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首页> 外文期刊>Infection and immunity >Identification of Brucella suis Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis
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Identification of Brucella suis Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis

机译：签名的转座子诱变鉴定在体外人巨噬细胞感染模型中影响细胞内存活的猪布鲁氏菌基因

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摘要

Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of Brucella virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D3-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the virBoperon and manB), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of Brucella for the different factors identified are discussed.
机译：布鲁氏菌属细菌是兼性的细胞内病原体，已发展出在专业和非专业吞噬细胞中生存和繁殖的能力。布鲁氏菌毒力的这一方面的遗传基础仍然知之甚少。为了确定新的毒力因子，我们将签名标记的转座子诱变技术（已主要用于动物模型中）适应了体外人类巨噬细胞感染模型。在12个库中，筛选了1,152株猪布鲁氏杆菌1330标签的mini-Tn5 Km2突变体文库，以观察细胞内存活情况以及在维生素D3分化的THP1细胞中的增殖情况。鉴定出18个突变体，并在THP1巨噬细胞和HeLa细胞中证实了它们的减毒作用。对于每个无毒突变体，克隆了包含转座子的基因组片段。转座子侧翼的基因组DNA序列使我们能够为所有失活的基因分配功能。转座子整合发生在14个不同的基因中，其中一些是已知的毒性基因，参与了细胞内存活或平滑脂多糖的生物合成（virBoperon和manB），从而验证了该模型。其他基因鉴定出编码基因表达调控中涉及的因子和生物合成或代谢途径中涉及的酶。讨论了布鲁氏菌的毒力对于不同因素的可能作用。

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