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Cited by 152Bacterial Genetics and BiotechnologyAntimicrobial Resistance in StaphylococcusTrace Elements in Health

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The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity

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Grim, Kyle P., San Francisco, Brian, Radin, Jana N., Brazel, Erin B., Kelliher, Jessica L., Párraga Solórzano, Paola K., Kim, Philip C., McDevitt, Christopher A., Kehl-Fie, Thomas E.·2017·mBio
DOI 10.1128/mbio.01281-17PMC PMC5666155
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Abstract

During infection, the host sequesters essential nutrients, such as zinc, to combat invading microbes. Despite the ability of the immune effector protein calprotectin to bind zinc with subpicomolar affinity, Staphylococcus aureus is able to successfully compete with the host for zinc. However, the zinc importers expressed by S. aureus remain unknown. Our investigations have revealed that S. aureus possesses two importers, AdcABC and CntABCDF, which are induced in response to zinc limitation. While AdcABC is similar to known zinc importers in other bacteria, CntABCDF has not previously been associated with zinc acquisition. Concurrent loss of the two systems severely impairs the ability of S. aureus to obtain zinc and grow in zinc-limited environments. Further investigations revealed that the Cnt system is responsible for the ability of S. aureus to compete with calprotectin for zinc in culture and contributes to acquisition of zinc during infection. The cnt locus also enables S. aureus to produce the broad-spectrum metallophore staphylopine. Similarly to the Cnt transporter, loss of staphylopine severely impairs the ability of S. aureus to resist host-imposed zinc starvation, both in culture and during infection. Further investigations revealed that together staphylopine and the Cnt importer function analogously to siderophore-based iron acquisition systems in order to facilitate zinc acquisition by S. aureus . Analogous systems are found in a broad range of Gram-positive and Gram-negative bacterial pathogens, suggesting that this new type of zinc importer broadly contributes to the ability of bacteria to cause infection. IMPORTANCE A critical host defense against infection is the restriction of zinc availability. Despite the subpicomolar affinity of the immune effector calprotectin for zinc, Staphylococcus aureus can successfully compete for this essential metal. Here, we describe two zinc importers, AdcABC and CntABCDF, possessed by S. aureus , the latter of which has not previously been associated with zinc acquisition. The ability of S. aureus to compete with the host for zinc is dependent on CntABCDF and the metallophore staphylopine, both in culture and during infection. These results expand the mechanisms utilized by bacteria to obtain zinc, beyond Adc-like systems, and demonstrate that pathogens utilize strategies similar to siderophore-based iron acquisition to obtain other essential metals during infection. The staphylopine synthesis machinery is present in a diverse collection of bacteria, suggesting that this new family of zinc importers broadly contributes to the ability of numerous pathogens to cause infection.

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Figures

FIG 1 S. aureus uses an AdcABC family member and a previously unknown Zn transporter to obtain Zn. (A and B) S. aureus containing the PadcA-YFP and Pcnt-YFP reporter plasmids was grown in NRPMI supplemented with 25 μM ZnSO4, 25 μM MnCl2, or 1 μM FeSO4 or all three metals (metal replete), and expression was assessed by measuring fluorescence. (A) Wild-type S. aureus was grown in medium lacking the indicated metal, and expression of adcA and the cnt loci was assessed. n ≥ 3. *, P < 0.05 relative to metal-replete medium via two-way analysis of variance with Dunnett’s posttest. (B) Expression of adcA and the cnt loci was assessed in Δzur, Δfur, and ΔmntR mutants and compared to wild-type (WT) bacteria following growth in metal-replete NRPMI. n = 3. *, P < 0.05 relative to wild type under the same growth condition via two-way analysis of variance with Dunnett’s posttest. (C) Wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were incubated in NRPMI supplemented with 25 μM ZnSO4, 25 μM MnCl2, or 25 μM FeSO4, and growth was assessed by measuring optical density (OD600). n = 3. *, P < 0.05 compared to wild type via two-way analysis of variance with Dunnett’s posttest. #, P < 0.05 compared to ΔadcA ΔcntA mutant in metal-depleted medium via two-way analysis of variance with Dunnett’s posttest. (D and E) Wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants containing the indicated plasmids (vc, vector control) were incubated in NRPMI supplemented with 25 μM MnCl2 and 25 μM FeSO4 or not, and growth was assessed by measuring optical density (OD600). n = 3. *, P < 0.05 for the indicated comparison via two-way analysis of variance with Tukey’s posttest. Error bars in all panels show standard errors of the means.
FIG 1 S. aureus uses an AdcABC family member and a previously unknown Zn transporter to obtain Zn. (A and B) S. aureus containing the PadcA-YFP and Pcnt-YFP reporter plasmids was grown in NRPMI supplemented with 25 μM ZnSO4, 25 μM MnCl2, or 1 μM FeSO4 or all three metals (metal replete), and expression was assessed by measuring fluorescence. (A) Wild-type S. aureus was grown in medium lacking the indicated metal, and expression of adcA and the cnt loci was assessed. n ≥ 3. *, P < 0.05 relative to metal-replete medium via two-way analysis of variance with Dunnett’s posttest. (B) Expression of adcA and the cnt loci was assessed in Δzur, Δfur, and ΔmntR mutants and compared to wild-type (WT) bacteria following growth in metal-replete NRPMI. n = 3. *, P < 0.05 relative to wild type under the same growth condition via two-way analysis of variance with Dunnett’s posttest. (C) Wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were incubated in NRPMI supplemented with 25 μM ZnSO4, 25 μM MnCl2, or 25 μM FeSO4, and growth was assessed by measuring optical density (OD600). n = 3. *, P < 0.05 compared to wild type via two-way analysis of variance with Dunnett’s posttest. #, P < 0.05 compared to ΔadcA ΔcntA mutant in metal-depleted medium via two-way analysis of variance with Dunnett’s posttest. (D and E) Wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants containing the indicated plasmids (vc, vector control) were incubated in NRPMI supplemented with 25 μM MnCl2 and 25 μM FeSO4 or not, and growth was assessed by measuring optical density (OD600). n = 3. *, P < 0.05 for the indicated comparison via two-way analysis of variance with Tukey’s posttest. Error bars in all panels show standard errors of the means.CC BY · PubMed Central
FIG 2 AdcABC and CntABCDF are Zn importers. Wild-type (WT) S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were grown in rich medium, and the intracellular metal content was analyzed utilizing ICP-MS. (A) Zn; (B) Mn; (C) Fe; (D) Co; (E) Ni; (F) Cu. n ≥ 2 or more. Error bars in all panels show standard errors of the means. *, P < 0.05 compared to wild type via one-way analysis of variance with Dunnett’s posttest.
FIG 2 AdcABC and CntABCDF are Zn importers. Wild-type (WT) S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were grown in rich medium, and the intracellular metal content was analyzed utilizing ICP-MS. (A) Zn; (B) Mn; (C) Fe; (D) Co; (E) Ni; (F) Cu. n ≥ 2 or more. Error bars in all panels show standard errors of the means. *, P < 0.05 compared to wild type via one-way analysis of variance with Dunnett’s posttest.CC BY · PubMed Central
FIG 3 The CntABCDF importer enables S. aureus to resist CP-imposed starvation. (A) Wild-type S. aureus containing either the PadcA-YFP or the Pcnt-YFP reporter plasmid was grown in the presence of various concentrations of CP, and fluorescence was assessed. n ≥ 3. *, P < 0.05 compared to untreated bacteria via one-way analysis of variance with Dunnett’s posttest. (B) Wild-type (WT) S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were incubated in the presence of increasing concentrations of CP, and growth was assessed by measuring optical density (OD600). *, P < 0.05 compared to wild type at the same CP concentration via two-way analysis of variance with Dunnett’s posttest. n ≥ 3. (C and D) Wild-type S. aureus and ΔadcA ΔcntA and ΔcntA mutants containing the indicated plasmids (vc, vector control) were incubated in the presence of CP, and growth was assessed by measuring optical density. n ≥ 3. *, P < 0.05 for the indicated comparison via two-way analysis of variance with Tukey’s posttest. (E) Wild-type S. aureus containing either the PadcA-YFP or the Pcnt-YFP reporter plasmid was incubated in the presence or absence of 480 μg/ml of either wild-type CP, the ΔMn/Zn site mutant, or the ΔZn site mutant, and fluorescence was assessed. n ≥ 3. *, P < 0.05 compared to expression in the absence of CP via one-way analysis of variance with Dunnett’s posttest. (F) Wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were incubated in the presence or absence of 480 μg/ml of either WT CP, the ΔMn/Zn site mutant, or the ΔZn site mutant, and growth was assessed by measuring optical density (OD600). n ≥ 3. *, P < 0.05 compared to wild type via two-way analysis of variance with Dunnett’s posttest. Error bars in all panels show standard errors of the means.
