To gain a better insight of how Cu ions toxify cells, metabolomic analyses were performed in S. aureus strains that lack the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop^-). Exposure of the cop^- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.

The glucose gluconate tab contains glucose v gluconate growth for Fig 3. Bacterial strains were grown at 37 °C in a microtiter plate containing a chemically defined medium with 11 mM glucose or 11 mM gluconate as a primary carbon source. 

The 13C ion counts tab contains the 13C counts utilized for Figure 2 and Figure s1. Metabolic analyses were conducted using biological triplicates. Overnight cultures were diluted into fresh TSB to OD₆₀₀ 0.1 and grown until OD₆₀₀ of 0.5 in a flask. At this point, 5 mL of culture was transferred to individual 30 mL capacity culture tubes and exposed to 0 or 5 µM Cu(II) for three hours. Samples for the metabolite profiling were prepared as described previously (1). Briefly, 1 mL of the cells were pelleted, washed once with PBS, and resuspended in 1 mL of Methanol:Acetonitrile:Water (2:2:1) solution. Cells were lysed by bead beating (2 cycles, 40 s each, 6.0 m s-1) using a FastPrep homogenizer (MP Biomedicals) and 0.1-mm silica glass beads (MP Biomedicals). Samples were centrifuged twice at 14,500 rpm at 4 °C for 2 minutes and supernatant was retained. The supernatant was filtered with nylon membrane syringe filters (13 mm, 0.22 μm, Fisherbrand) and samples were stored at -80 °C until metabolite analysis was performed.

Carbon labeling experiments were performed using D-Glucose-¹³C₆ (389374, Sigma-Aldrich) using the cop^- strain. Overnight cultures were diluted to an OD₆₀₀ of 0.1 in 1.5 mL of fresh glucose-free TSB supplemented with 11 mM glucose in 10-mL capacity culture tube. Cells were cultured to an OD₆₀₀ of 0.5 cells, pelleted, washed twice with PBS, and resuspended in 1.5 mL of glucose-free TSB supplemented with 11 mM ¹³C₆ glucose. Cells were cultured for 15, 30 or 60 additional minutes before samples were collected and metabolites were extracted as described above.

Samples were analyzed at the metabolomics core of the Cancer Institute of New Jersey. HILIC separation was performed on a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA) with an XBridge BEH Amide column (150 mm × 2.1 mm, 2.5 μm particle size, Waters, Milford, MA) using a gradient of solvent A (95%:5% H₂O:acetonitrile with 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4) and solvent B (20%:80% H₂O:acetonitrile with 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4). The gradient was 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B. The flow rate was 300 μL min^-1. The column temperature was set to 25 °C. The autosampler temperature was set to 4 °C, and the injection volume was 5 μL. MS scans were obtained in negative mode with a resolution of 70,000 at m/z 200, in addition to an automatic gain control target of 3 x 106 and m/z scan range of 72 to 1000. Metabolite data were obtained using the MAVEN software package (mass accuracy window: 5 ppm).

The chemical compliment tab contains the data used to generate Figure S8. Growth of the cop^- strain in liquid chemically defined media with and without 8 µM Cu(II). The complimented media was supplemented with 50 µM of uridine, tryptophan, guanosine, and nicotinamide mononucleotide (NMN). Bacterial strains were grown at 37 °C in a microtiter plate containing a chemically defined medium with 11 mM glucose as a primary carbon source.

The Wt apt defined media tab contains the data used to generate Figure S3. Growth of the cop^- and cop^- apt::tetM strains in liquid chemically defined media with and without 30 µM Cu(II). Bacterial strains were grown at 37 °C in a microtiter plate containing a chemically defined medium with 11 mM glucose as a primary carbon source.

The pEPSA5_apt overexpression tab contains the data used to generate Figure S6. Growth of the cop- strain containing pEPSA5, pEPSA5_apt, or pEPSA5_prs in TSB-Cm media with and without Cu(II). Overnight cultures in TSB-Cm were back diluted to an optical density of 0.001 in media containing Cm and 1% xylose Cu(II) was added at the indicated concentrations. Culture optical densities were measured after 18 hours of growth.

The acnA activity tab contains the data used to generate Figure S5. The cop^- and cop^- apt::tetM strains have similar aconitase (AcnA) activity after growth with Cu(II). The cop^- and cop^- apt::tetM strains were cultured in TSB media with and without 5 µM Cu(II) before the activity was monitored in cell-free lysates.

