Measurement of Accumulation of Antibiotics toStaphylococcus aureusin Phagosomes

AbstractStaphylococcus 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.