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Molecular Pathogenesis

Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters

Jyoti Velayudhan, Joyce E. Karlinsey, Elaine R. Frawley, Lynne A. Becker, Margaret Nartea, Ferric C. Fang
S. M. Payne, Editor
Jyoti Velayudhan
aDepartment of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA
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Joyce E. Karlinsey
bDepartment of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA
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Elaine R. Frawley
aDepartment of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA
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Lynne A. Becker
aDepartment of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA
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Margaret Nartea
aDepartment of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA
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Ferric C. Fang
aDepartment of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA
bDepartment of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA
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S. M. Payne
Roles: Editor
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DOI: 10.1128/IAI.01022-13
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  • FIG 1
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    FIG 1

    Relative H2O2 resistance of wild-type and mutant S. Typhimurium strains. Wild-type, cyaY (A, B, and C), stm3944 (A and C), stm3944 cyaY (A and C), and yggX and cyaY yggX (B and C) mutant strains were cultured in the presence of H2O2. (A and B) Strains were inoculated at a density of 106 CFU ml−1 in fresh LB with (open symbols) or without (closed symbols) 1 mM H2O2. Growth was monitored in a Bioscreen C microplate reader with agitation at 37°C. Data shown are means ± standard deviations from at least three independent experiments. (C) Strains were grown in LB at 37°C to an OD600 of ∼0.5 and then treated with 10 mM H2O2 for 2 h with aeration before percent survival was determined. The medians are indicated by horizontal bars. P values were calculated by comparison of the results for the mutants to those for the wild type using the Wilcoxon rank-sum test. Survival T = 2 h, survival at 2 h; *, significant difference between Salmonella cyaY (P < 0.05) and stm3944, stm3944 cyaY, yggX, and cyaY yggX (P < 0.005) mutants.

  • FIG 2
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    FIG 2

    Intracellular free iron concentrations of an S. Typhimurium fur mutant overexpressing STM3944. (A) Strains JV119 and JV120 were grown in LB to an OD600 of ∼1.0, and desferrioxamine-chelatable free iron concentrations were determined by EPR spectroscopy. Representative EPR spectra for an S. Typhimurium fur mutant containing the pBAD-Cm vector (JV119) or pBAD-Cm::stm3944 (JV120) normalized to cell density are shown. (B) Quantitation of intracellular free iron concentrations. Data are means ± 1 standard deviation from three independent experiments. *, concentration significantly different from that of the wild type (P = 0.01, Student's t test).

  • FIG 3
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    FIG 3

    Iron-sulfur cluster activity in wild-type and mutant S. Typhimurium strains under basal conditions and following H2O2 stress. (A) Basal iron-sulfur cluster enzyme activity was assayed in wild-type and cyaY, yggX, cyaY yggX, and iscA mutant strains. Aconitase (Acn), serine deaminase (Sda) and NADH dehydrogenase I (NdhI) activities were measured in mutant strains grown in LB to an OD600 of ∼1.0 and normalized to wild-type activity. Data shown are means ± 1 standard deviation from at least three independent experiments. *, activity significantly different from that of the wild type (P < 0.05, Student's t test); #, activity significantly different from that of the wild-type, cyaY mutant, or yggX mutant strain (P < 0.05, Student's t test). (B) Relative Sda, Acn, and NdhI activity was assayed following 15 min exposure to 0.5, 4, or 8 mM H2O2. For each assay, activity was normalized to the enzymatic activity in the absence of H2O2, which is displayed as 100%.

