Measurement of Small Molecule Accumulation into Diderm Bacteria

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 (OM), while for the Gram-indeterminate Mycobacteria, it is known as the “myco” membrane. Although different in composition, both the OM and mycomembrane are key structures that restrict the passive permeation of small molecules into bacterial cells. Although 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 bacterial chloro–alkane penetration assay (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 Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of 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 measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.

Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay

“…The chloroalkane penetration assay has been successfully applied to measure cytosolic and nuclear delivery for a large range of drug modalities including small molecules, peptides, natural products, and biotherapeutics. − In this work, we report the first-ever application of CAPA to measuring protein delivery by LNPs. CAPA allowed us to compare cytosolic delivery to more common measurements of total cellular uptake and functional delivery.…”

“…44 CAPA has also been applied in 3D deep tissue models and in bacteria. 45,46 To the best of our knowledge, CAPA has not been used to measure cytosolic penetration of proteins delivered using LNPs. Here, we apply CAPA to measure the cytosolic delivery of a functional protein using LNPs, comparing CAPA data to independent measurements of total cellular uptake and functional delivery.…”

“…Recently, we developed an assay called the chloroalkane penetration assay (CAPA). − Our initial work applied CAPA to measure the cytosolic delivery of cell-penetrant peptides, , and since then, we and others have adapted CAPA for measuring cytosolic delivery of DNA-encoded libraries, − peptoids, , small molecules including bivalent chemical degraders and protein inhibitors, , antisense oligonucleotides, , siRNAs, oligochalcogenides, , and cell-penetrant streptavidins . CAPA has also been applied in 3D deep tissue models and in bacteria. , To the best of our knowledge, CAPA has not been used to measure cytosolic penetration of proteins delivered using LNPs. Here, we apply CAPA to measure the cytosolic delivery of a functional protein using LNPs, comparing CAPA data to independent measurements of total cellular uptake and functional delivery.…”

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.…”

Cytoplasmic Accumulation and Permeability of Antibiotics in Gram Positive and Gram Negative Bacteria Visualized in Real-Time via a Fluorogenic Tagging Strategy

“…Finally, the fluorogenic assay was applied to compare the intracellular accumulation of ciprofloxacin and ampicillin with related pharmacophores in two types of bacteria. Current limitations of this assay are the requirements to express an RNA aptamer and append a tag to the compound, which is similar to the CAPA assay originally developed for mammalian cells but recently applied to bacteria. , However, our use of fluorogenic tags is a distinguishing feature and permits real-time analysis of drug permeability with no washing or cell fixation. MS-based methods to study permeability of bacteria, a human intestine model (Caco2 monolayer assay), and artificial membranes (PAMPA) do not require tags and the latter two are standard assays in industry .…”