Wei Guo-Xian, Bobek Libuse A
Department of Oral Biology, University at Buffalo, the State University of New York, 109 Foster Hall, 3435 Main Street, Buffalo, NY 14214-3092, USA.
Antimicrob Agents Chemother. 2005 Jun;49(6):2336-42. doi: 10.1128/AAC.49.6.2336-2342.2005.
MUC7 12-mer-L exhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-L and its all-D-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 microM concentration in clarified saliva and unclarified saliva, MUC7 12-mer-D demonstrated 94 versus 64% killing, respectively; MUC7 12-mer-L showed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the three L peptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-D and 23, 60, and 75% degradation of MUC7 12-mer-L, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 microM peptide concentrations, MUC7 12-mer-D and 12-mer-L caused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.
MUC7 12聚体-L在低离子强度缓冲液中表现出强大的体外抗真菌活性。在本研究中，我们研究了MUC7 12聚体-L及其全D-氨基酸异构体，以及Hsn5 12聚体（P113）和蛙皮素-II在有或无蛋白酶抑制剂混合物（PIC，其中包括EDTA）或单独EDTA存在的情况下，在人澄清和未澄清唾液中的抗念珠菌活性和稳定性。在唾液中不存在PIC或EDTA的情况下，只有MUC7肽表现出显著的杀念珠菌活性。在澄清唾液和未澄清唾液中浓度为100 microM时，MUC7 12聚体-D的杀菌率分别为94%和64%；MUC7 12聚体-L为57%和32%；Hsn5 12聚体为16%和0%；蛙皮素-II无杀菌作用。向任何一种唾液中添加PIC或EDTA都会使所有肽的抗真菌活性增强，尽管程度不同。综上所述，结果表明EDTA（一种金属依赖性蛋白酶抑制剂和/或二价阳离子螯合剂）主要通过螯合唾液中存在的二价阳离子（已知其会抑制肽的抗真菌活性）来增强所有四种肽的抗真菌活性，而PIC通过抑制所有类型的蛋白酶，使三种L肽的活性增强程度超过单独使用EDTA所能达到的程度。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳监测的肽在唾液中的稳定性显示，MUC7 12聚体-D未降解，而MUC7 12聚体-L、Hsn5 12聚体和蛙皮素-II分别有23%、60%和75%的降解。细胞毒性试验确定，在肽浓度为100 microM时，MUC7 12聚体-D和12聚体-L在磷酸盐缓冲盐水中引起3.5%和4.3%的溶血，对HOK-16B细胞系（源自正常人口腔角质形成细胞）无毒性。总之，MUC7 12聚体肽似乎是在口腔念珠菌病体内模型中研究抗真菌活性的优秀候选物。
