Fig 4. Formation of VHH-functionalized curli fibers by EcN-derived strain PBP8.

(a) Plasmid map of synthetic curli operon and domain map of CsgA-VHH fusion constructs. (b) Idealized schematic of curli fiber export and self-assembly mechanism. (c) Representative scanning electron micrograph demonstrating the formation of curli fibers by CsgA-VHH-expressing PBP8 (CsgA-αgp900-2, scale bar = 1 μm). (d) Congo Red (CR) binding assays of induced cultures of PBP8 bearing plasmids encoding various CsgA-VHH constructs. CR binding is indicative of the formation of curli protein fibers. Data corresponds to A₄₉₀ measurements normalized to the CsgA positive control, and is presented as mean ± SEM. One-way ANOVA (P < 0.0001) was performed to test presence of difference between conditions. All CsgA constructs exhibited P-values < 0.0001 compared to the “No CsgA” control, as calculated by Welch’s t-test.