Fig 1.
Hyaluronic acid expression by Streptococcus pyogenes promotes nasal infection in B6HLA mice.
(A) S. pyogenes constructs streaked onto TSA + 5% sheep blood agar plates. The plate figure are representative images of the wild-type MGAS8232, ΔhasA mutant and the hasA complemented strain. (B) B6HLA mice were administered ~1×108 CFUs of S. pyogenes MGAS8232 wildtype, ΔhasA, or ΔhasA +hasA strains intranasally and sacrificed 48 h later. Data points represent CFUs from cNTs of individual B6HLA mice. Horizontal bars represent the geometric mean. Significance was determined by Kruskal Wallis one-way ANOVA with Dunn’s multiple comparisons test (****, P < 0.0001; **, P < 0.01). The horizontal dotted line indicates the theoretical limit of detection. (C) Heat-map of cytokine responses in cNTs of B6HLA mice during S. pyogenes infection. Data shown represent normalized median cytokine responses from cNTs (n ≥ 3 per group). (D) Immunohistochemistry of infected cNTs at 24 h post-infection with wildtype S. pyogenes MGAS8232 and the ΔhasA mutant. Sections were stained with α-S. pyogenes (red), αB220 (blue), αCD3 (green), and αLy6G (white) antibodies. Panels are a close-up view from the boxed section. Arrows indicate regions with internalized S. pyogenes.
Table 1.
Bacterial strains and plasmids used in this study.
Fig 2.
The S. pyogenes HA capsule inhibits host cell invasion but promotes survival from neutrophil-mediated killing.
Binding of S. pyogenes to wells pre-coated with 1 μg of human ECM components (A) fibronectin and (B) collagen type IV. (C) Adhesion of S. pyogenes to D562 pharyngeal epithelial cells. Confluent cell monolayers were cultured with S. pyogenes (MOI of 100) for 2 h at 37°C + 5% CO2. Cells were washed with PBS and lysed with Triton X-100 for enumerating remaining adherent bacteria. (D) Internalization of S. pyogenes into D562 cells. Confluent D562 cells were cultured with S. pyogenes (MOI of 100) for 2 h at 37°C + 5% CO2 followed by 1 h in media supplemented with 100 μg mL-1 of gentamycin. Bars represent mean CFUs ± SEM and each dot represents a biological replicate. Statistical differences were evaluated by unpaired t-test (A–C) (**, P < 0.01; ****, P < 0.0001) or (D) one-way ANOVA (*, P < 0.05; ****, P < 0.0001). (E) Whole human blood survival assay. Heparinized blood from human donors were inoculated with ~103 CFUs of S. pyogenes MGAS8232 at 37°C with rotation for 3 h. Data points represent geometric mean CFUs ± SD at each timepoint (n ≥ 3). Statistical significance was determined using one-way ANOVA with Friedman test (***, P < 0.001). (F) Neutrophil survival assay. Neutrophils were isolated from human blood by density centrifugation and inoculated with opsonized S. pyogenes at a MOI of 10. Surviving bacteria were enumerated after 60 mins at 37°C with rotation and calculated as the difference between survival in the no neutrophil control and in the presence of neutrophils. Each data point represents S. pyogenes CFUs from an individual donor. Data shown are the means of percent survival ± SD. Statistical analyses were performed using one-way ANOVA with Kruskal-Wallis test (*, P < 0.05).
Fig 3.
Early clearance of the HA capsule-deficient mutant from murine nasal turbinates is due to enhanced susceptibility to neutrophil-mediated killing.
(A) Schematic outline for in vivo depletion of neutrophils with injections of 250 μg (500 μg total) of αLy6G or isotype control rat IgG2a 24 h prior to and 24 h post-intranasal challenge with 108 CFUs of S. pyogenes wildtype or ΔhasA mutant strains. (B) Representative flow cytometric analyses of nasal and blood innate immune cells from the neutrophil depletion experiments at 48 h. Flow plots show live cells that were negative for CD4, CD45R and CD19, and gates were set on Ly6G+ and F4/80- cells for neutrophils, and Ly6G- and F4/80+ for macrophage populations. Percentage of innate immune cell populations from either nasal cell extracts (C) or blood (D) for the indicated treatment groups as percentage of live cells. Data points represent individual mice and the bars represent the mean. Significance was determined by Mann-Whitney test (*, P < 0.05) (E) Neutrophil effects on S. pyogenes survival in the nasopharynx. Data points represent CFUs from cNTs of individual mice 24 and 48 h post-infection. Horizontal bars represent the geometric mean. The horizontal dotted line indicates limit of detection. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons (*, P < 0.05; **, P < 0.01; ****, P < 0.0001). Fig 3A was created using Biorender.com.
Fig 4.
Hyaluronic acid capsule expression by Streptococcus pyogenes promotes skin infection in B6HLA mice.
B6HLA mice were administered ~5 × 107 CFUs of wildtype S. pyogenes MGAS8232, or the hasA mutant, or the ΔhasA + hasA complemented strain by intradermal injections in each hind flank. (A) Weights of B6HLA mice at 24, 48, and 72 h following skin challenge. Data is represented as a percentage of day 0 weight. Data points represent the weight means ± SEM (n ≥ 5). (B) Skin lesion areas of mice at 24, 48, and 72 h after skin challenge. Data points represent individual lesion areas (2 per mouse), and the bars represent the mean. Significance was determined by two-way ANOVA with Geisser’s Greenhouse correction and Dunnett’s multiple comparisons test (*, P < 0.05, **, P < 0.01, ***, P < 0.001;) for panels A and B. (C) Data points represent CFUs from individual infected skin lesions from mice at 72 h. Horizontal bars represent the geometric mean. Significance was determined by one-way ANOVA with Kruskal-Wallis test (****, P < 0.0001; **, P < 0.01). The horizontal dotted line indicates the theoretical limit of detection. (D) Representative skin lesion images from B6HLA mice 72 h following skin challenge.
Fig 5.
The hyaluronic acid capsule is important for resisting neutrophil-mediated killing in the skin.
B6HLA mice were administered ~5 × 107 CFUs of wildtype S. pyogenes MGAS8232 or the ΔhasA or ΔhasA + hasA complemented strain intradermally in each hind flank. Mice received αLy6G or rat IgG2a isotype control antibodies intraperitoneally 24 h preceding and 24 h after skin infections. (A) Weights of B6HLA mice at 24, 48, and 72 h following S. pyogenes skin challenge. Data is represented as a percentage of day 0 weight. Data points represent the weight means ± SEM (n ≥ 3). (B) Skin lesion areas of mice following at 24, 48, and 72 h after skin challenge. Data points represent individual lesion areas (2 per mouse), and the bars represent the mean. (C) Data points represent CFUs from individual infected skin lesions from mice at 72 h. Horizontal bars represent the geometric mean. The horizontal dotted line indicates limit of detection. Significance was determined by two-way ANOVA with Geisser’s Greenhouse correction and Dunnett’s multiple comparisons test (*, P < 0.05, **, P < 0.01, ***, P < 0.001;) for panels A and B, or one-way ANOVA with Kruskal Wallis (***, P < 0.001; ****, P < 0.0001) for panel C. (D) Representative skin lesion images from B6HLA mice 72 h following skin challenge.