A Metabolic‐Tag‐Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria**

Abstract

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

Measurement of Accumulation of Antibiotics toStaphylococcus aureusin Phagosomes of Live Macrophages

“…Treatment of cells with azide-modified antibiotics (azAbx) post infection was expected to result in the reaction between the DBCO and azAbx via strain promoted alkyne-azide cycloaddition (SPAAC). 25 A chase step with an azide-modified fluorophore imprints a signal that is reflective of unmodified DBCO epitopes. Cellular fluorescence measurement should then enable a fluorescent, live-cell analysis of antibiotic accumulation, whereby cellular fluorescence inversely correlates with antibiotic accumulation levels.…”

Measurement of Accumulation of Antibiotics to Staphylococcus aureus in Phagosomes of Live Macrophages**

“…To measure the accumulation of antibiotics, we combined an assay our laboratory recently developed aimed at systematically assessing how flexibility and size of large biopolymers impact their ability to reach the peptidoglycan (PG) surface of S. aureus cells [17] with one that we developed to measure the permeability of molecules into mycobacteria. [18] In this new iteration (Figure 1B), a strained alkyne dibenzocyclooctyne (DBCO) is metabolically installed within the PG of S. aureus. DBCO-tagged bacterial cells are then incubated with macrophages to promote their uptake.…”

Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli

“…Our group has recently described methods to interrogate the accumulation of molecules onto the surface of Gram-positive bacteria and past the outer membrane of diderm bacteria using a combination of click chemistry , and HaloTag . Herein, we sought to establish a luciferase-based assay to determine the accumulation of molecules to the cytosol of Gram-negative bacteria in real time.…”

“…15,16 Critically, these methods often fail to report on whether the molecules arrive within the cytoplasmic space and, instead, provide information on the total association of the molecules with the target cells. 17 Our group has recently described methods to interrogate the accumulation of molecules onto the surface of Gram-positive bacteria and past the outer membrane of diderm bacteria using a combination of click chemistry 18,19 and HaloTag. 20 Herein, we sought to establish a luciferase-based assay 21 to determine the accumulation of molecules to the cytosol of Gram-negative bacteria in real time.…”