Liposomal Therapy Attenuates Dermonecrosis Induced by Community-Associated Methicillin-Resistant Staphylococcus aureus by Targeting α-Type Phenol-Soluble Modulins and α-Hemolysin

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), typified by the pulse-field type USA300, is an emerging endemic pathogen that is spreading rapidly among healthy people. CA-MRSA causes skin and soft tissue infections, life-threatening necrotizing pneumonia and sepsis, and is remarkably resistant to many antibiotics. Here we show that engineered liposomes composed of naturally occurring sphingomyelin were able to sequester cytolytic toxins secreted by USA300 and prevent necrosis of human erythrocytes, peripheral blood mononuclear cells and bronchial epithelial cells. Mass spectrometric analysis revealed the capture by liposomes of phenol-soluble modulins, α-hemolysin and other toxins. Sphingomyelin liposomes prevented hemolysis induced by pure phenol-soluble modulin-α3, one of the main cytolytic components in the USA300 secretome. In contrast, sphingomyelin liposomes harboring a high cholesterol content (66 mol/%) were unable to protect human cells from phenol-soluble modulin-α3-induced lysis, however these liposomes efficiently sequestered the potent staphylococcal toxin α-hemolysin. In a murine cutaneous abscess model, a single dose of either type of liposomes was sufficient to significantly decrease tissue dermonecrosis. Our results provide further insights into the promising potential of tailored liposomal therapy in the battle against infectious diseases.

Keywords: Anti-toxin therapy; CA-MRSA; Dermonecrosis; Liposomes; Phenol-soluble modulins; Skin and soft tissue infections; USA300; α-Hemolysin.

Fig. 1

Liposomal treatment decreased necrosis of human cells induced by the secretome of S. aureus USA300. (a) Human red blood cells (RBCs) were incubated with increasing concentrations of sphingomyelin (Sm) and cholesterol:sphingomyelin (Ch:Sm) liposomes (≥3 bacterial filtrates were tested per condition, RBCs were isolated from ≥3 different donors). (b-d) Human cells were incubated with sodium Tyrode's buffer (vehicle), bacterial supernatants and Sm or Ch:Sm liposomes (both 300 μg/ml, or as indicated) for 1 h. (b, c, e) The release of lactate dehydrogenase (LDH) of challenged human cells was determined. (b) PBMCs were isolated from 5 different donors and 6 bacterial filtrates were tested per condition (one-way ANOVA Dunn's multiple comparisons test, ∗∗∗∗p < .0001, ns = not significant). (c) Human bronchial epithelial 16HBE14o- cells (HBE) were incubated with 6 bacterial filtrates per condition (one-way ANOVA Dunn's multiple comparisons test, ∗∗∗∗p < .0001, ns = not significant). (d) Laser scanning micrographs of HBE cells stained with CellTracker Orange. HBE cells were treated with sodium Tyrode's buffer (vehicle), bacterial supernatant (supernat), supernat and Sm liposomes (supernat+Sm), and supernatant with Ch:Sm liposomes (supernat+Ch:Sm) (representative images from 3 bacterial filtrates per condition). Scale bar = 50 μm. (e) USA300 supernatants (50 μl) pre-incubated with Sm, Ch:Sm liposomes (300 and 600 μg/ml), or vehicle and subsequently depleted of liposomes, were applied to human bronchial epithelial HBE cells for 1 h (≥3 bacterial filtrates were tested per condition, one-way ANOVA Dunn's multiple comparisons test, ∗∗∗∗p < .0001, ∗∗∗p < .001, ns = not significant). Error bars (a-c, e), mean ± s.e.m.

Fig. 2

Liposomes neutralized major S. aureus USA300 virulence factors and attenuated dermonecrosis in a murine cutaneous abscess model. (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the USA300 supernatant (supernat) and pellets (p.) of vehicle, sphingomyelin (Sm) and cholesterol:sphingomyelin (Ch:Sm) liposome-treated supernatant revealed a strong protein band in p. Ch:Sm (25–37 kDa) indicated by an arrow (representative data of ≥3 bacterial supernatants per condition). (b) Western blotting with an anti-α-hemolysin antibody (anti-Hla antibody) identified the ~35 kDa protein band of p. Ch:Sm (arrow) as α-hemolysin (representative data of ≥2 bacterial supernatants per condition). (c) Human red blood cells were treated with purified phenol-soluble modulin α3 (PSM-α3, 50 μg/ml) and Tyrode's buffer (vehicle), Sm or by Ch:Sm liposomes (both liposomes 300 μg/ml) (n ≥ 3, two independent experiments, ∗p < .05). Error bars, mean ± s.e.m. (d-e) Sm or Ch:Sm liposomes (80 mg/kg) or vehicle were applied 1 h after the injection of 5 × 10⁷ CFU/injection USA300 in the back of CD-1 mice (vehicle and Sm treatment: n ≥ 9, four independent experiments; Ch:Sm treatment n = 8, two independent experiments, box and whiskers plot). (d) Dermonecrosis (Kruskal-Wallis and Dunn's multiple comparisons test, ∗∗p < .01, ∗p < .05, ns = not significant) and (e) CFU/abscess were evaluated 72 h after infection. (f-g) Sm or Ch:Sm liposomes (80 mg/kg) or vehicle were applied 1 h after the injection of 5 × 10⁷ CFU/injection bioluminescent USA300 in the back of CD-1 mice and the disease progress was monitored up to 10 days post-infection (one experiment, number of mice in brackets, error bars, mean ± s.e.m.). (f) Abscess size measurements (two-way ANOVA, Dunnett's multiple comparisons test, compared to vehicle, ∗∗∗∗p < .0001, ∗∗∗p < 0.001, ∗∗p < .01, ∗p < .05). (g) Bacterial loads indicated by luminescence counts.