Article Figures & Data
Figures
- FIG 1
Streptococcus pneumoniae killing of cardiomyocytes at a low MOI requires contact. (A) Cell viability (left graph) and total cell count (right graph) of noninfected (white bars) or TIGR4-infected (black bars) HL-1 cells were determined by trypan blue exclusion assay. (B) HL-1 cardiomyocyte death was measured by LDH release from cells infected by live, heat-killed, or ethanol-killed TIGR4. (C) Bacterial toxicity determined by LDH release in a Transwell permeable support system (n = 3). Errors bars represent standard errors of the means. Statistical analyses were done with t test (A) and 1-way analysis of variance (ANOVA) with Holm-Sidak correction for multiple comparison (B and C). ****, P ≤ 0.0001.
- FIG 2
Streptococcus pneumoniae invades and multiplies inside HL-1 cardiomyocytes. (A) Kinetics of the interaction between TIGR4 and HL-1 cardiomyocytes. Associated or internalized bacterial loads were determined by CFU counts without or with gentamicin treatment, respectively. (B) Immunofluorescence microscopy on HL-1 cardiomyocytes after 1 h of infection by S. pneumoniae. Pneumococci were stained before (rhodamine [red]) and after (FITC [green]) permeabilization of the cell membrane using anti-serotype 4 antibody. (C) Representative TEM image showing intracellular S. pneumoniae inside HL-1 cardiomyocytes after 2 h of infection. Green arrowheads show cytoplasmic S. pneumoniae. Red arrowheads show multiple intravacuolar S. pneumoniae organisms. (D) Intracellular multiplication was assayed using the BrdU incorporation assay. S. pneumoniae was stained with anti-serotype 4 antibody (red) after gentamicin treatment and a BrdU (green) pulse. DAPI staining shows nuclei. Scale bars, 10 μm.
- FIG 3
Clathrin-mediated endocytosis is required for intracellular S. pneumoniae-mediated killing. Shown is the interaction between HL-1 cardiomyocytes and TIGR4 in the presence of the designated endocytosis inhibitors. (A) Adhesion was measured 30 min after infection and invasion was measured 1 h after infection followed by 1 h of gentamicin treatment. (B) TIGR4-mediated HL-1 cell death in the presence of designated endocytosis inhibitors was measured using LDH release (n = 3). Errors bars represent standard errors of the means. Statistical analyses were done with t test (methyl-beta-cyclo-dextrin [MβCD] and PitStop 2) and 1-way ANOVA with Holm-Sidak correction for multiple comparison (cytochalasin D and genistein) (A) and t test (B). ***, P ≤ 0.001; ****, P ≤ 0.0001. Endocytosis inhibitors were used at the following concentrations: MβCD, 10 mM; cytochalasin D, 10 μM; genistein, 200 μM; and PitStop 2, 30 μM.
- FIG 4
Streptococcus pneumoniae interacts with cardiomyocytes independently of the canonical pathways. Shown are the interactions between HL-1 cardiomyocytes and wild-type TIGR4 (WT) and its derivative choline binding protein A (ΔcbpA) and cholinekinase (ΔlicA) mutants alone (A) or in the presence of the designated neutralizing antibodies (1 μg/ml) (B). Adhesion was measured 30 min after infection, and invasion was measured 1 h after infection followed by 1 h of gentamicin treatment (n = 3). Errors bars represent standard errors of the means. Statistical analyses were done with t tests (A) and 1-way ANOVA with Holm-Sidak correction for multiple comparison (B). **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG 5
Intracellular hydrogen peroxide produced by S. pneumoniae is required to induce cytotoxicity. (A and B) HL-1 death was measured by assaying LDH release induced by pyruvate oxidase (ΔspxB) or pneumolysin (Δply) TIGR4 mutants (A) or in the presence of extracellular (catalase, 10 U/ml) or extra- and intracellular (Tempol, 3 mM) antioxidants (B). (C) LDH release induced after 6 h of infection by S. pneumoniae TIGR4 and S. pneumoniae D39. (D) Kinetics of H2O2 produced by TIGR4 and D39 during infection. (E) TIGR4 and D39 adhesive and invasive abilities (n = 3). Errors bars represent standard errors of the means. Statistical analyses were done with 1-way ANOVA with Holm-Sidak correction for multiple comparison (A) and t test (B, C, and D). *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Catalase was used at 10 U/ml and Tempol at 3 mM.
- FIG 6
Tempol attenuates cardiac microlesion formation in vivo without modifying bacterial burden. BALB/cJ mice were challenged using 1.0 × 103 CFU/ml of TIGR4 in the presence or absence of Tempol (20 mg/kg). Tempol was administered a second time 24 h after infection. (A) Visualization of microlesion formation in the presence or absence of Tempol. S. pneumoniae was stained with anti-serotype 4 antibody (green), and sections were counterstained with DAPI (blue). Scale bar, 250 μm. (B) Microlesions were enumerated by counting foci of S. pneumoniae in three nonconsecutive stained heart sections separated by at least 50 μm. (C) Final heart burden at the time of sacrifice (T0 + 30 h) was measured by colony counts. (D) Bacteremia was assayed every 6 h by tail bleed. Dotted lines represent detection limits (n = 5 per condition). Error bars represent standard errors of the means. Statistical analyses were done with t tests (B and C) and 2-way ANOVA with Holm-Sidak correction for multiple comparison (D). *, P < 0.05.
Additional Files
Supplemental material
- Supplemental file 1 -
Table S1. Strains used in this study. Table S2. Primers used in this study. Fig. S1. Streptococcus pneumoniae invades HL-1 cardiomyocytes through clathrin-mediated endocytosis. Fig. S2. ΔspxB and Δply mutants are not impaired in adhesion and invasion of HL-1. Fig. S3. Inflammatory response of HL-1 upon Streptococcus pneumoniae infection.
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- Supplemental file 2 -
Supplemental materials and methods.
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- Supplemental file 1 -
