Fig. 5: Testing recombinant VHHs for diagnostics and therapeutics.
From: Bacterial cell surface characterization by phage display coupled to high-throughput sequencing
Flow cytometry of P. aeruginosa stained with selected rVHHs, anti-Human IgG1-PE secondary antibody (0.5 µg per well), and the nuclear stain SYTO9. Events were gated by FSC/SSC (Supplementary Fig. 30) and SYTO9+ events were interpreted as bacterial cells. Each rVHH was used to stain antigen+ cells (orange) and antigen– cells (blue). The rVHH+ gate was drawn for fluorescence above the 99th percentile of the antigen– cells; the percent of rVHH+ antigen+ cells is shown in the corner of each plot. The rightmost panel(s) shows the same scheme for cells stained with free IgG1-Fc. a rVHHs targeting the flagellar hook-basal body apparatus. PAK ΔflhA does not produce any part of the flagellar apparatus. PAK ΔfliC ΔfleN produces multiple flagellar hook-basal bodies but no flagellar filament. rVHH per well: D2, 0.073 µg; E1, 0.226 µg; E2, 0.271 µg; F1, 0.201 µg. Additional rVHHs shown in Supplementary Fig. 31. b–d rVHHs targeting efflux-associated outer membrane porins OprM (H), OprJ (I), or multiple efflux porins (J). PAO1 “Δefflux” lacks all Resistance Nodular Division (RND)-type multidrug efflux systems. The antigen-positive strains (“+mexAB +oprM”, etc.) are Δefflux but express the indicated efflux pump in trans from plasmid mpMQ72. All cells were induced overnight with 1% arabinose. b. rVHHs targeting OprM. rVHH per well: A6, 9.7 µg; H5, 9.2 µg; C6, 3.4 µg; IgG1-Fc, 8.8 µg. Additional rVHHs shown in Supplementary Fig. 33. c rVHHs targeting OprJ. rVHH per well: D9, 24.0 µg; E9, 16.7 µg; F9, 5.2 µg; IgG1-Fc, 8.8 µg. Additional rVHHs shown in Supplementary Fig. 34. d rVHHs targeting multiple efflux-associated porins. rVHH per well: 10 µg; IgG1-Fc, 8.8 µg. Additional rVHHs shown in Supplementary Fig. 36. Raw data for panel (e) is available at https://doi.org/10.5281/zenodo.12826667.
