476 S. aureus virulence factors enhance vaccinia virus infection in human keratinocytes

Orthopoxvirus infections are on the rise worldwide. In addition, a smallpox bioterrorism event would necessitate ring vaccination with vaccinia virus (VV). This would pose a significant threat to susceptible groups, such as patients with atopic dermatitis (AD). Subjects with AD, exposed to VV, can develop a serious complication called eczema vaccinatum (EV). As part of the Atopic Dermatitis Research Network, we have shown that a similar viral complication, called eczema herpeticum, is more common in AD subjects who are colonized with Staphylococcus aureus (S. aureus). We hypothesized that S. aureus virulence factors (toxins, superantigens [SAgs], proteases and lipases) would promote infectivity of epidermal cells to VV. Differentiated primary human foreskin keratinocytes (PHFK) were treated with supernatants (1 μg [total protein]/mL) from three S. aureus strains (USA300, HG003, and RN4220), that vary in their secreted virulence factors. Two days post-treatment, PHFK were infected with 40 plaque forming units of fluorescent VV (Western Reserve strain). Viral infectivity and spread were monitored by fluorescent microscopy and plaque assays. Treatment with S. aureus supernatants significantly enhanced VV infection, as demonstrated by increased number of plaques. Supernatants from USA300, which produced the greatest amounts of SAgs and cytotoxins, resulted in the greatest number of plaques (p=0.0065, n=6). To clarify which virulence factors are most critical, future experiments will utilize mutant strains lacking specific virulence factors to determine their relative contribution to the increased VV susceptibility we observed. Our findings suggest that S. aureus virulence factors, commonly observed in S. aureus AD isolates, may greatly enhance the permissiveness of skin epithelial cells to VV infectivity and spread. This is likely a key contributor to the risk of EV in AD subjects.