Abstract
A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.
Highlights
A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus
An earlier study from our laboratories showed that most strains of E. faecalis adhered to a substrate of type I collagen when bacteria were grown at elevated temperatures (46 °C), a condition that retarded growth, but not when grown at 37 °C
We report the identification of a gene, ace, encoding a MSCRAMM, Ace, which may be the agent responsible for the E. faecalis adhesion to collagen
Summary
Identification of E. faecalis Ace in a Microbial Genome Data Base— The amino acid sequence comprising the minimal collagen-binding region (residues 151–318) of the S. aureus collagen adhesin, Cna [4, 5], was used to search for homologous sequences in the Microbial Genome Database at the National Center for Biotechnology Information. A sequence alignment and model of the Ace A domain was returned by the ExPASy server composed of 145 amino acid residues based on the structure of Cna 151–318. Cloning and Construction of Expression Plasmids—The nucleotide sequence encoding the Ace A or AϩB domains (Fig. 1a) was obtained by PCR using a thermocycler (Perkin-Elmer Cetus 480) and chromosomal DNA [20] from E. faecalis strain EF1 as the template. FITC-labeled bacteria were first incubated with antiAce A domain IgG for 1 h at 37 °C before addition of the mixture to the collagen-coated wells. The slowest flow rate (1 l/min) specified for the instrument was employed Even at this rate, the association and dissociation of the recombinant Ace A domain protein with the collagen-coated surface were too rapid to be quantitated. The negative reciprocal of the slope yields the dissociation constant, KD, and the x axis intercept is equivalent to the number of sites, n, in collagen at which the MSCRAMM protein binds
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