Molecular Characterization of the Fragilysin Pathogenicity Islet of Enterotoxigenic Bacteroides fragilis

Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.

产肠毒素脆弱拟杆菌（Bacteroides fragilis）菌株可分泌一种细胞外金属蛋白酶毒素，命名为脆弱毒素（fragilysin），该毒素对肠上皮细胞具有细胞致病活性，并可在结扎肠袢中诱导液体分泌与组织损伤。本研究报道，脆弱毒素的编码基因位于一个小型遗传元件之中，该元件被称为脆弱毒素致病岛（pathogenicity island）。脆弱拟杆菌VPI 13784的致病岛全长6033 bp，其末端附近带有近乎完美的12 bp正向重复序列。对另外两株产肠毒素菌株的致病岛末端进行跨端测序，并结合针对另外20株产肠毒素菌株的聚合酶链式反应（PCR）分析，结果显示该致病岛插入于脆弱拟杆菌染色体的特定位点。三株非产毒菌株的整合位点包含一段17 bp的GC富集序列，该序列未在产毒菌株中出现，可作为染色体整合的靶序列。除脆弱毒素编码基因外，本研究还鉴定出一个开放阅读框（open reading frame），其编码的预测蛋白在分子量与结构特征上与脆弱毒素高度相似。推导得到的氨基酸序列与脆弱毒素的一致性为28.5%、相似性为56.3%，且带有近乎完全一致的锌结合基序与甲硫氨酸转角区域。