FIG 3 The CntABCDF importer enables S. aureus to resist CP-imposed starvation. (A) Wild-type S. aureus containing either the PadcA-YFP or the Pcnt-YFP reporter plasmid was grown in the presence of various concentrations of CP, and fluorescence was assessed. n ≥ 3. *, P < 0.05 compared to untreated bacteria via one-way analysis of variance with Dunnett’s posttest. (B) Wild-type (WT) S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were incubated in the presence of increasing concentrations of CP, and growth was assessed by measuring optical density (OD600). *, P < 0.05 compared to wild type at the same CP concentration via two-way analysis of variance with Dunnett’s posttest. n ≥ 3. (C and D) Wild-type S. aureus and ΔadcA ΔcntA and ΔcntA mutants containing the indicated plasmids (vc, vector control) were incubated in the presence of CP, and growth was assessed by measuring optical density. n ≥ 3. *, P < 0.05 for the indicated comparison via two-way analysis of variance with Tukey’s posttest. (E) Wild-type S. aureus containing either the PadcA-YFP or the Pcnt-YFP reporter plasmid was incubated in the presence or absence of 480 μg/ml of either wild-type CP, the ΔMn/Zn site mutant, or the ΔZn site mutant, and fluorescence was assessed. n ≥ 3. *, P < 0.05 compared to expression in the absence of CP via one-way analysis of variance with Dunnett’s posttest. (F) Wild-type S. aureus and ΔadcA, ΔcntA, and ΔadcA ΔcntA mutants were incubated in the presence or absence of 480 μg/ml of either WT CP, the ΔMn/Zn site mutant, or the ΔZn site mutant, and growth was assessed by measuring optical density (OD600). n ≥ 3. *, P < 0.05 compared to wild type via two-way analysis of variance with Dunnett’s posttest. Error bars in all panels show standard errors of the means.CC BY · PubMed Central
FIG 4 Staphylopine enables S. aureus to resist Zn starvation by functioning as a zincophore. (A) Wild-type (WT) S. aureus and ΔcntKLM and ΔadcA ΔcntKLM mutants were incubated in NRPMI supplemented with 25 μM ZnSO4, 25 μM MnCl2, and 25 μM FeSO4 as indicated, and growth was assessed by measuring optical density (OD600). (B) Wild-type S. aureus and ΔcntA, ΔcntKLM, ΔadcA ΔcntA, and ΔadcA ΔcntKLM mutants were incubated in various concentrations of CP, and growth was assessed by measuring optical density (OD600). (C) Wild-type S. aureus and the ΔadcA ΔcntA mutant were incubated in the presence or absence of 480 μg/ml of either wild-type CP, the ΔMn/Zn site mutant, or the ΔZn site mutant, and growth was assessed by measuring optical density (OD600). (D) Supernatant was harvested from wild-type S. aureus or the ΔcntA or ΔcntKLM mutant following growth in defined medium lacking Zn. The supernatant was then assessed for the ability to rescue the growth of wild-type S. aureus or ΔcntA, ΔcntKLM, ΔadcA ΔcntA, and ΔadcA ΔcntKLM mutants when incubated in the presence of CP. Growth in the presence of the various supernatants and CP was assessed by measuring the OD600. (A to D) n ≥ 3. *, P < 0.05 compared to wild type in the same supernatant treatment via two-way analysis of variance using Dunnett’s posttest. Error bars show standard errors of the means. (D) #, P < 0.05 compared to wild-type supernatant via two-way analysis of variance using Dunnett’s posttest.
FIG 4 Staphylopine enables S. aureus to resist Zn starvation by functioning as a zincophore. (A) Wild-type (WT) S. aureus and ΔcntKLM and ΔadcA ΔcntKLM mutants were incubated in NRPMI supplemented with 25 μM ZnSO4, 25 μM MnCl2, and 25 μM FeSO4 as indicated, and growth was assessed by measuring optical density (OD600). (B) Wild-type S. aureus and ΔcntA, ΔcntKLM, ΔadcA ΔcntA, and ΔadcA ΔcntKLM mutants were incubated in various concentrations of CP, and growth was assessed by measuring optical density (OD600). (C) Wild-type S. aureus and the ΔadcA ΔcntA mutant were incubated in the presence or absence of 480 μg/ml of either wild-type CP, the ΔMn/Zn site mutant, or the ΔZn site mutant, and growth was assessed by measuring optical density (OD600). (D) Supernatant was harvested from wild-type S. aureus or the ΔcntA or ΔcntKLM mutant following growth in defined medium lacking Zn. The supernatant was then assessed for the ability to rescue the growth of wild-type S. aureus or ΔcntA, ΔcntKLM, ΔadcA ΔcntA, and ΔadcA ΔcntKLM mutants when incubated in the presence of CP. Growth in the presence of the various supernatants and CP was assessed by measuring the OD600. (A to D) n ≥ 3. *, P < 0.05 compared to wild type in the same supernatant treatment via two-way analysis of variance using Dunnett’s posttest. Error bars show standard errors of the means. (D) #, P < 0.05 compared to wild-type supernatant via two-way analysis of variance using Dunnett’s posttest.CC BY · PubMed Central
FIG 5 The Cnt-StP system is used to resist host-imposed metal starvation during infection. C57BL/6 mice were retroorbitally infected with ~1 × 107 CFU of wild-type (WT) S. aureus or ΔadcA, ΔcntA, ΔcntKLM, ΔadcA ΔcntA, or ΔadcA ΔcntKLM mutant, and weight (A) and bacterial burdens in the liver (B), heart (C), and kidneys (D) were assessed. (A) * and #, P < 0.05 relative to mice infected with wild-type S. aureus for ΔadcA ΔcntA and ΔadcA ΔcntKLM mutants, respectively, via two-way analysis of variance with Dunnett’s posttest. Error bars show standard errors of the means. (B to D) *, P < 0.05 compared to mice infected with wild-type S. aureus via Mann-Whitney U test.
FIG 5 The Cnt-StP system is used to resist host-imposed metal starvation during infection. C57BL/6 mice were retroorbitally infected with ~1 × 107 CFU of wild-type (WT) S. aureus or ΔadcA, ΔcntA, ΔcntKLM, ΔadcA ΔcntA, or ΔadcA ΔcntKLM mutant, and weight (A) and bacterial burdens in the liver (B), heart (C), and kidneys (D) were assessed. (A) * and #, P < 0.05 relative to mice infected with wild-type S. aureus for ΔadcA ΔcntA and ΔadcA ΔcntKLM mutants, respectively, via two-way analysis of variance with Dunnett’s posttest. Error bars show standard errors of the means. (B to D) *, P < 0.05 compared to mice infected with wild-type S. aureus via Mann-Whitney U test.CC BY · PubMed Central
FIG 6 Bacteria possess a variety of StP-like synthesis loci. Diagrams of the 6 families of StP synthesis loci identified by neighborhood network analysis. See Table S1 in the supplemental material for a complete list of the loci identified. Abbreviations: NBD, nucleotide-binding domain; TMD, transmembrane domain; DH, dehydrogenase; NAS, nicotianamine synthase.
FIG 6 Bacteria possess a variety of StP-like synthesis loci. Diagrams of the 6 families of StP synthesis loci identified by neighborhood network analysis. See Table S1 in the supplemental material for a complete list of the loci identified. Abbreviations: NBD, nucleotide-binding domain; TMD, transmembrane domain; DH, dehydrogenase; NAS, nicotianamine synthase.CC BY · PubMed Central

