurs B. Endocytosis and intracellular transport of protein toxins. Biochem Soc Trans. 1993 Aug;21(3):707–711. doi: 10.1042/bst0210707. [DOI] [PubMed] [Google Scholar]
  30. Saxena S. K., O'Brien A. D., Ackerman E. J. Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes. J Biol Chem. 1989 Jan 5;264(1):596–601. [PubMed] [Google Scholar]
  31. Sixma T. K., Aguirre A., Terwisscha van Scheltinga A. C., Wartna E. S., Kalk K. H., Hol W. G. Heat-labile enterotoxin crystal forms with variable A/B5 orientation. Analysis of conformational flexibility. FEBS Lett. 1992 Jun 29;305(2):81–85. doi: 10.1016/0014-5793(92)80869-i. [DOI] [PubMed] [Google Scholar]
  32. Sixma T. K., Pronk S. E., Kalk K. H., Wartna E. S., van Zanten B. A., Witholt B., Hol W. G. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature. 1991 May 30;351(6325):371–377. doi: 10.1038/351371a0. [DOI] [PubMed] [Google Scholar]
  33. Speirs J. I., Stavric S., Konowalchuk J. Assay of Escherichia coli heat-labile enterotoxin with vero cells. Infect Immun. 1977 May;16(2):617–622. doi: 10.1128/iai.16.2.617-622.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stein P. E., Boodhoo A., Tyrrell G. J., Brunton J. L., Read R. J. Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli. Nature. 1992 Feb 20;355(6362):748–750. doi: 10.1038/355748a0. [DOI] [PubMed] [Google Scholar]
  35. Tamura M., Nogimori K., Murai S., Yajima M., Ito K., Katada T., Ui M., Ishii S. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry. 1982 Oct 26;21(22):5516–5522. doi: 10.1021/bi00265a021. [DOI] [PubMed] [Google Scholar]
  36. Waddell T., Head S., Petric M., Cohen A., Lingwood C. Globotriosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2. Biochem Biophys Res Commun. 1988 Apr 29;152(2):674–679. doi: 10.1016/s0006-291x(88)80091-3. [DOI] [PubMed] [Google Scholar]
  37. Weinstein D. L., Jackson M. P., Samuel J. E., Holmes R. K., O'Brien A. D. Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. J Bacteriol. 1988 Sep;170(9):4223–4230. doi: 10.1128/jb.170.9.4223-4230.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yutsudo T., Honda T., Miwatani T., Takeda Y. Physicochemical characterization of A and B subunits of Shiga toxin and reconstitution of holotoxin from isolated subunits. Microbiol Immunol. 1987;31(3):189–197. doi: 10.1111/j.1348-0421.1987.tb03083.x. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES