Research ArticleInfectious Disease

Staphylococcus aureus α toxin potentiates opportunistic bacterial lung infections

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Science Translational Medicine  09 Mar 2016:
Vol. 8, Issue 329, pp. 329ra31
DOI: 10.1126/scitranslmed.aad9922
  • Fig. 1. S. aureus promotes Gram-negative infection.

    (A) Survival of mice infected with S. aureus (5e7 CFU) (Sa), P. aeruginosa (1e5 CFU) (Pa), or S. aureus + P. aeruginosa. (B) S. aureus or P. aeruginosa CFU recovered from the lungs of mice infected as described above at various time points. (C) P. aeruginosa CFU recovered from the spleens of mice after infection with P. aeruginosa or S. aureus + P. aeruginosa at various time points. (D) Survival of mice infected with S. aureus (5e7 CFU), K. pneumoniae (5e1 CFU) (Kp), or S. aureus + K. pneumoniae. (E) S. aureus or K. pneumoniae CFU recovered from the lungs of mice infected as described above at various time points. (F) K. pneumoniae CFU recovered from the spleens of mice after infection with K. pneumoniae or S. aureus + K. pneumoniae at various time points. (G) Survival of mice infected with S. aureus (5e7 CFU), A. baumannii (6e5 CFU) (Ab), or S. aureus + A. baumannii. (H) S. aureus or A. baumannii CFU recovered from the lungs of mice infected as described above at various time points. (I) A. baumannii CFU recovered from the spleens of mice after infection with A. baumannii or S. aureus + A. baumannii at various time points. Significance was determined by log-rank test (A, D, and G) or analysis of variance (ANOVA) followed by Dunnett’s test (B, C, E, F, H, and I). ΦP < 0.02, compared to mixed infection; #P < 0.0001, P. aeruginosa versus S. aureus + P. aeruginosa (P. aeruginosa) and P < 0.03, S. aureus + P. aeruginosa (P. aeruginosa) versus S. aureus + P. aeruginosa (t = 4 hours) (P. aeruginosa); and *P < 0.03 mixed infection (Gram-negative) versus Gram-negative. Data are representative of at least three independent experiments. Exact P values in table S1.

  • Fig. 2. Pathogen-specific mAbs reduce disease severity.

    (A) Survival of mice treated with MEDI3902 (15 mg/kg) or c-IgG at the indicated times before or after infection with S. aureus (5e7 CFU) + P. aeruginosa (1e5 CFU). (B) S. aureus or P. aeruginosa CFU recovered from the lungs of infected (24 hours, S. aureus + P. aeruginosa) mice treated with MEDI3902 (15 mg/kg) or c-IgG 24 hours before infection. (C) Survival of mice treated with MEDI4893* (15 mg/kg) at the indicated times before or after infection with S. aureus + P. aeruginosa. (D) S. aureus or P. aeruginosa CFU recovered from the lungs of infected (24 hours, S. aureus + P. aeruginosa) mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hours before infection. Significance was determined by log-rank test (A and C) or Mann-Whitney test (B and D). *P < 0.0001, #P = 0.0105. Data are representative of at least three independent experiments (A to D).

  • Fig. 3. AT is sufficient for potentiation of P. aeruginosa.

    (A) Survival of mice infected with S. aureus mutants and P. aeruginosa. (B) Survival of mice infected with S. aureus (5e7 CFU), Δhla S. aureus, Δhla S. aureus comp:hla, AT (0.05 μg), or ATH35L (0.05 μg) and P. aeruginosa (1e5 CFU). (C) P. aeruginosa CFU recovered from lungs of mice 24 hours after infection with S. aureus (5e7 CFU), Δhla S. aureus, AT (0.05 μg), or ATH35L (0.05 μg) and P. aeruginosa (1e5 CFU). (D) S. aureus CFU recovered from lungs of mice 24 hours after infection with S. aureus (5e7 CFU) and Δhla S. aureus and P. aeruginosa (1e5 CFU). (E) S. aureus or P. aeruginosa CFU recovered from the lungs or spleen of infected (24 hours, S. aureus + P. aeruginosa) humanized mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hours before infection. Significance was determined by Mann-Whitney test (C to E). Data are representative of at least three (A to D) or two (E) independent experiments.

  • Fig. 4. Macrophages and neutrophils contribute to clearance of S. aureus.

    (A) S. aureus CFU recovered from the lungs of control [phosphate-buffered saline (PBS)]– or AM (clodronate)–depleted mice 24 hours after infection with S. aureus (5e7 CFU). (B) S. aureus CFU recovered from the lungs of control (c-IgG)– or neutrophil (anti-Ly6G)–depleted mice 24 hours after infection with S. aureus (5e7 CFU). (C) S. aureus CFU recovered from the lungs of infected (S. aureus 5e7 CFU, 24 hours) mice treated with MEDI4893* or c-IgG 24 hours before infection. (D) Numbers of AMs in the lungs of naïve mice or mice treated with MEDI4893* or c-IgG 24 hours before infection with S. aureus (5e7 CFU) for 4 or 24 hours. (E) Numbers of neutrophils in the lungs of naïve mice or mice treated with MEDI4893* or c-IgG 24 hours before infection with S. aureus (5e7 CFU) for 4 or 24 hours. (F) Numbers of inflammatory monocytes in the lungs of naïve mice or mice treated with MEDI4893* or c-IgG 24 hours before infection with S. aureus (5e7 CFU) for 4 or 24 hours. (G) Fluorescence-activated cell sorting (FACS) analysis of S. aureus association with macrophages in the lungs of MEDI4893* (15 mg/kg)– or c-IgG–treated (24 hours prior) mice infected with S. aureus (5e7 CFU, 4 hours). (H) FACS analysis of S. aureus association with neutrophils in the lungs of MEDI4893* (15 mg/kg)– or c-IgG–treated (24 hours prior) mice infected with S. aureus (5e7 CFU, 4 hours). (I) Confocal images of CD11c+ (green) cells containing S. aureus (yellow, identified by red arrows) recovered from BALF of MEDI4893* (15 mg/kg)– or c-IgG–treated (24 hours prior) mice infected with S. aureus (5e7 CFU, 4 hours). n = 3 images per animal, 3 animals per experiment, 3 experiments. Scale bars, 4 μm. Significance was determined by Mann-Whitney test. Data are representative of at least three independent experiments (A to H).

  • Fig. 5. NK cell function is altered by AT.

    (A) AT induced cytotoxicity of human neutrophils, NK cells, and PBMCs over 4 hours of incubation, measured by live/dead staining. (B) FACS analysis of ADAM10 expression on human PMNs (polymorphonuclear leukocyte), PBMCs and NK cells. MFI, mean fluorescence intensity. (C) In vitro stimulation of IFN-γ release from human NK cells by IL-12 in the presence of increasing amounts of AT. (D) Percent of NK cells expressing IFN-γ in the lungs of MEDI4893* (15 mg/kg)– or c-IgG–treated (24 hours prior) mice infected with S. aureus (5e7 CFU, 24 hours). (E) Percent of CD3+ cells expressing IFN-γ in the lungs of MEDI4893* (15 mg/kg)– or c-IgG–treated (24 hours prior) mice infected with S. aureus (5e7 CFU, 4 hours). (F) Numbers of NK cells in the lungs of mice 24 hours after treatment with c-IgG or anti-NK1.1 antibody. (G) S. aureus CFU recovered from the lungs of control (c-IgG)– or NK cell (anti-NK1.1)–depleted mice 24 hours after infection with S. aureus (5e7 CFU). (H) IFN-γ levels in BALF 24 hours after infection with S. aureus + P. aeruginosa in control (c-IgG)– or NK cell (anti-NK1.1)– depleted mice. Significance was determined by Mann-Whitney test (D to H). Data are representative of at least two independent experiments.

  • Fig. 6. AT prevents lysosomal acidification.

    (A) Confocal images of RAW cells infected with S. aureus [multiplicity of infection (MOI) of 10, 1 hour] in the presence of MEDI4893* or c-IgG. (B) FACS analysis of acidification of the bacterial microenvironment in macrophages infected with S. aureus in the presence of c-IgG, MEDI4893*, or Δhla S. aureus. (C) Confocal images of lysosomal acidification (red) in hPBMCs infected with Δhla S. aureus (MOI 10, 1 hour) in the presence of AT (1 μg/ml) or ATH35L (1 μg/ml); DNA (bacterial and eukaryotic) is labeled blue. (D) Calpain activation in RAW cells treated with AT or ATH35L for 1 hour at the indicated concentrations. (E) Confocal images of lysosomal acidification (red) in hPBMCs infected with S. aureus (MOI 10, 1 hour) in the presence of calpain inhibitor (100 μM) or dimethyl sulfoxide (DMSO); DNAs (both bacterial and eukaryotic) are labeled blue. (F) Percent of S. aureus killed after a 1-hour incubation with RAW cells (MOI 1) in the presence of MEDI4893* (10 μg/ml) or c-IgG. (G) Percent of S. aureus killed after a 1-hour incubation with RAW cells (MOI 1) in the presence of calpain inhibitor (100 μM) or DMSO. (H) Percent of S. aureus killed after a 1-hour incubation with hPBMCs (MOI 1) in the presence of MEDI4893* (10 μg/ml) or c-IgG. (I) Percent of S. aureus killed after a 1-hour incubation with human PMNs (MOI 1) in the presence of MEDI4893* (10 μg/ml) or c-IgG. Percent killed calculated as a ratio of (innocula − recovered CFU)/innocula. Significance was determined by t test (B and E to H). Data are representative of at least three independent experiments. Scale bars, 4 μm (A, C, and E).

  • Fig. 7. Macrophage processing of P. aeruginosa is altered by AT.

    (A) P. aeruginosa CFU recovered from the lungs of control (PBS)– or AM (clodronate)–depleted mice 24 hours after infection with P. aeruginosa (1e5 CFU). (B) P. aeruginosa CFU recovered from the lungs of control (c-IgG)– or neutrophil (anti-Ly6G)–depleted mice 24 hours after infection with P. aeruginosa (1e5 CFU). (C) P. aeruginosa CFU recovered from the lungs of control (IgG)– or NK cell (anti-NK1.1)–depleted mice 24 hours after infection with P. aeruginosa (1e5 CFU). (D) Percent of P. aeruginosa killed after 1 hour incubation with hPBMCs (MOI 1) in the presence of AT (0.1 μg/ml) or ATH35L (0.1 μg/ml). (E) Percent of K. pneumoniae killed after 1-hour incubation with hPBMCs (MOI 1) in the presence of AT (0.1 μg/ml) or ATH35L (0.1 μg/ml). (F) Confocal images of human macrophages infected with P. aeruginosa or S. aureus + P. aeruginosa (1:10 ratio) for 1 hour. Blue, DNA; magenta, actin; green, P. aeruginosa; yellow, LysoTracker. (G) Confocal images of human macrophages infected with S. aureus + P. aeruginosa (1:10 ratio) for 1 hour in the presence of MEDI4893* (10 μg/ml) or c-IgG. Scale bars, 4 μm. (H) Survival of macrophage-depleted (clodronate) mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hours before infection with S. aureus (5e7 CFU) + P. aeruginosa (1e5 CFU). Significance was determined by Mann-Whitney test (A to C), t test (D and E), or log-rank test (G). Data are representative of at least three independent experiments.

  • Fig. 8. S. aureus enhances Gram-negative proliferation through the activity of AT.

    (A) Gram-negative mono-infection. (B) Mixed infection.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/329/329ra31/DC1

    Materials and Methods

    Fig. S1. MEDI4893* protects against mixed infection with diverse S. aureus strains.

    Fig. S2. MEDI4893* prevents mortality associated with mixed infection of S. aureus and either K. pneumoniae or A. baumannii.

    Fig. S3. AT potentiates infection with either K. pneumoniae or A. baumannii.

    Fig. S4. Mixed infection does not result in excessive tissue damage or inflammation.

    Fig. S5. Immune cell populations in the lung.

    Fig. S6. AT neutralization increases phagocytosis of S. aureus by AMs.

    Fig. S7. AT reduces phagocytosis of S. aureus by AMs.

    Fig. S8. Anti-NK1.1 depletion of NK cells.

    Fig. S9. AT prevents colocalization of S. aureus with acidic lysosomes.

    Fig. S10. AT reduces lysosomal acidification in human cells.

    Fig. S11. Contribution of immune cells to the clearance of P. aeruginosa or K. pneumoniae from the lung.

    Fig. S12. Confocal imaging analysis of P. aeruginosa internalization by hPBMCs.

    Fig. S13. Flow cytometry gating strategy and isotype controls.

    Table S1. Exact P values for CFU data in Fig. 1.

    Table S2. Bacterial strains.

    Table S3. Source data for all figures (Excel).

  • Supplementary Material for:

    Staphylococcus aureus α toxin potentiates opportunistic bacterial lung infections

    Taylor S. Cohen, Jamese J. Hilliard, Omari Jones-Nelson, Ashley E. Keller, Terrence O'Day, Christine Tkaczyk, Antonio DiGiandomenico, Melissa Hamilton, Mark Pelletier, Qun Wang, Binh An Diep, Vien T. M. Le, Lily Cheng, JoAnn Suzich, C. Kendall Stover, Bret R. Sellman*

    *Corresponding author. E-mail: sellmanb{at}medimmune.com

    Published 9 March 2016, Sci. Transl. Med. 8, 329ra31 (2016)
    DOI: 10.1126/scitranslmed.aad9922

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. MEDI4893* protects against mixed infection with diverse S. aureus strains.
    • Fig. S2. MEDI4893* prevents mortality associated with mixed infection of S. aureus and either K. pneumoniae or A. baumannii.
    • Fig. S3. AT potentiates infection with either K. pneumoniae or A. baumannii.
    • Fig. S4. Mixed infection does not result in excessive tissue damage or inflammation.
    • Fig. S5. Immune cell populations in the lung.
    • Fig. S6. AT neutralization increases phagocytosis of S. aureus by AMs.
    • Fig. S7. AT reduces phagocytosis of S. aureus by AMs.
    • Fig. S8. Anti-NK1.1 depletion of NK cells.
    • Fig. S9. AT prevents colocalization of S. aureus with acidic lysosomes.
    • Fig. S10. AT reduces lysosomal acidification in human cells.
    • Fig. S11. Contribution of immune cells to the clearance of P. aeruginosa or K. pneumoniae from the lung.
    • Fig. S12. Confocal imaging analysis of P. aeruginosa internalization by hPBMCs.
    • Fig. S13. Flow cytometry gating strategy and isotype controls.
    • Table S1. Exact P values for CFU data in Fig. 1.
    • Table S2. Bacterial strains.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 Source data for all figures (Excel).