A highly conserved two-gene operon is crucial for lipoarabinomannan localization, pathogenesis, and cell envelope function in <i>Mycobacterium abscessus</i>

<i>Mycobacterium abscessus</i> is an emerging threat, causing infections that are difficult to treat due to intrinsic resistance to most antibiotics. Determinants of <i>M. abscessus</i> physiology and pathogenesis remain poorly understood, hampering therapeutic development. Here, we show that in <i>M. abscessus</i>, the <i>lprg-mfs</i> operon is essential for virulence in macrophages and in mice. Loss of <i>lprg-mfs</i> in <i>M. abscessus</i> causes accumulation of the glycolipid lipoarabinomannan (LAM) on the cell surface and in culture supernatant suggesting that this system participates in LAM import. This contrasts with its proposed role in <i>M. tuberculosis</i> where <i>lprg-mfs</i> has been implicated in the export of various lipids. Consistent with altered lipid distribution, the <i>lprg-mfs</i> mutant displays severe defects in mycomembrane permeability, fluidity, and integrity, and expression of <i>mfs</i> alone restores only a subset of these phenotypes, revealing a surprising uncoupling of envelope fluidity and permeability. Using a suppressor screen to further investigate factors that control the distribution of lipoarabinomannan we find that a point mutation in the unannotated gene <i>MAB_0995</i> can fully or partially complement all deletion mutant phenotypes. Our data also show that lipoarabinomannan in the mycomembrane is dynamically regulated in response to environmental conditions, including hypoxia and macrophage infection. Together, these findings redefine the role of LprG/Mfs in mycobacterial cell envelope homeostasis and reveal unexpected plasticity in mycomembrane lipid regulation in <i>M. abscessus</i>.