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

<i>Mycobacterium tuberculosis</i> uses several ESX type VII protein secretion systems for pathogenesis. <i>M. tuberculosis</i> ESX-5 is only partially characterized because it is essential for growth in standard lab culture conditions. To circumvent ESX-5 essentiality, we made an <i>M. tuberculosis</i> strain in which the central ESX-5 membrane component EccD₅ can be conditionally depleted. Here, we use this strain to demonstrate that <i>M. tuberculosis</i> requires the ESX-5 secretion system to grow using specific carbon sources <i>in vitro</i>, to grow in cultured macrophages, and to replicate and disseminate in aerosol-infected mice. <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i> on glycerol, suggesting that the main function of ESX-5 <i>in vitro</i> is to export nutrient transporters to the outer membrane. Depletion of EccD₅ in acutely infected mice caused clearance of <i>M. tuberculosis</i> from lung tissues, demonstrating the critical importance of ESX-5 activity during infection. Our findings suggest that ESX-5 promotes <i>M. tuberculosis</i> pathogenesis by mediating export of outer membrane proteins that enable nutrient acquisition.IMPORTANCE<i>Mycobacterium tuberculosis</i> 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 <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i> from lung tissues. Our results demonstrate that the <i>M. tuberculosis</i> ESX-5 system is a critical virulence factor and suggest that ESX-5 is a strong candidate for antitubercular drug development.