  • FIG 4
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    FIG 4

    Reactivation of aconitase activity following damage by H2O2. Strains grown in LB to an OD600 of ∼1.0 were treated with 4 mM H2O2 for 15 min. Spectinomycin and the iron chelator DTPA were included to block new protein synthesis and to inhibit iron transport from the extracellular medium, respectively. Catalase was added (at 0 min) to terminate the H2O2 stress, and aconitase reactivation was measured at the indicated time intervals after the termination of H2O2 stress. Activities were normalized to the activity of the untreated control (prior to H2O2 exposure), which was set to 100%. (A) Aconitase reactivation in wild-type S. Typhimurium, the cyaY mutant, the yggX mutant, and the cyaY yggX mutant. (B) Aconitase reactivation in wild-type S. Typhimurium, the cyaY mutant containing empty vector pBAD-Cm, and the cyaY mutant containing pBAD-Cm::cyaY. (C) Aconitase reactivation in wild-type S. Typhimurium, the yggX mutant containing empty vector pBAD-Kan, and the yggX mutant containing pBAD-Kan::yggX. Data shown are means ± 1 standard deviation from three independent experiments. *, reactivation levels significantly different from that for the wild type (P < 0.05, Student's t test); #, reactivation significantly different from that for the wild-type, cyaY mutant, and yggX mutant strains (P < 0.05, Student's t test).

  • FIG 5
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    FIG 5

    Relative nitric oxide resistance of wild-type and mutant S. Typhimurium strains. Growth of the wild type and the cyaY, yggX, and cyaY yggX mutant strains was monitored in the presence of the NO· donor Sper/NO. Strains were inoculated at a density of 106 CFU ml−1 in M9 minimal medium plus 0.2% gluconate with (open symbols) or without (closed symbols) 750 μM Sper/NO. Growth was monitored in a Bioscreen C microplate reader with agitation at 37°C. Data shown are means ± 1 standard deviation from at least three independent experiments.

  • FIG 6
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    FIG 6

    Virulence of wild-type S. Typhimurium and isogenic mutant derivatives in a murine model of systemic infection. (A) Seven-week-old C3H/HeN mice (n = 10) were injected intraperitoneally with 1 × 103 to 2 × 103 CFU of wild-type or isogenic mutant strains. Mice were monitored daily for signs of illness, and moribund animals were euthanized. Data are representative of those from at least two or more reproducible, independent experiments. P values were calculated using the log-rank Mantel-Cox test. (B) Seven-week-old C3H/HeN mice (n = 5) were injected intraperitoneally with 2 × 103 total CFU of a 1:1 mixture of wild-type and isogenic mutant bacteria. All animals were euthanized at 8 days postinfection, and the competitive index of the remaining mutant bacteria to the remaining wild-type bacteria was calculated for both livers and spleens. The cyaA, yggX, and iscA mutants each displayed a statistically significant (P < 0.05) competitive disadvantage in the liver compared to the wild type, though the yggX defect (confidence interval = 0.72) was minor compared to the cyaY (confidence interval = 0.19) and iscA (confidence interval = 0.41) mutations. In the spleen, only cyaY and iscA mutants displayed statistically significant (P < 0.05) competitive disadvantages of 0.41 and 0.39, respectively. P values were calculated by the Wilcoxon rank-sum test.

  • FIG 7
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    FIG 7

    Hypothetical model of interplay between CyaY, YggX, and STM3944 in iron-sulfur cluster repair and protection against iron-induced toxicity. Under conditions of mild and severe oxidative stress, prior to the recruitment of the SoxRS-dependent YggX protein, damage to labile iron-sulfur clusters requires continuous turnover by CyaY, which is shown in complex with the cysteine desulfurase IscS. Extrusion of iron released from oxidized clusters is facilitated by the STM3944 protein. Once recruited, YggX may serve as a sink for iron released from damaged iron-sulfur clusters and promote the repair of damaged clusters independently of CyaY.

