The Mycomembrane Differentially and Heterogeneously Restricts Antibiotic Permeation

The recalcitrance of Mycobacterium tuberculosis to antibiotic treatment has been broadly attributed to the impermeability of the organism’s outer mycomembrane. However, the studies that support this inference have been indirect or reliant on bulk population measurements. We previously developed the Peptidoglycan Accessibility Click-Mediated AssessmeNt (PAC-MAN) method to covalently trap azide-modified small molecules in the peptidoglycan cell wall of live mycobacteria after they have traversed the mycomembrane. Using PAC-MAN, we now show that the mycomembrane differentially restricts access to fluorophores and antibiotic derivatives. Mycomembranes of both M. tuberculosis and the model organism Mycobacterium smegmatis discriminate between divergent classes of antibiotics as well as between antibiotics within a single family, the fluoroquinolones. By analyzing subpopulations of M. tuberculosis and M. smegmatis, we also found that some fluorophores and vancomycin are heterogeneously restricted by the mycomembrane. Our data indicate that the mycomembrane is a molecule- and cell-specific barrier to antibiotic permeation.