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Dietary zinc and the control of Streptococcus pneumoniae infection

Eijkelkamp, Bart A., Morey, Jacqueline R., Neville, Stephanie L., Tan, Aimee, Pederick, Victoria G., Cole, Nerida, Singh, Prashina P., Ong, Cheryl-Lynn Y., Gonzalez de Vega, Raquel, Clases, David, Cunningham, Bliss A., Hughes, Catherine E., Comerford, Iain, Brazel, Erin B., Whittall, Jonathan J., Plumptre, Charles D., McColl, Shaun R., Paton, James C., McEwan, Alastair G., Doble, Philip A., McDevitt, Christopher A.·2019·PLOS Pathogens

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Fig 1 The effect of Zn restriction on animal survival, bacterial burden and tissue Zn abundance. (A) Survival of Zn-restricted or Zn-replete mice following intranasal challenge with 1 × 107 CFU of S. pneumoniae strain D39. Data represent analyses of seven mice with statistical analyses performed using a Log-rank (Mantel-Cox) test. Enumeration of S. pneumoniae from host niches, nasopharynx (B), pleural lavage (C), lung (D), blood (E), following intranasal challenge with 1 × 107 CFU of strain D39 (n ≥ 7). Colonisation was examined at 24 and 36 hrs post challenge in Zn-restricted or Zn-replete mice. Data represent the mean (± S.E.M.) of four independent experiments with statistical analyses performed by a one-way ANOVA. Determination of murine niche Zn abundance, nasopharynx (F), pleural lavage (G), lung (H), blood serum (I), prior to infection, or 24 hrs or 36 hrs post infection in both dietary groups (n ≥ 5) by ICP-MS. Data represent the mean (± S.E.M.) of four independent experiments with statistical analyses performed by a one-way ANOVA. Spatial distribution of Zn in the lungs of naïve Zn-restricted (J), or infected Zn-restricted (K) mice, and naïve Zn-replete (L) or infected Zn-replete (M) mice at 36 hrs post challenge. These data are representative elemental maps of murine tissue sections from analyses of at least three distinct murine samples (n ≥ 3) by laser-ablation-ICP-MS with quantitated regions of interest (ROI) highlighted. Mean (± S.E.M.) elemental data for the distinct tissue sections is presented in S2 Table. The scale bar represents a heat map (blue to red) for the intensity of Zn from 0 to 10.0 μg.g-1.
Fig 1 The effect of Zn restriction on animal survival, bacterial burden and tissue Zn abundance. (A) Survival of Zn-restricted or Zn-replete mice following intranasal challenge with 1 × 107 CFU of S. pneumoniae strain D39. Data represent analyses of seven mice with statistical analyses performed using a Log-rank (Mantel-Cox) test. Enumeration of S. pneumoniae from host niches, nasopharynx (B), pleural lavage (C), lung (D), blood (E), following intranasal challenge with 1 × 107 CFU of strain D39 (n ≥ 7). Colonisation was examined at 24 and 36 hrs post challenge in Zn-restricted or Zn-replete mice. Data represent the mean (± S.E.M.) of four independent experiments with statistical analyses performed by a one-way ANOVA. Determination of murine niche Zn abundance, nasopharynx (F), pleural lavage (G), lung (H), blood serum (I), prior to infection, or 24 hrs or 36 hrs post infection in both dietary groups (n ≥ 5) by ICP-MS. Data represent the mean (± S.E.M.) of four independent experiments with statistical analyses performed by a one-way ANOVA. Spatial distribution of Zn in the lungs of naïve Zn-restricted (J), or infected Zn-restricted (K) mice, and naïve Zn-replete (L) or infected Zn-replete (M) mice at 36 hrs post challenge. These data are representative elemental maps of murine tissue sections from analyses of at least three distinct murine samples (n ≥ 3) by laser-ablation-ICP-MS with quantitated regions of interest (ROI) highlighted. Mean (± S.E.M.) elemental data for the distinct tissue sections is presented in S2 Table. The scale bar represents a heat map (blue to red) for the intensity of Zn from 0 to 10.0 μg.g-1.CC BY · PubMed Central
Fig 2 Co-localisation of S. pneumoniae and Zn in murine lung tissue. Spatial distribution of Zn in the lungs of infected Zn-replete (A) or naïve Zn-replete (C) mice at 36 hrs post challenge. These data are representative elemental maps of murine tissue sections from at least two distinct murine samples by laser-ablation-ICP-MS with quantitated regions of interest (ROI) highlighted. The scale bar represents a heat map (blue to red) for the intensity of Zn from 0 to 20.0 μg.g-1. Spatial distribution of S. pneumoniae pVA838-GFP fluorescence in the lungs of infected Zn-replete (B) or naïve Zn-replete (D) mice at 36 hrs post challenge. The data are representative murine tissue sections from at least two distinct murine samples analysed by fluorescence microscopy with regions of interest (ROI) highlighted. The scale bar represents a heat map (black to green) for the relative fluorescence intensity.
