The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines

Abstract

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Keywords: SrrAB two-component system; Staphylococcus aureus; cysteine disulfide bond; sensor histidine kinase.

Fig. 1.

Domain architecture and regulation of SrrB activity by the PAS domain. (A) Domain architecture of SrrA and SrrB proteins. SrrB is a membrane-bound histidine kinase with extracellular Cache domain, transmembrane HAMP domain, intracellular PAS domain, and DHp-CA catalytic domain. SrrA is predicted to be an OmpR-like response regulator with receiver domain and winged helix-turn-helix DNA binding domain. (B) Autophosphorylation of SrrB HAMP-PAS-DHp-CA and (C) DHp-CA. (D) Kinetics of autophosphorylation of SrrB HAMP-PAS-DHp-CA and DHp-CA regions. K_m, V_max, and K_cat values were calculated by fitting the kinetics data to the Michaelis–Menten equation in GraphPad software. The errors represent the error of fitting the model to the data. Phosphotransfer of (E) SrrB HAMP-PAS-DHp-CA and (F) DHp-CA to full-length SrrA. Experiments were performed in triplicate, and representative gels are shown.

Fig. 2.

The SrrB histidine kinase activity is sensitive to the cysteine redox state. (A) The crystal structure of oxidized (-DTT) SrrB catalytic DHp-CA region refined to 2.0 Å (PDB ID code 6PAJ). The phosphorylated histidine residue (H369) is colored in green, and the cysteine residues are in yellow. The missing electron densities for residues 517 to 544 are shown as a dashed line. (Bottom) Top-down view of the DHp-CA region. Each monomer is shown in gray and blue, respectively. The 2Fo-Fc electron density map of the disulfide bond between C464 and C501 is shown in gray wire at contour level 1σ. (B) A structure-based sequence alignment of E. coli EnvZ (PDB ID code 4KP4; NCBI accession no. 584579776), T. maritima HK853 (PDB ID code 3DGE; NCBI accession no. 251836869), and S. aureus SrrB (NCBI accession no. 123003464) is shown with conserved N, G1, F, G2, and G3 box residues underlined. The residues 517 to 544, which are missing in the SrrB crystal structure, are colored in red, and residues forming the ATP-lid are in bold. (C) SDS/PAGE gel of SrrB Cyto showing redox regulation of the intramolecular disulfide bond by DTT. DTT (0 to 1 mM) was added to the reaction buffer containing SrrB Cyto, run on 12% SDS/PAGE gel, and stained with Coomassie blue. (D) Redox-dependent autophosphorylation of SrrB Cyto. Phosphorylated SrrB Cyto was quantified by using ImageJ and plotted in SigmaPlot. The autophosphorylation reactions of reduced and oxidized SrrB Cyto were done in triplicate.

Fig. 3.

Alignment of catalytic DHp-CA region of SrrB from various Staphylococcus species. (A) The phylogenetic tree was generated using the full-length SrrB sequence. The five Staphylococcus species containing conserved cysteine residues are in bold. (B) Sequence alignment of the catalytic DHp-CA region of SrrB showing the presence of the conserved cysteine residues. The two conserved cysteines are highlighted in yellow. The S. epidermidis sequence is shown for comparison.

Fig. 4.

SrrB cysteine mutations disrupt anaerobic biofilm formation and TSST-1 expression under low O₂ conditions. (A) Biofilm formation of the srrAB chromosomal deletion of LAC (ΔsrrAB) and complementation with srrAB mutants. Biofilm formation was quantified as the ratio of the crystal violet absorbance at 570 nm to cell density (absorbance determined at 590 nm). The data summarize six independent experiments (**P < 0.0001; *P < 0.0005). (B and C) Exoprotein profiles (Top) and Western blot analyses of TSST-1 protein expression (Bottom) from S. aureus wild-type MN8, ΔsrrAB mutant, and complemented strains: ΔsrrAB+pRMC2, ΔsrrAB+pRMC2::srrAB, and ΔsrrAB+pRMC2::srrAB_C501A. Concentrated supernatants from the indicated strains grown in brain heart infusion medium under (B) aerobic conditions for 8 h or (C) low O₂ conditions for 24 h were loaded onto 12% SDS/PAGE gels. Molecular mass markers on the left lane are indicated in kilodaltons, and the location of TSST-1 and the second Ig-binding protein (Sbi) are shown by black arrows (Lower). The TSST-1 Western blots were done in duplicate for aerobic conditions and triplicate in low O₂ conditions. A representative SDS/PAGE gel and Western blot of TSST-1 is shown.

Fig. 5.

SrrB cysteine mutation increases susceptibility to endocarditis development and lethality in rabbits infected with S. aureus MN8 strain. (A) The survival curve of rabbits infected with S. aureus MN8 wild-type, ΔsrrAB, and complementation strains: ΔsrrAB + pRMC2, ΔsrrAB + pRMC2::srrAB, and ΔsrrAB + pRMC2::srrAB_C501A. (B) The size of vegetations excised from dissected aortic valves of animals infected with each MN8 strain. (C) A representative picture of the dissected aortic valve from animals infected with each MN8 strain. Circles show where vegetations were formed. (Original magnification: 2×.) (D) Bacterial count obtained from vegetations of the animals infected with each MN8 strain. Four animals were used for the MN8 wild-type control group, and six animals each were used for the remaining strains. Statistical significance was determined by Student’s unpaired t test (vegetation size and CFU; **P < 0.0005; *P < 0.01).

Fig. 6.

Model of SrrAB two-component system regulation by redox. Under oxidizing conditions, terminal oxidases cause accumulation of oxidized menaquinone (MenQ). Under anerobic conditions (or terminal oxidase mutants), there is an accumulation of reduced menaquinone. The pool of reduced MenQ can reduce the SrrB cysteines directly or indirectly via a PAS domain/ligand complex to increase SrrB kinase activity. A putative redox-sensitive ligand binding to the PAS domain is depicted by the red square (see Discussion for details).