Molecular Probes for the Determination of Subcellular Compound Exposure Profiles in Gram-Negative Bacteria

The Gram-negative cell envelope presents a formidable barrier to xenobiotics, and achieving sufficient compound exposure inside the cell is a key challenge for the discovery of new antibiotics. To provide insight on the molecular determinants governing compound exposure in Gram-negative bacteria, we developed a methodology leveraging a cyclooctyne-based bioorthogonal probe to assess compartment-specific compound exposure. This probe can be selectively localized to the periplasmic or cytoplasmic compartments of Gram-negative bacteria. Once localized, the probe is used to test azide-containing compounds for exposure within each compartment by quantifying the formation of click-reaction products by mass spectrometry. We demonstrate this approach is an accurate and sensitive method of determining compartment-specific compound exposure profiles. We then apply this technology to study the compartment-specific exposure profiles of a small panel of azide-bearing compounds with known permeability characteristics in Gram-negative bacteria, demonstrating the utility of the system and the insight it is able to provide regarding compound exposure within intact bacteria.