Harris T O, Grossman D, Kappler J W, Marrack P, Rich R R, Betley M J
Department of Bacteriology, University of Wisconsin-Madison 53706.
Infect Immun. 1993 Aug;61(8):3175-83. doi: 10.1128/iai.61.8.3175-3183.1993.
This study examined the emetic activity of several staphylococcal enterotoxin type A and B (SEA and SEB, respectively) mutants that had either one or two amino acid residue substitutions. New sea gene mutations were constructed by site-directed mutagenesis; gene products were obtained with glycine residues at position 25, 47, 48, 81, 85, or 86 of mature SEA. Culture supernatants from Staphylococcus aureus RN4220, or derivatives containing either sea or a sea mutation, were analyzed for the ability to stimulate proliferation of murine splenocytes, as determined by incorporation of [3H]thymidine. Culture supernatants containing SEA-N25G (a SEA mutant with a substitution of glycine for the asparagine residue at position 25), SEA-F47G, or SEA-L48G did not stimulate T-cell proliferation, unlike supernatants containing the other substitution mutants. Purified preparations of SEA-N25G had weak activity and those of SEA-F47G and SEA-L48G had essentially no activity in the T-cell proliferation assay. All mutants except SEA-V85G, which was degraded by monkey stomach lavage fluid in vitro, were tested for emetic activity. SEA-C106A and two SEB mutants, SEB-D9N/N23D and SEB-F44S (previously referred to as BR-257 and BR-358, respectively), whose construction and altered immunological properties have been reported previously, were also tested in the emetic assay. Each mutant was initially administered intragastrically at doses of 75 to 100 micrograms per animal; if none of the animals responded, the dose was increased four-to fivefold. SEA-F47G, SEA-C106A, and SEB-D9N/N23D were the only mutants that did not induce vomiting at either dose tested; these three mutants had reduced immunological activity. However, there was not a perfect correlation between immunological and emetic activities; SEA-L48G and SEB-F44S retained emetic activity, although they had essentially no T-cell-stimulatory activity. These studies suggest that these two activities can be dissociated.
本研究检测了几种分别具有一个或两个氨基酸残基替换的A型和B型葡萄球菌肠毒素（分别为SEA和SEB）突变体的催吐活性。通过定点诱变构建了新的sea基因突变体；在成熟SEA的第25、47、48、81、85或86位获得了带有甘氨酸残基的基因产物。分析了金黄色葡萄球菌RN4220或含有sea或sea突变体的衍生物的培养上清液刺激小鼠脾细胞增殖的能力，通过[3H]胸苷掺入法进行测定。与含有其他替换突变体的上清液不同，含有SEA-N25G（一种在第25位用甘氨酸替换天冬酰胺残基的SEA突变体）、SEA-F47G或SEA-L48G的培养上清液不刺激T细胞增殖。纯化的SEA-N25G制剂活性较弱，而SEA-F47G和SEA-L48G制剂在T细胞增殖试验中基本无活性。除SEA-V85G在体外被猴胃灌洗液降解外，所有突变体均进行了催吐活性测试。SEA-C106A以及两个SEB突变体SEB-D9N/N23D和SEB-F44S（之前分别称为BR-257和BR-358，其构建和改变的免疫特性已在之前报道）也进行了催吐试验。每个突变体最初以每只动物75至100微克的剂量经胃内给药；如果没有动物出现反应，则将剂量增加四至五倍。SEA-F47G、SEA-C106A和SEB-D9N/N23D是在所测试的两种剂量下均未诱导呕吐的仅有的突变体；这三个突变体的免疫活性降低。然而，免疫活性与催吐活性之间并非完全相关；SEA-L48G和SEB-F44S虽然基本没有T细胞刺激活性，但仍保留催吐活性。这些研究表明这两种活性可以分离。
