Wiegert, P. C. | bioRxiv Science

bioRxiv2025

The Mycobacterium tuberculosis ESX-5 secretion system enables carbon source utilization and growth in mice

Mycobacterium tuberculosis uses several ESX type VII protein secretion systems for pathogenesis. M. tuberculosis ESX-5 is poorly characterized because it is essential for growth in standard lab culture conditions. To circumvent ESX-5 essentiality, we made an M. tuberculosis strain in which the central ESX-5 membrane component EccD5 can be conditionally depleted. Here, we use this strain to demonstrate that M. tuberculosis requires the ESX-5 secretion system to grow using specific carbon sources in vitro, to grow in cultured macrophages, and to replicate and disseminate in aerosol-infected mice. M. tuberculosis requires ESX-5 to use glycerol or glucose as the sole carbon source. Use of glycerol and glucose also depends on the outer membrane protein PPE51. We show that M. tuberculosis requires ESX-5 activity for outer membrane export and surface exposure of PPE51. Expression of the outer membrane porin MspA enabled growth of ESX-5 deficient M. tuberculosis on glycerol, suggesting that the main function of ESX-5 in vitro is to export nutrient transporters to the outer membrane. Importantly, depletion of EccD5 in acutely infected mice resulted in clearance of M. tuberculosis from lung tissues, demonstrating the critical importance of ESX-5 activity during infection. Our findings suggest that ESX-5 promotes M. tuberculosis pathogenesis by mediating export of outer membrane proteins that enable nutrient acquisition. ImportanceMycobacterium tuberculosis ESX type VII secretion systems play important roles in pathogenesis, but the functions of ESX-5 are not well characterized because it is essential for growth in standard lab culture conditions. We used a strain that conditionally expresses a central membrane component of the ESX-5 secretion apparatus to determine how ESX-5 impacts growth in lab cultures and in a mouse infection model. We found that M. tuberculosis requires ESX-5 to grow using several carbon sources and to grow in the lungs of infected mice. Inhibiting production of the ESX-5 secretion system in mice also led to clearance of M. tuberculosis from lung tissues. Our results demonstrate that the M. tuberculosis ESX-5 system is a critical virulence factor and suggest that ESX-5 is a strong candidate for anti-tubercular drug development.

microbiology↗

bioRxiv2023

Mycobacterium tuberculosis requires conditionally essential metabolic pathways for infection

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 functional 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. AUTHOR SUMMARYMycobacterium tuberculosis causes 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 of M. tuberculosis mutants 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 that M. tuberculosis also requires during infection. We find that M. tuberculosis mutants 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 kill M. tuberculosis. Overall, our work reveals multiple metabolic pathways that M. tuberculosis requires during infection, which could be pursued as new targets for development of anti-tubercular drugs.

microbiology↗