Identification of a Burkholderia mallei polysaccharide gene duster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant
Identification of a Burkholderia mallei polysaccharide gene duster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant
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DOI：
10.1006/mpat.2000.0430
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发表时间：
2001-05-01
影响因子：
3.8
通讯作者：
Woods, DE
中科院分区：
文献类型：
--
作者：
DeShazer, D;Waag, DM;Woods, DE
我们对马氏伯克氏菌的毒力因子知之甚少，我们采用减法杂交鉴定了马氏伯克氏菌中存在的遗传决定因素，而泰国伯克氏菌是一种非致病性土壤微生物。三个减法杂交产物被定位到一个基因位点，编码参与荚膜多糖的生物合成、输出和易位的蛋白质。我们在荚膜基因簇的一端鉴定了一个插入序列（IS407A），并证明它在B. mallei中具有功能。采用免疫印迹法和酶联免疫吸附法（ELISA）检测突变体与假马利氏伯克霍尔德菌表面多糖抗体的反应性。免疫金电镜显示mallei ATCC 23344（亲本菌株）表面存在荚膜，但在mallei DD3008（荚膜突变体）和泰国芽孢杆菌表面没有荚膜。令人惊讶的是，泰国芽孢杆菌也有一部分荚膜基因簇。ATCC 23344对仓鼠和小鼠均有高毒力，而DD3008对两种动物均无毒力。本研究的结果表明，在两种腺体感染的动物模型中，马来芽孢杆菌的荚膜多糖是产生疾病所必需的，是一个主要的毒力因子。
Little is known about the virulence factors of Burkholderia mallei, the etiologic agent of glanders, We employed subtractive hybridization to identify genetic determinants present in B. mallei but not in Burkholderia thailandensis, a non-pathogenic soil microbe. Three subtractive hybridization products were mapped to a genetic locus encoding proteins involved in the biosynthesis, export and translocation of a capsular polysaccharide. We identified an insertion sequence (IS407A) at one end of the capsule gene cluster and demonstrated that it was functional in B. mallei. Mutations were introduced in the B. mallei capsular gene cluster and the corresponding mutants were examined for their reactivity with antibodies raised against Burkholderia pseudomallei surface polysaccharides by immunoblotting and ELISA. Immunogold electron microscopy demonstrated the presence of a capsule on the surface of B. mallei ATCC 23344 (parental strain) but not on B. mallei DD3008 (capsule mutant) or B. thailandensis. Surprisingly, B. thailandensis also harboured a portion of the capsule gene cluster. ATCC 23344 was highly virulent in hamsters and mice, but DD3008 was avirulent in both animal models. The results presented here demonstrate that the capsular polysaccharide of B. mallei is required for production of disease in two animal models of glanders infection and is a major virulence factor.