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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

Staphylococcus aureus(S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enablesS. aureusto potentially escape host immune responses and survive the action of antibiotics. The elevated tolerance ofS. aureusto small molecule antibiotics is likely to be multifactorial. We pose that there may be contributions related to permeation into phagosome, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics in phagocytic vacuoles. Bioorthogonal reactive handles were metabolically anchored within the surface ofS. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of labeledS. aureuscells, we were able to specifically analyze the accumulation levels of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of differences between the permeability of antibiotics to extra- and intracellularS. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

The surfaces of most Gram-positive bacterial cells, including that of Staphylococcus aureus (S. aureus), are heavily decorated with proteins that coordinate cellular interactions with the extracellular space. In S. aureus, sortase A is the principal enzyme responsible for covalently anchoring proteins, which display the sorting signal LPXTG, onto the peptidoglycan (PG) matrix. Considerable efforts have been made to understand the role of this signal peptide in the sortase-mediated reaction. In contrast, much less is known about how the primary structure of the other substrate involved in the reaction (PG stem peptide) could impact sortase activity. To assess the sortase activity, a library of synthetic analogs of the stem peptide that mimic naturally existing variations found in the S. aureus PG primary sequence were evaluated. Using a combination of two unique assays, we showed that there is broad tolerability of substrate variations that are effectively processed by sortase A. While some of these stem peptide derivatives are naturally found in mature PG, they are not known to be present in the PG precursor, lipid II. These results suggest that sortase A could process both lipid II and mature PG as acyl-acceptor strands, which has not been previously described.

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 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 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.