Staphylococcus aureus α-toxin modulates skin host response to viral infection

Abstract

Background: Patients with atopic dermatitis (AD) with a history of eczema herpeticum have increased staphylococcal colonization and infections. However, whether Staphylococcus aureus alters the outcome of skin viral infection has not been determined.

Objective: We investigated whether S aureus toxins modulated host response to herpes simplex virus (HSV) 1 and vaccinia virus (VV) infections in normal human keratinocytes (NHKs) and in murine infection models.

Methods: NHKs were treated with S aureus toxins before incubation of viruses. BALB/c mice were inoculated with S aureus 2 days before VV scarification. Viral loads of HSV-1 and VV were evaluated by using real-time PCR, a viral plaque-forming assay, and immunofluorescence staining. Small interfering RNA duplexes were used to knockdown the gene expression of the cellular receptor of α-toxin, a disintegrin and metalloprotease 10 (ADAM10). ADAM10 protein and α-toxin heptamers were detected by using Western blot assays.

Results: We demonstrate that sublytic staphylococcal α-toxin increases viral loads of HSV-1 and VV in NHKs. Furthermore, we demonstrate in vivo that the VV load is significantly greater (P < .05) in murine skin inoculated with an α-toxin-producing S aureus strain compared with murine skin inoculated with the isogenic α-toxin-deleted strain. The viral enhancing effect of α-toxin is mediated by ADAM10 and is associated with its pore-forming property. Moreover, we demonstrate that α-toxin promotes viral entry in NHKs.

Conclusion: The current study introduces the novel concept that staphylococcal α-toxin promotes viral skin infection and provides a mechanism by which S aureus infection might predispose the host toward disseminated viral infections.

FIG 1

Staphylococcal α-toxin, but not superantigens, enhances viral load in NHKs. NHKs were pretreated with indicated S aureus superantigens and α-toxin, followed by VV (MOI, 0.1) and HSV-1 (MOI, 0.1) inoculations. A and B, The viral mRNA expression levels of HSV-1 (Fig 1, A) and VV (Fig 1, B) were evaluated by using real-time PCR. C, Viral plaque formation was determined by using the viral plaque assay. The left panel shows crystal violet staining of viral plaques; the right panel shows quantitative results of viral plaques. All data are presented as mean ± SEM values. Data from 1 representative experiment of 3 independent experiments performed are shown. **P < .01 and ***P < .001.

FIG 2

Sublytic α-toxin is the major staphylococcal virulent factor enhancing viral load in keratinocytes. A, Western blot showing α-toxin and TSST-1 protein expression in Hla-WT and Hla-KO S aureus culture supernatants. B, Viral gene expression in NHKs pretreated with supernatants collected from sham, Hla-WT, and Hla-KO S aureus cultures. Data are presented as mean ± SEM values. Data from 1 representative experiment of 3 independent experiments performed are shown. **P < .01 and ***P < .001.

FIG 3

VV load is greater in murine skin inoculated with Hla-WT S aureus strain than with Hla-KO S aureus strain. A, Representative picture of murine skin lesions at day 7 of VV inoculation. B, Quantitative results of the sizes of murine skin lesions at day 7 of VV inoculation. Data are presented as mean ± SEM values. C, Representative images of VV immunofluorescent staining of murine skin lesion biopsy specimens taken at day 7 after VV inoculation (magnification ×40). D, VV immunofluorescent intensity. Data are expressed as mean ± SEM values. MFI, Mean fluorescence intensity.

FIG 4

Silencing ADAM10 expression blocks sublytic α-toxin’s effect on viral enhancement. A, Western blot showing ADAM10 protein level and α-toxin in indicated conditions. β-actin was used as a loading control. B, HSV-1 mRNA expression in indicated conditions. C, VV mRNA expression in indicated conditions. Data are presented as mean ± SEM values. Data from 1 representative experiment of 3 independent experiments performed are shown.

FIG 5

H35A mutated α-toxin has no viral enhancement effect. NHKs were treated with indicated concentrations of wild-type (WT) α-toxin and H35A mutated α-toxin for 20 hours followed by 24 hours of viral incubation. Cells were then harvested for Western blot assay and real-time PCR. A, Western blot showing α-toxin in indicated conditions. β-actin was used as a loading control. B and C, HSV-1 (Fig 5, B) and VV (Fig 5, C) mRNA expression in indicated conditions. Data are presented as mean ± SEM values. Data from 1 representative experiment of 3 independent experiments performed are shown.

FIG 6

α-Toxin promotes viral entry into keratinocytes. A, VV luciferase activity was measured in α-toxin–pretreated NHKs after incubation with VV luciferase at an MOI of 10 for 1 hour. B, NHKs were pretreated with or without α-toxin (20 ng/mL) for 20 hours, and cells were then subjected to 100 μmol/L acyclovir treatment for 2 hours. Finally, HSV-1 (MOI, 0.05) was added into the cells for additional 5- and 10-hour incubations. Intracellular HSV-1 DNA copies were evaluated by using real-time PCR. Data are presented as mean ± SEM values. Data from 1 representative experiment of 3 independent experiments performed are shown.