Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus

Recent advances in the design of genetically encoded redox biosensors, such as redox-sensitive GFP (roGFP) have facilitated the real-time imaging of the intracellular redox potential in eukaryotic cells at high sensitivity and at spatiotemporal resolution. To increase the speciﬁcity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulﬁde (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential ( E BSH ) in the human pathogen Staphylococcus aureus . Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in S. aureus using the microplate reader. In particularly, we provide details for determination of the E BSH changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and ﬂuorescence in bacteria. • Brx-roGFP2 is speciﬁc for measurements of the bacillithiol redox potential in Staphylococcus aureus cells • Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements


Specifications
Immunology and Microbiology More specific subject area

Redox biology of pathogenic bacteria
Method name Brx-roGFP2 biosensor measurement method Name and reference of original method Original reference for Brx-roGFP2 biosensor construction, measurements and application in S. aureus : V.V. Loi

Resource availability
All resources including software, hardware and materials necessary to reproduce the method are described in the Methods details.

Method overview
Staphylococcus aureus is an important human pathogen that frequently encounters reactive oxygen species (ROS) by activated macrophages and neutrophils during infections. To survive under oxidative stress, S. aureus utilizes the low-molecular-weight (LMW) thiol bacillithiol (BSH), which serves as glutathione (GSH) surrogate to maintain the intracellular redox balance [1] .
In eukaryotes, redox changes lead to oxidation of GSH to glutathione disulfide (GSSG) resulting in a decreased GSH/GSSG redox ratio and an increased GSH redox potential ( E GSH ). Genetically encoded redox-sensitive GFPs (roGFPs) are powerful tools to measure dynamic E GSH changes in realtime at high sensitivity and spatiotemporal resolution in living cells and various organelles [2][3][4] . To increase their specificity towards the GSH/GSSG redox pair, roGFP2 probes have been fused to human glutaredoxin (Grx1). The Grx1-roGFP2 fusion allows specific equilibration between the GSH/GSSG and roGFP2 red / roGFP2 ox redox couples [3][4][5] . Oxidation of roGFP2 influences the spectral properties of the chromophore [4] . In reduced roGFP2, the fluorescence intensity at the 405 nm excitation maximum is low, while intensity at 488 nm excitation maximum is high. Disulfide bond formation between Cys147 and Cys204 of roGFP2 leads to ratiometric changes of the fluorescence intensities at the 405 nm and 488 nm excitation maxima [3] . The 405/488 nm excitation ratio is calculated as oxidation degree (OxD) of the biosensor which reflects the intracellular E GSH in eukaryotic cells [ 3 , 4 ]. We have previously constructed a Brx-roGFP2 fused biosensor to monitor BSH redox potential ( E BSH ) changes during the growth, under oxidative stress and after antimicrobial treatments in the wild type and different mutant backgrounds that are impaired in redox homeostasis ( Fig. 1 ) [6][7][8][9] . The Brx-roGFP2 biosensor is highly specific for bacillithiol disulfide (BSSB) in vitro and responds differentially to H 2 O 2 and HOCl in vivo [6] . In this work, we provide the methodological details of E BSH measurements using the microplate reader that are related to our previous publication [6] . The applications of Brx-roGFP2 expressing cells are focused on injection assays with oxidants and OxD measurements during the growth [6] . For each sample, fully reduced and oxidized controls have to be included which are used for normalization of the OxD values. In the following sections, the detailed protocol is described regarding S. aureus growth, harvesting and measurements of the Brx-roGFP2 biosensor response. The method is applicable also for other bacteria to measure intrabacterial redox changes at high spatiotemporal resolution without cell disruption. Biosensor and control strains 1. Staphylococcus aureus COL expressing pRB473-brx-roGFP2 [6] 2. Staphylococcus aureus COL pRB473 empty plasmid used as blank [6] Experimental Procedures