Fig 2 Co-localisation of S. pneumoniae and Zn in murine lung tissue. Spatial distribution of Zn in the lungs of infected Zn-replete (A) or naïve Zn-replete (C) mice at 36 hrs post challenge. These data are representative elemental maps of murine tissue sections from at least two distinct murine samples by laser-ablation-ICP-MS with quantitated regions of interest (ROI) highlighted. The scale bar represents a heat map (blue to red) for the intensity of Zn from 0 to 20.0 μg.g-1. Spatial distribution of S. pneumoniae pVA838-GFP fluorescence in the lungs of infected Zn-replete (B) or naïve Zn-replete (D) mice at 36 hrs post challenge. The data are representative murine tissue sections from at least two distinct murine samples analysed by fluorescence microscopy with regions of interest (ROI) highlighted. The scale bar represents a heat map (black to green) for the relative fluorescence intensity.CC BY · PubMed Central
Fig 3 The effect of host dietary intervention on pneumococcal metal ion homeostasis. Pneumococcal expression levels of czcD (A), phtE (B) and psaA (F) were determined following isolation from the lungs, blood or pleural lavage (PL) of Zn-restricted and Zn-replete mice (n ≥ 6). Gene expression levels are the difference, in fold-change, of pneumococci from the Zn-replete mice as compared to pneumococci from the Zn-restricted mice. Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed using Student’s t-test. The proliferation of pneumococcal Zn acquisition (ΔadcAΔadcAII) and Zn efflux (ΔczcD) mutants was compared to that observed for wild-type pneumococci in the lungs (C), pleural lavage (D) and blood (E) at 36 hrs post challenge (n ≥ 5). Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test.
Fig 3 The effect of host dietary intervention on pneumococcal metal ion homeostasis. Pneumococcal expression levels of czcD (A), phtE (B) and psaA (F) were determined following isolation from the lungs, blood or pleural lavage (PL) of Zn-restricted and Zn-replete mice (n ≥ 6). Gene expression levels are the difference, in fold-change, of pneumococci from the Zn-replete mice as compared to pneumococci from the Zn-restricted mice. Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed using Student’s t-test. The proliferation of pneumococcal Zn acquisition (ΔadcAΔadcAII) and Zn efflux (ΔczcD) mutants was compared to that observed for wild-type pneumococci in the lungs (C), pleural lavage (D) and blood (E) at 36 hrs post challenge (n ≥ 5). Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test.CC BY · PubMed Central
Fig 4 The effect of dietary Zn on the innate immune response. Quantitative ELISA analyses of IL-6, in the lungs (A) and blood (D), and IL-1β, in the lungs (B), in Zn-restricted and Zn-replete mice prior to or 36 hrs post infection. The data represent the mean (± S.E.M.) of at least four independent biological replicates with statistical analyses performed using a one-way ANOVA. Transcriptional profiling of the innate immune response for CCL2, in the lungs (C) and blood (F), and IL-1β, in blood (E), in Zn-restricted and Zn-replete mice (n ≥ 6) prior to or 36 hrs post infection by qRT-PCR. Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed using a one-way ANOVA. Examination of cell abundances (percentage of live cells of total cells analysed) for alveolar macrophages, in the lungs (G), monocytes, in the lungs (H) and blood (J), neutrophils, in the lungs (I) or blood (K) of Zn-restricted and Zn-replete mice prior to (naïve) or 36 hrs post infection by flow-cytometry (n ≥ 5). Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed using a one-way ANOVA.
