Fig 1.
PmSLP-1 exhibits characteristic features of a Slam-dependent surface lipoprotein.
(A) A model showing the proposed translocation of PmSLP-1 by Slam to the surface of E. coli. Surface exposed PmSLP-1 would be susceptible to cleavage by proteinase K. (B) Representative Western blot analysis for the proteinase K shaving assay. E. coli expressing either PmSLP-1 with Slam or PmSLP-1 alone were treated with proteinase K. Presence of PmSLP-1 remained after proteinase K treatment was detected using anti-FLAG antibodies (top panel). Proteins were stained with ponceau S to show the amount of sample loaded per lane and detect cell lysis (bottom panel). n = 3 independent experiments. (C) Schematic representation of the domain organization and the solved crystal structure of PmSLP-1 at 2.0 Å. The signal peptide (SP) and anchoring peptide (AP) were excluded in the construct used for structural study. The His tag was removed by thrombin cleavage during protein purification process. The inset shows the electron density at the interface between the handle domain and the barrel domain (contour level = 1.5 sigma).
Fig 2.
PmSLP-1 binds and forms stable complex with bovine complement factor I.
(A) A representative SDS-PAGE analysis of the serum pull-down assay showing the interaction between PmSLP-1 and bovine FI. Transferrin binding protein B (TbpB) and empty FLAG resin were used as negative controls. Samples were analyzed under non-reducing conditions (top panel). Western blot analysis of the pull-down samples under reducing conditions. The heavy chain of bovine FI was detected with anti-human FI antibodies (bottom panel). (B) A representative SDS-PAGE analysis of the pull-down experiment where PmSLP-1 was incubated with various animal sera; samples were analyzed under non-reducing conditions (top panel). Western blot analysis of the pull-down samples under reducing conditions (bottom panel). The heavy chain of bovine FI was detected with anti-human FI antibodies. (C-F) Representative biolayer interferometry sensorgrams and saturation curves showing binding of biotinylated bovine FI to PmSLP-115 and PmSLP-195. PmSLP-1 was present at various concentration as indicated in the sensorgrams (solid-coloured lines), and a 1:1 binding model was used to fit the association and dissociation data (red). The saturation curves were derived from the steady state values of the corresponding sensorgrams. (G) Binding kinetics and steady state binding constants for biotinylated bovine FI with wild type PmSLP-115 and PmSLP-195. The kon, koff, and binding constants represent three independent experiments and are presented as mean ± SD.
Fig 3.
Cryo-EM structure of PmSLP-1 in complex with bovine factor I.
(A) Cryo-EM map of PmSLP-1 in complex with bovine FI (top and side views), suggesting a dimer of the protein complex (left). The protomers are shown in different colours. The density of the complex monomer was obtained via symmetry expansion and local refinement; the estimated local resolution of the map is shown (middle). The atomic model of the protein complex is docked into the cryo-EM density map (right), with PmSLP-1 shown in yellow and bovine FI shown in blue. The color scheme is maintained throughout. (B) Atomic model of PmSLP-1:FI complex in cartoon representation. The top left inset shows an overlay between PmSLP-1 from the cryo-EM complex (in yellow) structure and the solved crystal structure of PmSLP-1 (in dark purple); the red circle highlights the short helix that is missing in the cryo-EM structure. The bottom left inset depicts the hydrophobic contacts between PmSLP-1 and FI. The right insets showcase the residues that form salt bridges between PmSLP-1 and FI. (C) Electrostatic potential maps at the interface between PmSLP-1 and bovine FI with the interface footprint outlined; red = negative, blue = positive.
Fig 4.
Validating the interface between PmSLP- 1 and bovine factor I.
(A) Inter-protein cross-links (dotted lines) are mapped on the atomic model of the protein complex. The cross-linked lysine residues and the Cα-Cα distance between each lysine pairs are indicated. (B) Ten solvent-exposed residues on PmSLP-1 were mutated and assessed for their effects on the binding of PmSLP-1 to bovine FI. The dissociation constant (KD) for each mutant was obtained via biolayer interferometry assay (n = 3) and compared with the KD of the wild-type PmSLP-115. NS = not statistically significant. The mutated residues are then mapped on the structure of the protein complex. The cryo-EM structure of PmSLP-1:FI is shown as white surface. The interfacing residues on PmSLP-1 and FI, identified through PISA analysis, are coloured yellow and light blue, respectively. The residues on PmSLP-1 selected for mutagenesis studies are labelled in the lower right panel. The residues annotated with an asterisk (*) are also identified as interfacing residues. While red indicates mutations that causes loss of binding to FI, light red indicates those with reduced binding to FI. Other residues selected for mutation but are not part of the complex interface are coloured purple and pink. Purple indicates mutations with reduced binding to FI, and pink highlights mutations with no significant change to the binding affinity with FI.
Fig 5.
PmSLP-1 activates factor I and promotes cleavage of C3b and C4b.
(A-B) FI-mediated degradation of C3b (A) and C4b (B) were evaluated in the presence of PmSLP-1 or the native fluid-phase cofactors (factor H and C4BP). The * in panel (B) indicates that human FI was used instead of bovine FI as there appeared to be a species incompatibility between human C4BP and bovine FI that resulted in much diminished FI-mediated cleavage of C4b to C4c and C4d in the neighboring lane to the left. (C) Protocol used for testing the effects of PMSF on FI. PMSF is added to either the pre-formed PmSLP-1:FI complex (1) or FI alone (2). After 1h incubation with PMSF, PmSLP-1 is added to group (2) to allow for complex formation. The substrates, C3b or C4b, are then added to the mixture, and the reaction is stopped by the addition of 2X sample buffer. Active PMSF is depicted as a red star, whereas inactive (i.e., water-hydrolysed) PMSF is shown as a blank star. (D-E), FI degradation of C3b (D) and C4b (E) in the presence of PmSLP-1. Lane 1 and 2 in both panels correspond to the treatment described in panel (C). N = 3 biologically independent experiments. (F) Detailed view of the catalytic triad of bovine FI. The cryo-EM map is depicted as clear surface with black outline.
Fig 6.
Expression of PmSLP-1 on E. coli cell surface is sufficient to mediate serum resistance.
(A-B) E. coli cells expressing PmSLP-1, Slam, Slam + PmSLP-1, or empty vectors were grown in LB media supplemented with either heat-inactivated (A) or normal bovine serum (B). Bacterial growth at 37oC was monitored for 3 hours and OD600 readings were recorded every 5 minutes. All values plotted represented the mean ± SD from three biologically independent experiments, each of which includes two technical replicates.
Fig 7.
Proposed mechanism of PmSLP-1-mediated immune evasion in P. multocida.
PmSLP-1 present on the surface of P. multocida hijacks complement factor I from the host. The binding of PmSLP-1 to FI induces a conformational change, locking FI in the active state. The PmSLP-1-bound FI cleaves C3b and C4b into their inactive forms in a similar manner observed when factor H or C4BP is present. In the presence of PmSLP-1, the FI-mediated processing of C3b halts after the production of iC3b, whereas the cleavage of C4b proceeds to the C4c and C4d formation. In both cases, the effective clearance of C3b and C4b molecules allows the bacteria to avoid killing from all three complement pathways.