Ito Tatsuro, Ichinosawa Takahiro, Shimizu Takehiko
Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Nihon University Research Institute of Oral Science, Chiba, Japan.
PLoS One. 2017 Apr 10;12(4):e0175483. doi: 10.1371/journal.pone.0175483. eCollection 2017.
Streptococcus mutans, the major causative agent of dental caries, adheres to tooth surfaces via the host salivary glycoprotein-340 (gp340). This adherence can be competitively inhibited by peptides derived from the SspA/B adhesins of Streptococcus gordonii, a human commensal microbe that competes for the same binding sites. Ssp(A4K-A11K), a double-lysine substituted SspA/B peptide analogue, has been shown to exhibit superior in vitro binding affinity for a gp340-derived peptide (SRCRP2), suggesting that Ssp(A4K-A11K) may be of clinical interest. In the present work, we tested the inhibitory effects of Ssp(A4K-A11K) on adherence and biofilm formation of S. mutans by reconstructing an artificial oral environment using saliva-coated polystyrene plates and hydroxyapatite disks. Bacterial adherence (adherence period: 1 h) was assessed by an enzyme-linked immunosorbent assay using biotinylated bacterial cells. Biofilm formation (periods: 8, 11, or 14 h) was assessed by staining and imaging of the sessile cells, or by recovering biofilm cells and plating for cell counts. The pH values of the culture media were measured as a biofilm acidogenicity indicator. Bactericidality was measured by loss of optical density during culturing in the presence of the peptide. We observed that 650 μM Ssp(A4K-A11K) significantly inhibited adherence of S. mutans to saliva-coated polystyrene; a similar effect was seen on bacterial affinity for SRCRP2. Ssp(A4K-A11K) had lesser effects on the adherence of commensal streptococci. Pretreatment of polystyrene and hydroxyapatite with 650 μM Ssp(A4K-A11K) significantly attenuated biofilm formation, whether tested with glucose- or sucrose-containing media. The SspA/B peptide's activity did not reflect bactericidality. Strikingly, pH in Ssp-treated 8-h (6.8 ± 0.06) and 11-h (5.5 ± 0.06) biofilms showed higher values than the critical pH. Thus, Ssp(A4K-A11K) acts by inhibiting bacterial adherence and cariogrnic biofilm formation. We further consider these results in the context of the safety, specificity, and stability properties of the Ssp(A4K-A11K) peptide.
变形链球菌是龋齿的主要致病菌，它通过宿主唾液糖蛋白340（gp340）附着于牙齿表面。这种附着可被戈登链球菌SspA/B黏附素衍生的肽竞争性抑制，戈登链球菌是一种人类共生微生物，会竞争相同的结合位点。Ssp(A4K - A11K)是一种双赖氨酸取代的SspA/B肽类似物，已被证明对gp340衍生肽（SRCRP2）具有优异的体外结合亲和力，这表明Ssp(A4K - A11K)可能具有临床应用价值。在本研究中，我们通过使用唾液包被的聚苯乙烯平板和羟基磷灰石圆盘重建人工口腔环境，测试了Ssp(A4K - A11K)对变形链球菌附着和生物膜形成的抑制作用。使用生物素化细菌细胞通过酶联免疫吸附测定法评估细菌附着（附着时间：1小时）。通过对固着细胞进行染色和成像，或通过回收生物膜细胞并进行平板计数来评估生物膜形成（时间：8、11或14小时）。测量培养基的pH值作为生物膜产酸性指标。在肽存在的情况下培养期间通过光密度损失来测量杀菌活性。我们观察到650μM Ssp(A4K - A11K)显著抑制变形链球菌对唾液包被聚苯乙烯的附着；对细菌与SRCRP2的亲和力也有类似作用。Ssp(A4K - A11K)对共生链球菌的附着影响较小。用650μM Ssp(A4K - A11K)预处理聚苯乙烯和羟基磷灰石，无论在含葡萄糖还是含蔗糖的培养基中测试，均显著减弱生物膜形成。SspA/B肽的活性与杀菌活性无关。令人惊讶的是，经Ssp处理8小时（6.8±0.06）和11小时（5.5±0.06）的生物膜中的pH值高于临界pH值。因此，Ssp(A4K - A11K)通过抑制细菌附着和致龋生物膜形成发挥作用。我们还结合Ssp(A4K - A11K)肽的安全性、特异性和稳定性来进一步考虑这些结果。
