His-ΔNarG and His-ΔFnBPA polypeptides were used as internal negative and positive controls, respectively. Since the His-ΔSCOR

and His-ΔIspD polypeptides remained insoluble in the E. coli cytoplasm, these proteins could not be purified in non-denaturing conditions and could unfortunately not be included in the verification. In the ELISA assay, the His-ΔCoa and His-ΔEbh polypeptides interacted with the same immobilized target molecules (upper panel of Figure QNZ solubility dmso 3B) as those of the corresponding Ftp library clones (upper panel of Figure 3A). The His-ΔPurK polypeptide bound to Fn but interacted poorly with Fg, whereas His-ΔUsp showed only a low level interaction with Fn. Similarly as the negative Compound C control polypeptide His-ΔNarG, the His-ΔFnBPA and His-ΔPBP polypeptides showed no binding to Fn or Fg in the ELISA. In the SPR analysis, the His-ΔPurK, His-ΔCoa, and His-ΔUsp polypeptides bound to immobilized Fg whereas the His-ΔFnBPA, His-ΔPurK, and see more His-ΔEbh polypeptides showed affinity to Fn similarly as did the cell free growth media of corresponding Ftp library clones tested by ELISA (Figure 3A). In contrast to the ELISA results, the His-ΔEbh polypeptide reacted also with Fg in the SPR analysis. The His-ΔPBP polypeptide and the negative control

peptide His-ΔNarG showed no binding properties in the SPR analysis. However, the SPR results mainly confirmed the results obtained with culture supernatants of Ftp clones. The affinity constants obtained in the SPR analysis are shown in Table 2. Table 2 SPR analysis of His6-polypeptides Polypeptide KD to Fn (M) * KD to Fg (M) * His-ΔNarG 0,77 Coproporphyrinogen III oxidase 0,72 His-ΔFnBPA 5,24 × 10 -6 0,31 His-ΔEbh 0,02 1,25 × 10 -6 His-ΔCoa < 0† 1,80 × 10 -7 His-ΔPurK 4,43 × 10 -7 5,39 × 10 -6 His-ΔUsp 0,35 6,45 × 10 -6 His-ΔPBP 0,36 0,13 * the steady state affinity constants (KD) of the seven analytes tested are shown in molar concentrations; values shown in bold indicate high affinity for the indicated ligand (Fn or Fg). † affinity was not measurable since all values were negative Discussion S. aureus NCTC 8325, the parental strain of the prophage-cured

S. aureus NCTC 8325-4 used for construction of the extracelluar secretion library, carries 22 of the genes encoding the 24 surface proteins implicated in adhesion and all the 13 genes for the secretable proteins implicated in immune response evasion as recently described by McCarthy and Lindsay [41]. According to the literature, only eight of these proteins have been reported to bind Fn and/or Fg and five interact with the ECM. Cna, the only collagen-binding protein in the list of adhesins, is not present in S. aureus NCTC 8325-4 [41]. Taking into consideration the above data and the fact that we deliberately screened for binding to only a few model targets of S. aureus, the yield from our Ftp library was very satisfying.