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Genetic Requirement for ADAM10 in Severe Staphylococcus aureus Skin Infection

2012; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês

10.1038/jid.2011.462

1523-1747

Naoko Inoshima, Yang Wang, Juliane Bubeck Wardenburg,

Antimicrobial Peptides and Activities

colony forming unit C-terminal fragment Staphylococcus aureus is a leading cause of human skin infections, contributing to disease in both healthy and immunocompromised individuals, also complicating burn and surgical wound sites and lesions of atopic dermatitis (Lowy, 1998Lowy F.D. Staphylococcus aureus infections.N Engl J Med. 1998; 339: 520-532Crossref PubMed Scopus (4597) Google Scholar; Ong and Leung, 2010Ong P.Y. Leung D.Y.M. The infectious aspects of atopic dermatitis.Immunol Allergy Clin North Am. 2010; 30: 309-321Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Host defense against staphylococcal skin infection is multifaceted, relying on local immunological control through TH17 and IL-1β–driven recruitment of neutrophils in addition to the protective actions of antimicrobial peptides and physical properties of the cutaneous barrier (Miller and Cho, 2011Miller L.S. Cho J.S. Immunity against Staphylococcus aureus cutaneous infections.Nat Rev Immunol. 2011; 11: 505-518Crossref PubMed Scopus (282) Google Scholar). Pathogen virulence in staphylococcal skin infection is likewise multifactorial (Weidenmaier et al., 2010Weidenmaier C. McLoughlin R.M. Lee J.C. The zwitterionic cell wall teichoic acid of Staphylococcus aureus provokes skin abscesses in mice by a novel CD4+ T-cell-dependent mechanism.PLoS ONE. 2010; 5: e13227Crossref PubMed Scopus (31) Google Scholar), relying in part on the action of α-hemolysin, a pore-forming cytotoxin secreted by almost all strains of S. aureus. Hla is required for dermonecrotic changes during infection, also contributing to abscess size (Kennedy et al., 2010Kennedy A.D. Bubeck Wardenburg J. Gardner D.J. et al.Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infection in a mouse model.J Infect Dis. 2010; 202: 1050-1058Crossref PubMed Scopus (267) Google Scholar). Immunization strategies targeting Hla protect against dermonecrosis (Kennedy et al., 2010Kennedy A.D. Bubeck Wardenburg J. Gardner D.J. et al.Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infection in a mouse model.J Infect Dis. 2010; 202: 1050-1058Crossref PubMed Scopus (267) Google Scholar); however, the molecular mechanism by which the toxin causes pathological disturbance of the epithelial barrier is ill understood. We recently identified the zinc-dependent metalloprotease ADAM10 as the cellular receptor for Hla (Wilke and Bubeck Wardenburg, 2010Wilke G.A. Bubeck Wardenburg J. Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury.Proc Natl Acad Sci USA. 2010; 107: 13473-13478Crossref PubMed Scopus (321) Google Scholar). ADAM10 regulates epithelial function through its ability to cleave E-cadherin, severing the protein-based adherens junction tether between adjacent cells (Maretzky et al., 2005Maretzky T. Reiss K. Ludwig A. et al.ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation.Proc Natl Acad Sci USA. 2005; 102: 9182-9187Crossref PubMed Scopus (531) Google Scholar). ADAM10 knockout mice exhibit embryonic lethality (Hartmann et al., 2002Hartmann D. de Strooper B. Serneels L. et al.The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts.Hum Mol Genet. 2002; 11: 2615-2624Crossref PubMed Google Scholar), whereas conditional skin knockout of ADAM10 during gestation or in the postnatal epidermis results in marked dysregulation of epithelial differentiation and barrier function (Weber et al., 2011Weber S. Niessen M.T. Prox J. et al.The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling.Development. 2011; 138: 495-505Crossref PubMed Scopus (124) Google Scholar). To examine the contribution of ADAM10 to skin infection, we generated conditional knockout mice in which exon 3 of ADAM10 is flanked by loxP sites (Tian et al., 2008Tian L. Wu X. Chi C. et al.ADAM10 is essential for proteolytic activation of Notch during thymocyte development.Int Immunol. 2008; 20: 1181-1187Crossref PubMed Scopus (77) Google Scholar), excised in the presence of a Cre recombinase expressed under control of the keratin 14 promoter (Vasioukhin et al., 1999Vasioukhin V. Degenstein L. Wise B. et al.The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin.Proc Natl Acad Sci USA. 1999; 96: 8551-8556Crossref PubMed Scopus (467) Google Scholar). Topical application of tamoxifen to a 1-cm2 skin area for 5 days leads to localized ADAM10 genomic excision (Supplementary Figure S1a and b online), abrogating epidermal ADAM10 expression (Figure 1a, ADAM10−/−). In contrast to littermate controls, ADAM10−/− mice did not develop dermonecrotic lesions following subcutaneous infection with 3 × 107 colony forming units (CFUs) of the epidemic S. aureus USA300/LAC (Figure 1b and c). Abscess size was reduced in ADAM10−/− mice (Figure 1d), however, lesional bacterial recovery was unaltered (Supplementary Figure S1c online). Download .pdf (.11 MB) Help with pdf files Supplementary Information The ability of ADAM10 to cleave E-cadherin suggested that Hla may use its receptor to cause epithelial barrier injury, not merely to facilitate binding. Toxin treatment of A431 keratinocytes led to upregulation of cell-associated metalloprotease activity measured in a fluorogenic substrate assay; the non-pore-forming HlaH35L mutant did not elicit this response (Figure 1e). Enhanced metalloprotease activity correlated with E-cadherin