Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

Abstract

By combining X-ray crystallography and modeling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbors an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbors two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development

“…S1). This CafA-independent but sucrose-dependent biofilm development is attributed to FimA, which explains the independent ability of FimA to bind polysaccharides in vitro (15), as well as the resistance of coaggregation to the polyclonal antibody raised against FimA.…”

“…More recently, following the sequencing of the Actinomyces MG1 genome, genetic and biochemical work done in our laboratory revealed that the type 2 fimbriae are composed of a shaft protein FimA and a tip fimbrillin FimB that are genetically linked together in a fimbrial gene cluster encoding the fimbria-specific sortase (11). Gene deletion experiments led us to conclude that the receptormediated coaggregation may require the shaft protein FimA but not the tip protein FimB (13,15). Although our in vitro experiments suggested a direct interaction between recombinant FimA of Actinomyces with surface receptors of oral Streptococcus (15), it was rather unusual that the polyclonal antibodies we raised against FimA (or another antiserum that was generated by the Cisar laboratory against the type 2 fimbria (20), which cross-reacts with FimA) do not prevent this receptor-mediated coaggregation process (Fig.…”

“…This interbacterial interaction, which is critical for the development of the oral biofilm (19), is attributed to the shaft fimbrillin FimA because its absence abrogated bacterial coaggregation, biofilm formation, and hemaglutination (13). Indeed, structural studies revealed that FimA contains three IgG-like modules that are commonly found in Gram-positive pilins (15), and the recombinant FimA protein was able to bind to the surface of epithelial cells and S. oralis as well as to asialofetuin, a glycoprotein that contains the RPSs for Actinomyces interaction (15). Paradoxically, whereas antibodies raised against the purified type 2 fimbriae have been reported to block bacterial coaggregation (20), polyclonal antibodies directed against recombinant FimA or FimB did not display this inhibitory activity (Fig.…”

“…To examine how CafA is incorporated into the type 2 fimbriae, we analyzed fimbrial assembly by IEM accordingly (15). In these experiments, Actinomyces cells were incubated with specific antibodies (α-CafA and α-FimA), followed by labeling of antibody-bound cells with IgG-conjugated gold particles, and were viewed with a transmission electron microscope.…”

“…Similarly, the fimB-fimA-srtC2 gene cluster specifies the type 2 fimbria, which is assembled from the shaft fimbrillin FimA and the tip fimbrillin FimB (11,13). Although all fimbrillins harbor a CWSS, the shaft fimbrillins (FimA and FimP) also contain an N-terminal pilin motif harboring a conserved lysine residue, which participates directly in the cross-linking reaction that joins each pilin subunit to another (14,15). According to our current model, using type 2 fimbriae as an example (16), the pilus-specific sortase SrtC2 joins FimB and FimA by cross-linking the threonine residue of the FimB LPXTG motif to the lysine residue of the FimA pilin motif.…”