Fig 4 The effect of dietary Zn on the innate immune response. Quantitative ELISA analyses of IL-6, in the lungs (A) and blood (D), and IL-1β, in the lungs (B), in Zn-restricted and Zn-replete mice prior to or 36 hrs post infection. The data represent the mean (± S.E.M.) of at least four independent biological replicates with statistical analyses performed using a one-way ANOVA. Transcriptional profiling of the innate immune response for CCL2, in the lungs (C) and blood (F), and IL-1β, in blood (E), in Zn-restricted and Zn-replete mice (n ≥ 6) prior to or 36 hrs post infection by qRT-PCR. Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed using a one-way ANOVA. Examination of cell abundances (percentage of live cells of total cells analysed) for alveolar macrophages, in the lungs (G), monocytes, in the lungs (H) and blood (J), neutrophils, in the lungs (I) or blood (K) of Zn-restricted and Zn-replete mice prior to (naïve) or 36 hrs post infection by flow-cytometry (n ≥ 5). Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed using a one-way ANOVA.CC BY · PubMed Central
Fig 5 The role of Zn in phagocyte-mediated killing of S. pneumoniae. (A) Intracellular Zn in PMNs from Zn-restricted or Zn-replete mice examined using FluoZin-3 AM and flow-cytometry (n ≥ 5). Relative fluorescence was corrected to levels observed in the phagocytic cells in Zn-restricted mice. Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed by Student’s t-test. (B) Survival of wild-type S. pneumoniae D39 in THP-1 monocyte-derived macrophages (n = 5) and human PMNs (n = 3) in the presence of Zn [50 μM Zn-pyrithione (Zn-pyr)] supplementation, by comparison with unsupplemented phagocytic cells. Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test. (C) Expression levels of czcD, phtE and psaA in S. pneumoniae in THP-1 monocyte-derived macrophages (n = 5) and human PMNs (n = 3) with or without 50 μM Zn treatment. Expression levels represent the fold-change difference between pneumococci in the Zn-supplemented phagocytic cells compared to untreated cells. Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test. Survival of S. pneumoniae mutant strains defective in Zn efflux (ΔczcD) (D) or Zn uptake (ΔadcAΔadcAII) (E) in THP-1 monocyte-derived macrophages (n = 5) and human PMNs (n = 3), compared to the wild-type strain. Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test.
Fig 5 The role of Zn in phagocyte-mediated killing of S. pneumoniae. (A) Intracellular Zn in PMNs from Zn-restricted or Zn-replete mice examined using FluoZin-3 AM and flow-cytometry (n ≥ 5). Relative fluorescence was corrected to levels observed in the phagocytic cells in Zn-restricted mice. Data represent the mean (± S.E.M.) of two independent experiments with statistical analyses performed by Student’s t-test. (B) Survival of wild-type S. pneumoniae D39 in THP-1 monocyte-derived macrophages (n = 5) and human PMNs (n = 3) in the presence of Zn [50 μM Zn-pyrithione (Zn-pyr)] supplementation, by comparison with unsupplemented phagocytic cells. Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test. (C) Expression levels of czcD, phtE and psaA in S. pneumoniae in THP-1 monocyte-derived macrophages (n = 5) and human PMNs (n = 3) with or without 50 μM Zn treatment. Expression levels represent the fold-change difference between pneumococci in the Zn-supplemented phagocytic cells compared to untreated cells. Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test. Survival of S. pneumoniae mutant strains defective in Zn efflux (ΔczcD) (D) or Zn uptake (ΔadcAΔadcAII) (E) in THP-1 monocyte-derived macrophages (n = 5) and human PMNs (n = 3), compared to the wild-type strain. Data represent the mean (± S.E.M.) of three independent experiments with statistical analyses performed by Student’s t-test.CC BY · PubMed Central

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Metadata

Authors
Eijkelkamp, B. A., Morey, J. R., Neville, S. L., Tan, A., Pederick, V. G., Cole, N., Singh, P. P., Ong, C.-L. Y., Gonzalez de Vega, R., Clases, D., Cunningham, B. A., Hughes, C. E., Comerford, I., Brazel, E. B., Whittall, J. J., Plumptre, C. D., McColl, S. R., Paton, J. C., McEwan, A. G., Doble, P. A., & McDevitt, C. A.
Venue
PLOS Pathogens
Published
2019-08-22
Volume
15
Issue
8
Pages
e1007957
ISSN
1553-7374
Cited by
64
References
13
Language
en
Funders
  • National Health and Medical Research Council (1080784, 1122582, 1071659, 1142695)
  • Australian Research Council (DP170100036, DP170102102, FT170100006, DP190102361)
  • Garnett Passe and Rodney Williams Memorial Foundation
  • Deutsche Forschungsgemeinschaft (417283954)

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