, 2004). The isdA, isdB, and isdH loci are all monocistronic, controlled by Fur, and their products have buy RAD001 been proposed to have a number of functions. In particular, all three proteins have been suggested to be involved in a cascade of heme binding and transfer from the host, to the IsdC uptake and degradation system (Mack et al., 2004). As well as a potential
function in iron acquisition, IsdA has also been shown to bind multiple host protein ligands as an adhesin, including those associated with the nasal epithelia (Clarke et al., 2004, 2009). In fact, IsdA is required for nasal colonization in an animal model, and vaccination with IsdA can prevent colonization (Clarke & Foster, 2006). IsdA also renders S. aureus more hydrophilic, making the cells more resistant to skin fatty acids and is necessary for survival on human skin (Clarke et al., see more 2007). IsdB has been shown to also bind human platelets (Visai et al., 2009; Miajlovic et al., 2010), and IsdH is
involved in evasion of the host innate immune response (Visai et al., 2009). The Isd proteins have differing roles in animal models (Clarke & Foster, 2006; Torres et al., 2006; Cheng et al., 2009; Kim et al., 2010) and have been proposed to be good potential candidates for a vaccine (Stranger-Jones et al., 2006) and monoclonal antibody approaches (Kim et al., 2010). IsdB in particular has been the subject of considerable effort (Brown et al., 2009; Ebert et al., 2010; Harro et al., 2010). As the Isd proteins have been suggested to act in concert with iron acquisition, in this study we report experiments to establish the combined role of IsdA, IsdB, and IsdH in cellular physiology and pathogenesis. All strains used in this study are listed in Table 1. All cells were grown in iron-limited
chemically defined, metal limitation medium with metal replaced (CLR; Horsburgh et al., 2001a, b). CLR medium consists of CL (which contains 400 μM magnesium sulfate) without glucose and replete with the following metals added at 0.2 μM final concentration: calcium chloride, copper sulfate, manganese chloride, nickel sulfate, molybdenum sulfate, and zinc sulfate. Prior to the addition of metal ions, divalent cations were removed by treatment with Chelex-100 (Sigma Aldrich). To further deplete available Tacrolimus (FK506) iron, 20 μM dipyridyl was added to all CLR cultures. CLR was supplemented with different heme- and iron-containing molecules; hemoglobin 5 μg mL−1 (Sigma Aldrich), hemin 50 μg mL−1 (Sigma Aldrich), and iron-loaded lactoferrin at the concentrations indicated. The lactoferrin (Sigma Aldrich) was iron-loaded using the protocol described previously (Clarke & Foster, 2008). When included, antibiotics were added at the following concentrations: erythromycin 5 μg mL−1, lincomycin 25 μg mL−1, kanamycin 50 μg mL−1, neomycin 50 μg mL−1, and tetracycline 5 μg mL−1. Cells were grown at 37 °C, with shaking at 250 r.p.m. in a volume of 50 mL in a 250-mL flask. E.