SummaryMycobacterium abscessus is an emerging pathogen that is increasingly recognized as a relevant cause of human lung infection in cystic fibrosis patients. This highly antibiotic-resistant mycobacterium is an exception within the rapidly growing mycobacteria, which are mainly saprophytic and non-pathogenic organisms. M. abscessus manifests as either a smooth (S) or a rough (R) colony morphotype, which is of clinical importance as R morphotypes are associated with more severe and persistent infections. To better understand the molecular mechanisms behind the S/R alterations, we analysed S and R variants of three isogenic M. abscessus S/R pairs using an unbiased approach involving genome and transcriptome analyses, transcriptional fusions and integrating constructs. This revealed different small insertions, deletions (indels) or single nucleotide polymorphisms within the non-ribosomal peptide synthase gene cluster mps1-mps2-gap or mmpl4b in the three R variants, consistent with the transcriptional differences identified within this genomic locus that is implicated in the synthesis and transport of Glyco-Peptido-Lipids (GPL). In contrast to previous reports, the identification of clearly defined genetic lesions responsible for the loss of GPL-production or transport makes a frequent switching back-and-forth between smooth and rough morphologies in M. abscessus highly unlikely, which is important for our understanding of persistent M. abscessus infections.

Mycobacterium abscessus is the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies of M. abscessus proteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and functional characterization of an arylamine N-acetyltransferase (NAT) from M. abscessus [(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significant N-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid and p-aminosalicylate. The enzyme is endogenously expressed and functional in both the rough and smooth M. abscessus morphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related to Nocardia farcinica NAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme from M. abscessus and provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance of M. abscessus.