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bioRxiv · 10.1101/2025.10.28.685107

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

Block, A. M.·Nair, R. R.·Meikle, V.·Wiegert, P. C.·White, D. W.·Zhang, L.·Niederweis, M.·Tischler, A. D.
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Abstract

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.

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Block, A. M., Nair, R. R., Meikle, V., Wiegert, P. C., White, D. W., Zhang, L., Niederweis, M., Tischler, A. D.. 2025-10-28. The Mycobacterium tuberculosis ESX-5 secretion system enables carbon source utilization and growth in mice. https://doi.org/10.1101/2025.10.28.685107

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