Complement regulators were allowed to adsorb to the Borrelia surface and bound proteins were subsequently eluted with acidified 0.1 M glycine.

The wash and the eluate fraction were analyzed for the presence of CFH and FHL-1 by OICR-9429 in vitro Western blotting. As shown in Fig 3, FHL-1, but not CFH could be AZD2281 detected in the eluate fraction indicating that B. garinii ST4 PBi specifically interact with FHL-1. Figure 3 Detection of bound complement regulators by B. garinii ST4 PBi. After incubation of spirochetes with NHS-EDTA, bound proteins were eluted. The wash (w) and the eluate (e) fraction were separated by SDS-PAGE. The last wash and eluate fraction were subjected to SDS-PAGE and

separated proteins were blotted on nitrocellulose. CFH and FHL-1 were visualised using a polyclonal goat anti-factor CFH antiserum (Calbiochem). It is shown that B. garinii ST4 PBi is able to bind FHL-1 on its selleck chemical membrane. Accessibility and surface exposure of CFH/FHL-1 binding proteins of B. garinii ST4 PBi In order to identify FHL-1 binding proteins produced by B. garinii ST4 PBi and to determine whether these proteins are exposed to the extracellular space, spirochetes were treated with increasing concentrations of proteinase K or trypsin and proteolysis was detected by ligand affinity blotting. Cell lysates obtained after protease treatment were separated by SDS-PAGE, transferred to nitrocellulose and the respective proteins were detected. As shown

in Fig 4, four distinct binding Methane monooxygenase proteins could be detected in untreated serum-resistant B. garinii ST4 PBi. Treatment with proteinase K at the lowest concentration resulted in the complete elimination of CFH/FHL-1 binding. Upon treatment with trypsin, degradation was only achieved at a concentration of 100 μg/μl. As expected, the intracellular protein flagellin was resistant to trypsin and proteinase K treatment, even at the highest concentration. These data demonstrate that B. garinii ST4 PBi produced up to four surface-exposed CFH/FHL-1 binding proteins, in the range of 19-26 kDa. This is in concordance to the findings of McDowell et al, where B. garinii ST4 PBi expressed a 20.5 and 26 kDa protein that were found to interact with CFH [33]. The CspA orthologs tested in this study are in the range of 25-27 kDa, the smaller proteins detected appear to belong to the Erp protein family. Figure 4 Accessibility of CFH/FHL-1 binding proteins of B. garinii ST4 PBi by different proteases. Spirochetes of B. garinii ST4 PBi were incubated with either proteinase K or trypsin at concentrations of 12.5 to 100 μg/ml or in buffer without any protease (0). After 1 h of incubation, cells were lysed by sonication as described in Materials and Methods.