The SigD regulon of Mycobacterium abscessus determines cell envelope composition and antibiotic susceptibility

A major determinant of the exceptional intrinsic resistance of M. abscessus is the lipid-rich cell envelope, yet the regulatory systems that remodel envelope-associated pathways remain poorly defined. Here, we determine the σ D regulon in M. abscessus and establish its role in cell envelope homeostasis and intrinsic resistance to hydrophobic antibiotics. RNA-Seq analysis of a MabΔ sigD mutant identified 447 differentially expressed genes, while ChIP-Seq mapped 72 σ D binding sites and defined a conserved promoter motif (GTAACA/G-N 16 -CGAT). Using a combination of σ D binding, motif orientation and expression data, we identified a core set of directly regulated genes, distinct from what was previously observed in M. tuberculosis , many of which encode proteins involved in envelope-associated functions. These include loci involved in trehalose polyphleate (TPP) biosynthesis, the antigen 85 complex and peptidoglycan remodeling enzymes. Deletion of sigD resulted in a significant reduction in TPPs in the cell envelope and an increase in ethidium bromide accumulation. Consistent with these changes, loss of σ D selectively sensitized M. abscessus to hydrophobic antibiotics, including rifampicin and tigecycline. Deletion of mmpL10 , which is required for transport of TPP precursors, recapitulated the drug sensitivity of MabΔ sigD , implicating envelope composition as a key effector of the phenotype. Expression of the σ D regulon further increased during starvation and in response to SDS, isoniazid, and ethambutol, mediated by degradation of RsdA, consistent with a role in stress-responsive envelope adaptation. Together, these findings demonstrate σ D is active during logarithmic growth in rich media where it regulates the expression of envelope-associated genes that influence envelope permeability and basal level susceptibility to hydrophobic antibiotics; its activity further increases in response to cell envelope stress, presumably promoting envelope remodeling to counteract damage.