Abstract Some of the most dangerous bacterial pathogens (Gram-negative and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous molecules. For Gram-negative bacteria, this second exterior membrane is known as the outer membrane, while for the Gram-indeterminate Mycobacteria, it is known as the ‘myco’ membrane. Although different in composition, both the outer membrane and mycomembrane are key structures that restrict the passive permeation of small molecules into bacterial cells. While it is well-appreciated that such structures are principal determinants of small molecule permeation, it has proven to be challenging to assess this feature in a robust and quantitative way or in complex, infection-relevant settings. Herein, we describe the development of the B acterial C hloro- A lkane P enetration A ssay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of molecules into model Gram-negative and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, respectively, and into the human pathogen M. tuberculosis. Directing the localization of the HaloTag protein to either the cytoplasm or periplasm of bacteria enabled a compartmental analysis of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of molecules into host cell-internalized E. coli and M. tuberculosis, a critical capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile, compartment-specific measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.

Compartment-Specific Measurement of Small Molecule Accumulation into Diderm Bacteria