- Title
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NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes
- Authors
- Leseigneur, C., Boucontet, L., Duchateau, M., Pizarro-Cerda, J., Matondo, M., Colucci-Guyon, E., Dussurget, O.
- Source
- Full text @ Elife
(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 72 hpi (n=48). (B) Bacterial burden in zebrafish larvae upon intravenous injection with 104 S. aureus containing the empty vector (pSD1) or the NADK knockdown strain (NADK sgRNA). For each strain, CFU was determined in living larvae (open circles) or dead larvae (filled circles) 0, 24, and 48 hpi. (C) Representative fluorescence confocal images of transgenic mpx:GFP zebrafish larvae uninfected (CTRL), or intravenously injected with 104 S. aureus containing the empty vector (pSD1) or the NADK knockdown strain (NADK sgRNA) at 12 hpi. Maximum intensity Z-projection images (2 μm serial optical sections) of bacteria (red) and neutrophils (green). Scale bars, 25 μm. Insets are shown at higher magnification on the right panels for head and caudal regions. Scale bars, 10 μm. Neutrophils containing NADK knockdown bacteria can be seen in the caudal inset. (D) Representative fluorescence confocal images of transgenic mpx:GFP zebrafish larvae uninfected (CTRL), or intravenously injected with 104 S. aureus containing the empty vector (pSD1) or the NADK knockdown strain (NADK sgRNA) at 24 hpi, showing neutrophils (green). White arrows indicate the injection site. Scale bars, 500 μm (E) Number of neutrophils in uninfected zebrafish larvae (CTRL) or in zebrafish larvae intravenously injected with 104 S. aureus containing the empty vector (pSD1) or the NADK knockdown strain (NADK sgRNA) at 24 hpi. Comparison of data was performed using one-way analysis of variance (***p<0.001, ****p<0.0001).
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(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 untreated or treated with N-acetylcysteine (NAC). Bars indicate standard errors of the means of biological replicates (n=4). Comparison of data was performed using two-ways analysis of variance (*p<0.05, **p<0.01, ****p<0.0001). (B–C) Representative images of RAW264.7 macrophages infected with S. aureus/pSD1 or S. aureus/NADK sgRNA strains and analyzed 6 hpi by confocal fluorescence microscopy using antibodies to label S. aureus (Cy5, red) and LAMP-1 (FITC, green). Nuclei were labeled with DAPI (blue). (B) shows untreated macrophages. (C) shows macrophages treated with NAC. Insets are shown at higher magnification. Images are representative of three independent experiments. (D) Cell death of RAW264.7 macrophages uninfected (NI), uninfected and treated with Triton X-100 (lysis control), or 6 hpi with S. aureus strains containing the empty vector (pSD1) or the ppnK knockdown strain (NADK sgRNA). Bars indicate standard errors of the means of biological replicates. Comparison of data was performed using two-ways analysis of variance (*p<0.05, **p<0.01, ****p<0.0001). Data are representative of at least three independent experiments.
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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 for 6 hr to increasing concentrations of H2O2 relative to the untreated condition. (B) Percentage of growth of the S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) exposed for 6 hr to H2O2 and catalase alone or in combination, relative to the untreated condition. Data shown are representative of three independent experiments. Bars indicate the standard error of the means of biological replicates. Comparison of data was performed using one-way analysis of variance (ns: nonsignificant, *p<0.05, ***p<0.001, ****p<0.0001). (C) Bacterial cell death of the S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) untreated or exposed for 30 min to 0.05 mg/mL lysostaphin and 5 mg/mL lysozyme. Bacterial permeability was assessed using the CellTox Green cytotoxicity assay. Bars indicate standard errors of the means of biological replicates. Comparison of data was performed using two-ways analysis of variance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Data are representative of at least three independent experiments.
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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. Voronoi treemaps were generated to visualize proteins more abundant in S. aureus/pSD1 than in the knockdown strain at three hierarchical levels according to KEGG database: top level (A), second level (B), third level (C). Colors represent functional categories. Category size based on LFQ intensity corresponds to differences in protein abundance. (D) Whole bacterial cell lysates and culture supernatants were analyzed by immunoblotting using antibodies against alpha-hemolysin and EF-Tu. (E) Quantification of the Hla immunoblots normalized to corresponding EF-Tu protein levels in the pellet. (F) A Christie-Atkins-Munch-Peterson test was performed by streaking the S. aureus strain containing the empty vector (pSD1) and the ppnK knockdown strain (NADK sgRNA) perpendicularly to the S. aureus RN4220 strain that produces only beta-hemolysin (vertical streak) on sheep blood agar. Clearing zones indicate hemolysis.
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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 RT-PCR using oligonucleotides specific to gyrA, RNAIII, bsaA, and agrA genes, respectively. (D) Relative gene expression was normalized to gyrA transcript levels. (E) S. aureus ROS levels were quantified using the DCFH2-DA assay. Comparison of data was performed using t-test (****p<0.0001). Data are representative of at least three independent experiments.
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