The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule.
Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay.
Hufnagle
W O
WO
Department of Bacteriology, University of Wisconsin-Madison 53706.
Tremaine
M T
MT
Betley
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000378:metabolism; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000737:chemistry*; D005798:Genes, Bacterial / Q000235:genetics; D005779:Immunodiffusion; D007369:Interferon Inducers / Q000737:chemistry*; D008213:Lymphocyte Activation; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D013211:Staphylococcus aureus / Q000235:genetics*; D013601:T-Lymphocytes
D004768:Enterotoxins; D007369:Interferon Inducers; C026066:enterotoxin A, Staphylococcal