CN113430158A - 香叶醇在制备促进铜绿假单胞菌3oc12-hsl信号分子合成制剂中的应用 - Google Patents

香叶醇在制备促进铜绿假单胞菌3oc12-hsl信号分子合成制剂中的应用 Download PDF

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CN113430158A
CN113430158A CN202110685800.1A CN202110685800A CN113430158A CN 113430158 A CN113430158 A CN 113430158A CN 202110685800 A CN202110685800 A CN 202110685800A CN 113430158 A CN113430158 A CN 113430158A
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谢小保
李文茹
曾桃花
张志清
施庆珊
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Abstract

本发明公开了香叶醇在促进铜绿假单胞菌3OC12‑HSL信号分子合成制剂中的应用。本发明发现香叶醇轻微抑制铜绿假单胞菌PAO1菌株的生长，但能够显著促进该菌3OC12‑HSL信号分子的合成。因此可用于促进铜绿假单胞菌3OC12‑HSL信号分子合成制剂中的应用。

Description

香叶醇在制备促进铜绿假单胞菌3OC12-HSL信号分子合成制剂 中的应用
技术领域：
本发明属于有害微生物防控技术领域，具体涉及香叶醇在制备促进铜绿假单胞菌3OC12-HSL信号分子合成制剂中的应用。
背景技术：
铜绿假单胞菌(Pseudomonas aeruginosa)是一种条件致病菌，极易引起烧伤部位、呼吸道、尿道感染，对健康人影响不大，但会危及免疫功能低下的患者和囊肿性纤维化患者的生命。PA对大多数抗生素极易产生耐药，可以通过基因突变或获得外源性耐药基因产生耐药，或者由于环境刺激导致基因和蛋白质表达发生短暂的变化，产生适应性耐药。铜绿假单胞菌的感染率一直居高不下，难以根除，而且抗生素的大量使用，导致多重耐药铜绿假单胞菌的传播，使铜绿假单胞菌的防治任务更加艰巨，因此急需寻找新的防控策略，抑制铜绿假单胞菌的群体感应成为了研究热点。群体感应(quorum sensing,QS)是细菌个体之间的一种密度依赖性交流机制，铜绿假单胞菌的生物膜、绿脓菌素、弹性蛋白酶等毒力因子的产生都受到该菌群体感应系统的调控。抑制群体感应可以抑制该菌的毒性和致病力，但对其生长影响不大，因而不会为耐药菌提供有利的选择压力。因此，群体感应抑制剂的研发对解决细菌耐药性问题具有重要意义。
铜绿假单胞菌的群体感应系统有三个，分别是las、rhl和pqs系统。该菌las系统由lasI和lasR组成，rhl系统由rhlI和rhlR组成，pqs系统由pqsABCDE、pqsH和pqsR组成，其中lasI、rhlI、pqsABCDE和pqsH是信号分子合成酶的编码基因，分别编码3OC12-HSL、C4-HSL、HHQ和PQS信号分子，lasR、rhlR和pqsR是相应信号分子受体蛋白的编码基因。las、rhl和pqs三个群体感应系统，共同调控着铜绿假单胞菌很多毒力因子的产生，包括胞外蛋白酶LasA、弹性蛋白酶LasB、绿脓菌素和生物膜等。
香叶醇是一种非环单萜类化合物，是玫瑰油和香茅油的主要成分之一，常温下是无色至黄色的油状液体，具有温和的玫瑰花香味，常用来制作日用香精和食用香精。
发明内容：
本发明的目的是提供一种香叶醇在制备促进铜绿假单胞菌3OC12-HSL信号分子合成制剂中的应用。
本发明通过实验表明，香叶醇会轻微抑制铜绿假单胞菌PAO1菌株的生长，但能够显著促进该菌3OC12-HSL信号分子合成。
因此，本发明的目的是提供香叶醇在制备促进铜绿假单胞菌3OC12-HSL信号分子合成制剂中的应用。
所述的铜绿假单胞菌优选为铜绿假单胞菌(Pseudomonas aeruginosa)PAO1。
所述的3OC12-HSL信号分子为该菌las群体感应系统的信号分子，由信号分子合成酶LasI合成。
所述的香叶醇，其在铜绿假单胞菌培养液中的浓度为0.313-2.5μL/mL。
本发明发现香叶醇在浓度为0.313-2.5μL/mL时会轻微抑制铜绿假单胞菌PAO1菌株的生长，但能够显著促进该菌3OC12-HSL信号分子的合成。因此可用于促进铜绿假单胞菌3OC12-HSL信号分子合成制剂中的应用。
附图说明：
图1是香叶醇作用下铜绿假单胞菌PAO1的生长曲线；
图2是香叶醇对铜绿假单胞菌群体感应系统关键基因及其调控的毒力基因表达的影响。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
PAO1菌[铜绿假单胞菌(Pseudomonas aeruginosa)PAO1]悬液的制备：将指数生长期的PAO1培养液取样，离心，用PBS缓冲液洗涤一次，重悬于PBS中，并将菌浓度稀释至108CFU/mL，得到PAO1菌悬液。
1.香叶醇对铜绿假单胞菌PAO1生长的影响实验
分别向试管中加入LB培养基、香茅醛，并接入指数生长期的PAO1菌悬液，总体积均为10mL，使PAO1的菌浓度均为106CFU/mL，香叶醇的浓度分别为0(对照)、0.313μL/mL、0.625μL/mL、1.25μL/mL和2.5μL/mL。将几个实验组分别取样加入到自动生长曲线测定仪(Bioscreen C)专用的蜂窝培养板中，每孔加入350μL的培养液，每个实验组三个平行。将蜂窝培养板置于自动生长曲线测定仪中，37℃振荡培养3天，每小时测定一次OD600。以OD600为纵坐标，培养时间为横坐标，绘制香茅醛作用下PAO1的生长曲线，以此研究香叶醇对PAO1生长的影响。结果如图1所示。
2.香叶醇对铜绿假单胞菌群体感应系统关键基因及其调控的毒力基因表达的影响实验
向50mL无菌LB液体培养基中加入处于对数生长期的PAO1菌悬液，使菌液终浓度为106CFU/mL。加入香叶醇，使得终浓度为0(对照组三个生物学重复分别命名为A1、A2、A3)和1.25μL/mL(实验组三个生物学重复分别命名为B1、B2、B3)；将各组置于37℃、180rpm条件下培养5h；离心并收集菌体，-80℃速冻后备用。
使用Trizol(Thermo公司)的试剂盒提菌体总RNA。提取后用超微量分光光度计(Implen,Munich,德国)检测RNA的纯度。每份RNA样品的A260/A280值应该在1.8～2.0之间。使用PrimeScript RTMaster Mix试剂盒(Takara，大连，中国)及ETC 811PCR仪(北京东胜创新生物科技有限公司进行反转录及实时荧光定量PCR扩增。q-PCR反应体系采用Takara的SYBR Premix Ex TaqII(Tli RNaseH Plus)(Code No.RR820A)，PCR程序为95℃预变性30s；95℃变性5s，60℃退火34s，40个循环；根据GenBank网站上已发布10个基因序列，利用Primer Premier 5.0软件设计出进行q-PCR的引物，同时将16SrRNA基因作为内参基因，引物序列参数见表1。
表1 实时荧光定量PCR使用的基因及其引物序列
Figure BDA0003124601540000041
Figure BDA0003124601540000051
实验结果：
不同浓度香叶醇作用下铜绿假单胞菌PAO1的生长曲线结果如图1所示。实验结果显示，香叶醇对PAO1细胞的生长具有浓度依赖性抑制作用，香叶醇浓度越高，抑菌作用越强。0.313、0.625、1.25和2.5μL/mL的香叶醇均轻微抑制PAO1的生长，尤以2.5μL/mL的香叶醇处理组抑菌活性最高。2.5μL/mL香叶醇处理组在对数生长期的细菌增殖速率明显低于其它组，但它的对数生长期比其它组长，稳定期很短，细菌过了延滞期后一直在缓慢增殖，然后进入衰亡期，导致33-41h这段时间2.5μL/mL香叶醇处理组的菌浓反而高于其它香叶醇处理组。总之，0.313、0.625、1.25和2.5μL/mL的香叶醇均轻微抑制了PAO1细胞的生长。
用1.25μL/mL香叶醇处理PAO1细胞5h之后，其群体感应系统关键基因和相关毒力基因的转录水平如图2所示。las系统的信号分子合成酶编码基因lasI的表达上调，信号分子受体蛋白编码基因lasR的表达下调。rhl系统的信号分子受体蛋白编码基因rhlR的表达下调，信号分子合成酶编码基因rhlI的表达水平变化不明显。pqs系统的信号分子合成酶编码基因pqsA的表达下调，信号分子受体蛋白编码基因pqsR的表达上调。毒力基因lasA、lasB、phzM和chiC的表达水平都显著下调，toxA、pslB和pelF的表达水平下调不明显。由此可见，香叶醇能够抑制铜绿假单胞菌的群体感应系统las和rhl的信号分子受体蛋白编码基因的表达，并抑制pqs系统信号分子合成酶编码基因的表达，进而抑制了这三个群体感应系统的调控通路，抑制了由这三个群体感应系统调控的毒力因子的产生，从而能够控制铜绿假单胞菌的毒力和致病力。
综上的实验结果表明，低浓度的香叶醇轻微抑制铜绿假单胞菌PAO1菌株的生长(图1)，但能够显著促进该菌3OC12-HSL信号分子合成。

Claims (4)

1.香叶醇在制备促进铜绿假单胞菌3OC12-HSL信号分子合成制剂中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的铜绿假单胞菌为铜绿假单胞菌(Pseudomonas aeruginosa)PAO1。
3.根据权利要求1所述的应用，其特征在于，所述的3OC12-HSL信号分子为该菌las群体感应系统的信号分子，由信号分子合成酶LasI合成。
4.根据权利要求1、2或3所述的应用，其特征在于，所述的香叶醇，其在铜绿假单胞菌培养液中的浓度为0.313-2.5μL/mL。
CN202110685800.1A 2021-06-21 2021-06-21 香叶醇在制备促进铜绿假单胞菌3oc12-hsl信号分子合成制剂中的应用 Active CN113430158B (zh)

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WO2022122051A1 (zh) * 2021-06-21 2022-06-16 广东省科学院微生物研究所(广东省微生物分析检测中心) 香叶醇在制备促进铜绿假单胞菌3oc 12-hsl信号分子合成制剂中的应用

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