Dubreuil J D, Kostrzynska M, Austin J W, Trust T J
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
J Bacteriol. 1990 Sep;172(9):5035-43. doi: 10.1128/jb.172.9.5035-5043.1990.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein.
使用酸性甘氨酸缓冲液从胎儿弯曲杆菌菌株中提取的S层蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，不同菌株的主要S层蛋白亚基分子量在90,000至140,000范围内。电子显微镜观察发现，野生型菌株VC1、VC119、VC202和VC203上存在倾斜的S层晶格，间距约为5.6 nm（γ = 75度）。还检测了胎儿弯曲杆菌VC119的三个变体，它们产生的主要S层亚基蛋白分子量与亲本不同。每个变体产生的倾斜晶格在形态上与亲本无法区分。氨基末端序列分析表明，VC119亲本及其变体的S层蛋白在第18位残基之前的序列相同，且该序列与其他三个野生型胎儿弯曲杆菌分离株的S层蛋白共有的前16个N末端残基不同但相关。用针对VC119蛋白制备的抗血清和针对胎儿弯曲杆菌84-40 LP制备的抗血清进行的Western免疫印迹分析（Z.Pei、R.T.Ellison、R.V.Lewis和M.J.Blaser，《生物化学杂志》263:6416 - 6420，1988年）表明，胎儿弯曲杆菌菌株能够产生至少四种不同抗原特异性的S层蛋白。用针对VC119 S层蛋白的抗血清进行免疫电子显微镜观察表明，胎儿弯曲杆菌培养物中含有具有免疫反应性倾斜S层晶格的细胞以及不结合抗体的倾斜S层晶格细胞。这表明胎儿弯曲杆菌S层蛋白会发生抗原变异。热变性实验表明，胎儿弯曲杆菌表面暴露的S层表位赋予的抗原性对热具有异常的抗性，热稳定性似乎归因于S层高度有序的晶格结构。对纯化的VC119 S层蛋白进行蛋白酶消化显示，存在一个对胰蛋白酶、胰凝乳蛋白酶和内蛋白酶Glu-C有抗性的结构域，其表观分子量为110,000，携带S层蛋白的大部分表位，以及一个小的酶敏感结构域。对胰蛋白酶和胰凝乳蛋白酶有抗性的多肽共享一个与完整S层蛋白的N末端序列不同的重叠序列。
