DOI: 10.5061/dryad.8w9ghx3s0
title: Data for: Copper ions inhibit pentose phosphate pathway function in Staphylococcus aureus

description: 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.
description: 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 OD<sub>600</sub> 0.1 and grown until OD<sub>600
 </sub>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-<sup>13</sup>C<sub>6</sub> (389374,
 Sigma-Aldrich) using the <em>cop<sup>-</sup></em>
 strain. Overnight cultures were diluted to an OD<sub>600</sub>
 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
 OD<sub>600</sub> of 0.5 cells, pelleted, washed twice with
 PBS, and resuspended in 1.5 mL of glucose-free TSB supplemented with 11 mM
 <sup>13</sup>C<sub>6</sub> 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% H<sub>2</sub>O:acetonitrile with 20 mM
 acetic acid, 40 mM ammonium hydroxide, pH 9.4) and solvent B (20%:80%
 H<sub>2</sub>O: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<sup>-1</sup>. 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 <em>cop</em><sup>-</sup> 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 <em>cop</em><sup>-</sup> and
 <em>cop<sup>-</sup> apt::tetM</em> 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. <br> The
 pEPSA5_apt overexpression tab contains
 the data used to generate Figure S6. Growth of the
 <em>cop</em>- strain containing pEPSA5,
 pEPSA5_<em>apt</em>, or pEPSA5_<em>prs </em>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
 <em>cop</em><sup>-</sup> and
 <em>cop<sup>-</sup> apt::tetM</em> strains have
 similar aconitase (AcnA) activity after growth with Cu(II). The
 <em>cop</em><sup>-</sup> and
 <em>cop<sup>-</sup> apt::tetM</em> 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<em> cop</em><sup>-</sup>
 and <em>cop<sup>-</sup> apt::tetM</em> 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 (A<sub>600</sub>) 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 CuSO<sub>4 </sub>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
 <em>cop</em><sup>-</sup> and
 <em>cop</em><sup>-</sup>
 <em>pglS::Tn</em> 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
 <em>cop<sup>-</sup></em><sup>
 </sup>strain carrying pSK_CRISPi vectors were cultures overnight and
 diluted into 5 mL of fresh TSB-Cm to OD<sub>600</sub> 0.1 in
 30 mL culture tubes. The cells were cultured until an
 OD<sub>600</sub> of 0.5 before the addition of 0 or 100 ng
 mL<sup>-1</sup> 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 <em>in vivo</em> assays,
 the <em>cop<sup>-</sup></em><sup>
 </sup>strain carrying the pEPSA5_<em>prs</em> vector was
 cultured overnight in TSB-Cm. The was then diluted to an
 OD<sub>600</sub> 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<strong>.</strong> Measurements were carried
 out using UV-STAR microplates (Greiner Bio-One) and Varioskan LUX
 Multimode Microplate Reader (Thermo Scientific). <em>in vitro
 </em>Prs activity assays contained 0.07 mg
 mL<sup>-1</sup> 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 10<sup>7</sup> cfu of <em>S.
 aureus</em> 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 10<sup>6</sup> cfu of <em>S. aureus</em> 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 <em>S.
 aureus</em> plates (BD Biosciences) The PRPP
 levels tab was used to generate the
 data in Figure 7B. The
 <em>cop<sup>-</sup></em><sup>
 </sup>and <em>cop- apt::tetM</em> strains vector were
 grown overnight in TSB and diluted to an OD<sub>600</sub> of
 0.1 in TSB medium.  Cells were cultured until an OD<sub>600
 </sub>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.  
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