NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes
- Institut Pasteur, Université Paris Cité, CNRS UMR6047, Unité de Recherche Yersinia, France
- Institut Pasteur, Université Paris Cité, CNRS UMR3738, Unité Macrophages et Développement de l’Immunité, France
- Institut Pasteur, Université Paris Cité, CNRS USR2000, Unité de Spectrométrie de Masse pour la Biologie, Plateforme de protéomique, France
Figures
Figure 1 with 2 supplements
Nicotinamide adenine dinucleotide kinase (NADK) promotes S. aureus virulence in zebrafish.
(A) Survival of zebrafish larvae uninfected (CTRL) or intravenously injected at 60 hpf with 104 S. aureus containing the empty vector (pSD1) or the NADK knockdown strain (NADK sgRNA) between 0 and …
Figure 1—figure supplement 1
Nicotinamide adenine dinucleotide kinase (NADK) is important for S. aureus growth.
(A) Total RNAs of S. aureus containing the empty vector (pSD1) or the NADK knockdown strain (NADK sgRNA) were analyzed by RT-PCR using oligonucleotides specific to the target gene ppnK or the …
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Figure 1—figure supplement 1—source data 1
NADK transcript levels (A).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig1-figsupp1-data1-v1.pdf
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Figure 1—figure supplement 1—source data 2
NADK protein levels (B).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig1-figsupp1-data2-v1.pdf
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Figure 1—figure supplement 1—source data 3
NADK operon transcript levels (E).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig1-figsupp1-data3-v1.pdf
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Figure 1—figure supplement 1—source data 4
RluA transcript levels (F).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig1-figsupp1-data4-v1.pdf
Figure 1—figure supplement 2
Nicotinamide adenine dinucleotide kinase (NADK) contributes to S. aureus virulence in zebrafish.
(A) The number of pSD1 (gray) or NADK sgRNA (blue) S. aureus was monitored in zebrafish larvae after intravenous injection with 5.103 bacteria. For each strain, CFU was determined 0, 6, 24, and 48 …
Figure 2
Nicotinamide adenine dinucleotide kinase (NADK) promotes S. aureus survival and cytotoxicity in macrophages.
(A) Percentage of growth of the S. aureus strains containing the empty vector (pSD1) or the ppnK knockdown strain (NADK sgRNA) at time 0, and 1, 2, 4, and 6 hpi of RAW264.7 macrophages left …
Figure 3
Nicotinamide adenine dinucleotide kinase (NADK) protects S. aureus from antimicrobial defense compounds.
Bacterial growth was monitored at OD600nm in BHI broth at 37°C. (A) Percentage of growth of the S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) exposed …
Figure 4 with 1 supplement
Nicotinamide adenine dinucleotide kinase (NADK) promotes expression of S. aureus virulence determinants.
The S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) were grown in BHI broth at 37°C. (A–C) Whole bacterial cell lysates were analyzed by LC-MS/MS. …
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Figure 4—source data 1
Alpha-hemolysin protein levels (D).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig4-data1-v1.pdf
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Figure 4—source data 2
Hemolysis levels (F).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig4-data2-v1.pdf
Figure 4—figure supplement 1
Nicotinamide adenine dinucleotide kinase (NADK) promotes expression of S. aureus virulence determinants.
The S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) were grown at 37°C in BHI broth supplemented with H2O2. (A–C) Whole bacterial cell lysates were …
Figure 5
Nicotinamide adenine dinucleotide kinase (NADK) is required for AgrA expression and S. aureus redox control.
The S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) were grown aerobically in BHI broth at 37°C for 4 hr. (A–C) RNA was extracted and subjected to …
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Figure 5—source data 1
RNAIII transcript levels (A).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig5-data1-v1.pdf
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Figure 5—source data 2
BsaA transcript levels (B).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig5-data2-v1.pdf
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Figure 5—source data 3
AgrA transcript levels (C).
- https://cdn.elifesciences.org/articles/79941/elife-79941-fig5-data3-v1.pdf
Author response image 1
NADK protects S. aureus USA300 from hydrogen peroxide toxicity.
