Fält I C, Mills D, Schweda E K, Timmis K N, Lindberg A A
Department of Immunology, Microbiology, Pathology and Infectious Diseases, Karolinska Institute, Huddinge Hospital, Sweden.
Microb Pathog. 1996 Jan;20(1):11-30. doi: 10.1006/mpat.1996.0002.
The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced. All derivatives but one (SL3235) stably inherited the new trait. Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS. Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccharide with the following structure, [formula: see text] demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core.
构建了含有编码痢疾志贺氏菌1型脂多糖（LPS）O抗原生物合成的rfp和rfb基因座的TN501耐汞转座子，并将其导入鼠伤寒沙门氏菌和都柏林沙门氏菌的aroA突变体中。在五个重组菌株中，产生了同源LPS和由沙门氏菌脂质A核心和志贺氏菌O抗原组成的杂合LPS。除一个菌株（SL3235）外，所有衍生物都稳定地遗传了新性状。使用对沙门氏菌或志贺氏菌O抗原具有特异性的差异标记抗体混合物进行免疫荧光显微镜检查，结果表明单个细菌产生了两种类型的LPS。通过甲基化分析和核磁共振光谱对纯化LPS轻度水解得到的多糖进行了定性和定量分析，结果显示，基于1,4,5-三-O-乙酰基-2,3-二-O-甲基-L-鼠李糖醇（沙门氏菌重复单元）和鼠李糖醇（志贺氏菌重复单元）的相对比例，分离多糖的高分子量部分中沙门氏菌与志贺氏菌O抗原重复单元的比例在1.3:1至8.4:1之间变化。通过构建仅编码痢疾志贺氏菌1型O抗原一个重复单元合成的突变rfp-rfb基因簇，并将其导入粗糙型沙门氏菌菌株中，研究了志贺氏菌O抗原与沙门氏菌核心的连接位点。这种杂交生物体产生了具有以下结构的多糖，[分子式：见正文]，表明痢疾志贺氏菌1型O抗原与沙门氏菌核心中亚末端D-葡萄糖单元的O-4位相连。