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  • TABLE 1

    Bacterial strains, plasmids, and primers

    Strain, plasmid, or primerGenotype, relevant characteristics, or sequenceaSource or reference
    Strains
        ATCC 14028sSalmonella enterica serovar Typhimurium ATCC 14028s (WT)ATCC
        KLM001ATCC 14028s fur::bla32
        JV119ATCC 14028s fur::bla/pBAD18-CmThis study
        JV120ATCC 14028s fur::bla/pBAD18-Cm::STM3944This study
        EF434ATCC 14028s/pHR105/pBAD18-KanThis study
        EF435ATCC 14028s/pHR105/pJK714This study
        JV122ATCC 14028s cyaY::FRT-kanThis study
        JK1266ATCC 14028s STM3944::FRTThis study
        JK1264ATCC 14028s STM3944-cyaY::FRT-catThis study
        JV125ATCC 14028s yggX::FRT-catThis study
        JK1206ATCC 14028s cyaY::FRT-kan yggX::FRT-catThis study
        JV129ATCC 14028s iscA::FRT-catThis study
        JV131ATCC 14028s WT/pBAD18-KanThis study
        JV132ATCC 14028s yggX::FRT-cat/pBAD18-KanThis study
        JV133ATCC 14028s yggX::FRT-cat/pBAD18-Kan::yggXThis study
        JV134ATCC 14028s WT/pBAD18-CmThis study
        JV135ATCC 14028s cyaY::FRT-kan/pBAD18-CmThis study
        JV136ATCC 14028s cyaY::FRT-kan/pBAD18-Cm::cyaYThis study
        JK1193ATCC 14028s WT/pRB3This study
        JK1194ATCC 14028s cyaY::FRT-kan/pRB3This study
        JK1196ATCC 14028s yggX::FRT-cat/pRB3This study
        JK1213ATCC 14028s cyaY::FRT-kan/pJK707This study
        JK1216ATCC 14028s yggX::FRT-cat/pJK710This study
        JK1259ATCC 14028s Δedd::FRT-kanThis study
    Plasmids
        pKD3bla FRT cat FRT PS1 PS2 ori6K31
        pKD4bla FRT kan FRT PS1 PS2 ori6K31
        pKD46bla araC-ParaB γ β exo oriR101 repA101(Ts)
        pCP20bla cat cI857 λPr flp PSC101 ori(Ts)31
        pBAD18-CmaraC ParaBAD cat33
        pBAD18-Cm::cyaYpBAD18-Cm::cyaYThis study
        pBAD18-Cm::stm3944pBAD18-Cm::stm3944This study
        pBAD18-KanaraC ParaBAD kan33
        pBAD18-Kan::yggXpBAD18-Kan::yggXThis study
        pRB3par RK2 bla stable low-copy-number cloning vector35
        pJK707−750 to −1 ATG stm3944 and cyaY coding region in pRB3This study
        pJK710−361 bp upstream and yggX coding region in pRB3This study
        pHR105cat ParaBAD-feoAB ori/p15A43
        pJK714pBAD18-Kan::stm3944This study
    Primers
        cyaYP1ACCTTTTCACCCGCTTGTTGCGTTGCCGCCTGCTCCAGCAGATCCCAGAAGGTTTCGCCGGTGTAGGCTGGAGCTGCTTCThis study
        cyaYP2CAGTGAATTTCATCGCCTGGCTGACGCCCTGTGGCTCACCATTGAAGAACGCCTCGATAGCATATGAATATCCTCCTTAGThis study
        JKP570-cyaY3-P2CGTTGCCGCCTGCTCCAGCAGATCCCAGAAGGTTTCGCCGCATATGAATATCCTCCTTAGThis study
        JKP568-STM3944-P1AAACCGTTTTGCGCACTTGATTAAACATTTGAAAAACGCCGTGTAGGCTGGAGCTGCTTCThis study
        JKP569-STM3944-P2TAAATAGCGCACCGGTGATAAGTATGACGGACGCGTTAACCATATGAATATCCTCCTTAGThis study
        yggXP1GTCGATGAAGAGGATGACTTATGAGCAGAACGATTTTTTGTACTTACCTGCAACGCGACGGTGTAGGCTGGAGCTGCTTCThis study
        yggXP2AGCGTCATTCAGCAACGGGCCTATTTTTTATCTTCCGGCGTATAACCTTCGATATGAACGCATATGAATATCCTCCTTAGThis study
        iscAP1ATGTCGATTACACTTAGCGACAGTGCAGCAGCGCGAGTTAATACCTTCCTGGCCAACCGTGTGTAGGCTGGAGCTGCTTCThis study
