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. 1996 May;64(5):1569–1576. doi: 10.1128/iai.64.5.1569-1576.1996

Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice.

A R Melton-Celsa 1, S C Darnell 1, A D O'Brien 1
PMCID: PMC173963  PMID: 8613362

Abstract

The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from mouse small intestine and colon and that growth in small-intestine mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incubation with human colonic mucus. In contrast, culture supernatants from LB-grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc or SLT-IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxicity activity. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Bockemühl J., Aleksić S., Karch H. Serological and biochemical properties of Shiga-like toxin (verocytotoxin)-producing strains of Escherichia coli, other than O-group 157, from patients in Germany. Zentralbl Bakteriol. 1992 Jan;276(2):189–195. doi: 10.1016/s0934-8840(11)80005-8. [DOI] [PubMed] [Google Scholar]
  3. Burgess B. J., Roberts L. M. Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga-like toxin I A subunit is not essential for cytotoxicity. Mol Microbiol. 1993 Oct;10(1):171–179. doi: 10.1111/j.1365-2958.1993.tb00913.x. [DOI] [PubMed] [Google Scholar]
  4. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  5. Fontaine A., Arondel J., Sansonetti P. J. Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox- mutant of Shigella dysenteriae 1. Infect Immun. 1988 Dec;56(12):3099–3109. doi: 10.1128/iai.56.12.3099-3109.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fraser M. E., Chernaia M. M., Kozlov Y. V., James M. N. Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 A resolution. Nat Struct Biol. 1994 Jan;1(1):59–64. doi: 10.1038/nsb0194-59. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Griffin P. M., Ostroff S. M., Tauxe R. V., Greene K. D., Wells J. G., Lewis J. H., Blake P. A. Illnesses associated with Escherichia coli O157:H7 infections. A broad clinical spectrum. Ann Intern Med. 1988 Nov 1;109(9):705–712. doi: 10.7326/0003-4819-109-9-705. [DOI] [PubMed] [Google Scholar]
  9. Hii J. H., Gyles C., Morooka T., Karmali M. A., Clarke R., De Grandis S., Brunton J. L. Development of verotoxin 2- and verotoxin 2 variant (VT2v)-specific oligonucleotide probes on the basis of the nucleotide sequence of the B cistron of VT2v from Escherichia coli E32511 and B2F1. J Clin Microbiol. 1991 Dec;29(12):2704–2709. doi: 10.1128/jcm.29.12.2704-2709.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ito H., Terai A., Kurazono H., Takeda Y., Nishibuchi M. Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome. Microb Pathog. 1990 Jan;8(1):47–60. doi: 10.1016/0882-4010(90)90007-d. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Jemal C., Haddad J. E., Begum D., Jackson M. P. Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis. J Bacteriol. 1995 Jun;177(11):3128–3132. doi: 10.1128/jb.177.11.3128-3132.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Lindgren S. W., Melton A. R., O'Brien A. D. Virulence of enterohemorrhagic Escherichia coli O91:H21 clinical isolates in an orally infected mouse model. Infect Immun. 1993 Sep;61(9):3832–3842. doi: 10.1128/iai.61.9.3832-3842.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lindgren S. W., Samuel J. E., Schmitt C. K., O'Brien A. D. The specific activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia coli differ when measured by Vero cell cytotoxicity but not by mouse lethality. Infect Immun. 1994 Feb;62(2):623–631. doi: 10.1128/iai.62.2.623-631.1994. [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. Paton A. W., Bourne A. J., Manning P. A., Paton J. C. Comparative toxicity and virulence of Escherichia coli clones expressing variant and chimeric Shiga-like toxin type II operons. Infect Immun. 1995 Jul;63(7):2450–2458. doi: 10.1128/iai.63.7.2450-2458.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Paton A. W., Paton J. C., Heuzenroeder M. W., Goldwater P. N., Manning P. A. Cloning and nucleotide sequence of a variant Shiga-like toxin II gene from Escherichia coli OX3:H21 isolated from a case of sudden infant death syndrome. Microb Pathog. 1992 Sep;13(3):225–236. doi: 10.1016/0882-4010(92)90023-h. [DOI] [PubMed] [Google Scholar]
  19. Paton A. W., Paton J. C., Manning P. A. Polymerase chain reaction amplification, cloning and sequencing of variant Escherichia coli Shiga-like toxin type II operons. Microb Pathog. 1993 Jul;15(1):77–82. doi: 10.1006/mpat.1993.1058. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Schmidt H., Montag M., Bockemühl J., Heesemann J., Karch H. Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples. Infect Immun. 1993 Feb;61(2):534–543. doi: 10.1128/iai.61.2.534-543.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schmitt C. K., McKee M. L., O'Brien A. D. Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escherichia coli strains are responsible for the antigenic heterogeneity of the O157:H- strain E32511. Infect Immun. 1991 Mar;59(3):1065–1073. doi: 10.1128/iai.59.3.1065-1073.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sixma T. K., Kalk K. H., van Zanten B. A., Dauter Z., Kingma J., Witholt B., Hol W. G. Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin. J Mol Biol. 1993 Apr 5;230(3):890–918. doi: 10.1006/jmbi.1993.1209. [DOI] [PubMed] [Google Scholar]
  25. Strockbine N. A., Jackson M. P., Sung L. M., Holmes R. K., O'Brien A. D. Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1. J Bacteriol. 1988 Mar;170(3):1116–1122. doi: 10.1128/jb.170.3.1116-1122.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Strockbine N. A., Marques L. R., Holmes R. K., O'Brien A. D. Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli. Infect Immun. 1985 Dec;50(3):695–700. doi: 10.1128/iai.50.3.695-700.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Takeda Y., Kurazono H., Yamasaki S. Vero toxins (Shiga-like toxins) produced by enterohemorrhagic Escherichia coli (verocytotoxin-producing E. coli). Microbiol Immunol. 1993;37(8):591–599. doi: 10.1111/j.1348-0421.1993.tb01681.x. [DOI] [PubMed] [Google Scholar]
  28. Tesh V. L., Burris J. A., Owens J. W., Gordon V. M., Wadolkowski E. A., O'Brien A. D., Samuel J. E. Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice. Infect Immun. 1993 Aug;61(8):3392–3402. doi: 10.1128/iai.61.8.3392-3402.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tesh V. L., O'Brien A. D. The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol Microbiol. 1991 Aug;5(8):1817–1822. doi: 10.1111/j.1365-2958.1991.tb00805.x. [DOI] [PubMed] [Google Scholar]
  30. Tesh V. L., Ramegowda B., Samuel J. E. Purified Shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages. Infect Immun. 1994 Nov;62(11):5085–5094. doi: 10.1128/iai.62.11.5085-5094.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wadolkowski E. A., Burris J. A., O'Brien A. D. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7. Infect Immun. 1990 Aug;58(8):2438–2445. doi: 10.1128/iai.58.8.2438-2445.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wadolkowski E. A., Sung L. M., Burris J. A., Samuel J. E., O'Brien A. D. Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin type II. Infect Immun. 1990 Dec;58(12):3959–3965. doi: 10.1128/iai.58.12.3959-3965.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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