Growth of the USA300 strain containing the empty plasmid (USA300 pSD1, grey) and the NADK knockdown strain (USA300 sgRNA, blue) was monitored at OD600nm in BHI broth at 37°C (NT, open circles) or in …
Author response image 2
Reactive oxygen species contribute to S. aureus growth defect upon NADK knockdown.
Aerobic growth of the Xen36 strain containing the empty plasmid (pSD1, grey) and the NADK knockdown strain (NADK sgRNA, blue) was monitored at OD600nm in BHI broth at 37°C (NT, open circles) or in …
Tables
Table 1
Relative abundance of a subset of protective proteins differentially expressed by S. aureus pSD1 and NADK sgRNA strains.
| Uniprot | Protein | Description | Log2R* | P† |
|---|---|---|---|---|
| Q2FV54 Q2G000 Q2G261 Q2FZZ3 Q2G0D9 Q2G280 P0A086 Q2FVL7 Q2G0E0 Q2FYU7 | OatA Trx2 SodM - GraS - MsrA2 - GraR KatA | O-acetyl transferase Thioredoxin 2 Superoxide dismutase [Mn/Fe] Thioredoxin domain-containing protein Sensor histidine kinase Peroxiredoxin Methionine sulfoxide reductase Thioredoxin domain-containing protein Response regulator protein Catalase | 3.07 1.79 1.68 1.59 1.31 1.26 1.10 1.02 1.00 0.59 | 1.78E-05 2.75E-05 2.11E-06 7.22E-04 4.00E-04 6.69E-07 2.25E-05 1.94E-03 2.23E-03 1.00E-05 |
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*
Log2R=Log2[pSD1]/[NADK sgRNA].
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†
Adjusted p-value.
Table 2
Relative abundance of a subset of virulence-related proteins differentially expressed by S. aureus pSD1 and NADK sgRNA strains.
| Uniprot | Protein | Description | Log2R* | P† |
|---|---|---|---|---|
| Q2FWM4 P0C818 Q2FVK2 P0C7Y1 Q2FUU5 Q2FVK1 Q2G1 × 0 Q2FWN9 Q2FVK3 Q2FWP0 Q2G2R8 | AgrA PsmA4 HlgC PsmA1 Lip1 HlgB Hly LukL2 HlgA LukL1 SspP | Accessory gene regulator protein Phenol-soluble modulin alpha 4 Gamma-hemolysin component C Phenol-soluble modulin alpha 1 Lipase Gamma-hemolysin component B Alpha-hemolysin Leukocidin-like protein 2 Gamma-hemolysin component A Leukocidin-like protein 1 Staphopain A | + + + + + 7.88 6.99 3.88 3.84 3.09 2.89 | NA NA NA NA NA 3.85E-10 3.85E-10 4.14E-08 1.47E-08 2.25E-06 4.08E-07 |
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*
Log2R=Log2[pSD1]/[NADK sgRNA]; +: protein detected in pSD1 strain and not detected from NADK sgRNA strain.
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†
Adjusted p-value: NA: not applicable.
Table 3
Strains and plasmids used in this study.
| Strain/plasmid | Relevant characteristics | Reference |
|---|---|---|
| Strains | ||
| E. coli strain | ||
| TOP10 | F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7,697 galU galK λ- rpsL(StrR) endA1 nupG | Invitrogen |
| S. aureus strains | ||
| Xen36 | Strain derived from a clinical isolate from a bacteremic patient | PerkinElmer, ATCC49525 |
| Xen36/pSD1 | Xen36 strain carrying plasmid pSD1 | Gelin et al., 2020 |
| Xen36/NADK sgRNA | Xen36 strain carrying plasmid pSD1 ppnK | Gelin et al., 2020 |
| RN4220 | Restriction-deficient strain derived from NCTC 8325–4 | Kreiswirth et al., 1983 |
| Plasmids | ||
| pSD1 | dCas9 ATc-inducible and sgRNA constitutive expression plasmid | Zhao et al., 2017 |
| pSD1 ppnK | pSD1 plasmid carrying sgRNA targeting S. aureus ppnK | Gelin et al., 2020 |
Table 4
Primers used in this study.