        iscAP2ACCACACTCATCTTTCACATTCGGGTTGGAGAATTTAAACCCTTCGTTCAGACCTTCTTTCATATGAATATCCTCCTTAGThis study
        cyaYpBADSacIATGCGAGCTCAGAAGCTGACGCTTCCCCTTThis study
        cyaYpBADSphIrevATGCGCATGCAGCGCTATTCTTCTGGCTCTThis study
        STM3944pBADSacIATGCGAGCTCTACACCAACAATAAAGACATThis study
        STM3944pBADSphIrevATGCGCATGCGCGTCAGCTTCTGGAACCGAThis study
        yggXpBADSacIATGCGAGCTCCGCCCCGGTCGATGAAGAThis study
        yggXpBADSphIrevATGCGCATGCCATAACTCACAGGCCGAATAThis study
        JKP497-cyaYA1AACCGGATCCCCGTCAGGCTGAACAGAGThis study
        JKP499-cyaYBTCCGGTGCCTGCGTTATGATGThis study
        JKP500-cyaYCTAACGCAGGCACCGGAATGAACGACAGTGAATTTCATCGThis study
        JKP501-cyaYDAACCAAGCTTAGCGAAAACTCACCTTTTCACThis study
        JKP505-yggXDAACCAAGCTTCTATTTTTTATCTTCCGGCGTAThis study
        JKP506-yggXFAACCGGATCCTAATCAGGAGATTTTCCTGGCGThis study
        JKP548-edd-P1GAAGCCTATCTTGCCCGCATTGAGCAGGCGAAAACCGCCAGTGTAGGCTGGAGCTGCTTCThis study
        JKP549-edd-P2CCTGCTCCGCACCCGACAGCTTTTCGCGCAGCGCGCCAAACATATGAATATCCTCCTTAGThis study
        JKP82-rpoD_FGTGAAATGGGCACTGTTGAACTGThis study
        JKP83-rpoD_RTTCCAGCAGATAGGTAATGGCTTCThis study
        JKP552-acnA_FTCCTGATGCAGGACTTTACCThis study
        JKP553-acnA_RGGGTTCACTTTCGACGTATCThis study
        JKP554-acnB_FACTCAGACGTTTTCCGTCAGThis study
        JKP555-acnB_RACGGATACCTTTCACACCACThis study
        JKP556-sdaA _FACGTCATTGACTGGGTGAACThis study
        JKP557-sdaA_RGTAAATATCCGGGCTGACTGThis study
        JKP558-sdaB_FCGTCCTTTATCCAGGATGTGThis study
        JKP559-sdaB_RTCTCATGCAGAGACAGGTTGThis study
        JKP560-tdcG_FTTCAAGCTCCCATACCGTAGThis study
        JKP561-tdcG_RAGCGATAGCGATCCGTAAAGThis study
        JKP562-dsdA_FACTCTCGCACGCTATTTCTGThis study
        JKP563-dsdA_RAACAAACAGTGGGTGACTGGThis study
        JKP564-nuoA_FTTGTGTTACTGGCTGGTCTGThis study
        JKP565-nuoA_RGCGTTGACGATTAGCGATACThis study
    • ↵a FRT, FLP recombination target; WT, wild type. All primer sequences are 5′-3′.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 -

      Fig. S1. Complementation of H2O2 resistance in cyaY and yggX mutant Salmonella strains. Fig. S2. Catalase activity of wild-type and mutant Salmonella strains. Fig. S3. Transcript levels for aconitase, serine deaminase, and NADH dehydrogenase I in wild-type and mutant Salmonella. Fig. S4. Aconitase activity is restored in vitro following the addition of DTT and iron. Fig. S5. 6-Phosphogluconate is inhibited by nitric oxide (NO·).

      PDF, 2.9M

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Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters
Jyoti Velayudhan, Joyce E. Karlinsey, Elaine R. Frawley, Lynne A. Becker, Margaret Nartea, Ferric C. Fang
Infection and Immunity Mar 2014, 82 (4) 1390-1401; DOI: 10.1128/IAI.01022-13

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Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters
Jyoti Velayudhan, Joyce E. Karlinsey, Elaine R. Frawley, Lynne A. Becker, Margaret Nartea, Ferric C. Fang
Infection and Immunity Mar 2014, 82 (4) 1390-1401; DOI: 10.1128/IAI.01022-13
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