Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B

Abstract

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. KEY POINTS: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells.

Keywords: Acinetobacter baumannii; Antimicrobial resistance; CsuA/B; Nanobody; Outer membrane vesicle; Pilus; Single-domain antibody; VHH.

Fig. 1

A. baumannii OMV llama immunization, serum response monitoring, and VHH isolation. a Pre-immune and immune (day 42) polyclonal serum IgGs from a llama immunized with OMVs from four A. baumannii strains were separated by protein G chromatography into heavy-chain-only IgG (G1; IgG2b) and conventional IgG (G2; IgG1) fractions. ELISA wells were coated with OMVs from each strain and binding of the G1 and G2 fraction polyclonal IgGs was detected with an HRP-conjugated anti-llama IgG. b Binding of VHHs isolated by panning a phage-displayed VHH library on OMVs. ELISA plates were coated with 3.8 μg/mL OMV from the indicated A. baumannii strain, probed with 10 μg/mL (~600–650 nM) of purified VHH monomer and detected with an HRP-conjugated anti-His₆ IgG

Fig. 2

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)

Fig. 3

Validation of VHH OMV81 binding to recombinant CsuA/B. a SPR sensorgrams from OMV81 and control antibodies flowed over immobilized CsuA/B, MUC1, and FC5 surfaces. Black lines show raw data, and red lines show 1:1 binding model fits. Reported affinities and kinetics are from 3 independent experiments. b ELISA responses from dilutions of VHH OMV81 and an anti-MUC1 VHH binding to immobilized recombinant CsuA/B. c, d Competitive ELISAs of recombinant CsuA/B and VHH OMV81. Microtiter wells were coated with c OMVs from 19606 and LAC-4 (3.8 μg/mL) or d recombinant CsuA/B (1 μg/mL) and treated with c 50 ng/mL (3.2 nM) or d 25 ng/mL (1.6 nM) VHH OMV81 pre-incubated for 30 min with increasing concentrations of recombinant CsuA/B. e ELISA responses from dilutions of VHH OMV81 and an anti-IGF1R VHH control (Alata et al. 2022) binding to coated A. baumannii OMVs. Error bars represent the mean ± SEM from n = 3 experimental replicates

Fig. 4

Binding of VHH OMV81 to the surface of A. baumannii cells. a A. baumannii cells were stained with 10 μg/mL (620 nM) of fluorescently labeled VHH OMV81 co-stained with Hoechst 33342. Bacteria were then fixed with formaldehyde and imaged on a glass coverslip. Hoechst fluorescence is shown in blue, and OMV81 fluorescence is shown in red/magenta. Scale bars (10 μm) are shown in white. b Whole cell ELISA of A. baumannii strains and VHH OMV81. A. baumannii and E. coli cells were grown overnight and heat killed. Bacterial samples with an OD₆₀₀ of 1.0 were adhered to microtiter wells and probed with 10 μg/mL (620 nM) OMV81. Error bars represent the mean ± SEM from n = 3 experimental replicates