The metal analysis tab contains the data used to generate Figure S4. The cop^- and cop^- apt::tetM strains were cultured in TSB media with and without 5 µM Cu(II) before cells were harvested and total cell-associated metal was determined by ICP-MS. cells were grown for 18 hours overnight in TSB before diluting them to an OD of 0.05 (A₆₀₀) in 7.5 mL of Chelex (Bio-Rad)-treated TSB in a 30 mL capacity culture tubes. Cells were allowed to grow with shaking for eight hours, before 0 or 5 µM CuSO₄ was added. Cultures were further incubated for 60 minutes. Pre-weighted metal-free 15 mL propylene tubes were used to pellet the cells using a prechilled tabletop centrifuge (Eppendorf, Hauppauge, NY). Pellets were washed three times with 10 mL of ice-cold PBS. All samples were kept at -80 °C or on dry ice until processing.  Cell pellets were acid digested and quantification was performed using an Agilent 7700 inductively coupled plasma mass spectrometer (Agilent, Santa Clara, CA). Data were acquired and analyzed using the Agilent Mass Hunter Workstation Software version A.01.02.

The Cop pglS tab contains the data used to generate Figure S2. The optical densities of cultures of the cop^- and cop^- pglS::Tn strains were recorded after 18 hours of static growth in TSB media with 0-1 mM Cu(II). Bacterial strains were grown at 37°C in a microtiter plate containing TSB media.

The qPCR hla prsA tab contains the data used to generate Figures S10 and Figure 6. To analyze RNA abundances corresponding to genes targeted by CRISPRi, the cop^- strain carrying pSK_CRISPi vectors were cultures overnight and diluted into 5 mL of fresh TSB-Cm to OD₆₀₀ 0.1 in 30 mL culture tubes. The cells were cultured until an OD₆₀₀ of 0.5 before the addition of 0 or 100 ng mL^-1 anhydrotetracycline (Atet). After an additional one-hour culture, five mL of cells were harvested by centrifugation, washed with PBS, and resuspended in 500 µL with RNA protect (QIAGEN). RNA extraction, cDNA synthesis, and transcript quantification (QuantStudio 3, Bio-Rad Laboratories Inc., Hercules, CA) were performed as previously described (2). 

The enzyme activity tab contains the data used to generate Figure S11 and Figure 7. For in vivo assays, the cop^- strain carrying the pEPSA5_prs vector was cultured overnight in TSB-Cm. The was then diluted to an OD₆₀₀ of 0.1 in 1.5 mL of TSB-Cm supplemented with 0.5% xylose. Cultures were grown as indicated for metabolomics, one mL of cells were collected by centrifugation and washed twice with PBS. Cells were resuspended in 1 mL of PBS and lysed by bead beating using 0.1-mm silica glass beads (MP Biomedicals). Cell debris was removed by centrifugation at 4˚C using a tabletop microcentrifuge and supernatants were concentrated using Vivaspin 500 (3,000K MWCO, PES, Sartorius). Prs activity was measured by monitoring AMP formation using a coupled enzyme system (3). For each extract, we subtracted the basal NADH oxidation activity (before addition R5P) from the R5P-dependent NADH oxidation. This was necessary due to the presence of many other enzymes that consume ATP and produce AMP. Measurements were carried out using UV-STAR microplates (Greiner Bio-One) and Varioskan LUX Multimode Microplate Reader (Thermo Scientific). in vitro Prs activity assays contained 0.07 mg mL^-1 Prs. Cu(I) was prepared using asorbic acid as described (2).

The pathogenesis data tab contains the data used to generate Figure 8. C57BL/6 mice, 6 weeks of age, were intranasally infected with 2-4 x 10⁷ cfu of S. aureus in 50 μL of PBS under anesthesia (ketamine and xylazine). Bacterial loads were enumerated at 24 h post-infection from bronchoalveolar lavage fluid (BALF) by washing the airway 3 times with 1 ml of PBS and homogenized lung tissue. Skin infection was initiated with 2-4 x 10⁶ cfu of S. aureus in 100 mL of PBS delivered subcutaneously. Flow cytometry was performed on fluorescently labelled cells from BALF as described elsewhere (4). Bacterial counts were quantified by using CHROMagar S. aureus plates (BD Biosciences)

The PRPP levels tab was used to generate the data in Figure 7B. The cop^- and cop- apt::tetM strains vector were grown overnight in TSB and diluted to an OD₆₀₀ of 0.1 in TSB medium.  Cells were cultured until an OD₆₀₀ of 0.5 and 0 or 5 µM Cu(II) was added. Cells were cultured for an additional three hours before collecting 0.5 mL of cells and washing them with PBS. Cells were resuspended in 1 mL of PBS and lysed by bead beating using 0.1-mm silica glass beads (MP Biomedicals). Cell debris was removed by centrifugation at 4˚C using a tabletop microcentrifuge. PRPP levels were measured using the PRPP ELISA Kit (Mybiosource) following manufacturer instructions. PRPP levels were standardized to protein concentration which was determined using Bradford reagent. 

1. Patel JS, Norambuena J, Al-Tameemi H, Ahn YM, Perryman AL, et al. 2021. ACS Infect Dis 7: 2508-21
2. Al-Tameemi H, Beavers WN, Norambuena J, Skaar EP, Boyd JM. 2020. Mol Microbiol
3. Koenigsknecht MJ, Fenlon LA, Downs DM. 2010. Microbiology (Reading, England) 156: 950-9
4. Parker D. 2018. Cytokine 107: 130-6