, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the genes required for replication within amoebae. To this end, we constructed and screened a transposon () insertion library of an subspcies clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to growth and survival in amoebae and macrophages, we generated a deletion mutant of that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.

SummaryMycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance-Nodulation-Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surfaceexposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid-growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non-tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/ functional information are also provided to explain the molecular basis underlying the proton-motive force driven lipid transport. Overall, this review emphasises the Janus-face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.