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. 2014 Dec 26;289(52):36263-74.
doi: 10.1074/jbc.M114.611020. Epub 2014 Nov 14.

Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation

Jong Gyu Lim  1 , Ye-Ji Bang  1 , Sang Ho Choi  2
Affiliations

Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation

Jong Gyu Lim et al. J Biol Chem. .

Abstract

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that reduce toxic peroxides. A new Vibrio vulnificus Prx, named Prx3, was identified and characterized in this study. Biochemical and mutational analyses revealed that Prx3 reduces H2O2, utilizing glutaredoxin 3 (Grx3) and glutathione (GSH) as reductants, and requires only N-terminal peroxidatic cysteine for its catalysis. These results, combined with the monomeric size of Prx3 observed under non-reducing conditions, suggested that Prx3 is a Grx3/GSH-dependent 1-Cys Prx and oxidized without forming intermolecular disulfide bonds. The prx3 mutation impaired growth in the medium containing peroxides and reduced virulence in mice, indicating that Prx3 is essential for survival under oxidative stress and pathogenesis of V. vulnificus. The Fe-S cluster regulator IscR activates prx3 by direct binding to a specific binding sequence centered at -44 from the transcription start site. The binding sequence was homologous to the Type 2 IscR-binding sequence, most likely recognized by the Fe-S clusterless apo-IscR in Escherichia coli. The iscR3CA mutant, chromosomally encoding the apo-locked IscR, exhibited 3-fold higher levels of activation of prx3 than the wild type and accumulated more IscR3CA protein in cells. The IscR-dependent activation of prx3 by aerobic growth and iron starvation was also associated with the increase in cellular levels of IscR protein. Taken together, the results suggested that IscR senses iron starvation as well as reactive oxygen species and shifts to the apo-form, which leads to the increase of cellular IscR and in turn prx3 expression, contributing to the survival and virulence of V. vulnificus during pathogenesis.

Keywords: Bacterial Pathogenesis; Gene Regulation; Iron Starvation; IscR; Oxidative Stress; Peroxiredoxin; Reactive Oxygen Species (ROS); Vibrio vulnificus.

