家蝇抗菌肽AMP-17对白色念珠菌菌丝的抑制作用

Inhibitory effect of housefly antimicrobial peptide AMP-17 on hyphae of Candida albicans

摘要:

【背景】AMP-17是从微生物诱导的家蝇转录组数据库筛选到的一条特异性高表达基因，采用原核表达体系获得其重组蛋白并证实了具有显著的抗菌效果，特别是对白色念珠菌具有较强的抗菌活性。【目的】研究抗菌肽AMP-17对白色念珠菌菌丝的抑制作用。【方法】采用微量液体稀释法测定AMP-17对11株白色念珠菌的最小抑菌浓度(minimal inhibitory concentration，MIC)；根据对AMP-17的敏感程度选取3株绘制生长曲线；通过光学显微镜观察并计数经AMP-17作用后白色念珠菌芽生孢子生成率及芽管形成率；倒置荧光显微镜观察白色念珠菌酵母相向菌丝相转化及以菌丝相为起点AMP-17促进菌丝相转化为酵母相的情况。【结果】AMP-17对支气管肺泡灌洗液分离株16105的MIC为10 μg/mL，对粪便分离株16214的MIC为40 μg/mL，对其余9株白色念珠菌的MIC均为20 μg/mL；白色念珠菌经不同浓度的AMP-17作用后，各时间点的芽生孢子生成率均显著低于对照组，尤其是40 μg/mL的AMP-17组，芽生孢子生成率仅15%，显著低于阳性药物氟康唑；各实验组芽管形成率显著低于对照组，且芽管形成缓慢，培养6 h后芽管形成率仅为6%，形成的芽管长度较短，仅为菌体的1?2倍。镜下观察低浓度的AMP-17即可以完全抑制白色念珠菌菌丝的生长，并且对已经形成的菌丝有一定的生长抑制作用，高浓度的AMP-17则可使已形成的部分菌丝向酵母相转化。【结论】家蝇抗菌肽AMP-17可抑制白色念珠菌菌丝生长。

Abstract:

[Background] AMP-17 is a specific and highly expressed gene screened from the database of housefly transcriptional group induced by microorganisms. Its recombinant AMP-17 protein was obtained by utilizing the prokaryotic expression system and proved to have significant antimicrobial effect, especially for Candida albicans. [Objective] To investigate the inhibitory effect of AMP-17 on the hyphae of Candida albicans. [Methods] The minimum inhibitory concentration (MIC) of AMP-17 on 11 strains of Candida albicans were determined by microdilution method; Three strains of Candida albicans were selected to plot the growth curve according to their sensitivity to AMP-17; The blastospore production rate and germ tube formation rate of Candida albicans after AMP-17 were observed and counted by optical microscope; The transformation of Candida albicans yeast phase to mycelial phase and AMP-17 promoted the transformation of mycelial phase into yeast phase with mycelial phase as the starting point were observed by fluorescent inverted microscope. [Results] The MIC of AMP-17 to the strain isolated from BALF 16105 was 10 μg/mL, the MIC to the strain isolated from fecal 16214 was 40 μg/mL, and the MIC to the other 9 strains of Candida albicans were 20 μg/mL. After the action of AMP-17 at different concentrations, the rate of blastospore formation at each time point was significantly lower than that of the control group, especially in the AMP-17 group of 40 μg/mL, the rate of blastospore formation was only 15%, obviously lower than that of fluconazole, a positive drug. The rate of germ tube formation in each experimental group were obviously lower than that in the control group, and the germ tube formation was slow. After 6 h of culture, the germ tube formation rate was only 6%, and the germ tube was short, which was only 1?2 times of thallus. Low concentration of AMP-17 could completely inhibit the growth of Candida albicans hyphae, and inhibit the growth of already formed hyphae to some extent, while high concentration of AMP-17 could transform some formed hyphae into yeast phase by microscopes. [Conclusion] Housefly antimicrobial peptide AMP-17 can inhibit the growth of Candida albicans hyphae.