以表皮葡萄球菌LuxS为靶点的反义锁核酸对生物被膜的影响  

Influence of Antisense LNA Targeted Lux S Transcription on Biofilm Formation in Staphylococcus epidermidis

作　　者：管翠萍[1,2] 何芳[1,2] 卢金霞[1,2] 冯峰[1,2] 周学章[1,2] GUAN Cui-Ping HE Fang LU Jin-Xia FENG Feng ZHOU Xue-Zhang(Key Laboratory of the Ministry of Education for the Conservation and Utilization of Special Biological Resources of Western China, Ningxia University, Yinchuan 750021, China College of Life science, Ningxia University, Yinchuan 750021, China)

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室,银川750021 [2]宁夏大学生命科学学院,银川750021

出　　处：《农业生物技术学报》2017年第2期291-298,共8页Journal of Agricultural Biotechnology

基　　金：宁夏自然科学基金(No.NZ15019)

摘　　要：细菌生物被膜的形成是导致细菌耐药性升高的主要原因,表皮葡萄球菌(Staphylococcus epidermidis)作为条件致病菌,一旦形成生物被膜后会粘附在医疗器械和病灶上造成反复感染,难以治疗。以细菌群体感应系统作为抑制生物被膜形成的研究靶点近年来受到广泛关注。S-核糖基高半胱氨酸酶(S-ribosyl homocysteine lyase,Lux S)是合成自诱导信号分子2(autoinducer-2,AI-2)的关键酶,构成了细菌内依赖Lux S基因产物的AI-2群体感应系统(AI-2/Lux S)。本研究以生物被膜形成初期为时间点,利用反义锁核酸技术阻断表皮葡萄球菌内AI-2/Lux S群体感应系统中的关键酶Lux S基因的表达,比较该基因被阻断前后对其他相关产膜基因的表达、Al-2信号分子的活性以及生物被膜形成能力等方面的影响。结果表明,针对Lux S m RNA序列设计的两条反义锁核酸序列LNA81和LNA352,相比而言,LNA81的抑制效果更佳,其他与被膜形成的相关基因除σ因子B(sigma factor B,sig B)和细胞间粘附因子AB(intercellular adhesion AB,ica AB)表达量上调外,附属调节子A(staphylococal accessory regulator A,sar A)、聚集相关蛋白(accumulation-associated protein,aap)、纤维蛋白原结合蛋白(fibrinogen-binding protein,fbe)和自溶素E(autolysin E,atl E)的表达量均下调,AI-2信号分子活性在一定时间内与对照组相比有所降低。由此说明,Lux S基因表达被部分抑制后会影响AI-2信号分子的活性以及其他被膜相关基因的表达,最终导致生物被膜形成能力减弱。本研究为深入探讨AI-2/Lux S群体感应系统在表皮葡萄球菌生物被膜形成过程中所起的调控作用提供了理论依据,同时也为抗产膜型表皮葡萄球菌形成的感染提供更多有效的潜在靶点和治疗方案。Bacterial biofilm is the leading cause of bacterial drug resistance increases. Staphylococcus epidermidis is an opportunistic pathogen, once it formed the biofilm, it will adhere to the surfaces of medical device and make the diseases recurrent and difficult to cure. Quorum sensing system has been recently regarded as the research target to suppress the bacterial biofilm formation. S-ribosyl homocysteine lyase （LuxS） is the key enzyme for the synthesis of autoinducer-2 （AI-2）, they constitute the AI-2/LuxS quorum sensing system in bacteria. In this study, the biofilm formed in the early stage was chosen as the experiment subject, then antisense locked nucleic acid （LNA） technology was used to block the expression of LuxS gene in S. epidermidis AI-2/LuxS quorum sensing system, the inhibitory effect of 2 antisense locked oligonucleotides named LNA81 and LNA352 was compared by the transcription level of LuxS gene, Using real-time PCR measured the relative expression quantity of biofilm formation related genes, including sigma factor B （sigB）, intercellular adhesion AB （icaAB）, staphylococal accessory regulator A （sarA）, accumulation-associated protein （aap）, fibrinogen-binding protein （/be） and autolysin E （atlE）. AI-2 signaling molecule activity was measured by Vibrio harveyi bioluminescence assay, and the formation and characteristic parameters of biofilm were analyzed by confocal laser scanning microscope （CLSM） and image structure analyzer （ISA） software. Results showed that between the 2 antisense locked oligonucleotides, LNA81 and LNA352, 100 nmol/L LNA81 mixed with 2.5 μL RNAiMAX transfection reagent exhibited better inhibition in expression of LuxS through targeting LuxS mRNA, it could get a 43% inhibition ratio in LuxS transcription at 6 h. When the transcription of LuxS was inhibited partially, the transcript level of gene sigB and icoAB increased, sarA, aap, foe and atlE decreased. The activity of AI-2 signaling molecule was decreased in 9 h compared to control

关 键 词：反义锁核酸 表皮葡萄球菌 生物被膜 群体感应系统 

分 类 号：S852.6[农业科学—基础兽医学]