(A) Measurements of Brx-roGFP2 biosensor responses in S. aureus during the growth in vivo
The Brx-roGFP2 biosensor was cloned under control of the xylose-inducible promoter P XylR into plasmid pRB473, which was transduced into S. aureus COL wild type and various mutant strains [ 6 , 7 ].
For measurements of the Brx-roGFP2 biosensor response during the growth, S. aureus COL pRB473brx-roGFP2 was cultivated in LB medium with 1% xylose and cells harvested during different times along the growth (exponential growth phase, transition to stationary phase and later stationary phase) [6] . The methods details for bacterial growth, harvesting and measurements are as follows: Preparation of S. aureus cells expressing the Brx-roGFP2 biosensor 1. Growth of the overnight cultures of S. aureus COL-pRB473-brx-roGFP2 (biosensor strain) and S. aureus COL-pRB473 (control strain with empty plasmid used as blank) in 20 ml LB medium with 1 % xylose and 10 μg/ml Cm in a shaking water bath at 37 °C for 16 h. 2. Inoculation of the overnight cultures into fresh LB medium with 1 % xylose and 10 μg/ml Cm to an optical density at 540 nm (OD 540 ) of 0.1. Cultivation of S. aureus strains under vigorous agitation at 37 °C for sampling at different time points along the growth. 3. Sampling of 3 × 10 ml each at the 3, 4, 5 and 6 hour time points after inoculation.
• One sample is treated with 10 mM NEM to block free thiols of Brx-roGFP2 expressed in S. aureus cells. NEM alkylation is required to avoid changes in biosensor oxidation in S. aureus cells during sample processing, which can also lead to stress exposure in S. aureus . NEM is a membranepermeable thiol-trapping reagent, which rapidly alkylates accessible thiols in intact cells in vivo and in protein extracts after cell lysis [11][12][13][14] . Concentrations of 10-20 mM NEM were previously used to freeze successfully the redox state of roGFP2 biosensors expressed in HeLa cells and Plasmodium falciparum in vivo [ 2 , 15 ]. Thus, we applied 10 mM NEM to block free thiols of Brx-roGFP2 inside S. aureus cells before sample harvesting and fluorescence measurements.

Setup the microplate reader for measurements of Brx-roGFP2 fluorescence
Measurements of roGFP2 fluorescence can be performed using various microplate readers that are equipped with 405 and 488 nm excitation filters and a 510 nm emission filter. Due to recommendation by various roGFP2 users, we applied the CLARIOstar microplate reader and the related software version 5.20 R5. Here we provide the comprehensive protocol of previous Brx-roGFP2 measurements. Thus, the description is related to the CLARIOstar reader used in our lab to generate previous results [6] . First, the temperature is set to 37 °C and the new measurement protocol is created under "Test Protocols" based on the following parameters:  [16] , the BSH redox potential ( E BSH ) can be calculated using to the Nernst equation (2) : (2)

(B) Measurements of Brx-roGFP2 responses in S. aureus after exposure to oxidants in vivo
For measurements of the Brx-roGFP2 biosensor response after exposure to oxidants, S. aureus COL pRB473-brx-roGFP2 cells are harvested from the LB overnight culture. Cells are transferred to minimal medium and filled into the microplate wells. Oxidants are directly injected into the microplate wells with the biosensor cells. The methods details are as follows:

Preparation of S. aureus cells expressing the Brx-roGFP2 biosensor for injection assays.
1. Growth of the overnight cultures of S. aureus COL-pRB473-brx-roGFP2 (biosensor strain) and COL-pRB473 (strain with empty plasmid used as blank) in 20 ml LB medium with 1 % xylose and 10 μg/ml Cm in a shaking water bath at 37 °C for 16 h. 2. Sampling of 20 ml cells by centrifugation at 8500 rpm at 25 °C. 3. Washing and dilution of cells in BMM with 1% xylose and 10 μg/ml Cm, adjustment to an OD 500 of 2 to start injection assays. directly into the wells to the 190 μl samples and continue Brx-roGFP2 measurement immediately. 5. Export and analyze the data using the software MARS version 3.10 as described in A).

(C) Measurements of Brx-roGFP2 biosensor responses by BSSB in vitro
The specific response of purified Brx-roGFP2 protein is analyzed after exposure to BSSB in comparison to other LMW thiol disulfides in vitro (e.g. cystine, GSSG, MSSM) [6] . The concentrations of the LMW thiol disulfides are in the physiological range as determined in previous studies using monobromobimane derivatisation of LMW thiols [17] . The methods details are as follows: 1. Prepare stock solutions for biosensor reduction and oxidation including 100 mM DTT, 50 mM Dia and 10-fold stock of LMW thiol disulfides (BSSB, GSSG, cystine) on ice. 2. Reduce purified Brx-roGFP2 protein with 10 mM DTT for 20 min on ice, equilibrate Micro Bio-Spin TM 6 columns with 500 μl reaction buffer and load 75 μl of reduced Brx-roGFP2 to the column to remove excess DTT. Process protein sample as fast as possible to avoid air-oxidation. 3. Measure Brx-roGFP2 concentration by Nanodrop20 0 0 (Thermofisher) and calculate concentration in μM based on the molecular weight (MW) and extinction coefficients. ( http://web.expasy.org/ protparam/ ). The amino acid sequence of Brx-roGFP2 is available in Fig. S1  5. Prepare the microplate reader for measurements using the instrument setup parameters as described in A) with modifications: Temperature setting 25 °C, settling time 0.2. Note : The temperature is set to 25 °C to avoid air-oxidation of the purified Brx-roGFP2 biosensor. For scanning the fluorescence excitation spectrum, select the measurement mode "Spectral scanning", the scanning range from 360 to 500 nm and the bandwidth of 10 nm using 510 nm as emission filter. Example results for injection assays after BSSB treatment of purified Brx-roGFP2 biosensor are shown in Fig. 2 and in the previous publication [6] .

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.