Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis
Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis
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DOI：
10.1128/iai.01240-06
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发表时间：
2007-03-01
影响因子：
3.1
通讯作者：
Wren, B. W.
中科院分区：
文献类型：
--
作者：
Cuccui, J.;Easton, A.;Wren, B. W.
类鼻疽伯克霍尔德氏菌是类鼻疽病的病原体，在东南亚和澳大利亚北方是一种重要的人类病原体，目前还没有疫苗。使用鼻内BALB/c小鼠感染模型筛选了一组892个双签名标记的突变体的毒力。一种新的DNA标签微阵列鉴定出33个突变体在脾脏中被减毒，而6个在肺和脾脏中都被减毒。脾减毒突变体中的转座子插入位点揭示了参与荚膜多糖生物合成和DNA复制和修复的几个阶段的基因，推定的氧化还原酶，ABC转运蛋白和脂蛋白，这可能是重要的细胞间传播。发现在肺和脾中被鉴定为缺失的六种突变体在涉及SOS反应和DNA修复的recA中插入;还发现亮氨酸、苏氨酸、对氨基苯甲酸的假定营养缺陷型和在aroB中插入导致芳香族化合物营养缺陷型的突变体。在这项研究中开发的改进的签名标记的诱变方法被用来有效地识别减毒突变体，从这个高致病性的物种，并可应用于其他生物。
Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine, p-aminobenzoic acid, and a mutant with an insertion in aroB causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the aroB mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.