Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center and School of Dentistry, New Orleans, LA 70119, USA.
Mol Oral Microbiol. 2010 Aug;25(4):293-304. doi: 10.1111/j.2041-1014.2010.00579.x.
Innate and adaptive immunity are considered critical to protection against mucosal candidal infections. Among innate anti-Candida mechanisms, oral and vaginal epithelial cells have antifungal activity. The mechanism is fungistatic, acid-labile and includes a requirement for cell contact by intact, but not necessarily live, epithelial cells. The purpose of this study was to use the acid-labile property to further characterize the effector moiety. Surface material extracted from phosphate-buffered saline (PBS) -treated, but not acid-treated, epithelial cells significantly inhibited the growth of Candida blastoconidia in a dose-dependent manner which was abrogated by prior heat and protease treatment. Proteins extracted from PBS-treated cells bound blastoconidia and hyphae more intensely than those from acid-treated cells. Proteins from PBS-treated cells eluted from Candida revealed two unique bands of approximately 33 and 45 kDa compared with acid-treated cells. Mass spectrometry identified these proteins as Annexin-A1 and actin, respectively. Oral epithelial cells stained positive for Annexin-A1, but not actin. Western blots showed reduced Annexin-A1 in proteins from acid-treated epithelial cells compared with those from PBS-treated epithelial cells. Lastly, it was demonstrated that immunoprecipitation of Annexin-A1 from proteins extracted from PBS-treated oral epithelial cells resulted in abrogation of inhibitory activity. Taken together, these results indicate that Annexin-A1 is a strong candidate for the epithelial cell anti-Candida effector protein.
先天免疫和适应性免疫被认为是对抗黏膜念珠菌感染的关键。在先天抗念珠菌机制中，口腔和阴道上皮细胞具有抗真菌活性。其机制是抑菌、酸不稳定，包括完整但不一定存活的上皮细胞的细胞接触要求。本研究旨在利用酸不稳定特性进一步表征效应部分。从磷酸盐缓冲盐水 (PBS) 处理但未用酸处理的上皮细胞中提取的表面材料以剂量依赖性方式显著抑制芽生孢子的生长，而先前的热和蛋白酶处理则消除了这种抑制作用。与酸处理的细胞相比，从 PBS 处理的细胞中提取的蛋白质与芽生孢子和菌丝的结合更为强烈。从 PBS 处理的细胞洗脱的蛋白质与酸处理的细胞相比，显示出约 33 和 45 kDa 的两个独特条带。质谱鉴定这些蛋白质分别为膜联蛋白 A1 和肌动蛋白。口腔上皮细胞对膜联蛋白 A1 呈阳性染色，但对肌动蛋白呈阴性染色。Western blot 显示，与 PBS 处理的上皮细胞相比，酸处理的上皮细胞中的膜联蛋白 A1 减少。最后，证明从 PBS 处理的口腔上皮细胞中提取的蛋白质中免疫沉淀膜联蛋白 A1 可消除抑制活性。总之，这些结果表明膜联蛋白 A1 是上皮细胞抗念珠菌效应蛋白的有力候选者。
