Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
Antimicrob Agents Chemother. 2012 Mar;56(3):1548-56. doi: 10.1128/AAC.05100-11. Epub 2012 Jan 3.
Porphyromonas gingivalis is a bacterial pathogen associated with chronic periodontitis that results in destruction of the tooth's supporting tissues. The major virulence determinants of P. gingivalis are its cell surface Arg- and Lys-specific cysteine proteinases, RgpA/B and Kgp. Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in saliva and gingival crevicular fluid, is believed to play an important role in innate immunity. In this study, bovine milk LF displayed proteinase inhibitory activity against P. gingivalis whole cells, significantly inhibiting both Arg- and Lys-specific proteolytic activities. LF inhibited the Arg-specific activity of purified RgpB, which lacks adhesin domains, and also inhibited the same activity of the RgpA/Kgp proteinase-adhesin complexes in a time-dependent manner, with a first-order inactivation rate constant (k(inact)) of 0.023 min(-1) and an inhibitor affinity constant (K(I)) of 5.02 μM. LF inhibited P. gingivalis biofilm formation by >80% at concentrations above 0.625 μM. LF was relatively resistant to hydrolysis by P. gingivalis cells but was cleaved into two major polypeptides (53 and 33 kDa) at R(284) to S(285), as determined by in-source decay mass spectrometry; however, these polypeptides remained associated with each other and retained inhibitory activity. The biofilm inhibitory activity of LF against P. gingivalis was not attributed to direct antibacterial activity, as LF displayed little growth inhibitory activity against planktonic cells. As the known RgpA/B and Kgp inhibitor N-α-p-tosyl-l-lysine chloromethylketone also inhibited P. gingivalis biofilm formation, the antibiofilm effect of LF may at least in part be attributable to its antiproteinase activity.
牙龈卟啉单胞菌是一种与慢性牙周炎相关的细菌病原体，可导致牙齿支持组织的破坏。牙龈卟啉单胞菌的主要毒力决定因素是其细胞表面的精氨酸和赖氨酸特异性半胱氨酸蛋白酶 RgpA/B 和 Kgp。乳铁蛋白（LF）是一种 80kDa 的铁结合糖蛋白，存在于唾液和牙龈沟液中，被认为在先天免疫中发挥重要作用。在这项研究中，牛乳铁蛋白显示出对牙龈卟啉单胞菌全细胞的蛋白酶抑制活性，显著抑制精氨酸和赖氨酸特异性蛋白水解活性。LF 抑制缺乏粘附结构域的纯化 RgpB 的精氨酸特异性活性，并且以时间依赖性方式抑制 RgpA/Kgp 蛋白酶-粘附复合物的相同活性，一级失活速率常数（k(inact)）为 0.023 min(-1)，抑制剂亲和力常数（K(I)）为 5.02 μM。LF 在浓度高于 0.625 μM 时可抑制超过 80%的牙龈卟啉单胞菌生物膜形成。LF 相对抵抗牙龈卟啉单胞菌细胞的水解，但在 R(284)到 S(285)处被切割成两个主要多肽（53 和 33 kDa），如源内衰减质谱法确定；然而，这些多肽仍然彼此相关并保持抑制活性。LF 对牙龈卟啉单胞菌生物膜的抑制活性不是归因于直接的抗菌活性，因为 LF 对浮游细胞的生长抑制活性很小。由于已知的 RgpA/B 和 Kgp 抑制剂 N-α-对甲苯磺酰-L-赖氨酸氯甲基酮也抑制了牙龈卟啉单胞菌生物膜的形成，因此 LF 的抗生物膜作用至少部分归因于其抗蛋白酶活性。
