Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis. A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with

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... GM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci. A KO mutant of the MAB_0855 gene, designated herein as mmpL8MAB, had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mmpL8MAB mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8MAB. Systematic lipid analysis revealed that MmpL8MAB was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8MAB in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.