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Streptococcus mutans Strains Harboring Collagen-binding Adhesin
Y. Sato1,2,*,
K. Okamoto1,
A. Kagami1,
Y. Yamamoto1,
T. Igarashi3 and
H. Kizaki1,2
1 Department of Biochemistry and
2 Oral Health Science Center, Tokyo Dental College, 2-2, Masago 1-chome, Mihama-ku, Chiba City, 261-8502 Japan; and
3 Department of Oral Microbiology, Showa University School of Dentistry, Japan;
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Figure 1. Alignment of Cnm CBD with CBDs from previously identified collagen-binding adhesins. All 5 sequences were numbered from the initiation codon of the precursor proteins. The putative CBD identified from the Cnm sequence was aligned with CBDs from S. aureus, E. faecium, S. equi, and E. faecalis, with the use of the DNASIS-Mac program. Identical amino acid residues are indicated as letters on a gray background.
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Figure 2. Binding of recombinant putative CBD of Cnm protein to immobilized ECM proteins. (A) Strain ZAXF was grown in the presence of 2 x 10–3% arabinose as an inducer, and the crude cell-free extract was prepared as described in the text. Binding of recombinant Cnm protein to immobilized ECM proteins, collagen type I, fibronectin, and laminin was indicated as a function of protein concentration (1–10 µg in 20 µL of PBS with 0.1% BSA) of the extracts applied to the wells. BSA was used as a negative control. (B) The extract contained 10 µg protein from strain ZAXF cells grown in the absence of arabinose (uninduced ZAXF), and those from strains pBAD (as a negative control) and SBP6 (a strain expressing another histidine-tagged protein as a negative control) cells grown in the presence of 2 x 10–3% arabinose were also used as controls for binding of recombinant Cnm protein to collagen type I and laminin. (C) Biotin-labeled strains Z1 and 05A02 whole cells were examined for binding to the ECM proteins as described in the text.
We measured relative binding by monitoring absorbance at 490 nm following the peroxidase reaction for 3 min in the recombinant assays and for 4 min in whole-cell assays with o-phenylenediamine, and H2O2 was terminated with the addition of 2 M H2SO4. All OD490 nm values were corrected for the responses of peroxidase activities with the respective ECM proteins. Data points represent the means of OD490 nm values ± standard deviation from more than 3 independent experiments.
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Figure 3. Presence of the cnm gene in a population of S. mutans. HindIII-digested chromosomal DNA fragments from reference strains, including strain UA159, and several natural isolates in addition to strain Z1 were analyzed by Southern hybridization under high stringency conditions, with the cnm gene fragment as a probe. Asterisked are the cnm gene-positive strains. Binding assays of the strains to collagen/laminin were carried out as in Fig. 2 . Data points represent the means of OD490 nm values ± standard deviation from 4 (collagen) or 3 (laminin) independent experiments.
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Figure 4. The DNA nucleotide and deduced amino acid sequences of the cnm gene. (The DDBJ-EMBL-GenBank nucleotide sequence databases accession number is AB102689.) Underlines indicate, respectively, the N-terminal sequence determined from isolated Cnm protein, CBD homologous sequence, putative B repeats domain, and LPXTG motif.
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Journal of Dental Research, Vol. 83, No. 7,
534-539 (2004)
DOI: 10.1177/154405910408300705
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