家蝇Phormicin A、B的抗菌作用及抑制白色念珠菌机制研究

The rise of drug resistance of pathogens caused by increasing antibiotic abuse is seriously endangering people's health. There is an innate immune molecule called antimicrobial peptide. Due to its multi-Targets inhibits function, high antimicrobial activity and low drug resistance, antimicrobial peptide has the potential to become a new type of peptide antibiotic with characters of high efficiency, low toxicity and being able to kill drug-resistant pathogens. In our previous unpublished work, we used Escherichia coli, Staphylococcus aureus and Candida albicans to stimulate the third instar larvae of Musca Domestica seperately, and performed the Transcriptome Sequencing. It turned out that two new members of the phormicin family in Musca domestica, Phormicin A and Phormicin B, may have different or new functions because Phormicin A expression was significantly up-regulated while Phormicin B expression was significantly down-regulated after the stimulation. Different from the published report saying that phormicin in Phormia terranovae is only antibacterial to gram-positive bacterium (G+), the Trace liquid dilution method experiment has proved that the prokaryotic expression protein of Phormicin A and B in Musca Domestica can resist the growth of Candida albicans in vitro. The Minimal inhibitory concentration (MIC) was obtained and the Bacteriostatic curve also draw out. Thus, in this research, we will explore Phormicin A and B’s antimicrobial activity and spectrum of Musca domestica in vitro and in vivo. And then we will investigate the antimicrobial mechanism on Candida albicans of Phormicin A and B. Aspects of Pore forming, Biofilm forming, Growth and proliferation activity, Nucleic acid and protein synthesis ability, Virulence gene expression and Drug resistance gene expression would be focused. This research will provide theoretical foundation and strong technical support for the study and development of new antimicrobial drugs.

抗生素滥用导致诸多病原菌耐药性增加。天然免疫分子抗菌肽多靶点抑菌，不易耐药，具备开发为高效、低毒、杀耐药病原菌的新型肽类抗生素的潜力。白色念珠菌为条件致病菌，感染免疫低下人群，可以生物膜形式附着于体内组织或医疗装置及手术器械，难于根除。前期研究显示，大肠杆菌、金黄色葡萄球菌、白色念珠菌刺激家蝇3龄幼虫并测序转录组，发现防御素家族新成员家蝇Phormicin A，B。刺激后A表达显著上调，B下调。这提示二者或具不同功能。与文献报道绿蝇Phormicin只抗革兰氏阳性菌不同，微量液体稀释法实验表明家蝇A，B原核表达蛋白可抑制白色念珠菌生长。我们测得二者MIC并绘制抑菌曲线。本研究将在体内、外进一步探究其对G+、G-、真菌的抗菌谱系。在形成穿孔、形成生物膜、生长增殖、核酸和蛋白合成、毒力基因、耐药基因表达方面，探究其对白色念珠菌的抑菌机制，从而为研发新型抗菌药物提供理论基础和技术支持。

白色念珠菌为临床常见条件致病菌，因其可以形成生物膜而难于根除，导致多种感染。抗菌肽被认为是具有潜力的下一代新型抗生素用来治疗耐药菌感染。本课题从家蝇3龄幼虫中筛选出具有广谱抗菌活性的Phormicin A,B。通过原核表达证实其具有抑制白色念珠菌，金黄色葡萄球菌等的活性。制备了多克隆抗体。通过人工合成多肽的方法合成了Phormicin A,B及其成熟肽Phormicin C,D。测定了其抗菌谱系。证实其可以抑制MRSA生物膜的形成。探究了Phormicin A,B的表达模式，发现A在早期发挥作用，B在晚期发挥作用。发现在N端对Phormicin 成熟肽进行山梨酸修饰，可以显著增强其抑制白色念珠菌的活性。Phormicin C-NS 可以抑制白色念珠菌生物膜的形成并减弱其生物膜内菌的活力和菌体保有量。转录组测序分析发现， Phormicin C-NS 肽通过抑制白色念珠菌菌丝特异性基因HWP1,Ece1p和黏附基因ALS3的表达，来抑制其由酵母态向菌丝态转化以及白色念珠菌生物膜的形成，为白色念珠菌的治疗提供新的候选多肽。相关结果已表SCI论文1篇（International Journal of Biological Macromolecules；Q1,IF 6.953），北图核心论文3篇，科技核心论文1篇。此外，还有2篇SCI论文正在审稿中（1篇Under Review，International Journal of Biological Macromolecules，IJBIOMAC-D-22-01363，IF 6.953, 另1篇已投稿）。