Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice

Abstract

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Keywords: ACKR1; DARC; HlgAB; LukED; Staphylococcus aureus; endothelial cells; leukocidin.

Figure 1:. LukED and HlgAB cause lethal vascular collapse.

(A) Survival curve of Swiss-Webster mice infected intravenously with 2–3 × 10⁷ CFU of the indicated S. aureus Newman strains (n=7–8 mice per group). See also Figure S1A–D. (B, C) Survival curve of Swiss-Webster mice injected intravenously with purified toxin (n=4 mice per group). See also Figure S1F, G. (D) Quantification of LukD levels in kidneys and hearts of C57BL/6J mice 96 hours post infection (hpi) with 2.5 × 10⁷ CFU of the WT (n=14) or ΔlukED (n=3) S. aureus Newman by ELISA. See also Figure S1E. Representative photographs (E) and H&E staining (F) of ears from Swiss-Webster mice challenged with toxin. Scale bar indicates 100 μM. See also Figure S1H. (G) Hematocrit and (H) temperature change in toxin challenged Swiss Webster mice (n = 5–12 mice per group). (I) Intravital imaging of livers of Swiss Webster mice before and after toxin challenge. Evans blue in red is vascular fluid; Hoechst in blue is nuclei. Scale bars are 80 μM, arrows indicate vessels depleted of fluid. See also Video S1. (J) Evans blue content in organs of toxin challenged Swiss Webster mice without perfusion (n = 7–12 mice per group). See also Figure S1I. Data shown are pooled from (A, B, C, D, G, H, J) or representative of (E, F, I) at least two independent experiments. Where relevant, means ± SEM are shown. Sa = S. aureus. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 (A, B, C, Mantle-Cox test; G, H, J, one-way analysis of the variance (ANOVA) with Tukey’s correction).

Figure 2:. LukED and HlgAB target endothelial DARC to cause lethality during challenge.

(A) Survival curve of toxin challenged C57BL/6J WT and Darc^−/− mice (n = 4–5 mice per group). See also Table S1. (B) Hematocrit of toxin challenged mice (n=3 mice per group). (C) Evans blue content in lungs (left) and ears (right) of toxin challenged WT and Darc^−/− mice (n = 3–11 mice per group). (D) Survival curves of toxin challenged bone marrow chimeric mice (n = 4–6 mice per group). See also Figure S2A–D. (E) DARC staining of endothelial cells from the skin of Darc^fl/fl and Darc^fl/fl Tie2 Cre mice. See also Figure S2F–H. (F) Survival curve of toxin challenged Darc^fl/fl and Darc^fl/fl Tie2 Cre mice (n = 4–5 mice per group). KO=Darc^−/−, bone marrow chimeras annotated as donor → recipient. Data shown are pooled from (A, B, C, D, F), or representative of (E) at least two independent experiments. Where relevant, means ± SEM are shown. ** p<0.01, *** p<0.001.

Figure 3:. LukED and HlgAB target primary human endothelial cells in vitro.

(A) Propidium Iodide (PI) uptake by HPMECs transfected with the indicated plasmid after exposure to 0.3 μM of toxin (10 μg/ml) for 3 hours. (B) Immunoblot for DARC and (C) qPCR for DARC transcript in HPMEC after exposure to human whole blood +/− cyclohexamide. (D) PI uptake by HPMEC cells after treatment with human whole blood and exposure to 0.6 μM of toxin (20 μg/ml). (E) Images of and (F) PI uptake by HPMEC cells after transfection with siRNA, treatment with human whole blood, and exposure to 0.6 μM of toxin. Data are pooled from (A, C, D, F) or representative of (B, E) at least three independent experiments. Where relevant, means ± SEM are shown. * p<0.05, ** p<0.01, *** p<0.001 (A, D, F, ANOVA with Tukey’s correction; C, two-tailed student’s t-test).

Figure 4:. LukED and HlgAB target nonhematopoietic DARC to cause lethality during infection.

Survival curves of C57BL/6J (WT) and Darc^−/− mice infected with 2–3.5 × 10⁷ CFU of the indicated strains of S. aureus Newman (A), 1–2.7 × 10⁷ CFU of S. aureus USA500 BK2395 (B), and 0.9–1.3 × 10⁷ CFU of S. aureus LAC (C) (n = 18–24 mice per group). See also Figure S4A, B. (D) Survival curve of bone marrow chimeric mice infected with 1–2 × 10⁷ CFU WT S. aureus Newman (n = 8–9 mice per group). See also Figure S2E. (E) Scoring of kidney pathology from 72–96 hpi of WT and Darc^−/− mice infected with 2–3.5 × 10⁷ CFU of S. aureus Newman. (F) H&E staining of hearts and (G) Serum levels of analytes in mice at 96 hpi with 2.5 × 10⁷ CFU of S. aureus Newman. Scale bar in (F) is 1 mm, 100 μm in the magnified images. See also Figure S4C–F. “Baseline” in (G) is the average values for WT and DARC KO mice mock infected (mock infected WT and DARC KO mice had similar values of all analytes measured, See also Table S2). BUN = Blood Urea Nitrogen, ALP = Alkaline Phosphatase. Sa = S. aureus, KO = Darc^−/−. Data are pooled from (A-E, G) or representative of (F) at least two independent experiments. Where relevant, means ± SEM are shown. * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001 (A, B, C, D Mantel-Cox test, E, two-tailed linear-by-linear association test, G, one-tailed student’s t-test).