Devine D A, Marsh P D, Percival R S, Rangarajan M, Curtis M A
Leeds Dental Institute, University of Leeds, Clarendon Way, Leeds LS2 9LU, UK.
MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St Bartholomew's and the Royal London School of Medicine and Dentistry, 32 Newark St, Whitechapel, London E1 2AA, UK.
Microbiology (Reading). 1999 Apr;145 ( Pt 4):965-971. doi: 10.1099/13500872-145-4-965.
This study investigated the ability of anaerobic periodontal bacteria to inactivate and resist killing by antimicrobial peptides through production of extracellular proteases. Antibacterial activities of peptides were assessed in a double-layer agarose diffusion assay, and MICs and MBCs were determined in broth microdilution assays. Culture supernates of Porphyromonas gingivalis and Prevotella spp. inactivated mastoparan, magainin II and cecropin B whilst Gram-positive oral supragingival bacteria had no effect. Inactivation was prevented by protease inhibitors and was unaffected by 45% human serum. Purified proteases from the periodontopathogen Porph. gingivalis inactivated peptides [cecropin B, brevinin, CAMEL (cecropin A 1-7 + melittin 2-9), mastoparan] as would be predicted from the amino acid sequences of the peptides and the known bond specificities of these Arg-x and Lys-x enzymes. MALDI-TOF MS revealed that inactivation of cecropin B by Porph. gingivalis protease was due to specific cleavage of the molecule. Inactivation of cecropin B by proteases took 10-15 min. Paradoxically, MICs of cecropin B against Porph. gingivalis and Prevotella intermedia were low, while Prevotella nigrescens was resistant, suggesting that production of proteases alone is insufficient to protect Porph. gingivalis and Prev. intermedia from the action of antimicrobial peptides. Thus, antimicrobial peptides could be developed as therapeutic agents targeted against specific periodontal pathogens.
本研究调查了厌氧性牙周细菌通过产生细胞外蛋白酶使抗菌肽失活并抵抗被其杀灭的能力。在双层琼脂糖扩散试验中评估了肽的抗菌活性，并在肉汤微量稀释试验中测定了最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。牙龈卟啉单胞菌和普雷沃菌属的培养上清液可使马蜂毒素、蛙皮素II和天蚕素B失活，而革兰氏阳性口腔龈上细菌则无此作用。蛋白酶抑制剂可阻止失活，且45%的人血清对此无影响。从牙周病原体牙龈卟啉单胞菌中纯化的蛋白酶可使肽（天蚕素B、铃蟾肽、骆驼毒素（天蚕素A 1-7 + 蜂毒肽2-9）、马蜂毒素）失活，这与肽的氨基酸序列以及这些精氨酸-x和赖氨酸-x酶已知的键特异性所预测的结果一致。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）显示牙龈卟啉单胞菌蛋白酶使天蚕素B失活是由于该分子的特异性切割。蛋白酶使天蚕素B失活需10 - 15分钟。矛盾的是，天蚕素B对牙龈卟啉单胞菌和中间普雷沃菌的MIC较低，而变黑普雷沃菌具有抗性，这表明仅产生蛋白酶不足以保护牙龈卟啉单胞菌和中间普雷沃菌免受抗菌肽的作用。因此，抗菌肽可被开发为针对特定牙周病原体的治疗药物。
