爱媛类芽孢杆菌抗菌肽对白色念珠菌生物膜的抑制作用机制

Antimicrobial Mechanism of Antimicrobial Peptide from Paenibacillus ehimensis against Candida albicans Biofilms

研究爱媛类芽孢杆菌抗菌肽对白色念珠菌生物膜的抑制作用,采用微量二倍稀释法和时间-杀菌曲线测定抑制效果,显微镜观察对白色念珠菌芽管和菌丝形成的影响,2,3-二(2-甲氧基-4-硝基-5-磺酸基苯基)-2H-四唑-5-甲酰苯胺法研究对生物膜、预成型生物膜形成和成熟生物膜清除率的影响,荧光探针观察对生物膜结构和膜内菌体状态的影响,平板涂布法和实时聚合酶链式反应测定生物膜内活菌数量和相关基因表达水平。结果表明,抗菌肽对白色念珠菌的最小抑菌浓度为8.28 AU/mL。抗菌肽能降低白色念珠菌的芽管形成率,阻止菌丝的生成,使其以酵母形式存在。抗菌肽对生物膜的形成和清除产生影响,能在较短时间内破坏生物膜的结构完整性,导致菌体受损和死亡,减少膜内菌落数量。在基因水平上,抗菌肽可降低多个生物膜形成相关的基因(如ALS1、ALS3、HWP1、EFG1、ECE1和UME6)的表达水平,从而抑制生物膜的形成。研究证明抗菌肽能够有效抑制白色念珠菌生物膜形成、成熟膜清除、成膜基因表达等,结果可为白色念珠菌新型抑菌物质的开发奠定理论基础,为食品病原微生物污染防治和食品质量安全保障提供依据。

This study investigated the inhibitory effect of antimicrobial peptide from Paenibacillus ehimensis on the biofilms of Candida albicans by the microdilution and time-kill curve assays.Microscopic observation was conducted to assess the impact of the peptide on the formation of C.albicans germ tube and hyphae.Additionally,the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT)assay was employed to investigate the influence on the formation of C.albicans biofilms,pre-formed biofilms,and the clearance rate of mature biofilms.Fluorescent probes were utilized to observe the alteration in the structure of biofilms and the status of intracellular fungal entities.The number of viable cells in biofilms was measured by the spread plate method,and real-time polymerase chain reaction(PCR)was employed to determine the expression levels of genes associated with biofilm formation.The results showed that the minimum inhibitory concentration of the antimicrobial peptide against C.albicans was 8.28 AU/mL.This peptide reduced the rate of germ tube formation in C.albicans,and prevented the formation of hyphae,making it exist in the form of yeasts.Furthermore,the antimicrobial peptide influenced both the formation and eradication of biofilms and disrupted the structural integrity of biofilms after a short duration,leading to damage and even death of C.albicans,and reducing cell counts in biofilms.The antimicrobial peptide reduced the expression levels of multiple genes related to biofilm formation(such as ALS1,ALS3,HWP1,EFG1,ECE1 and UME6),therefore inhibiting biofilm formation.The above findings demonstrated that antimicrobial peptide effectively inhibited the biofilm formation,mature biofilm clearance and the expression of biofilm-forming genes in C.albicans.This study lays a theoretical foundation for the development of novel antimicrobial agents against C.albicans and provides a basis for the prevention and control of foodborne pathogen contamination to ensure food quality and safety.