Figure 3. Characterization of translocation-competent derivative proteins and the expressing strains using a protective monoclonal antibody, Mab166.

(A) Secretion of PcrV and PcrV::EZ derivatives that contain a linker within a predicted epitope region. V-proteins released into the bacterial culture supernatant under the induced growth condition (+NTA) were detected with Mab166 as a probe. Concentrated supernatant was titrated (2-fold serial dilution) for immunoblot analysis. (B) Retention of the protective epitope analyzed by using a hemolysis-based protection assay. Sheep erythrocytes were infected with ΔpcrV mutants in the presence of 10 µg Mab166. The conformational-epitope regions of Mab166 are underlined. (C) Localization of PcrV on the bacterial cell surface detected by immunofluorescence microscopy. PcrV was probed either with rabbit polyclonal IgG alone (shown in green, top panels) or with polyclonal IgG and Mab166 (shown in red and green, respectively; lower panels). Bacterial cells were stained with DAPI (shown in blue).