The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

Abstract

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure-function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular "Velcro-like" mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Keywords: Ag43; structural biology; urinary tract infection; virulence factor.

Fig. 1.

Domain organization and structure of the Ag43a passenger domain (α^43a). (A) Schematic showing the arrangement of the protein domains: signal peptide (SP), passenger domain (α-domain), autochaperone domain (AC), and translocator domain (β-domain). (B) Ribbon representation of the secondary-structure elements of the α^43a β-helix with β-strands and loops colored in blue and green, respectively. Loop 1 and loop 2 protruding from the β-spine are colored in red, and the four β-hairpins are shown in black. (C) Top view of the β-helix showing the internal side-chain stacking. Residues are shown in stick representation. (D) Stereoview of the 2F_o − F_c electron density map contoured at 1σ of the cross-section of the α^43a β-helix (residues ⁹⁰L–¹⁰⁸H).

Fig. 2.

α^43a–α^43a interactions. (A) Self-association of α^43a molecules in trans configuration showing interfaces 1 and 1′. A close-up view of the packing interface is shown; each interface contains nine hydrogen bonds [N29–T256 (two hydrogen bonds), N60–T256, N60–T237, D79–T237, N96–R200, T97–R200, T98–R200, G115–R200] and a salt bridge between the R59 and E216 side chains. (B) Close-up view of the packing interface of modeled α^43b molecules indicating the predicted hydrogen-bond interactions (D29–T255, R59–N215, N60–T236, N60–T255, D79–T236, S78–N215, T97–R199, G114–R199). The α^43b interface residues that are not conserved in α^43a are colored in magenta.

Fig. 3.

Heterologous association of Ag43a–Ag43b adhesins. (A) Modeled heterologous head-to-tail association of α^43a and α^43b, showing close-up views of interfaces 1 and 1′ and indicating the predicted interactions. Interface 1: eight hydrogen bonds [N29–T255, R59–N215, N60–T236, N60–T255, D79–T236, N96–R199, G115–R199 (two hydrogen bonds)]; interface 1′: six hydrogen bonds (D29–T256, D60–T237, N60–T256, D79–T237, G96–R200, G114–R200); and a salt bridge between the R59 and E216 side chains. (B–E) Ag43a–Ag43b interaction drives E. coli cell aggregation. Fluorescence micrographs of E. coli expressing (B) Ag43a (green) or Ag43b (red), (C) pBAD/Myc-His A (green) or pBAD/Myc-His A (red), (D) Ag43a (green) or pBAD/Myc-His A (red), and (E) pBAD/Myc-His A (green) or Ag43b (red). Panels are representative images from two independent experiments.

Fig. 4.

Functional characterization of Ag43a mutants. E. coli aggregation profiles of strains expressing (A) ag43a 7 loop mutant (N29G, R59G, N60G, S78G, D79G, N96G, T98G) or (B) ag43a hairpin deletion mutants ΔH1, ΔH2, and ΔH1,H2. Mutants were compared with isogenic control strains containing ag43a WT or empty vector. Ag43a protein production at the bacterial cell surface was examined in all Ag43a-overexpressing cultures using an anti-Ag43a polyclonal serum by (A and B, Insets) heat release of α^43a from the cell surface and Western blot analysis or (C) immunofluorescence labeling and confocal laser microscopy. α^43a was detected in strains containing ag43a-encoding plasmids (WT and mutants) but not the empty vector control (−).

Fig. 5.

Model of a molecular “Velcro-like” mechanism of bacterial cell aggregation mediated by Ag43a self-assembly in a head-to-tail conformation. (A) Close-up view of the self-associating α^43a molecules rotated 90°. The α^43a functional domains are predicted to interact with the β-translocating domain (depicted as gray cylinders) using the foot of the L, which will position the long stem of the protein almost perpendicular to the cell surface, making the interacting surfaces (shown in yellow and orange) easily accessible to neighboring molecules. Mutation of the interacting residues in interface 1 (red arrows) completely abolishes Ag43-mediated cell aggregation. (B) Loss of the overall L shape in α^43a likely changes the positioning of the long stem of the protein relative to the cell surface, from an easily accessible perpendicular arrangement to a nearly parallel layout, compromising the ability of α^43a to interact with Ag43 molecules on adjacent bacterial cells.