Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves

Abstract

Aims: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states.

Methods and results: Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential.

Conclusion: Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.

Keywords: Staphylococcus aureus; Endocarditis; Fibrinogen; Platelets; von Willebrand factor.

Figure 1

Spontaneous endocarditis in mice (A) Proportion of mice that developed endocarditis after injection of 2 × 10⁶ colony forming units Staphylococcus aureus Newman in young (10–15 weeks of age) and old mice (>18 months). (B) Gram staining of an aortic valve without endocarditis. (C) Aortic valve endocarditis in an old mouse. Staining: haematoxylin and eosin (H&E), Martius, Scarlet, and Blue (MSB) (stains fibrin red, collagen blue), von Willebrand factor (VWF) immunostaining, and Gram staining. (D) Same stainings of human S. aureus endocarditis.

Figure 2

New endocarditis model to measure early bacterial adhesion in different risk states (A) Experimental set-up: fluorescent-labelled Staphylococcus aureus (3 × 10⁷ CFUs) was injected intravenously in C57Bl/6 mice and a small catheter was inserted in the carotid artery and advanced beyond the aortic valve. To create cardiac valve damage, the catheter was left in place for 5–30 min (depending on the experiment). To mimic cardiac valve inflammation the valvular endothelium was locally activated through infusion of histamine (200 mM at 10 μL/min) for 5 min. Mice were immediately sacrificed and 200 μM thick aortic valve sections were analysed with confocal microscopy. (B) 3D reconstruction of an aortic valve (isolectin staining, green), with S. aureus adhering (red). (C) Quantification of bacterial adhesion in sham operated mice vs. mice with a catheter (5 min damage) vs. mice with catheter + 5 min histamine infusion (inflammation). Results represent log-transformed volumes of single mice. (D–H) Immune-fluorescent imaging of aortic valves (n = 14) for (A) von Willebrand factor (VWF), (B) P-selectin, (C) VE-cadherin, (D) adhering CD45+ cells, (E) adhering platelets (stained with anti-Gp1b antibodies). Mean ± standard deviation range is given *P < 0.05, **P < 0.01, two-tailed Student;s t-test. (G–J) Representative images of (G) VE-cadherin, (H) VWF/CD31, (I) platelets, and (J) CD45/CD105 stainings.

Figure 3

Studying mature endocarditis vegetations. Staphylococcus aureus was injected intravenously in C57Bl/6 mice and cardiac valve damage (30 min) or inflammation (5 min histamine infusion) was induced. Afterwards, the catheter was removed and mice were monitored for 3 days to see if endocarditis developed. (A–I) Proportion of mice developing endocarditis after infection with three different strains: S. aureus Newman, USA 300, and a clinical endocarditis strain; (A–C) in the inflammation-induced model with 2 × 10⁶ CFUs, (D–F) in the damaged-induced model with 2 × 10⁶ CFUs and (G–I) in the damaged-induced model with 2 × 10⁷ CFUs. *P < 0.05, **P < 0.01, Fisher’s exact. Number of mice used is indicated. (J) Gram staining of the aortic valve showing growing vegetations at Day 0–3 after surgery (S. aureus Newman). (K) Scanning electron microscopy of aortic valve endocarditis with S. aureus Newman. (L) Echocardiographic imaging of endocarditis lesion causing severe aortic regurgitation (S. aureus Newman).

Figure 4

Mechanisms of bacterial adhesion in the damage-induced endocarditis model (15 min of damage by a transaortic catheter). (A) Adhesion of Staphylococcus aureus Newman in wild type (Vwf^+/+) vs. VWF knockout mice (Vwf^−/−), (B) in platelet depleted vs. control mice, and (C) in ancrod treated vs. control mice. (D–F) Adhesion of different S. aureus Newman mutants to damage aortic valves compared with wild type bacteria; (D) Sortase A (ΔsrtA), (E) von Willebrand Factor binding protein (Δvwb), and (F) Clumping factor A (ΔclfA). Results represent log-transformed volumes in single mice. Mean ± standard deviation, *P < 0.05, two-tailed Student’s t-test.

Figure 5

Mechanisms of bacterial adhesion in the inflammation-induced endocarditis model (5 min of histamine infusion through transaortic catheter). (A) Adhesion of Staphylococcus aureus Newman in wild type (Vwf^+/+) vs. VWF knockout mice (Vwf^−/−), (B) in platelet depleted, and (C) in ancrod treated mice. (D–F) Adhesion of different S. aureus Newman mutants to damage aortic valves compared with wild type (WT) S. aureus; (D) Sortase A (ΔsrtA), (E) von Willebrand Factor binding protein (Δvwb), and (F) Clumping factor A (ΔclfA). Results represent log-transformed volumes in single mice. (G) Flow chamber experiment measuring the adhesion of WT S. aureus Newman, or ΔsrtA to resting or activated endothelial cells (shear rate 1000 s⁻¹) in the presence or absence of platelets. (H) Adhesion of WT S. aureus Newman to aortic valves of mice after local infusion via an aortic catheter of saline or S. aureus alpha-toxin (0.5 mg/mL). Mean ± standard deviation, *P < 0.05, ***P < 0.005, two-tailed unpaired Student’s t-test.

Figure 6

Detailed confocal imaging of early endocarditis lesions. Fluorescent-labelled Staphylococcus aureus Newman, fibrinogen, and platelet-staining antibodies were injected in vivo and the endothelium was counterstained with isolectin and DAPI. (A and B) Damage induced endocarditis, with (A) 3D reconstruction of an early endocarditis lesion. Different pictures show the same lesion: first panel showing all components, second after removing the platelet layer, and the third after removal of fibrin layer showing only bacteria and endothelium. (B) Detailed 2D microscopy images. (C and D) Inflammation-induced endocarditis: (C) 3D reconstruction, (D) 2D images.

Figure 7

Scanning electron microscopy of early endocarditis lesions. (A–C) Early lesion on a damaged aortic valve, showing fibrin (Fi) deposition on damaged endothelium and secondary platelet and bacterial adhesion. (D–I) Early lesion on an inflamed heart valve, showing (E) a platelet rich vegetation (PV) that adheres to intact endothelial cells (EC). (F–G) Activated platelets (AP) are incorporated into the vegetation. (H and I) Neutrophils (Neu) adhere to the activated endothelium. (J and K) Normal valves.

Take home figure

Differential mechanisms of S. aureus adhesion to damaged or inflamed heart valves.