| Primer | Sequence (5’–3’) |
|---|---|
| RT-ppnK-F | GTGACTCCAAGTCTAATGCC |
| RT-ppnK-R | ATTTTTCAACTTCATGAGGTAACC |
| RT-gyrA-F | GTGTTATCGTTGCTCGTG |
| RT-gyrA-R | CGGTGTCATACCTTGTTC |
| RT-RNAIII-F | AGTTTCCTTGGACTCAGTGCT |
| RT-RNAIII-R | AGGGGCTCACGACCATACTT |
| RT-bsaA-F | GCGAAGAAGCAGCTCAAAAC |
| RT-bsaA-R | CCTTCGCGATCCACTAAAAA |
| RT-agrA-F | GCCCTCGCAACTGATAATCC |
| RT-agrA-R | CACCGATGCATAGCAGTGTTC |
| RT-rluA-F | CCGCGAGAAATACCGAGTGT |
| RT-rluA-R | TCTCCCACAATTGGATGCCC |
| RT-operon-F1 | TGACTTGCTTAAAAAGCACACTG |
| RT-operon-R1 | ACGAGCATTTGTCCTACTTCAGA |
| RT-operon-F2 | AACCGTTGAAGAAACATTCGACA |
| RT-operon-R2 | GACGCTTGTTCACCAACTTCA |
| RT-operon-F3 | CATCGTTTGGAAAGAGCGGC |
| RT-operon-R3 | GGCATTAGACTTGGAGTCACCT |
| RT-operon-F4 | ACGTGTGCACGATTCTTTCAT |
| RT-operon-R4 | ATGGCGCTCACTGTCTTCT |
| RT-operon-F5 | AGTTCATTTGCATACGGGACG |
| RT-operon-R5 | ACGCTCTTTTTCATCTGTGTTCA |
| RT-operon-F6 | TAACTTGTGCGATGACGGTGG |
| RT-operon-R6 | TTCCAATCACAATCCCCATCAA |
| RT-operon-F7 | ACGTTGATGAATTGAAGCAAGAG |
| RT-operon-R7 | ACTTTAGCGACACCAAAAGCA |
| RT-operon-F8 | TCAAGTGGCGTTACAGGTGA |
| RT-operon-R8 | TTCAAATACCGCCAACGCAT |
Additional files
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Supplementary file 1
Proteins more abundant in the S. aureus pSD1 strain than in the NADK sgRNA strain after growth in BHI broth.
- https://cdn.elifesciences.org/articles/79941/elife-79941-supp1-v1.xlsx
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Supplementary file 2
Relative abundance of a subset of protective proteins differentially expressed by S. aureus pSD1 and NADK sgRNA strains exposed to hydrogen peroxide.
- https://cdn.elifesciences.org/articles/79941/elife-79941-supp2-v1.docx
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Supplementary file 3
Proteins more abundant in the S. aureus pSD1 strain than in the NADK sgRNA strain after growth in BHI broth supplemented with hydrogen peroxide.
- https://cdn.elifesciences.org/articles/79941/elife-79941-supp3-v1.xlsx
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Supplementary file 4
Relative abundance of a subset of protective proteins differentially expressed by S. aureus pSD1 and NADK sgRNA strains upon zebrafish infection.
- https://cdn.elifesciences.org/articles/79941/elife-79941-supp4-v1.docx
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Supplementary file 5
Proteins more abundant in the S. aureus pSD1 strain than in the NADK sgRNA strain upon zebrafish infection.
- https://cdn.elifesciences.org/articles/79941/elife-79941-supp5-v1.xlsx
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Supplementary file 6
Relative abundance of a subset of virulence-related proteins differentially expressed by S. aureus pSD1 and NADK sgRNA strains exposed to hydrogen peroxide.
- https://cdn.elifesciences.org/articles/79941/elife-79941-supp6-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79941/elife-79941-mdarchecklist1-v1.docx
