Quantitative Imaging of Human Hemoglobin Binding to <i>Staphylococcus aureus</i> With Single‐Molecule Resolution

Staphylococcus aureus is a widespread human pathogenic bacterium that poses an increasingly serious threat to public health worldwide, due to the emergence and spread of antibiotic-resistant strains. A key virulence mechanism is the acquisition of iron from the heme group of human host's hemoglobin, initiated by hemoglobin binding to receptors exposed on the bacterial surface. Although this interaction is a promising target for new antimicrobials, quantitative data on hemoglobin capture by single bacteria are lacking. In this work, single-molecule localization microscopy is used to directly quantify the number of hemoglobin molecules captured by individual S. aureus cells and resolve their nanoscale spatial distribution at the sub-cellular level. This quantitative approach provides a methodological framework to monitor, with single-molecule sensitivity, changes in surface density of functional receptors in response to iron limitations. This piece of information, in turn, is critical to define the overall efficacy of antimicrobials targeting bacterial receptors. As proof of this concept, we develop a photo-activated molecular system based on hemoglobin for targeted photodynamic inactivation of S. aureus.