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  1. World Health Organization . 2014. Antimicrobial resistance global report on surveillance. World Health Organization, Geneva, Switzerland.

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Metadata

Authors
Grim, K. P., San Francisco, B., Radin, J. N., Brazel, E. B., Kelliher, J. L., Párraga Solórzano, P. K., Kim, P. C., McDevitt, C. A., & Kehl-Fie, T. E.
Affiliations
  • University of Illinois Urbana-Champaign (US)
  • Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
  • The University of Adelaide (AU)
  • Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2017-11-08
Volume
8
Issue
5
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.01281-17
PMC
PMC5666155
OpenAlex
W2767136855
Cited by
152
References
77
Citations by year
2026: 19 · 2025: 18 · 2024: 17 · 2023: 13 · 2022: 15 · 2021: 22 · 2020: 18 · 2019: 21 · 2018: 9
FWCI
8.89
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Adelaide Research & Scholarship (AR&S) (University of Adelaide) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by)
  • Repositorio Institucional de la Universidad de las Fuerzas Armadas ESPE (Universidad de las Fuerzas Armadas ESPE) (submittedVersion, other-oa)
  • Greater South Information System
Funders
  • Institute for Genomic Biology Post Doctoral Fellowship
  • HHS | National Institutes of Health (K22 AI104805, R01 AI118880)
  • Department of Health | National Health and Medical Research Council (1080784, 1122582)
  • Australian Research Council (DP150104515, DP170102102)
  • March of Dimes Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
  • Health Sciences › Nursing › Nutrition and Dietetics › Trace Elements in Health
Keywords
Staphylococcus aureus, metallophores, zinc uptake, nutritional immunity, calprotectin, zinc starvation, bacterial virulence
MeSH terms
Bacterial Proteins, Imidazoles, Staphylococcal Infections, Staphylococcus aureus, Zinc, Gene Expression Regulation, Bacterial, Siderophores, Membrane Transport Proteins, Leukocyte L1 Antigen Complex, Host-Pathogen Interactions

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