HssS activation by membrane heme defines a paradigm for 2-component system signaling in Staphylococcus aureus.

Strict management of intracellular heme pools, which are both toxic and beneficial, can be crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, heme is embedded in the membrane bilayer with its 2 protruding porphyrin propionates interacting with 2 conserved Arg94 and Arg163 that are located extracellularly. Mutagenesis of these arginines and of 2 highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic heme binding pocket abolished the ability of HssS to induce HrtBA synthesis. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation to induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper" mechanism could limit intracellular diffusion of exogenous heme in S. aureus, and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors. ImportanceIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphorelay mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the TM helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen sensing mechanisms as a prerequisite to the discovery of inhibitors.