Fig 1: Identification of CsuA/B as the target of VHH OMV81 and recombinant CsuA/B production. a A. baumannii OMVs (5 µg) were separated by gel electrophoresis, transferred to a PVDF membrane and probed with 10 µg/mL (620 nM) VHH OMV81. VHH binding was detected with HRP-conjugated anti-c-Myc IgG. Bands on a corresponding SDS-PAGE gel were excised, digested, and subjected to bottom-up nLC-MS/MS for protein identification (see Table 2). b, c Production of recombinant His6-tagged CsuA/B in E. coli. b SEC chromatogram and reducing (red) SDS-PAGE analysis of IMAC-purified and refolded CsuA/B. c Confirmation by intact mass analysis. Molecular weight profile of the deconvoluted protein mass spectrum. The expected average mass for amino acids 24–186 of CsuA/B, including an internal disulfide bond, a GA linker, and His6 tag, is 17,009.71 Da. d Detection of recombinant CsuA/B with VHH OMV81. Cell extract (~1 µg) from E. coli BL21(DE3) cells expressing CsuA/B (lanes 1, 3, and 5) and 100 ng of purified recombinant CsuA/B (lanes 2, 4, and 6) were separated by SDS-PAGE and transferred to a PVDF membrane. Western blotting with an HRP-conjugated anti-His6 IgG bound directly to recombinant CsuA/B that contains a C-terminal GA-His6 tag (lanes 1–2). VHH OMV81 bound the identical band in a separate blot following the addition of an HRP-conjugated anti-c-Myc IgG (lanes 5–6). No binding was observed with the HRP-conjugated anti-c-Myc IgG alone (lanes 3–4)