Mycobacterium tuberculosisrequires conditionally essential metabolic pathways for infection

Abstract

ABSTRACTNew drugs are needed to shorten and simplify treatment of tuberculosis caused byMycobacterium tuberculosis. Metabolic pathways thatM. tuberculosisrequires for growth or survival during infection represent potential targets for anti-tubercular drug development. Genes and metabolic pathways essential forM. tuberculosisgrowth in standard laboratory culture conditions have been defined by genome-wide genetic screens. However, whetherM. tuberculosisrequires these essential genes during infection has not been comprehensively explored because mutant strains cannot be generated using standard methods. Here we show thatM. tuberculosisrequires functional phenylalanine (Phe) andde novopurine and thiamine biosynthetic pathways for mammalian infection. We used a defined collection ofM. tuberculosistransposon (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 inpheA(Phe biosynthesis) orpurF(purine and thiamine biosynthesis) cause death ofM. tuberculosisin the absence of nutrient supplementationin vitroand strong attenuation in infected mice. Our findings show that Tn-seq with defined Tn mutant pools can be used to identifyM. tuberculosisgenes required during mouse lung infection. Our results also demonstrate thatM. tuberculosisrequires 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.AUTHOR SUMMARYMycobacterium tuberculosiscauses more than 10 million new cases of active tuberculosis (TB) disease and 1.6 million deaths worldwide each year. Individuals with active TB must take a combination of four antibiotics for a minimum of 6-9 months to cure the infection. New anti-tubercular drugs are needed to simplify TB treatment and combat drug resistance. Here, we describe a novel collection ofM. tuberculosismutants lacking metabolic pathways essential for growth in standard laboratory conditions. Using these mutants, a mouse infection model, and deep sequencing we identified those metabolic pathways thatM. tuberculosisalso requires during infection. We find thatM. tuberculosismutants that cannot synthesize purine nucleotides, riboflavin, or certain amino acids are unable to grow in mice. We also find that mutant strains which cannot synthesize purine nucleotides or the amino acid phenylalanine die rapidly in laboratory cultures without nutrient supplementation, suggesting that new drugs targeting these pathways would killM. tuberculosis. Overall, our work reveals multiple metabolic pathways thatM. tuberculosisrequires during infection, which could be pursued as new targets for development of anti-tubercular drugs.