Dual function of a tip fimbrillin of Actinomyces in fimbrial assembly and receptor binding

Abstract

Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly.

Fig. 1.

Requirements for polymerization of the type 1 fimbriae. (A) Schematic organization of the type 1 fimbrial gene cluster of A. oris MG-1 (WT). (B and C) M and W fractions were collected from MG-1 and its isogenic derivatives grown to early log phase. Equivalent protein samples were separated on a 4 to 12% Tris-glycine gradient gel and detected by immunoblotting with α-FimP (B) or α-FimQ (C). The positions of monomers (FimP/Q_M) and high-molecular-mass products (FimP/Q_HMW) of fimbrial polymerization and molecular mass markers are indicated.

Fig. 2.

The fimbria-specific sortase SrtC1 is required for the assembly of type 1 fimbriae. Actinomyces cells of various strains were immobilized on nickel grids and stained with a specific antiserum against FimP (α-FimP) (A to C) or FimQ (α-FimP) (D to F) and goat anti-rabbit IgG conjugated to 12-nm gold particles. Samples were viewed by transmission electron microscopy after being stained with 1% uranyl acetate. Scale bars, 0.2 μm.

Fig. 3.

Components of the type 1 fimbriae required for surface assembly. Bacterial cells were immobilized on nickel grids and stained with α-FimP (A, C, E, and G) or α-FimQ (B, D, F, and H) and goat anti-rabbit IgG conjugated to 12-nm gold particles. Samples were viewed by transmission electron microscopy after being stained with 1% uranyl acetate. Scale bars, 0.2 μm.

Fig. 4.

The tip fimbrillin FimQ is required for surface assembly of type 1 fimbriae. (A and B) Cells of ΔfimA or ΔfimQA mutants were stained 1% uranyl acetate before being analyzed by transmission electron microscopy. Cells of the ΔfimA or ΔfimQA mutant were subjected to immunogold labeling with α-FimP (C and D) or α-FimQ (E and F) and goat anti-rabbit IgG conjugated to 12-nm gold particles. Samples were viewed by transmission electron microscopy after being stained with 1% uranyl acetate. Scale bars, 0.2 μm.

Fig. 5.

Receptor recognition of A. oris type 1 fimbriae depends on FimQ. Strains were examined for aggregation of PRP-coated latex beads (magnification, ×1.25) and for reactivity with α-FimP, α-FimQ, and α-FimA by dot immunoblotting. The last antibody, which reacted similarly with all strains, was included as a spotting control.

Fig. 6.

Binding of recombinant protein FimQ to a PRP-coated surface. Recombinant His-tagged proteins at increasing concentrations were added to ELISA plates coated with PRPs. Binding was quantified by ELISA at an absorbance of 405 nm. The points represent the means, with error bars showing triplicate measurements.