CN105130963B - 一种新型群体感应抑制剂的分离提取、结构鉴定及其应用 - Google Patents
一种新型群体感应抑制剂的分离提取、结构鉴定及其应用 Download PDFInfo
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Abstract
本发明涉及一种由水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)中分离提取的具有群体感应淬灭活性的化合物及其应用。本发明是通过对海洋污泥来源的水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)进行发酵培养、分离纯化得到一种具有抑制紫色杆菌和铜绿假单胞菌群体感应系统活性的环二肽类单体化合物,其化学名称为环(L‑色氨酸‑L‑丝氨酸)。与传统抗菌药物的作用机制相比,本发明筛选到的活性化合物作用于一种新颖的抗菌靶点,只干扰细菌群体感应调控的致病行为,不影响细菌自身的生长,因此理论上不会产生细菌耐药性;化合物本身及其衍生物可以作为先导化合物指导开发新型抗菌药物应用于临床研究、农业生产等领域,对人类健康和农业发展具有重大的意义。
Description
技术领域
本发明属于微生物制药领域,具体涉及一种由海洋污泥来源的水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)的发酵培养、具有细菌群体感应抑制活性化合物的分离纯化、结构鉴定及其应用。
背景技术
传统的抗生素都是以细菌的细胞壁合成、蛋白质合成、叶酸合成、DNA的超螺旋等细菌的重要生命过程为靶点,直接杀死或者抑制微生物的生长来实现抗感染的目的,在这种生存压力下,细菌通过改变自身机制,很容易产生耐药性。解决细菌耐药性已成为人类面临的重大卫生挑战之一。近些年来研究发现,细菌群体感应(quorum sensing, QS)系统使作为单细胞生物的细菌,具备了部分类似于多细胞生物的功能,让细菌在应对环境挑战时,能协调一致,使细菌形成一种群体行为来有效的抵御外界环境压力、攻击宿主等。以细菌QS系统为靶标筛选的抗菌药物,与传统抗生素的作用机制完全不同,它不杀死细菌或抑制单个细菌的生长,只抑制细菌QS调控的致病行为,不易诱导耐药突变。
据统计,海洋微生物是天然活性物质的重要来源。海洋相对独特的高盐、高压、寡营养、低光照等复杂生态环境造就了海洋微生物独特的代谢机制,产生了众多的结构多样性和生物活性多样性的先导化合物。目前,已从海洋微生物中发现了大量的具有化学结构新颖、生物活性特异的化合物,其中有些已经作为药物广泛应用于临床。此外,海洋微生物资源丰富、易于培养,可通过人工发酵培养提供稳定的代谢产物,已成为一种重要的环境友好的微生物资源。
有关环(L-色氨酸-L-丝氨酸)化合物的文献报道较多,但是该化合物从水莱茵海默氏菌中分离得到未见有报道。本发明人研究得知,水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)(保藏编号为CCTCCNO:M2015245)液体发酵产物的甲醇提取物具有降低紫色杆菌紫色素产生的作用,进而对其化学成分进行了研究。研究发现所得化合物环(L-色氨酸-L-丝氨酸)在一定浓度范围内对紫色杆菌和铜绿假单胞菌都具有群体感应抑制活性,目前尚未见对该化合物的群体感应抑制活性的报道,因此市场上也尚未有与此相关的药物。
发明内容
本发明旨在提供一种由海洋污泥来源的水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)发酵提取物中分离纯化得到的具有抑制紫色杆菌和铜绿假单胞菌群体感应系统的活性化合物,该化合物在不抑制致病菌菌体生长的范围内能够显著的降低相关致病因子的表达,减弱致病菌的耐药性。
本发明所提供的活性化合物来源于海洋细菌水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02),该菌株于2015年04月23日保藏于中国武汉武汉大学的中国典型培养物保藏中心,保藏编号为CCTCCNO:M2015245;海洋污泥来源水莱茵海默氏菌QSI02经液体发酵、分离纯化得到具有细菌群体感应抑制活性的化合物环(L-色氨酸-L-丝氨酸),英文名称为:(3S,6S)-3-((1H-indol-3-yl)methyl)-6-(hydroxymethyl)piperazine-2,5-dione,分子式为C14H15N3O3,化学结构式如式。
本发明所述的活性化合物环(L-色氨酸-L-丝氨酸)的制备方法包括:海洋细菌水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)发酵培养与化合物的分离纯化两个步骤。将活化的菌种接种到海水培养基中进行摇床培养。发酵结束后,离心、真空低温浓缩至膏状,用甲醇浸提得甲醇提取物;将甲醇提取物用0.22 μm有机相滤膜过滤,采用紫外检测器(UV-900)与凝胶柱层析(Sephadex LH20)串联,以甲醇作为洗脱剂,分离为5个流份,对流份采用筛选模型进行活性检测,其中第4个活性洗脱流份再经过薄层层析制备,展开剂为水:正丁醇:乙酸乙酯=1:3:1,分离纯化得到具有抑制紫色杆菌和铜绿假单胞菌群体感应的单体化合物。
本发明涉及的活性化合物环(L-色氨酸-L-丝氨酸)具有抑制紫色杆菌和铜绿假单胞菌感染的作用;该化合物在0 ~ 0.3 mg/ml浓度范围内不抑制紫色杆菌CV026的菌体生长,但能显著的降低紫色杆菌紫色素的生成;并且随着浓度的逐渐增加,其抑制紫色素产生的作用越强;该化合物在0 ~ 0.4 mg/ml浓度范围内对铜绿假单胞菌PA01的菌体生长不产生影响,但能显著的降低铜绿假单胞菌群体感应调控的群集运动。
本发明所涉及的水莱茵海默氏菌QSI02(Rheinheimera aquimaris QSI02)菌株具有发酵培养条件可控,代谢产物产量稳定,化合物制备工艺过程简便的优点,同时所得化合物对群体感应抑制作用明显,并且环境友好。
附图说明
图1为本发明的活性化合物的活性筛选图。
图2为本发明不同含量的化合物对紫色杆菌CV026菌体生长的测定图。
图3为本发明不同含量的化合物对紫色杆菌紫色素产量的测定图。
图4为本发明化合物对铜绿假单胞菌PA01群集运动(swarming)现象的影响图。
具体实施方式
实施例1 化合物环(L-色氨酸-L-丝氨酸)的发酵生产及分离纯化。
1、发酵生产:海洋污泥来源水莱茵海默氏菌QSI02(Rheinheimera aquimarisQSI02)的发酵培养:按培养微生物的常规方法,取水莱茵海默氏菌QSI02(保藏编号是:CCTCCNO:M2015245)适量,接种到LB液体培养基(蛋白胨 10 g/L;酵母提取物 5 g/L;NaCl10 g/L,去离子水1 L,121℃,灭菌20 分钟)中,震荡培养12 h,温度28 ℃、转速160 rmp。
取上述培养12 h的水莱茵海默氏菌QSI02接种到装有2.5 L海水培养基(蛋白胨5g/L;酵母提取物 2 g/L;磷酸高铁0.1 g/L,0.22 μm微孔滤膜过滤的海水l L,121℃,灭菌20 分钟)的5 L锥形瓶中,接种量为4%,震荡培养24 h,温度28℃、转速160 rmp,获得发酵培养物。
2、浸膏的获得:上述发酵培养物用大型离心机将菌体离掉,并收集发酵液;然后将发酵液真空低温浓缩至膏状,用甲醇从膏状发酵物中浸提,真空浓缩得到粗提物浸膏, -4℃保存备用。
3、化合物的分离纯化:将上述获得的粗提物浸膏用甲醇溶解,再用0.22 μm有机相滤膜过滤,采用紫外检测器(UV-900)与凝胶柱层析(Sephadex LH20)串联,以甲醇作为洗脱剂,根据254 nm处紫外检测器检测结果洗脱分离为5个流份,对流份采用群体感应筛选模型进行活性检测,其中第4个活性洗脱流份再经过薄层层析制备,展开剂为水:正丁醇:乙酸乙酯=1:3:1,分离纯化得到单体化合物环(L-色氨酸-L-丝氨酸)。
4、化合物结构鉴定:综合利用紫外(UV)、红外(IR)、质谱(MS)、核磁共振谱(1H-NMR、13C-NMR、DEPT)等技术对化合物进行结构鉴定。紫外谱图在354 nm 、220 nm左右出现苯的特征吸收峰,推测该化合物可能含有苯环;红外谱图中给出羟基(3422 cm-1),氨基(3220cm-1),羰基(1638 cm-1)的官能团吸收;阳离子低分辨质谱在274处给出 [M+H]+峰,提示化合物的分子量为273,结合氢谱和碳谱分析,化合物的分子式为C14H15N3O3;1H-NMR、13C-NMR和DEPT谱数据提示该化合物中存在一个吲哚基团、一个羟基、两个酰胺基、两个亚甲基。综合上述所有数据,并与文献(J. Comb. Chem., 2006, 8(6): 915-922)比较,确定该化合物为环(L-色氨酸-L-丝氨酸)。
化合物1H-NMR数据:
1H-NMR (600MHz, DMSO-d 6, TMS, δ) δ H:10.88 (br s, 1H, 1-NH), 7.88(d,2H, 9-NH, 12-NH), 7.53(d, 1H, H-4 ), 7.32(d, 1H, H-7), 7.11(d, 1H, H-2), 7.04(m, 1H, H-6), 6.95(m, 1H, H-5), 4.90(m, 1H, 15-OH), 4.00(s, 1H, H-11), 3.67(s, 1H, H-8), 3.29-3.33(m, 1H, H-15a), 3.16-3.18(m, 2H, 14-CH2) , 3.03-3.05(m, 1H, H-15b)。
化合物13C-NMR数据:
13C-NMR (600MHz, CD3OH, TMS, δ) δ c:173.58, 174.51 (C=O, C-10, C-13),136.32(C-7a), 127.04 (C-3a), 127.45 (C-2), 122.31 (C-3), 118.44 (C-4), 119.46(C-5), 111.96 (C-7),122.17(C-6), 72.05 (C-11), 55.04(C-8), 62.49(C-15), 26.37(C-14).。
实施例2 化合物环(L-色氨酸-L-丝氨酸)的活性测试。
1、化合物环(L-色氨酸-L-丝氨酸)抑制紫色杆菌群体感应活性测试。
1.1 实验样品及实验方法:活性检测所用培养基为LB培养基,其固体培养基加入1.5%的琼脂。紫色杆菌的代谢产物紫色素是受群体感应系统严格调控的(Microbiology,1997, 143(12): 3703-3711),紫色杆菌CV026为信号分子缺失株,只有在加入外源性的信号分子己酰高丝氨酸内酯C6-HSL后,才能启动群体感应调控的紫色素的产生,当受到群体感应抑制因子的干扰时,则能抑制紫色素的产生,在琼脂板上表现出浑浊但不透明的晕圈。
紫色杆菌平板筛选方法:取20 ml 热融化的LB软琼脂培养基,待冷却至30℃左右,向其中加入200 µl 过夜培养的紫色杆菌CV026菌液、50 µl 信号分子C6-HSL(10 µmol/ml)及20 µl 卡那霉素(Kan, 50 mg/ml),轻轻混匀,倒入平板中,待凝固后,用打孔器打孔,将所分离提取物加入孔中(10 µl),并置于30℃恒温培养箱中培养20 h以上,观察现象。
紫色杆菌摇瓶筛选方法:挑取紫色杆菌CV026于新鲜的LB液体培养基中,30℃、160rpm培养至对数期;用新鲜LB液体培养基将上述菌液稀释至OD600nm=0.1,每瓶10 ml;分别加入不同体积的该化合物母液,并向培养基中分别加入50 μl C6-HSL和20 μl的卡那霉素,放入30℃、160 rmp的恒温摇床里培养24 h;以不加该化合物,但加有C6-HSL、卡那霉素和DMSO溶剂做对照。培养结束后,将菌体离心(转速4000 rmp,30 min),将上清液吸出,用1ml DMSO重新溶解,离心(4000 rmp,30 min),将上清液分别倒入已标记好的离心管中,然后分别依次取200 μl 上清液加入到96孔微板中,用酶标仪测585 nm的吸光度A585,以表征紫色杆菌紫色素的产量;第二次离心获得的沉淀,各用1 ml的无菌水重新悬浮菌体,然后依次取200μl菌体悬浮液加入到96孔微孔板中,用酶标仪测600 nm的吸光度OD600,以表征菌体生长密度的变化。
1.2 实验结果:紫色杆菌平板筛选:若该化合物为细菌群体感应抑制因子,则能够抑制紫色杆菌紫色素的产生,在琼脂平板上表现出浑浊但不透明的晕圈,筛选结果如图1所示:300 µl、250 µl、200 µl、150 µl、100 µl、50 µl的该化合物均能抑制紫色杆菌紫色素产生,且随着浓度的增加抑制效果增强。
紫色杆菌摇瓶筛选:以该化合物浓度为横坐标,以600 nm处的光吸收度为纵坐标,绘制紫色杆菌CV026在该化合物作用下24 h的菌体生长曲线(图2),该化合物在0 ~ 0.3mg/ml浓度范围内对紫色杆菌CV026的生长不产生影响;以该化合物浓度为横坐标,以585nm处的光吸收度为纵坐标,绘制紫色杆菌CV026在该化合物作用下24 h的紫色杆菌紫色素的产量(图3),该化合物在0 ~ 0.3 mg/ml浓度范围内能显著的降低紫色杆菌紫色素的产量,且随着浓度的增加,降低紫色杆菌紫色素产量的作用越明显,呈浓度依赖性,在加入100µl时降低量已经达到50%(图3)。
2、化合物环(L-色氨酸-L-丝氨酸)抑制铜绿假单胞菌群体感应活性测试。
2.1实验样品及实验方法:活性检测所用培养基为改良的swarming培养基:葡萄糖10 g;琼脂粉5 g;蛋白胨5 g;酵母浸粉2 g;溶于1 L 去离子水,121℃,灭菌20 min。铜绿假单胞菌是一种机会致病菌,可以引起急性和慢性感染,尤其是造成严重的囊肿性纤维化。铜绿假单胞菌毒力因子(生物被膜、绿脓菌素、群集、弹性蛋白 酶等)的表达均受到群体感应系统的调控。细菌的群集运动是一种细菌群体在半固体培养基表面快速、同步转移的复杂型运动(J. Bacteriol, 1999, 181(13): 4133-4136)。
群集运动(swarming)筛选方法:取4.8ml 热融化的swarming软琼脂培养基,待冷却至30℃左右,向其中加入200 µl 不同浓度的该化合物,轻轻混匀,倒入平板中,待凝固后,用打孔器打孔,将2 µl过夜培养的铜绿假单胞菌PA01(Pseudomonas aeruginosa PA01)(OD600nm=0.1)菌液加入孔中,37℃恒温培养24 h,观察现象。
2.2 实验结果:群集运动(swarming)筛选:若该化合物为细菌群体感应抑制因子,则能够抑制群体感应介导的毒力因子的表达,在琼脂平板上表现出群集运动减弱,筛选结果如图4所示:300 µl、100 µl的该化合物均能抑制铜绿假单胞菌的群集运动,且随着浓度的增加抑制效果增强。
3、结论:化合物环(L-色氨酸-L-丝氨酸)能够明显地抑制紫色杆菌和铜绿假单胞菌的群体感应活性,可作为先导化合物用于新型抗菌药物的研究以及治疗耐药性细菌引起的感染。
Claims (2)
1.一种式Ⅰ化合物的制备方法,所述方法包括如下步骤:
(1)发酵生产:海洋污泥来源水莱茵海默氏菌QSI02的发酵培养:按培养微生物的常规方法,取水莱茵海默氏菌QSI02适量,所述水莱茵海默氏菌QSI02的保藏编号是CCTCCNO:M2015245,接种到LB液体培养基中,震荡培养12h,温度28℃、转速160rmp,所述LB液体培养基为:蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,去离子水1L,121℃,灭菌20分钟;
取上述培养12h的水莱茵海默氏菌QSI02接种到装有2.5L海水培养基的5L锥形瓶中,接种量为4%,震荡培养24h,温度28℃、转速160rmp,获得发酵培养物;所述海水培养基为:蛋白胨5g/L,酵母提取物2g/L,磷酸高铁0.1g/L,0.22μm微孔滤膜过滤的海水lL,121℃,灭菌20分钟;
(2)浸膏的获得:上述发酵培养物用大型离心机将菌体离掉,并收集发酵液;然后将发酵液真空低温浓缩至膏状,用甲醇从膏状发酵物中浸提,真空浓缩得到粗提物浸膏,-4℃保存备用;
(3)化合物的分离纯化:将上述获得的粗提物浸膏用甲醇溶解,再用0.22μm有机相滤膜过滤,采用紫外检测器UV-900与凝胶柱层析Sephadex LH20串联,以甲醇作为洗脱剂,根据254nm处紫外检测器检测结果洗脱分离为5个流份,对流份采用群体感应筛选模型进行活性检测,其中第4个活性洗脱流份再经过薄层层析制备,展开剂为水:正丁醇:乙酸乙酯=1:3:1,分离纯化得到式Ⅰ化合物。
2.根据权利要求1所述制备方法制备的式Ⅰ化合物用于制备铜绿假单胞菌PA01群体感应活性抑制剂方面的用途,其特征在于该化合物在100μl和300μl时具有抑制铜绿假单胞菌PA01群集运动的活性。
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