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首页> 外文期刊>Journal of bacteriology >Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene ofHaemophilus ducreyi
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Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene ofHaemophilus ducreyi

机译：杜克伊嗜血杆菌脂寡糖半乳糖基转移酶II基因的克隆与鉴定

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Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes theN-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases ofHaemophilus influenzae, Haemophilus somnus,Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in theN-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.
机译： Duemyi嗜血杆菌是生殖器溃疡疾病类chan虫的病原体。脂寡糖（LOS）被认为是主要的毒力决定因素，并与 H的依从性有关。 ducreyi 角化细胞。菌株A77，是从巴黎收集的分离株，对血清敏感，对成纤维细胞的粘附性很差，并且在小菌落的形成上也很缺乏。结构分析表明，菌株A77的LOS缺乏在菌株35000HP LOS的 N -乙酰基乳糖胺部分中发现的半乳糖残基以及唾液酸取代。从 H。 ducreyi 35000HP基因组DNA文库中，通过单克隆抗体（MAb）3F11的免疫筛选鉴定了一个与A77缺陷互补的克隆，MAb可识别35000HP LOS菌株的 N -乙酰基乳糖胺部分。该克隆包含一个已测序的4 kb插入片段。鉴定出一个开放阅读框，其编码分子量为33,400的蛋白质。该蛋白与流感嗜血杆菌，嗜血嗜血杆菌，奈瑟氏球菌和溶血巴斯德氏菌的糖基转移酶具有同源性。推定的 H。将杜克雷伊糖基转移酶基因插入失活，并构建了35000HP菌株的同基因突变体。由该突变体产生的最复杂的LOS糖型在十二烷基硫酸钠-聚丙烯酰胺凝胶上具有与菌株A77的LOS相同的迁移率，并且缺乏3F11结合表位。结构研究证实，从突变体中分离出的LOS的最复杂糖型缺乏在35000HP LOS菌株的 N -乙酰基乳糖胺部分中发现的半乳糖残基。尽管以前发表的数据表明A77的血清敏感性表型是由于LOS突变引起的，但我们观察到互补的A77菌株保留了其血清敏感性表型，而半乳糖基转移酶突变体保留了其血清抗性表型。因此，菌株A77的血清敏感性不能归因于菌株A77的半乳糖基转移酶突变。

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