title: Real-Time Imaging of the Bacillithiol Redox Potential in the Human Pathogen Staphylococcus aureus Using a Genetically Encoded Bacilliredoxin-Fused Redox Biosensor.
abstract: Bacillithiol (BSH) is utilized as a major thiol-redox buffer in the human pathogen Staphylococcus aureus. Under oxidative stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolation, which can be reversed by bacilliredoxins (Brx). In eukaryotes, glutaredoxin-fused roGFP2 biosensors have been applied for dynamic live imaging of the glutathione redox potential. Here, we have constructed a genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus.
  
  
  
  The Brx-roGFP2 biosensor showed a specific and rapid response to low levels of bacillithiol disulfide (BSSB) in vitro that required the active-site Cys of Brx. Dynamic live imaging in two methicillin-resistant S. aureus (MRSA) USA300 and COL strains revealed fast and dynamic responses of the Brx-roGFP2 biosensor under hypochlorite and hydrogen peroxide (H2O2) stress and constitutive oxidation of the probe in different BSH-deficient mutants. Furthermore, we found that the Brx-roGFP2 expression level and the dynamic range are higher in S. aureus COL compared with the USA300 strain. In phagocytosis assays with THP-1 macrophages, the biosensor was 87% oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes, indicating that antibiotics do not cause oxidative stress in S. aureus. Conclusion and Innovation: This Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be applied for dynamic live imaging of redox changes in S. aureus and other BSH-producing Firmicutes. Antioxid. Redox Signal. 26, 835-848.
authors.affiliation: 1 Institute for Biology-Microbiology, Freie Universität Berlin , Berlin, Germany .
authors.forename: Vu Van
authors.initials: VV
authors.lastname: Loi
authors.affiliation: 2 Junior Research Group Pathoproteomics, Ernst-Moritz-Arndt-University of Greifswald , Greifswald, Germany .
authors.forename: Manuela
authors.initials: M
authors.lastname: Harms
authors.affiliation: 3 Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald , Greifswald, Germany .
authors.forename: Marret
authors.initials: M
authors.lastname: Müller
authors.affiliation: 1 Institute for Biology-Microbiology, Freie Universität Berlin , Berlin, Germany .
authors.forename: Nguyen Thi Thu
authors.initials: NTT
authors.lastname: Huyen
authors.affiliation: 4 School of Pharmacy, University of East Anglia , Norwich Research Park, Norwich, United Kingdom .
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Hamilton
authors.affiliation: 2 Junior Research Group Pathoproteomics, Ernst-Moritz-Arndt-University of Greifswald , Greifswald, Germany .
authors.forename: Falko
authors.initials: F
authors.lastname: Hochgräfe
authors.affiliation: 3 Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald , Greifswald, Germany .
authors.forename: Jan
authors.initials: J
authors.lastname: Pané-Farré
authors.affiliation: 1 Institute for Biology-Microbiology, Freie Universität Berlin , Berlin, Germany .
authors.forename: Haike
authors.initials: H
authors.lastname: Antelmann
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D003545:Cysteine / Q000031:analogs & derivatives* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; C543521:bacillithiol; D049452:Green Fluorescent Proteins; D003545:Cysteine; D005944:Glucosamine
keywords: Staphylococcus aureus; bacilliredoxin; bacillithiol; oxidative stress; redox biosensor; roGFP