cleavage, detectable by immunoblot analysis of E-cadherin precipitates from A431 lysates as the toxin-induced loss of full-length protein and C-terminal fragment (Ctf) accumulation produced by ADAM10-dependent cleavage (Figure 1f). Ctf accumulation was also observed in cultured primary keratinocytes upon toxin treatment (Supplementary Figure S1d online). These molecular events occurred at subcytolytic concentrations of Hla (10μg per ml, data not shown), wherein a disturbance of cell–cell interactions in an A431 monolayer was manifest as a loss of resistance to the passage of an electrical current using electrical cell–substrate impedance sensing (Figure 1g). These findings suggest that the principal role of the toxin–receptor complex may be to disrupt the epithelial barrier. Within 24hours following S. aureus infection, loss of E-cadherin surface expression in epidermal tissue overlying the infection was evident in control mice as compared with ADAM10−/− mice (Figure 1h). Inflammatory cell infiltration at the infection site was similar in both control and ADAM10−/− mice (Figure 1i, upper, arrows), however, epidermal loss was apparent overlying the abscess site only in control mice (Figure 1i, lower). Together with CFU analysis, these results indicate that the Hla–ADAM10 complex does not markedly alter the host response to infection or bacterial accumulation. Epithelial injury induced by the toxin–receptor complex likely contributes to tissue edema and increased lesional size observed in control mice (Figure 1d). These data raised the possibility that an enzymatic inhibitor of ADAM10 may protect the epithelium. Mice receiving systemic treatment with the ADAM10 inhibitor GI254023X (Ludwig et al., 2005Ludwig A. Hundhausen C. Lambert M.H. et al.Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules.Comb Chem High Throughput Screen. 2005; 8: 161-171Crossref PubMed Scopus (269) Google Scholar; 200mgkg−1 per day, 5 days) did not demonstrate dermonecrotic lesions following infection compared with mice treated with the DMSO vehicle (Figure 2a). Topical application of GI254023X also prevented skin breakdown (Figure 2b, 100mgkg−1 per day, 5 days). Both routes of treatment led to a reduction in abscess size (data not shown). In vitro GI254023X treatment preserved E-cadherin expression and epithelial barrier function in toxin-treated A431 cells (Figure 2c and d). These studies provide insight into pathogenesis of staphylococcal skin infection demonstrating a genetic requirement for ADAM10 expression to mediate epithelial barrier injury, which to our knowledge is previously unreported. The Hla–ADAM10 complex thereby disables the most innate host defense of the skin. These findings extend recent observations that demonstrate a role for ADAM10 in Hla-mediated injury to the lung epithelium and vascular endothelium (Inoshima et al., 2011Inoshima I. Inoshima N. Wilke G.A. et al.A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice.Nat Med. 2011; 17: 1310-1314Crossref PubMed Scopus (297) Google Scholar; Powers et alPowers M, Kim HK, Wang Y et al. ADAM10 mediates vascular injury induced by Staphylococcus aureus a-hemolysin. J Infect Dis (in press)Google Scholar), defining a generalized principle of toxin action in which Hla uses its cellular receptor as a means to bind host cells and cause barrier injury. As ADAM10 cleaves multiple cellular substrates, it is of considerable interest to now understand the potential role of these proteins in staphylococcal disease pathogenesis. These observations have important implications for S. aureus skin disease. First, Hla may facilitate an initial breach of intact skin to permit pathogen invasion. Second, ADAM10 polymorphisms that alter toxin binding or enzymatic activity may exist, impacting susceptibility to infection. Finally, in disease states characterized by a compromise of epithelial barrier function such as atopic dermatitis, S. aureus may potentiate or incite barrier disruption, contributing to pathology and facilitating niche establishment. Indeed, S. aureus is the most common bacterial pathogen complicating atopic dermatitis lesions (Ong and Leung, 2010Ong P.Y. Leung D.Y.M. The infectious aspects of atopic dermatitis.Immunol Allergy Clin North Am. 2010; 30: 309-321Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Studies implicating ADAM10 in atopic dermatitis pathogenesis (Maretzky et al., 2008Maretzky T. Scholz F. Koten B. et al.ADAM10-mediated E-cadherin release is regulated by proinflammatory cytokines and modulates keratinocyte cohesion in eczematous dermatitis.J Invest Dermatol. 2008; 128: 1737-1746Crossref PubMed Scopus (69) Google Scholar), together with our observations, suggest that the Hla–ADAM10 interaction may contribute to epidermal pathology over a continuum of staphylococcal skin disease ranging from acute infection to chronic barrier insults. Therapies impacting ADAM10 activity may therefore demonstrate broad clinical utility. We thank C Labno for microscopy support, T Li for immunohistochemistry support, and the Integrated Microscopy and Immunohistochemistry Facilities at the University of Chicago. This work was supported by the Departments of Pediatrics and Microbiology at the University of Chicago and the University of Chicago Institute for Translational Medicine (National Institutes of Health award UL1RR024999 from the National Center for Research Resources). The authors acknowledge membership in and support from the Region V ‘Great Lakes’ RCE (National Institutes of Health award 2-U54-AI-057153). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid