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  南方医科大学学报  2019, Vol. 39Issue (3): 344-350  DOI: 10.12122/j.issn.1673-4254.2019.03.13.
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杜艳丽, 韩宗利, 王湘雨, 万成松. 大肠杆菌O157:H7体内多聚磷酸盐的DAPI荧光定量检测[J]. 南方医科大学学报, 2019, 39(3): 344-350. DOI: 10.12122/j.issn.1673-4254.2019.03.13.
DU Yanli, HAN Zongli, WANG Xiangyu, WAN Chengsong. A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic Escherichia coli O157:H7[J]. Journal of Southern Medical University, 2019, 39(3): 344-350. DOI: 10.12122/j.issn.1673-4254.2019.03.13.

基金项目

国家自然科学基金(81371765);广东省自然科学基金(2018B030311063, 2014A030313312)

作者简介

杜艳丽,博士,讲师,E-mail: 50189172@qq.com

通信作者

通信作者:
万成松,博士,教授,020-61648304,E-mail: gzwcs@smu.edu.cn

文章历史

收稿日期:2018-10-09
大肠杆菌O157:H7体内多聚磷酸盐的DAPI荧光定量检测
杜艳丽 1, 韩宗利 2, 王湘雨 3,4, 万成松 3     
1. 深圳职业技术学院医护学院,广东 深圳 518036;
2. 北京大学深圳医院神经外科,广东 深圳 518036;
3. 南方医科大学公共卫生学院 BSL-3实验室,广东 广州 510515;
4. 深圳大学第一附属医院,深圳市第二人民医院消化内科,广东 深圳 518036
摘要: 目的 应用荧光剂DAPI探索一种直接定量大肠杆菌O157:H7无机多聚磷酸盐(polyP)的可靠方法。方法 提取并纯化大肠杆菌O157:H7野生株DNA,DAPI与DNA、polyP45结合,测定360 nm和415 nm激发光的发射光谱;应用共聚焦显微镜,观察细菌DAPI-DNA和DAPI-polyP复合物荧光,验证DAPI检测polyP的可行性;细菌液氮速冻溶解、-80 ℃速冻溶解、-20 ℃速冻溶解、60 ℃加热10 min、Triton x-100作用、室温下放置后倍比稀释涂平板,观察6种方法对细菌存活的影响;与DAPI作用后,测定荧光值,确定细胞膜通透性的最佳前处理方法;建立标准曲线,测定EHEC野生株、ppk1两个突变株polyP含量。结果 应用360 nm激发波长,DAPI-DNA最大发射波长为460 nm;应用415 nm激发波长,DAPI-polyP最大发射波长为550 nm;应用405 nm激发光,收集DAPI-DNA蓝色荧光(425~475 nm),应用488 nm激发光,收集DAPI-polyP绿色荧光(500~560 nm);最佳细菌前处理方法为-80 ℃速冻后室温下自然溶解;标准曲线为Y=1849X+127.5(R2=0.991),测定各株菌polyP量,其中野生株显着高于ppk1缺失突变株。结论 DAPI荧光定量检测方法具有直接、可靠、易于操作、定量检测等特点,可为进一步研究polyP在肠出血型大肠埃希菌O157:H7中的作用提供技术支持。
关键词: 无机多聚磷酸盐    DAPI染色    大肠杆菌O157:H7    
A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic Escherichia coli O157:H7
DU Yanli 1, HAN Zongli 2, WANG Xiangyu 3,4, WAN Chengsong 3     
1. Department Medical Technology and Nursing, Shenzhen Polytechnic Institute, Shenzhen 518036, China;
2. Department of Neurosurgery, Peking University Shenzhen Hospital, Shenzhen 518036, China;
3. Biosafety Level-3 Laboratory, School of Public Health, