CN114088946B - 转录调控因子WalR和MgrA的亚硝基化修饰在治疗金黄色葡萄球菌感染中的应用 - Google Patents
转录调控因子WalR和MgrA的亚硝基化修饰在治疗金黄色葡萄球菌感染中的应用 Download PDFInfo
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Abstract
本发明通过蛋白质组学联合质谱鉴定发现金黄色葡萄球菌内源产生的一氧化氮可以引起自身蛋白质的亚硝基化修饰。实验证明重要转录调控因子WalR和MgrA的亚硝基化修饰有潜力成为万古霉素耐药治疗的靶点。
Description
技术领域
本发明属于基因工程技术领域,具体涉及转录调控因子WalR和MgrA 的亚硝基化修饰在治疗金黄色葡萄球菌感染中的应用,可以为临床金黄色葡萄球耐药菌的治疗提供新的靶点。
背景技术
金黄色葡萄球菌(Staphylococcus aureus)是人类主要的机会性细菌病原体,自然界中广泛存在,其感染可能引发肺炎、心内膜炎、骨髓炎,甚至脓毒血症等多种严重疾病。金黄色葡萄球菌的感染治疗主要依赖抗生素,然而抗生素的使用以及滥用催生并富集了耐药性菌株,尤其是耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcusaureus,MRSA)在医院和社区的广泛感染与传播已成为最难解决的感染性问题。多年来,MRSA 菌株多重耐药性的报道也屡见不鲜。万古霉素作为临床上治疗MRSA感染的最后一道防线,其中度抗性(vancomycin-intermediate Staphylococcus aureus,VISA)及抗性菌株(vancomycin-resistant Staphylococcus aureus, VRSA)的出现,为金黄色葡萄球菌的感染治疗提出了巨大挑战。因此寻找新型抗菌药物的靶标对开发新药物具有至关重要的作用。
在环境与宿主体内的长期进化过程中,金黄色葡萄球菌发展出了非常灵敏完善的环境刺激应答机制,其致病性和耐药性发生、特异性基因表达、与宿主相互作用等行为受到细菌胞内一系列调控体系和复杂网络的严密调控。例如气体信号分子(gasotransmitters)就是其中一种典型的重要调控单元。目前被广泛研究和报道的气体信号分子主要有三种:一氧化氮 (NO),一氧化碳(CO)和硫化氢(H2S)。
其中NO是一种双原子亲脂性气体分子,在细胞内以自由基的形式存在,可自由透过细胞膜,高浓度下具有细胞毒性,低浓度时具有信号转导和生理调控功能。NO作为信号分子首先在真核生物中被发现,可由一氧化氮合成酶(nitric oxide synthases,NOS)内源产生,参与调节各种生理及免疫功能。不仅如此,NO在细菌体内也发挥着重要作用。一方面,外源高浓度NO(宿主来源或NO供体)对细菌具有杀伤作用;另一方面,细菌在应对宿主高浓度NO压力时,不仅进化出一系列NO感应和防御机制,有的自身也能产生少量NO,尤其在某些革兰氏阳性菌如金黄色葡萄球菌中也存在可以催化产生NO的一氧化氮合成酶。低浓度NO反而对细菌具有重要的生理调控作用,如保护细胞抵抗氧化压力以及提高细菌的耐药性和致病性等。
NO作为生物活性分子,能对蛋白质半胱氨酸残基的巯基进行亚硝基化修饰产生亚硝基化硫醇SNO,进而影响蛋白质的活性和功能。目前还没有关于细菌内源NO对自身蛋白组进行修饰的报道,因此我们利用蛋白质修饰组学的方法鉴定了金黄色葡萄球菌中可被其内源NO亚硝基化修饰的蛋白质靶点,希望能为金黄色葡萄球菌VISA感染以及其他细菌病原体的感染治疗提供新的靶标和策略。
发明内容
本发明通过蛋白质组学联合质谱鉴定发现金黄色葡萄球菌内源产生的一氧化氮可以引起自身蛋白质的亚硝基化修饰。实验证明重要转录调控因子WalR和MgrA的亚硝基化修饰有潜力成为万古霉素耐药治疗的靶点。
本发明首先构建了nos敲除菌株,以抑制蛋白质半胱氨酸位点发生亚硝基化修饰,然后利用iodoTMT(irreversible isobaric iodoacetyl Cys-reactive tandem masstag)对野生型菌株中发生SNO亚硝基化修饰的蛋白质进行同位素标记并富集,然后结合基于质谱的蛋白质组学方法分析,并将nos敲除菌株与野生型菌株进行对比,以鉴定金黄色葡萄球菌中可被细菌内源 NO亚硝基化修饰的蛋白质靶点。根据蛋白质修饰组学的鉴定结果,选择了两个金黄色葡萄球菌的重要全局调控因子WalR和MgrA作为研究对象。 WalR属于二元信号***WalK/WalR的应答调节蛋白,对自溶、细胞壁代谢、生物被膜形成等生理过程具有重要调控作用。WalR蛋白共含有C33 和C67两个半胱氨酸残基,均在信号接收结构域(receiver domain)上,其中C67被质谱鉴定可被NO修饰;MgrA蛋白也是金黄色葡萄球菌中一个重要的全局调控因子,只含有C12位的一个半胱氨酸,且被质谱鉴定可被NO修饰(图1)。本发明构建了被鉴定修饰位点半胱氨酸残基的单点突变菌株,并研究了其对万古霉素敏感性的变化。结果表明,WalR中的亚硝基化修饰位点C67和MgrA中的亚硝基化修饰位点C12在发生突变后,无法被亚硝基化,导致金黄色葡萄球菌的耐药性下降,可以作为万古霉素耐药治疗的靶点。
在此基础上,一方面,本发明提出了WalR的亚硝基化修饰和/或MgrA 的亚硝基化修饰作为靶点在制备治疗金黄色葡萄球菌感染的药物中的应用。
另一方面,本发明提出了将WalR的亚硝基化修饰位点和/或MgrA的亚硝基化修饰位点的半胱氨酸修饰为不含巯基的氨基酸的试剂在制备治疗金黄色葡萄球菌感染的药物或试剂盒中的应用。
在一些实施方案中,所述WalR的亚硝基化修饰位点为C67,所述 MgrA的亚硝基化修饰位点为C12。
另一方面,本发明提出了对WalR蛋白的亚硝基化修饰位点和/或MgrA 蛋白的亚硝基化修饰位点进行修饰以防止亚硝基化修饰的试剂在制备治疗金黄色葡萄球菌感染的药物或试剂盒中的应用。
在一些实施方案中,所述WalR蛋白的亚硝基化修饰位点为C67,所述MgrA蛋白的亚硝基化修饰位点为C12。
又一方面,本发明提出了一种治疗金黄色葡萄球菌感染的药物组合物或试剂盒,包括突变诱导剂和抗生素,所述突变诱导剂能够将WalR的亚硝基化修饰位点和/或MgrA的亚硝基化修饰位点的半胱氨酸修饰为不含巯基的氨基酸。
在一些实施方案中,所述WalR的亚硝基化修饰位点为C67,所述 MgrA的亚硝基化修饰位点为C12。
又一方面,本发明提出了一种治疗金黄色葡萄球菌感染的药物组合物或试剂盒,包括亚硝基化抑制剂和抗生素,所述亚硝基化抑制剂能够对 WalR蛋白的亚硝基化修饰位点和/或MgrA蛋白的亚硝基化修饰位点进行修饰以防止亚硝基化修饰。
在一些实施方案中,所述WalR蛋白的亚硝基化修饰位点为C67,所述MgrA蛋白的亚硝基化修饰位点为C12。
在一些实施方案中,所述抗生素为万古霉素。
本发明的有益效果:
NO作为信号分子发挥调控功能的分子机制主要在真核生物中被广泛研究和报道;而在细菌中,其内源产生的NO也被报道可以影响细菌的环境适应性、压力应答、抗性、毒力等多种细胞行为,但其发挥功能的具体分子机制研究甚少。
本发明在临床来源的万古霉素中度耐药菌株XN108以及甲氧西林敏感菌株NCTC8325中发现金黄色葡萄球菌nos的缺失导致对万古霉素敏感性增加,表明金黄色葡萄球菌内源NO在万古霉素耐药发生过程中扮演了重要角色。
本发明利用蛋白质修饰组学的方法鉴定金黄色葡萄球菌中可被细菌内源NO亚硝基化修饰的蛋白质靶点。经对蛋白质修饰组学的鉴定结果分析,选择其中的两个金黄色葡萄球菌重要全局调控因子WalR和MgrA作为研究对象,并在VISA菌株XN108中构建了被鉴定修饰位点半胱氨酸残基的单点突变菌株:walR(C67S)和mgrA(C12S)(半胱氨酸被取代为丝氨酸)。表型实验发现walR和mgrA的突变均导致对万古霉素敏感性增加,Triton X-100诱导时自溶能力增强,细胞壁厚度下降。表明金黄色葡萄球菌内源 NO对自身重要全局调控因子WalR的C67位半胱氨酸和MgrA的C12位半胱氨酸残基的亚硝基化修饰与万古霉素耐药性的发生具有重要关系。有望为金黄色葡萄球菌及其他细菌病原体的感染治疗提供新型药物靶点和策略。
附图说明
以下附图仅旨在于对本发明做示意性说明和解释,并不限定本发明的范围。其中:
图1.WalR(A)、MgrA(B)蛋白功能结构域示意图及半胱氨酸残基位点,C67和C12表示被质谱鉴定可被NO修饰的半胱氨酸位点。
图2VISA菌株XN108中nos突变菌株的Triton X-100诱导的自溶能力检测(A)和溶葡萄球菌酶(lysostaphin)诱导的自溶能力检测(B);
图3.通过透射电子显微镜观察到的VISA菌株XN108中nos突变菌株的细胞壁厚度(A)和细胞壁厚度比较(B)。
图4.VISA菌株XN108中walR(C67S)(A)和mgrA(C12S)(B)突变菌株在Triton X-100诱导时均表现为自溶能力均增强;
图5.通过透射电子显微镜观察到的VISA菌株XN108中walR(C67S) 和mgrA(C12S)突变菌株的细胞壁厚度(A)和细胞壁厚度比较(B)。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,并参照附图,对本发明作进一步的详细说明。
本发明实施例中使用的培养基包括:
LB(Luria-Bertani medium)液体培养基:10g胰蛋白胨,5g酵母提取物,10g NaCl,溶于超纯水,定容至1000ml,分装为200ml每瓶,高压灭菌(121℃,20min)。
LB固体培养基:于LB液体培养基中添加1.5%(W/V)的Agar A。
TSB(Tryptic Soy Broth)液体培养基:3%(W/V)TSB培养基粉末, BD公司(Becto,Dickinson and company)。高压灭菌(121℃,15min)。
TSB固体培养基:于TSB液体培养基中添加1.5%(W/V)的AgarA。
MH液体培养基:2.2%(W/V)MH培养基粉末,BD公司。用于金黄色葡萄球菌MIC值检测。高压灭菌(121℃,10min)。
本发明实施例中使用的主要试剂包括:
(1)分子克隆相关试剂
EasyTaqTMDNA Polymerase(TransGen,AP111)用于一般PCR检测;
HS DNA Polymerase(TaKaRa,DR010A)用于克隆操作中DNA片段的高保真扩增;
DNA连接酶:T4 DNA连接酶(Thermo,EL0012);
胶回收纯化试剂盒:GeneStar StarPrep Gel Extraction Kit StarPrep;
质粒小量试剂盒:GeneStar StarPrep Gel Extraction Kit StarPrep;
基因组抽提:使用GeneStar质粒小量试剂盒,仅P3换为苯酚:氯仿:异戊醇(25∶24∶1),其他不变;
电泳琼脂糖凝胶:1.0-2.0%(W/V)Agarose溶于1×TAE,煮沸冷凝;
50×TAE:2M Tris-Acetate,PH 8.5,100mM EDTA;
Gel-red染液:Gel-red母液1∶10000溶解于0.1M NaCl;
6×DNA loading buffer:36%甘油,0.05%二甲苯青FF,0.05%溴酚蓝, 30mMEDTA;
DNAladder:100bp,1kb(TransGen)。
(2)金黄色葡萄球菌表型实验相关试剂
溶葡萄球菌工作液(2X LSA):20μg/ml溶葡萄球菌酶(ABMI, LSPN-50),10mg/ml溶菌酶,20%(V/V)甘油)甘油,用于裂解金黄色葡萄球菌细胞壁;
50mM Tris-HCl(pH 7.5)缓冲液;
Triton X-100(Solarbio,T8200)。
本发明实施例中使用的主要仪器包括:
PCR仪(Biolab;Applied Biosystem),电泳槽,凝胶成像仪(Tanon1600),恒温水浴锅(上海精宏,DK-8D),恒温摇床(上海智诚),恒温培养箱(上海智诚),超净台,生物安全柜(LABCONCO),电转化仪(BioRad, MicroPulser),酶标仪(BioTek),分光光度计(Beckman,DU800),低温离心机(Eppendorf,Centrifuge 5430R;Beckman,microfuge 20R),常温离心机(Eppendorf,Centrifuge 5424),Step One Real-Time PCR仪(ABI),超低温冰箱(Thremo),常规冰箱(-20℃、-4℃),漩涡震荡仪,烘箱,灭菌锅等。
本发明实施例中使用的质粒为:
本发明实施例中使用的菌株包括:
本发明实施例中实验菌株的相关操作如下:
1菌株的保藏与培养条件
所有实验菌株验证无误后,均在18%的甘油中保藏在-80℃冰箱。一般情况下,大肠杆菌使用LB培养基进行培养。液体培养条件为37℃摇床,转速为220rpm;划线或涂布于LB固体培养基时培养条件为37℃恒温培养箱中平板倒置静止培养。金黄色葡萄球菌使用TSB培养基进行培养。液体培养条件为37℃摇床,转速为220rpm;划线或涂布于TSB固体培养基时培养条件为37℃恒温培养箱中平板倒置静止培养。根据细菌携带的质粒特性选择加入相应的抗生素。正常情况下,氨苄青霉素(Ampicillin)终浓度为150μg/ml,卡那霉素(Kanamycin)终浓度为50μg/ml,氯霉素 (Chloramphenicol)终浓度为15μg/ml。对金葡菌进行最小抑浓度检测时,使用MH培养基。进行基因敲除时,温度依据具体操作更改。
2大肠杆菌感受态制备
(1)取-80℃保藏的Trans T1菌株划线于LB空白平板培养基上,静置培养。
(2)挑单克隆于LB空白液体培养基中,37℃震荡培养过夜。
(3)取经过16-20小时培养的新鲜菌液,1∶100转接入含0.5%NaCl 的LB液体培养基的三角瓶中,220rpm,37℃培养约2-3小时至OD600 为0.3-0.5。
(4)5000g,4℃,15min离心收菌,弃上清,将离心管倒置数秒使残留的少量培养液流尽。
(5)使用等体积预冷的0.1M MgCl2-CaCl2(80mM MgCl2+20mM CaCl2)重悬,冰上放置约30min,中间轻柔颠倒混匀。
(6)5000g,4℃,15min离心收菌,弃上清,将离心管倒置数秒使残留的少量培养液流尽。
(7)每50ml初始菌液用2ml预冷的0.1M CaCl2重悬,并迅速将重悬后的细胞分装成100μl每管于灭菌的EP管中,-80℃贮存备用。
3大肠杆菌化学转化
(1)取-80℃冻存的感受态细胞静置于冰上融化,加入连接产物或质粒(外源DNA体积不大于感受态细胞体积的1/10),轻弹混匀,冰上静置 30min。
(2)将EP管置于42℃的水浴锅中热激45-60s,该过程不要摇动试管。
(3)立即将EP管转移到冰浴中静置2-3min。
(4)加入300-500μl LB无抗液体培养基,转移于37℃摇床复苏1h。
(5)将复苏后的细胞涂布于LB抗性平板,并倒置于37℃恒温箱培养至长出合适大小的菌落(通常12-16小时即可)。
4金黄色葡萄球菌感受态制备
(1)取-80℃保藏的金黄色葡萄球菌冻存菌株划线于TSB空白平板培养基上,静置培养。
(2)挑单克隆于TSB空白液体培养基中,37℃震荡培养过夜。
(3)取经过16-20小时培养的新鲜菌液,1∶100转接入经过滤灭菌的 TSB液体培养基的三角瓶中,220rpm,37℃培养约2-3小时至OD600为 0.3-0.6。
(4)在无菌条件下将细胞转移至50ml离心管中,冰上放置15min 后,5000g,4℃,15min离心收菌,弃上清,将离心管倒置数秒使残留的少量培养液流尽。
(5)使用等体积预冷的0.5M蔗糖重悬菌体,冰上放置约15min后, 5000g,4℃,15min离心收菌,弃上清。
(6)再次使用1/2体积预冷的0.5M蔗糖重悬菌体,冰上放置约30min 后,5000g,4℃,15min离心收菌,弃上清,将离心管倒置数秒使残留的少量培养液流尽。
(7)每50ml初始菌液用1ml预冷的0.5M蔗糖溶液重悬,并迅速将重悬后的细胞分装成100μl每管于EP管中,-80℃贮存备用。
5金黄色葡萄球菌电转化
(1)取-80℃冻存的感受态细胞静置于冰上融化,加入不大于感受态细胞体积的1/10体积待转化的质粒,轻弹混匀,冰上静置30min。
(2)轻轻将感受态细胞转移至电极杯中,电转仪电击,电转仪通常设置转化参数为:电压2.5kV,电容50μF,电阻200欧姆。
(3)电击之后,立即加入500μl TSB空白液体培养基,静置10min。
(4)将点击后的细胞转移至EP管中复苏1h,复苏温度通常为37℃,温敏型质粒如pBTs复苏温度为30℃。
(5)将复苏后的细胞涂布于TSB抗性平板,并倒置于37℃恒温箱培养至长出合适大小的菌落。
本发明实施例中质粒构建的一般方法包括:使用特异性引物对目的序列进行扩增,使用对应的限制性内切酶处理表达载体和片段,T4 DNA连接酶进行连接,连接产物转化至大肠杆菌克隆载体Trans T1,用对应载体引物进行PCR筛选阳性克隆并测序验证,抽提质粒转入目的菌株。金黄色葡萄球菌转入质粒时,先转化进RN4220进行修饰,再转入目的宿主菌株。
本发明实施例中使用的引物如下所示:
实施例1.nos敲除菌株的构建:
nos基因用于编码NOS蛋白(一氧化氮合成酶),由它产生NO,进而介导蛋白质半胱氨酸位点发生亚硝基化修饰。
本实施例采用目标基因无痕敲除技术,以温敏型质粒pBTs为载体构建同源重组质粒,方法如下:使用HS DNA Polymerase (TaKaRa,DR010A)从XN108基因组上,用bNOS-up-F和bNOS-up-R, bNOS-down-F和bNOS-down-R分别扩增nnos基因上下游同源臂片段,通过PCR进行overlap连接上下游得到组合片段,使用对应的快速限制性内切酶KpnI(Thermo,FD0524)和SacI(Thermo,FD1133)分别处理载体 pBTs和片段,回收纯化后使用T4 DNA连接酶(Thermo,EL0012)进行连接,连接产物转化至大肠杆菌Trans T1克隆载体,此时使用终浓度为150 μg/ml的氨苄青霉素LB培养基,37℃培养。使用EasyTaqTMDNAPolymerase (TransGen,AP111),利用载体引物pBTS-F和pBTS-R进行PCR筛选出带有正确质粒的阳性克隆并测序检验。抽提质粒先转化至RN4220进行修饰,再转化至XN108菌株,此时均使用终浓度为15μg/ml的氯霉素TSB 培养基,30℃培养。
该实施例中突变菌株的具体诱导及筛选步骤如下:
a)将含有同源重组质粒的目的菌株划线挑单克隆,TSB氯霉素(15 μg/ml)抗性液体培养基中,30℃过夜培养。
b)1:100转接至TSB无抗液体培养基,220rpm,42℃高温诱导同源重组,24小时转接一次,共转接3次。
c)TSB氯霉素抗性平板划线,42℃过夜培养。
d)挑单克隆至30℃TSB无抗液体培养基中,220rpm,30℃培养,24 小时转接一次,共转接2次。
e)取30℃培养的菌液,用无菌水稀释至合适梯度后涂布于含有脱水四环素(anhydrotetracycline,ATC,100ng/ml)的TSB固体平板上,稀释倍数以涂布后长出克隆数为100-500CFU为宜,一般稀释105-107倍即可, 30℃过夜培养。
f)从平板上挑单克隆到96孔板(TSB无抗液体培养基)中,200rpm, 30℃培养至细菌完全生长。
g)使用复制器转接至加有TSB氯霉素抗性液体培养基的96孔板,200 rpm,30℃培养至细菌完全生长。
h)两组96孔板中对照比较,选取无抗培养基中生长氯霉素抗性培养基中不生长的克隆,即为疑似突变株。
若没有疑似突变株,可从步骤(c)中的氯霉素平板上挑取单克隆重复步骤(d-h)。也可增加步骤(b)在42℃中的传代次数。
i)抽提疑似突变株的基因组,使用引物bNOS-up-F和bNOS-down-R 进行PCR检验并测序。
实施例2.可被细菌内源NO亚硝基化修饰的蛋白质靶点的鉴定:
该实施例利用iodoTMT(irreversible isobaric iodoacetyl Cys-reactivetandem mass tag)对发生SNO亚硝基化修饰的蛋白质进行同位素标记并富集,然后结合基于质谱的蛋白质组学方法分析,并将nos敲除菌株与野生型菌株进行对比,以鉴定金黄色葡萄球菌中可被细菌内源NO亚硝基化修饰的蛋白质靶点。iodoTMT标记(thermo,90100、90101、90102、90103) 和蛋白质组学均由景杰生物公司完成。
实施例3.氨基酸点突变菌株的构建:
菌株XN108中walR(C67S)和mgrA(C12S)单点突变菌株的构建,均采用目标基因无痕敲除技术,以温敏型质粒pBTs为载体构建同源重组质粒,方法如下:
使用HS DNA Polymerase(TaKaRa,DR010A)从XN108 基因组上,用walR-C67S-up-F和walR-C67S-up-R,walR-C67S-down-F和walR-C67S-down-R分别扩增walR基因第67位半胱氨酸(C67)上下游同源臂片段;用mgrA-C12S-up-F和mgrA-C12S-up-R,mgrA-C12S-down-F 和mgrA-C12S-down-R分别扩增mgrA基因第12位半胱氨酸(C12)上下游同源臂片段。分别通过PCR进行overlap连接上下游得到组合片段。 walR(C67S)的上下游组合片段使用快速限制性内切酶KpnI(Thermo, FD0524)和EcoRI(Thermo,FD0274)分别处理载体pBTs和片段; mgrA(C12S)的上下游组合片段使用快速限制性内切酶KpnI(Thermo,FD0524)和SalI(Thermo,FD0644)分别处理载体pBTs和片段。将酶切后的片段回收纯化后使用T4 DNA连接酶(Thermo,EL0012)进行连接,连接产物转化至大肠杆菌Trans T1克隆载体,此时使用终浓度为150μg/ml 的氨苄青霉素LB培养基,37℃培养。使用EasyTaqTMDNAPolymerase(TransGen,AP111),利用载体引物pBTS-F和pBTS-R进行PCR筛选出带有正确质粒的阳性克隆并测序检验。抽提质粒先转化至RN4220进行修饰,再转化至XN108菌株,此时均使用终浓度为15μg/ml的氯霉素TSB 培养基,30℃培养。
该实施例中突变菌株的具体诱导及筛选步骤同“实施例1.nos敲除菌株的构建”中的诱导及筛选步骤。使用引物walR-C67S-up-F和 walR-C67S-down-R,mgrA-C12S-up-F和mgrA-C12S-down-R分别对 walR(C67S)和mgrA(C12S)单点突变疑似菌株进行PCR检验并测序,突变后的walR和mgrA基因序列分别为SEQ ID NO:15和SEQ ID NO:16。
实施例4.抗生素敏感性测定
液体稀释法MIC值检测法:无抗平板划线培养18-24小时,挑6-10个 nos敲除菌株和氨基酸点突变菌株克隆分别重悬于1ml MH液体培养基中,调整测OD600值至OD0.4(菌落数约为2×108CFU/ml),取10μl菌液加入1990μl MH培养基中(稀释200倍,菌落数约为1×106CFU/ml),1∶1 接种到含有不同抗生素浓度梯度的MH培养基中(菌落数约为5×105 CFU/ml),使用96孔板培养24-48h后观察细菌生长情况。
实施例5.细菌自溶能力检测
(1)活化后的nos敲除菌株和氨基酸点突变菌株单克隆细菌培养液 1∶100转接至含有10m1 TSB培养基的三角瓶中,37℃,220rpm震荡培养至OD600≈0.8。
(2)离心收菌,Tris-HCl缓冲液洗细胞三次,适量含有0.2%(V/V) Triton X-100(Solarbio,T8200)或200ng/ml溶葡萄球菌酶(lysostaphin)(ABMI,LSPN-50)的Tris-HCl(pH 7.5)缓冲液重悬菌体,并使OD600 值一致。
(3)每隔30min使用酶标仪检测OD600值,并与初始值计算自溶率。
实施例6金黄色葡萄球菌细胞壁透射电子显微镜观察
(1)1200g离心收集培养过夜的nos敲除菌株和氨基酸点突变菌株,小心去除上清;
(2)用1ml pH7.2PBS重悬细菌两次,1200g离心再次收菌;
(3)沿管内壁缓慢加入1ml 2.5%戊二醛到细菌沉淀上进行样品的前固定(不可吹打悬浮),4℃固定12~24h。
(4)利用透射电子显微镜观察nos敲除菌株和氨基酸点突变菌株的细胞壁厚度。
结果
(1)nos突变导致金黄色葡萄球菌万古霉素敏感性增强
在构建的nos敲除株中,MIC结果显示nos突变导致万古霉素抗性下降(表1)。我们对该突变株进行了一些基础表型研究,发现其自溶能力明显增强(图2),细胞壁厚度有所下降(图3)。我们在万古霉素敏感的MSSA 菌株NCTC8325中也构建了nos敲除株,发现nos突变也可导致其对万古霉素抗性敏感性增加(MIC由2μg/mL降到1ug/mL)(表1)。这些结果表明金黄色葡萄球菌bNOS在万古霉素抗性发生过程中扮演了重要角色。
表1.nos突变菌株抗生素敏感性检测
(2)mgrA和walR突变均导致对万古霉素敏感性增加
为探究细菌内源NO引起的SNO亚硝基化修饰对靶蛋白生物学功能的影响,对获得的walR(C67S)和mgrA(C12S)的突变菌株进行了研究。表型实验发现walR和mgrA的突变均导致对万古霉素敏感性增加(表2),在Triton X-100诱导时均表现为自溶能力增强(图4),细胞壁厚度下降(图 5)。表明walR和mgrA的突变位点与万古霉素耐药性及细胞壁代谢调控具有重要关系。
表2.walR和mgrA突变菌株抗生素敏感性检测
以上实验表明,WalR和/或MgrA的亚硝基化修饰与万古霉素耐药性相关,当亚硝基化修饰被抑制后,耐药性降低。因此,WalR和/或MgrA 的亚硝基化修饰可以为临床金黄色葡萄球耐药菌的治疗提供新的靶点。通过诱导WalR和/或MgrA的亚硝基化修饰位点突变为不含巯基的氨基酸或将WalR和/或MgrA蛋白的亚硝基化修饰位点进行修饰,以防止所述亚硝基化修饰位点被亚硝基化修饰,可以降低金黄色葡萄球菌耐药性的方法。因此,通过施用突变诱导剂诱导WalR和/或MgrA的亚硝基化修饰位点突变为不含巯基的氨基酸或者通过施用亚硝基化抑制剂将WalR和/或MgrA 蛋白的亚硝基化修饰位点进行修饰,以防止所述亚硝基化修饰位点被亚硝基化修饰后,再施用抗生素可以用来治疗金黄色葡萄球菌感染,尤其是耐药金黄色葡萄球菌的感染。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一种提高金黄色葡萄球菌对抗生素敏感性的方法,包括通过施用突变诱导剂诱导WalR蛋白和/或MgrA蛋白的亚硝基化修饰位点突变为不含巯基的氨基酸,
其中,所述WalR蛋白的亚硝基化修饰位点为C67,所述MgrA蛋白的亚硝基化修饰位点为C12,突变后的walR基因和mgrA基因序列分别为SEQ ID NO:15和SEQ ID NO:16,所述抗生素为万古霉素。
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