Modulation of riboflavin biosynthesis and utilization in mycobacteria.
Modulation of riboflavin biosynthesis and utilization in mycobacteria.
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分枝杆菌核黄素生物合成和利用的调节。
DOI：
10.1101/2023.08.30.555301
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Mizrahi,Valerie
中科院分区：
文献类型：
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作者：
Chengalroyen,MelissaD;Mehaffy,Carolina;Lucas,Megan;Bauer,Niel;Raphela,MabuleL;Oketade,Nurudeen;Warner,DigbyF;Lewinsohn,DeborahA;Lewinsohn,DavidM;Dobos,KarenM;Mizrahi,Valerie
核黄素（维生素B2）是黄素辅酶FAD和FMN的前体，它们在细胞氧化还原代谢中起核心作用。虽然人类必须从饮食来源获得核黄素，但某些微生物，包括结核分枝杆菌（Mtb），可以重新生物合成核黄素。核黄素前体还涉及粘膜相关不变T（MAIT）细胞的活化，所述MAIT细胞识别衍生自与MHC-I样分子MR 1复合的核黄素生物合成途径的代谢物。为了研究核黄素及其途径中间产物在分枝杆菌代谢和生理中的生物合成和功能，我们通过诱导型CRISPR干扰构建了耻垢分枝杆菌（Msm）和结核分枝杆菌（Mtb）核黄素生物合成和利用基因的条件性敲除（hypomorphs）。使用这一全面的亚型面板，我们分析了基因沉默对生存能力的影响，对（其他）核黄素途径基因的转录，对途径蛋白质的水平，以及对核黄素本身。我们的研究结果表明：（i）尽管缺乏典型的转运蛋白，但Msm和Mtb在高浓度下都能同化外源核黄素;（ii）Msm中的鲁马嗪合酶活性存在功能冗余;（iii）RibA 2或ribF的沉默在Mtb中具有深刻的杀菌作用;和（iv）在Msm中，ribA 2沉默导致伴随的其他途径基因的敲除，与RibA 2和核黄素消耗偶联，并且也是杀菌的。除了它们在结核病的潜在药物靶点的遗传学验证中的用途之外，这种亚型的集合为将来的研究提供了有用的资源，所述研究调查途径中间体在分枝杆菌的MAIT细胞识别中的作用。重要信息核黄素的生物合成和利用途径，必需辅酶FMN和FAD的前体，在富含黄素的病原体结核分枝杆菌（Mtb）中特别感兴趣，由于两个重要的原因：（i）该途径包括潜在的结核病（TB）药物靶点，和（ii）核黄素生物合成途径的中间体提供了粘膜相关的不变T（MAIT）细胞的配体，MAIT细胞与TB发病机制有关。然而，核黄素途径在分枝杆菌中知之甚少，其缺乏运输这种维生素和调节黄素辅酶稳态的典型机制。通过有条件地破坏途径的每个步骤并评估对分枝杆菌活力和途径蛋白以及核黄素水平的影响，我们的工作提供了核黄素途径作为TB药物发现靶点的遗传验证，并为进一步探索核黄素生物合成，MAIT细胞活化和TB感染和疾病之间的关联提供了资源。
Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, includingMycobacterium tuberculosis(Mtb), can biosynthesize riboflavinde novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes inMycobacterium smegmatis(Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing ofribA2orribFis profoundly bactericidal in Mtb; and (iv) in Msm,ribA2silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.IMPORTANCEThe pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen,Mycobacterium tuberculosis(Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.