Department of Microbiology and Immunology, University of Minnesota, Twin Cities Campus, Minneapolis, Minnesota, United States of America.
PLoS Pathog. 2024 Mar 18;20(3):e1011663. doi: 10.1371/journal.ppat.1011663. eCollection 2024 Mar.
New drugs are needed to shorten and simplify treatment of tuberculosis caused by Mycobacterium tuberculosis. Metabolic pathways that M. tuberculosis requires for growth or survival during infection represent potential targets for anti-tubercular drug development. Genes and metabolic pathways essential for M. tuberculosis growth in standard laboratory culture conditions have been defined by genome-wide genetic screens. However, whether M. tuberculosis requires these essential genes during infection has not been comprehensively explored because mutant strains cannot be generated using standard methods. Here we show that M. tuberculosis requires the phenylalanine (Phe) and de novo purine and thiamine biosynthetic pathways for mammalian infection. We used a defined collection of M. tuberculosis transposon (Tn) mutants in essential genes, which we generated using a custom nutrient-rich medium, and transposon sequencing (Tn-seq) to identify multiple central metabolic pathways required for fitness in a mouse infection model. We confirmed by individual retesting and complementation that mutations in pheA (Phe biosynthesis) or purF (purine and thiamine biosynthesis) cause death of M. tuberculosis in the absence of nutrient supplementation in vitro and strong attenuation in infected mice. Our findings show that Tn-seq with defined Tn mutant pools can be used to identify M. tuberculosis genes required during mouse lung infection. Our results also demonstrate that M. tuberculosis requires Phe and purine/thiamine biosynthesis for survival in the host, implicating these metabolic pathways as prime targets for the development of new antibiotics to combat tuberculosis.
需要新的药物来缩短和简化由结核分枝杆菌引起的结核病的治疗。结核分枝杆菌在感染期间生长或存活所需的代谢途径代表了抗结核药物开发的潜在靶点。通过全基因组遗传筛选已经定义了结核分枝杆菌在标准实验室培养条件下生长所需的基因和代谢途径。然而，结核分枝杆菌在感染期间是否需要这些必需基因尚未得到全面探索，因为无法使用标准方法生成突变株。在这里，我们表明结核分枝杆菌需要苯丙氨酸（Phe）和从头嘌呤和硫胺素生物合成途径来进行哺乳动物感染。我们使用了在必需基因中生成的一组定义的结核分枝杆菌转座子（Tn）突变体，我们使用了一种定制的富含营养的培养基，并使用转座子测序（Tn-seq）来鉴定适应在小鼠感染模型中生存所需的多个中心代谢途径。我们通过单独的重新测试和互补来证实，pheA（苯丙氨酸生物合成）或 purF（嘌呤和硫胺素生物合成）的突变导致结核分枝杆菌在体外缺乏营养补充时死亡，并在感染的小鼠中强烈衰减。我们的发现表明，使用定义的 Tn 突变体池的 Tn-seq 可用于鉴定在小鼠肺感染期间需要的结核分枝杆菌基因。我们的结果还表明，结核分枝杆菌在宿主中生存需要苯丙氨酸和嘌呤/硫胺素生物合成，这表明这些代谢途径是开发新抗生素以对抗结核病的主要目标。
