HssS activation by membrane heme definesa paradigm for two-component system signaling in Staphylococcus aureus

Abstract

Strict management of intracellular heme pools, which are both toxic and beneficial,is 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 effluxtransporter 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, a heme-binding site is present in the HssS dimer between the membrane and extracellular domains. Heme is embedded in the membrane bilayer with its two protruding porphyrin propionates interacting with two conserved Arg94 and Arg163 that are located extracellularly. Single substitutions of these arginines and two highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic pocket limited the ability of HssS to induce HrtBA synthesis. Combination of the four substitutions abolished HssS activation. Wild-type (WT) HssS copurifiedwith heme from Escherichia coli, whereas heme binding was strongly attenuated in the variants. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation and induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper"mechanism could limit intracellular diffusionof exogenous heme in S. aureus and may serve as a paradigm for how effluxtransporters control detoxificationof exogenous hydrophobic stressors.