10.1016/j.tim.2011.01.007. skin, and gastrointestinal tract that also causes invasive disease, including skin and soft tissue infections (SSTI), bacteremia, sepsis, endocarditis, pneumonia, and osteomyelitis (1, 2). causes disease in healthy individuals, which most frequently manifests as purulent SSTIs (3). Invasive disease is usually associated with bloodstream contamination (BSI) that may develop into fulminant sepsis or endocarditis (4, 5). The treatment of infections has been complicated by the emergence and spread of antibiotic-resistant strains, designated methicillin-resistant (MRSA), that have developed resistance characteristics against many therapeutics (6, 7). Patients with indwelling catheters, endotracheal intubation, medical implantation of foreign bodies (prosthetic joints, implants, and heart valves), trauma, surgical procedures, hemodialysis, peritoneal dialysis, immunosuppressive Pdgfra or malignancy therapy, and diabetes, as well as individuals with increased age and low birth weight, are all at elevated risk of contamination (8, A-582941 9). These individual populations can be broadly classified into three groups: individuals with a breach of barrier function, enabling staphylococci to cause invasive infections (trauma, medical procedures); individuals with implants that serve as a protective market for staphylococci; and individuals with diminished innate immune defenses, most importantly patients with reduced opsonophagocytic killing of bacteria, which is usually mediated by neutrophil granulocytes (10, 11). The latter can occur in individuals with main immunodeficiency disorders, caused in patients with chronic granulomatous disease, for example, by mutations that impact the NADPH oxidase of neutrophil granulocytes to produce oxygen radicals for killing of staphylococci (12, 13). Much more frequently, however, diminished opsonophagocytic killing of staphylococci occurs in individuals with hematologic malignancies and/or anticancer chemotherapy (14), a patient populace where BSIs cause significant morbidity and mortality (15, 16). Cyclophosphamide is an alkylating agent that is utilized for the therapy of malignancies and autoimmune diseases and for bone marrow transplantation (17). Cyclophosphamide therapy induces neutropenia and leukopenia and is associated with increased risk for BSI (18, 19). Although cyclophosphamide-induced leukopenia in mice has been used extensively A-582941 as a preclinical model to assess therapeutic efficacy of antibiotics against bacterial infections, this model has heretofore not been used to analyze the virulence factors for BSI in patients with diminished capacity for opsonophagocytic killing (20, 21). Although there is a need for vaccines and immunotherapies that safeguard high-risk patients against contamination, several clinical trials for staphylococcal vaccines failed to meet their study endpoints (22). These trials have focused on single staphylococcal antigens that, during preclinical screening, were demonstrated to act as virulence factors for the pathogenesis of specific disease and, when used as subunit vaccines, elicited immune responses that protect immunocompetent animals (23,C27). Clinical trials for vaccines included capsular polysaccharides (CP types 5 and 8), -hemolysin (Hla), A-582941 coagulase (Coa), and the iron-regulated surface determinant B (IsdB) (28,C30). Here, we use the leukopenic mouse model to analyze variants lacking specific protective antigens to characterize targets for any staphylococcal vaccine A-582941 in malignancy patients. Using genetic vaccinology to derive vaccine targets, we report that a cocktail of four surface protein antigens (ClfA, FnBPB, SdrD, and SpAKKAA) provides protection against BSI in leukopenic mice. MATERIALS AND METHODS Animal care and regulatory compliance. All experiments involving the care and use of animals followed protocols that were examined, approved, and performed under the regulatory supervision of The University or college of Chicago’s Institutional Biosafety Committee and A-582941 the Institutional Animal Care and Use Committee. Animal care was managed by The University or college of Chicago Animal Resource Center, accredited by the American Association for Accreditation of Laboratory Animal Care and the Department of Health and Human Services (A3523-01). Animals were maintained in accordance with the applicable portions of the Animal Welfare Act and the (31). Veterinary care was under the direction of full-time resident veterinarians boarded by the American College of Laboratory Animal Medicine. CD-1 mice (strain code 022) were purchased from Charles River. Statistical analysis. Mouse survival was analyzed for significance using the two-tailed log rank test. The bacterial weight following contamination, represented as the log10 CFU/g of organ tissue, was analyzed with the Mann-Whitney test for statistical significance. Quantification of abscess formation was analyzed for statistical significance using the unpaired two-tailed Student test. Statistical analyses were performed using GraphPad Prism 4 software. All animal experiments were examined for reproducibility using either two or three impartial determinations. Bacterial strains, media, and growth conditions. Mutants harboring the mariner transposon in defined genes were transduced with bacteriophage.