Mitchison M, Bulach D M, Vinh T, Rajakumar K, Faine S, Adler B
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
J Bacteriol. 1997 Feb;179(4):1262-7. doi: 10.1128/jb.179.4.1262-1267.1997.
Immunity to leptospirosis is principally humorally mediated and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. The only protective antigen identified to date is the leptospiral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little is known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragment from the chromosome of serovar Copenhageni was cloned in pUC18 in Escherichia coli, after which flanking regions were cloned from a genomic library constructed in bacteriophage lambda GEM12. Sequence analysis identified four open reading frames which showed similarity to the rfbC, rfbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the assembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showed similarity with the rhamnosyltransferase genes of Shigella and Yersinia enterocolitica but not Salmonella. Comparison of deduced amino acid sequences showed up to 85% similarity of the leptospiral proteins with those of other gram-negative bacteria. Polyacrylamide gel electrophoresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira. Moreover, it could restore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexneri which was deficient in all four genes, thereby confirming the functional identification of a part of the leptospiral rfb locus.
钩端螺旋体病的免疫主要由体液介导，涉及巨噬细胞和中性粒细胞对钩端螺旋体进行调理吞噬作用。迄今为止确定的唯一保护性抗原是钩端螺旋体脂多糖（LPS），其生化特性类似于典型的革兰氏阴性LPS，但内毒素活性大大降低。关于钩端螺旋体LPS的结构知之甚少。从哥本哈根血清型染色体上分离出一个2.1kb的EcoRI片段，克隆到大肠杆菌的pUC18中，之后从构建于噬菌体λGEM12的基因组文库中克隆侧翼区域。序列分析确定了四个开放阅读框，它们与rfbC、rfbD、rfbB和rfbA基因相似，按此顺序转录，这些基因编码参与肠炎沙门氏菌、大肠杆菌和福氏志贺氏菌中LPS组装的dTDP-鼠李糖生物合成的四种酶。rfbCDBA基因座下游的另一个开放阅读框与志贺氏菌和小肠结肠炎耶尔森氏菌的鼠李糖基转移酶基因相似，但与沙门氏菌不同。推导的氨基酸序列比较显示，钩端螺旋体蛋白与其他革兰氏阴性菌的蛋白相似度高达85%。重组克隆的聚丙烯酰胺凝胶电泳鉴定出推定的RfbCDBA蛋白，而逆转录酶介导的PCR分析表明rfbCDBA基因簇在钩端螺旋体中表达。此外，它可以将正常的LPS表型恢复到福氏志贺氏菌一个明确的rfbB::Tn5突变体，该突变体在所有四个基因上都有缺陷，从而证实了钩端螺旋体rfb基因座一部分的功能鉴定。
