MmpL8_MAB controls <i>Mycobacterium abscessus</i> virulence and production of a previously unknown glycolipid family.

Abstract

<i>Mycobacterium abscessus</i> is a peculiar rapid-growing <i>Mycobacterium</i> (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as <i>Mycobacterium tuberculosis</i> A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an <i>M. abscessus</i> transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the <i>MAB_0855</i> and <i>Mycobacterium chelonae mmpL8</i> loci. A KO mutant of the <i>MAB_0855</i> gene, designated herein as <i>mmpL8</i>_<i>MAB</i> , had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the <i>mmpL8</i>_<i>MAB</i> 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 MmpL8_MAB Systematic lipid analysis revealed that MmpL8_MAB 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 MmpL8_MAB in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.