RESUMEN
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 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, 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 copurified with 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 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 phosphotransfer 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 transmembrane domains (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.
Asunto(s)
Proteínas Bacterianas , Hemo , Transducción de Señal , Staphylococcus aureus , Hemo/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/química , Regulación Bacteriana de la Expresión Génica , Sitios de Unión , Membrana Celular/metabolismoRESUMEN
Enterococcus faecalis is a commensal Gram-positive pathogen found in the intestines of mammals and is also a leading cause of severe infections occurring mainly among antibiotic-treated dysbiotic hospitalized patients. Like most intestinal bacteria, E. faecalis does not synthesize heme (in this report, heme refers to iron protoporphyrin IX regardless of the iron redox state). Nevertheless, environmental heme can improve E. faecalis fitness by activating respiration metabolism and a catalase that limits hydrogen peroxide stress. Since free heme also generates toxicity, its intracellular levels need to be strictly controlled. Here, we describe a unique transcriptional regulator, FhtR (named FhtR for faecalis heme transport regulator), which manages heme homeostasis by controlling an HrtBA-like efflux pump (named HrtBA Ef for the HrtBA from E. faecalis). We show that FhtR, by managing intracellular heme concentration, regulates the functional expression of the heme-dependent catalase A (KatA), thus participating in heme detoxification. The biochemical features of FhtR binding to DNA, and its interaction with heme that induces efflux, are characterized. The FhtR-HrtBA Ef system is shown to be relevant in a mouse intestinal model. We further show that FhtR senses heme from blood and hemoglobin but also from crossfeeding by Escherichia coli These findings bring to light the central role of heme sensing by FhtR in response to heme fluctuations within the gastrointestinal tract, which allow this pathogen to limit heme toxicity while ensuring expression of an oxidative defense system.IMPORTANCEEnterococcus faecalis, a normal and harmless colonizer of the human intestinal flora can cause severe infectious diseases in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote its resistance and its virulence. E. faecalis, which cannot synthesize heme, an essential but toxic metabolite, needs to scavenge this molecule from the host to respire and fight stress generated by oxidants. Here, we report a new mechanism used by E. faecalis to sense heme and trigger the synthesis of a heme efflux pump that balances the amount of heme inside the bacteria. We show in a mouse model that E. faecalis uses this mechanisms within the gastrointestinal tract.