Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice
Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice
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DOI：
10.1099/mic.0.029884-0
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发表时间：
2009-09-01
期刊：
影响因子：
2.8
通讯作者：
Sharma, Umender
中科院分区：
文献类型：
--
作者：
Awasthy, Disha;Gaonkar, Sheshagiri;Sharma, Umender
乙酰羟基酸合成酶（AHAS）是细菌支链氨基酸生物合成途径中的第一个酶。生物信息学分析显示，结核分枝杆菌基因组包含4个基因（ilvB1、ilvB2、ilvG和ilvX）编码AHAS的大催化亚基，而只发现了一个基因（ilvN或ilvH）编码AHAS的小调节亚基。为了了解AHAS在体外和体内生物生存中的生理作用，我们灭活了结核分枝杆菌的Delta i1vB1基因。突变菌株在C-6或C-2碳源下生长时，对所有三种支链氨基酸（异亮氨酸、亮氨酸和缬氨酸）都缺乏营养，这表明i1vB1基因产物是结核分枝杆菌的主要AHAS。培养基中这些支链氨基酸的消耗导致Delta ilvB1菌株在体外的生存能力丧失，导致10天后菌落形成单位减少4倍。在支链氨基酸浓度低于最佳浓度的巨噬细胞中培养的突变菌株的生存动力学没有显示出任何活力损失，这表明细胞内环境富含这些氨基酸，或者在这些条件下其他AHAS催化亚基发挥作用。此外，Delta ilvB1菌株在小鼠体内的生长动力学表明，尽管该突变株在体内表现出生长缺陷，但它可以在感染小鼠体内持续很长时间，因此可能是潜在的候选疫苗。
Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the Delta i1vB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C-6 or C-2 carbon sources, suggesting that the i1vB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the Delta ilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the Delta ilvB1 strain in mice indicated that although this Mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.