Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo

Abstract

Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection.

Figure 1. The regulatory input of Agr upon Rot directly influences bloodstream infection with S. aureus

(A-B) “Survival” of mice infected systemically with Newman wild type (WT), N=12 (A) and N=17 (B); Δagr, N=6; Δagr Δrot, N=6; or Δrot, N=12. Mice were injected with ~1×10⁷ CFU of the indicated strains via the retro-orbital venous plexus and monitored for “survival” over time as described in Experimental procedures. (C-D) Immunoblots of leukotoxins produced by the indicated strains. (−) Control indicates proteins isolated from gene deletion mutants of the particular leukotoxin probed in each panel. For LukE immunoblots an ΔhlgΔlukED or an ΔrotΔhlgΔlukED mutant was included due to cross reactivity of the anti-LukE antibody with subunits of γ-hemolysin. Statistically significant differences between curves were determined by Log-Rank (Mantel Cox) test and p values are shown (***, p≤0.0005).

Figure 2. Identification of virulence factors critical to S. aureus bacteremia-mediated death

(A-D) “Survival” of mice infected with Newman wild type (WT), Δrot, and rot/leukotoxin double mutants, all N=6. Mice were injected with ~1×10⁷ CFU of the indicated strains via the retro-orbital venous plexus and monitored for “survival” over time as described in Experimental procedures. Statistically significant differences between curves were determined by Log-Rank (Mantel Cox) test and p values are shown (*, p<0.05; **, p≤0.005).

Figure 3. LukED contributes to lethality in S. aureus bloodstream infection

(A-B) PCR and immunoblot confirmation of the Newman ΔlukED mutant and its complement ΔlukED∷lukED. For LukE immunoblots, an ΔhlgΔlukED mutant was included due to cross reactivity of the anti-LukE antibody with subunits of γ-hemolysin. (C-D) “Survival” curves of MSSA (strain Newman) and MRSA (USA500 strains) infected mice. Mice were injected with ~1×10⁷ CFU of Newman wild type (WT), N=6; ΔlukED, N=6; ΔlukED∷lukED, N=6; USA500 (BK2371 or BK2395) wild type (WT), N=10; or an isogenic ΔlukED mutant from each USA500 strain, N=10, via the retro-orbital venous plexus and monitored for “survival” over time as described in Experimental procedures. Statistically significant differences between “survival” curves were determined by Log-Rank (Mantel Cox) test and p values are shown (**, p≤0.005; ***, p≤0.0005).

Figure 4. LukED facilitates bacterial replication in vivo

(A) Gross pathology of abscess formation in kidneys of mice infected for 96 hours with Newman wild type (WT), ΔlukED, and ΔlukED∷lukED. White arrows point to abscesses in wild type and ΔlukED∷lukED infected organs. (B) Enumeration of CFU from infected kidneys. Ten mice per group were injected with ~1×10⁷ CFU of Newman wild type (WT), ΔlukED, and ΔlukED∷lukED. After 96 hours, kidneys were removed and homogenized, followed by plating of serial dilutions onto solid media for enumeration of bacterial burden. (C) IL-6 and GCSF levels in the serum of animals from Panel B. Statistical significance between CFU and Serum cytokine levels was determined by 1-way-ANOVA with Tukey’s multiple comparison test (**, p≤0.005; ***, p≤0.0005).

Figure 5. LukED kills phagocytes elicited to the site of infection ex vivo

(A) Profile of innate immune cells recruited to the peritoneum upon infection with S. aureus and subsequently intoxicated with the indicated amounts of LukE, LukD, or LukED. Representative FACS plots of cells in the live gate are shown. The graphical depiction represents the average percent viable PECs within the live gate. (B) Killing of primary murine neutrophils (CD11b⁺/Ly6G⁺) by LukED. Representative FACs plots are shown. The graphical depiction displays percent viable neutrophils relative to the total viable neutrophils recovered from unintoxicated controls (set to 100%). Statistical significance was determined by 1-way ANOVA with Tukey’s multiple comparison test (***, p≤0.0005).

Figure 6. LukED targets and kills neutrophils by damaging their plasma membrane

Intoxicated PECs were evaluated for viability, membrane damage and pore formation via (A) light microscopy at 40X magnification, (B) metabolic activity (CellTiter), (C) LDH release (CytotoxOne), and (D) ethidium bromide incorporation into cellular DNA. For microscopic imaging of intoxicated cells (A and D) a toxin dose of 5 μg/ml was used. For all other assays of viability, membrane integrity, and pore formation a dose response of equimolar ratios of LukE and LukD was added to cells and measurements made as described in the Experimental procedures.

Figure 7. LukED promotes S. aureus virulence in vivo via its potent toxicity toward neutrophils

(A) Determination of percent viable CD11b⁺ phagocytic leukocytes in S. aureus infected kidneys. (B) Immune cell profile (neutrophils; GR1⁺/CD11b^+, B lymphocytes CD3⁻/B220⁺, and T lymphocytes CD3⁺/B220⁻) of mice treated with a neutrophil depleting antibody (1A8) or an isotype control antibody (2A3) followed by systemic infection with wild type S. aureus. (C) “Survival” curves of mice treated with 2A3, 1A8, or untreated followed by systemic infection of ~1×10⁸ CFU wild type (WT), N=10 for 2A3 or 1A8 and N=6 for untreated; or a ΔlukED mutant, N=10 for 2A3 or 1A8 and N=6 for untreated. Statistically significant differences between “survival” curves were determined by Log-Rank (Mantel Cox) test and p values are shown (*, p<0.05; **, p≤0.005).