Low Kai Leng, Shui Guanghou, Natter Klaus, Yeo Wee Kiang, Kohlwein Sepp D, Dick Thomas, Rao Srinivasa P S, Wenk Markus R
NUS Graduate School for Integrative Sciences and Engineering, Yong Loo Lin Schoolof Medicine, Singapore.
J Biol Chem. 2010 Jul 9;285(28):21662-70. doi: 10.1074/jbc.M110.135731. Epub 2010 May 6.
Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in baker's yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.
在缺氧诱导的非复制性持续存在过程中，分枝杆菌以细胞内脂滴（LDs）的形式储存三酰甘油（TGs）。这些细菌具有表型耐药性，因此被认为是结核病治疗时间延长的原因。脂滴也与肺结核患者痰液中的杆菌及高毒力菌株有关。尽管在真核生物中与脂滴结合的蛋白质已得到充分表征，但在分枝杆菌中此类蛋白质的身份和功能尚未见报道。在此，我们鉴定出了五种蛋白质：Tgs1（BCG3153c）、Tgs2（BCG3794c）、BCG1169c、BCG1489c和BCG1721，它们仅与从缺氧非复制性卡介苗（BCG）中纯化的脂滴相关。在牛分枝杆菌卡介苗中破坏tgs1、tgs2、BCG1169c和BCG1489c基因表明，它们确实参与了TG代谢。我们还对BCG1721进行了表征，它是一种必需的双功能酶，能够根据代谢状态促进TG的积累和水解。过表达具有无活性脂肪酶结构域的BCG1721构建体的非复制性分枝杆菌表现出TG分解减弱和复苏后再生长的表型。此外，通过在面包酵母中的异源表达，这些分枝杆菌蛋白也与脂滴共定位，并补充了脂肪酶缺陷型酵母菌株，这表明真核和原核微生物中的中性脂质沉积和稳态在功能上是相关的。BCG1721在复苏后支持生长的已证明的功能作用，使这种新的与脂滴相关的因子成为治疗干预的潜在新靶点。
