Characterization of the mycomembrane permeability barrier

The recalcitrance of Mycobacterium tuberculosis to antibiotic treatment has been broadly attributed to the impermeability of the organism’s outer mycomembrane. The mycomembrane is covered with long chain fatty acids that make the cell envelope extremely hydrophobic as well as other surface lipids and proteins. It is largely presumed that the mycomembrane is a key factor in cellular impermeability for many compounds. However, the evidence supporting this hypothesis has been indirect and/or reliant on bulk population measurements. In this work we utilized labeling methods via alkyne-azide cycloaddition to track small fluorescent molecule accumulation directly past the mycomembrane. We were also able to use a similar workflow via the 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 of fluorophores and antibiotic derivatives through testing in M. tuberculosis and the model organism M. smegmatis. Mycomembranes of both M. tuberculosis and M. smegmatis discriminate between divergent classes of antibiotics as well as between antibiotics within a single family, the fluoroquinolones. By analyzing sub-populations of M. tuberculosis and M. smegmatis, we also found that some fluorophores and vancomycin are heterogeneously restricted by the mycomembrane. Our work has also confirmed increase in antibiotic activity alongside increased accumulation. Our data indicate that the mycomembrane is a molecule- and cell-specific barrier to antibiotic permeation.