Abstract
To generate nontoxic derivatives of Escherichia coli heat-labile enterotoxin (LT), site-directed mutagenesis has been used to change either the amino acid residues located in the catalytic site (M. Pizza, M. Domenighini, W. Hol, V. Giannelli, M. R. Fontana, M. M. Giuliani, C. Magagnoli, S. Peppoloni, R. Manetti, and R. Rappuoli, Mol. Microbiol. 14:51-60, 1994) or those located in the proteolytically sensitive loop that joins the A1 and A2 moieties of the A subunit (C. C. R. Grant, R. J. Messer, and W. J. Cieplack, Infect. Immun. 62:4270-4278, 1994; B. L. Dickinson and J. D. Clements, Infect. Immun. 63:1617-1623, 1995). In this work, we compared the in vitro and in vivo toxic properties and the resistance to protease digestion of the prototype molecules obtained by both approaches (LT-K63 and LT-R192G, respectively). As expected, LT-K63 was normally processed by proteases, while LT-R192G showed increased resistance to trypsin in vitro and was digested by trypsin only under denaturing conditions (3.5 M urea) or by intestinal proteases. No toxicity was detected with the LT-K63 mutant, even when 40 micrograms and 1 mg were used in the in vitro and in vivo assays, respectively. In marked contrast, LT-R192G showed only a modest (10-fold) reduction in toxicity in Y1 cells with a delay in the appearance of the toxic activity and had toxicity comparable to that of wild-type LT in the rabbit ileal loop assay. We conclude that mutagenesis of the active site generates molecules that are fully devoid of toxicity, while mutagenesis of the A1-A2 loop generates molecules that are resistant to trypsin in vitro but still susceptible to proteolytic activation by proteases other than trypsin, and therefore they may still be toxic in tissue culture and in vivo.
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- Clements J. D., Finkelstein R. A. Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures. Infect Immun. 1979 Jun;24(3):760–769. doi: 10.1128/iai.24.3.760-769.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clements J. D., Yancey R. J., Finkelstein R. A. Properties of homogeneous heat-labile enterotoxin from Escherichia coli. Infect Immun. 1980 Jul;29(1):91–97. doi: 10.1128/iai.29.1.91-97.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DE S. N. Enterotoxicity of bacteria-free culture-filtrate of Vibrio cholerae. Nature. 1959 May 30;183(4674):1533–1534. doi: 10.1038/1831533a0. [DOI] [PubMed] [Google Scholar]
- Dickinson B. L., Clements J. D. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect Immun. 1995 May;63(5):1617–1623. doi: 10.1128/iai.63.5.1617-1623.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Douce G., Turcotte C., Cropley I., Roberts M., Pizza M., Domenghini M., Rappuoli R., Dougan G. Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1644–1648. doi: 10.1073/pnas.92.5.1644. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grant C. C., Messer R. J., Cieplak W., Jr Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin. Infect Immun. 1994 Oct;62(10):4270–4278. doi: 10.1128/iai.62.10.4270-4278.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pizza M., Domenighini M., Hol W., Giannelli V., Fontana M. R., Giuliani M. M., Magagnoli C., Peppoloni S., Manetti R., Rappuoli R. Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis. Mol Microbiol. 1994 Oct;14(1):51–60. doi: 10.1111/j.1365-2958.1994.tb01266.x. [DOI] [PubMed] [Google Scholar]
- Pizza M., Fontana M. R., Giuliani M. M., Domenighini M., Magagnoli C., Giannelli V., Nucci D., Hol W., Manetti R., Rappuoli R. A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit. J Exp Med. 1994 Dec 1;180(6):2147–2153. doi: 10.1084/jem.180.6.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rappuoli R., Douce G., Dougan G., Pizza M. Genetic detoxification of bacterial toxins: a new approach to vaccine development. Int Arch Allergy Immunol. 1995 Dec;108(4):327–333. doi: 10.1159/000237176. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Sugii S., Tsuji T. Binding specificities of heat-labile enterotoxins isolated from porcine and human enterotoxigenic Escherichia coli for different gangliosides. Can J Microbiol. 1989 Jun;35(6):670–673. doi: 10.1139/m89-109. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]