Targeting of Alpha-Hemolysin by Active or Passive Immunization Decreases Severity of USA300 Skin Infection in a Mouse Model

MAJOR ARTICLE Targeting of Alpha-Hemolysin by Active or Passive Immunization Decreases Severity of USA300 Skin Infection in a Mouse Model Adam D. Kennedy,1 Juliane Bubeck Wardenburg,2,3 Donald J. Gardner,4 Daniel Long,4 Adeline R. Whitney,1 Kevin R. Braughton,1 Olaf Schneewind,2 and Frank R. DeLeo1 1 Laboratory of Human Bacterial Pathogenesis and 4Veterinary Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana; and Departments of 2Microbiology and 3Pediatrics, University of Chicago, Chicago, Illinois Staphylococcus aureus is a leading cause of infection in hospitals, as well as in the community. Communityassociated methicillin-resistant S. aureus (CA-MRSA) strains typically cause infection in otherwise healthy individuals and, therefore, may be considered to be highly virulent. This notion is supported by data from animal infection models, in which prominent CAMRSA strains, such as USA300, are more virulent than are traditional hospital-associated strains [1, 2]. The enhanced virulence phenotype of USA300 is likely to Received 30 December 2009; accepted 9 April 2010; electronically published 19 August 2010. Potential conflicts of interest: none reported. Financial support: Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), and Region V “Great Lakes” Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (NIH Award 1-U54-AI-057153 to J.B.W. and O.S.). Reprints or correspondence: Dr Frank R. DeLeo, Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, NIAID, NIH, 903 South 4th St, Hamilton, MT 59840 (). The Journal of Infectious Diseases 2010; 202(7):1050–1058  2010 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2010/20207-0009$15.00 DOI: 10.1086/656043 1050 • JID 2010:202 (1 October) • Kennedy et al be attributable in part to the relatively high expression of virulence factors, such as phenol soluble modulins and alpha-hemolysin (a-hemolysin, a-toxin, and Hla) [1]. Hla is a secreted pore-forming toxin that has cytolytic activity toward a variety of host cell types, including human keratinocytes, epithelial cells, and lymphocytes [3–8]. Expression of Hla is regulated, at least in part, by the 2-component agr and saeR/S signaltransduction systems [9]. Hla is lethal to animals, especially rodents and rabbits [10], and S. aureus strains that are deficien in hla have significantl reduced virulence in animal infection models [6, 11–18]. Despite this previous work, the role of Hla in the pathogenesis of CA-MRSA infections was unknown until recently. With use of USA300 and USA400 wild-type and isogenic hla-negative mutant (Dhla) strains, Bubeck Wardenburg and colleagues [6, 13] demonstrated that Hla is essential for pathogenesis in a mouse model of CA-MRSA pneumonia. Subsequent studies showed that vaccination against Hla protects mice from lethal USA300 or USA400 pneumonia [6, 13]. More recent studies by Bartlett et al [19] have Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. We tested the hypothesis that alphahemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Dhla) strains caused skin lesions in a mouse infection model that were significantl smaller than those caused by wild-type USA300 and Newman strains. Moreover, infection due to wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Dhla strains. Passive immunization with Hla-specifi antisera or active immunization with a nontoxigenic form of Hla significantl reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude that Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections. demonstrated that Hla elicits production of CXC chemokines by host cells during experimental S. aureus pneumonia, thereby promoting severe lung inflam ation. Inasmuch as CA-MRSA infections are primarily those affecting the skin and soft tissues, we determined the role played by Hla in a mouse model of USA300 skin infection and, in turn, tested whether a vaccine approach directed at Hla moderated disease severity. METHODS Figure 1. hla contributes to the pathogenesis of USA300 skin infections. Results are presented as mean values  standard error of the mean for all groups. The number in parenthesis is the number of mice in each group. A and B, Mouse abscess size monitored once per day after subcutaneous infection with 1 ⫻ 107 of the indicated bacteria. *P ! .05 versus wild-type USA300 (LAC) or Newman strains using a 2way analysis of variance (ANOVA) and Bonferroni’s post-test. C, Size of abscesses at maximum. *P ! .05 using an ANOVA and Dunnett’s posttest. PBS, phosphate buffered saline. nosorbent assay (ELISA), which was shown previously to protect against the effects of staphylococcal Hla [6]. Active immunization. Female Balb/c mice (4 weeks of age) were administered 20 mg of endotoxin free glutathione-S-transferase (GST)–HlaH35L in complete Freund’s adjuvant (CFA; Difco Laboratories) via intramuscular injection, followed by a boost with 20 mg of endotoxin free GST-HlaH35L in incomplete Freund’s adjuvant (IFA; Difco Laboratories) 10 days later. Animals were challenged with S. aureus 21 days after the initial vaccination and monitored once per day for 14 days. Serum samples were collected on day 20 (24 h prior to infection) to assess Hla-specifi antibody titer by ELISA. Anti-Hla ELISA. Blood was obtained from 5 control mice Hla and USA300 Skin Infections • JID 2010:202 (1 October) • 1051 S. aureus strains and culture conditions. S. aureus strains used in this study were characterized previously [13]. To prepare inocula, S. aureus was cultured to mid-exponential phase of growth, washed twice in Dulbecco’s phosphate-buffered saline (DPBS), and diluted in DPBS to the appropriate concentration. Inocula were stored on ice until used. Mouse skin infection model. We used a previously described mouse skin infection model [2, 20, 21]. Animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Shaved female Balb/c mice were anesthetized with isofluran and inoculated by subcutaneous injection in the right fl nk with 1 ⫻ 107 S. aureus in 50 mL of DPBS. Mice were weighed before inoculation, and mass and abscess formation (...truncated)