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. 1991 Mar;59(3):829–835. doi: 10.1128/iai.59.3.829-835.1991

Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.

L P Perera 1, J E Samuel 1, R K Holmes 1, A D O'Brien 1
PMCID: PMC258334  PMID: 1997433

Abstract

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.

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Selected References

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  1. Downes F. P., Barrett T. J., Green J. H., Aloisio C. H., Spika J. S., Strockbine N. A., Wachsmuth I. K. Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies. Infect Immun. 1988 Aug;56(8):1926–1933. doi: 10.1128/iai.56.8.1926-1933.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Endo Y., Tsurugi K., Yutsudo T., Takeda Y., Ogasawara T., Igarashi K. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur J Biochem. 1988 Jan 15;171(1-2):45–50. doi: 10.1111/j.1432-1033.1988.tb13756.x. [DOI] [PubMed] [Google Scholar]
  3. Gentry M. K., Dalrymple J. M. Quantitative microtiter cytotoxicity assay for Shigella toxin. J Clin Microbiol. 1980 Sep;12(3):361–366. doi: 10.1128/jcm.12.3.361-366.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hovde C. J., Calderwood S. B., Mekalanos J. J., Collier R. J. Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2568–2572. doi: 10.1073/pnas.85.8.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jackson M. P., Deresiewicz R. L., Calderwood S. B. Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits. J Bacteriol. 1990 Jun;172(6):3346–3350. doi: 10.1128/jb.172.6.3346-3350.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jackson M. P., Wadolkowski E. A., Weinstein D. L., Holmes R. K., O'Brien A. D. Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis. J Bacteriol. 1990 Feb;172(2):653–658. doi: 10.1128/jb.172.2.653-658.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnson W. M., Lior H., Bezanson G. S. Cytotoxic Escherichia coli O157:H7 associated with haemorrhagic colitis in Canada. Lancet. 1983 Jan 1;1(8314-5):76–76. doi: 10.1016/s0140-6736(83)91616-1. [DOI] [PubMed] [Google Scholar]
  8. Karmali M. A. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38. doi: 10.1128/cmr.2.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kozlov YuV, Kabishev A. A., Lukyanov E. V., Bayev A. A. The primary structure of the operons coding for Shigella dysenteriae toxin and temperature phage H30 shiga-like toxin. Gene. 1988 Jul 30;67(2):213–221. doi: 10.1016/0378-1119(88)90398-8. [DOI] [PubMed] [Google Scholar]
  10. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lingwood C. A., Law H., Richardson S., Petric M., Brunton J. L., De Grandis S., Karmali M. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J Biol Chem. 1987 Jun 25;262(18):8834–8839. [PubMed] [Google Scholar]
  13. May M. J., Hartley M. R., Roberts L. M., Krieg P. A., Osborn R. W., Lord J. M. Ribosome inactivation by ricin A chain: a sensitive method to assess the activity of wild-type and mutant polypeptides. EMBO J. 1989 Jan;8(1):301–308. doi: 10.1002/j.1460-2075.1989.tb03377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Montfort W., Villafranca J. E., Monzingo A. F., Ernst S. R., Katzin B., Rutenber E., Xuong N. H., Hamlin R., Robertus J. D. The three-dimensional structure of ricin at 2.8 A. J Biol Chem. 1987 Apr 15;262(11):5398–5403. [PubMed] [Google Scholar]
  15. Newland J. W., Strockbine N. A., Neill R. J. Cloning of genes for production of Escherichia coli Shiga-like toxin type II. Infect Immun. 1987 Nov;55(11):2675–2680. doi: 10.1128/iai.55.11.2675-2680.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. O'Brien A. O., Lively T. A., Chen M. E., Rothman S. W., Formal S. B. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (SHIGA) like cytotoxin. Lancet. 1983 Mar 26;1(8326 Pt 1):702–702. doi: 10.1016/s0140-6736(83)91987-6. [DOI] [PubMed] [Google Scholar]
  18. Perera L. P., Marques L. R., O'Brien A. D. Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay. J Clin Microbiol. 1988 Oct;26(10):2127–2131. doi: 10.1128/jcm.26.10.2127-2131.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Riley L. W., Remis R. S., Helgerson S. D., McGee H. B., Wells J. G., Davis B. R., Hebert R. J., Olcott E. S., Johnson L. M., Hargrett N. T. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med. 1983 Mar 24;308(12):681–685. doi: 10.1056/NEJM198303243081203. [DOI] [PubMed] [Google Scholar]
  20. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  21. Samuel J. E., Perera L. P., Ward S., O'Brien A. D., Ginsburg V., Krivan H. C. Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants. Infect Immun. 1990 Mar;58(3):611–618. doi: 10.1128/iai.58.3.611-618.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schlossman D., Withers D., Welsh P., Alexander A., Robertus J., Frankel A. Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes. Mol Cell Biol. 1989 Nov;9(11):5012–5021. doi: 10.1128/mcb.9.11.5012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scotland S. M., Smith H. R., Rowe B. Two distinct toxins active on Vero cells from Escherichia coli O157. Lancet. 1985 Oct 19;2(8460):885–886. doi: 10.1016/s0140-6736(85)90146-1. [DOI] [PubMed] [Google Scholar]
  24. Siegall C. B., Chaudhary V. K., FitzGerald D. J., Pastan I. Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin. J Biol Chem. 1989 Aug 25;264(24):14256–14261. [PubMed] [Google Scholar]
  25. Smith H. R., Scotland S. M. Vero cytotoxin-producing strains of Escherichia coli. J Med Microbiol. 1988 Jun;26(2):77–85. doi: 10.1099/00222615-26-2-77. [DOI] [PubMed] [Google Scholar]
  26. Strockbine N. A., Marques L. R., Newland J. W., Smith H. W., Holmes R. K., O'Brien A. D. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect Immun. 1986 Jul;53(1):135–140. doi: 10.1128/iai.53.1.135-140.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weinstein D. L., Jackson M. P., Perera L. P., Holmes R. K., O'Brien A. D. In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity. Infect Immun. 1989 Dec;57(12):3743–3750. doi: 10.1128/iai.57.12.3743-3750.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]

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