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Figures

FIGURE 1.
FIGURE 1.
Amino acid sequence relatedness of V. vulnificus Prx3 (VvPrx3) and members of the Prx5 subfamily. The amino acid sequences retrieved from the NCBI protein database (accession numbers: ADV89163 for V. vulnificus Prx3, NP_230994 for the V. vulnificus Prx3 homologue in V. cholerae, 1TP9_A for P. trichocarpa PrxD (PtPrxD), XP_002808799 for P. falciparum AOP (PfAOP), NM_012094 for human Prx5 (HsPrx5)) were aligned using the ClustalW2 program. Identical (asterisks), highly conserved (double dots), conserved (dots), and missing (dashes) sequences are indicated. All cysteine residues are shaded in gray. The positions of the peroxidatic (CP), second (C2), and resolving cysteines (CR) are indicated above the amino acid sequences. The active site PXXXTXXC motifs are indicated by an open box.
FIGURE 2.
FIGURE 2.
Peroxidase activities of Prx3 with H2O2. A, the peroxidase activities of Prx3 were determined by measuring NADPH oxidation in the reaction mixtures. The reaction mixture contained Prx3 (■), Grx3/GSH/GR (○), Prx3 and Grx3/GSH/GR (●), TrxA/TrR (▵), or Prx3 and TrxA/TrR (▴) in addition to NADPH. B and C, the Grx3/GSH-dependent peroxidase activities of Prx3 were determined by measuring the initial rates of NADPH oxidation (μm NADPH·min−1). B, the reaction mixture contained GSH/GR with (●) or without (○) Prx3 in addition to NADPH and various concentrations of Grx3 as indicated. C, the reaction mixture contained Grx3/GR with (●) or without (○) Prx3 in addition to NADPH and various concentrations of GSH as indicated. All of the reactions were initiated by adding 0.1 mm H2O2 to the reaction mixture. The S.E. values were too small to be shown by error bars. B and C, each solid line represents a fit of the data to a curve of exponential rise to maximum.
FIGURE 3.
FIGURE 3.
Peroxidase activities of wild-type and mutant Prx3. The peroxidase activities of wild-type Prx3, Prx3-C48S, and Prx3-C73S were determined by measuring the initial rates of NADPH oxidation (μm NADPH·min−1). The reaction mixture contained Grx3/GSH/GR and either Prx3 (●), Prx3-C48S (▴), or Prx3-C73S (■) in addition to NADPH. The reaction was initiated by adding 0.1 mm H2O2 to the reaction mixture. The S.E. values were too small to be shown by error bars. Each solid line represents a fit of the data to a curve of exponential rise to maximum.
FIGURE 4.
FIGURE 4.
SDS-PAGE and Western blot analyses of Prx3. A, Purified His-tagged Prx3 proteins were resolved on SDS-PAGE with (+) or without (−) 10 mm DTT as indicated. B, total proteins were isolated from the WT and prx3 mutant grown to an A600 of 0.3 and then resolved by non-reducing SDS-PAGE and immunoblotted using a rabbit anti-Prx3 antiserum. The positions of protein size markers (in kDa; Bio-Rad) are shown on the left of the gel. Arrowheads, two conformations of monomeric Prx3s.
FIGURE 5.
FIGURE 5.
Growth of the V. vulnificus strains under oxidative stress. The V. vulnificus strains were compared for their ability to grow in LBS broth supplemented without oxidant (A) or with 250 μm H2O2 (B) or 200 μm ONOO− (C) at final concentrations. Error bars, S.E. **, p < 0.005 relative to the wild type at the indicated time. WT (pJK1113), wild type; prx3 (pJK1113), prx3 mutant; prx3 (pJK1303), complemented strain.
FIGURE 6.
FIGURE 6.
Effect of prx3 mutation on mouse mortality. Seven-week-old specific pathogen-free female ICR mice were intragastrically infected with the WT or prx3 mutant at doses of 109 cfu.
FIGURE 7.
FIGURE 7.
Effect of IscR on the Pprx3 activity and sequence analysis of the prx3 regulatory region. A, Pprx3 activities were determined by primer extension of the RNAs isolated from the WT and the iscR mutant grown aerobically to an A600 of 0.3. Lanes C, T, A, and G, nucleotide sequencing ladders of pJK1402. The asterisk indicates the transcription start site of Pprx3. B, the transcription start site of Pprx3 is indicated by a bent arrow, and the positions of the putative −10 and −35 regions are underlined. The sequences for binding of IscR determined later in this study (Fig. 9) are presented as shaded boxes. The consensus sequences of the Type 2 IscR-binding site are indicated above the V. vulnificus DNA sequence. The ATG translation initiation codons and the putative ribosome-binding site (SD) are also indicated in boldface. W, A or T; Y, C or T; R, A or G; x, any nucleotide.
FIGURE 8.
FIGURE 8.
Specific binding of IscR to Pprx3. A 309-bp DNA fragment of the upstream region of Pprx3 was radioactively labeled and then used as a DNA probe. The radiolabeled probe DNA (5 nm) was mixed with increasing amounts of IscR, as indicated. For competition analysis, the same but unlabeled DNA fragment was used as a self-competitor DNA. Various amounts of the self-competitor DNA were added to a reaction mixture containing the 5 nm labeled DNA prior to the addition of 20 nm IscR. B, bound DNA; F, free DNA.
FIGURE 9.
FIGURE 9.
Sequences for binding of IscR to Pprx3. A 309-bp DNA fragment of the upstream region of Pprx3 was radioactively labeled and then used to as a DNA probe. The radiolabeled probe DNA (25 nm) was mixed with increasing amounts of IscR as indicated and then digested with DNase I. The regions protected by IscR are indicated by open boxes, and the nucleotides showing enhanced cleavage are indicated by black boxes. Lanes C, T, A, and G, nucleotide sequencing ladders of pJK1402. The transcription start site of Pprx3 (+1) and the putative −10 and −35 regions are indicated on the left of the nucleotide sequencing ladders.
FIGURE 10.
FIGURE 10.
Effects of mutations of three cysteines on the IscR activity. Total RNAs and proteins were isolated from the strains grown aerobically to an A600 of 0.3. A, the prx3 mRNA levels in the total RNA were determined by qRT-PCR analyses, and the prx3 mRNA level in the wild type was set to 1. B, protein samples were resolved by SDS-PAGE, and IscR or IscR3CA was detected by Western blotting using a rabbit anti-IscR antiserum. The positions of protein size markers (in kDa; Bio-Rad) are shown on the right of the gel. In the graph, the IscR protein levels were calculated based on the band intensities, and the IscR protein level in the wild type was set to 1. Error bars, S.E. **, p < 0.005 relative to WT. iscR, iscR mutant; iscR3CA, a strain expressing apo-locked IscR3CA.
FIGURE 11.
FIGURE 11.
Effects of oxidative stress and iron starvation on the IscR activity. Total RNAs and proteins were isolated from the wild type and iscR mutant grown anaerobically or aerobically to an A600 of 0.3 (A and B) or from the strains grown aerobically to an A600 of 0.3 after being exposed to various concentrations of DP for 10 min as indicated (C and D). A and C, the prx3 mRNA levels were determined by qRT-PCR analyses, and the prx3 mRNA level in the wild type grown aerobically was set to 1. B and D, protein samples were resolved by SDS-PAGE, IscR was detected by Western blotting, and results are presented as described in the legend to Fig. 10. In the graph, the IscR protein levels were calculated based on the band intensities, and the IscR protein level in the wild type grown aerobically was set to 1. Error bars, S.E. **, p < 0.005 relative to the strains grown anaerobically (A and B) or to the strains unexposed to DP (C and D). −, grown anaerobically; +, grown aerobically.
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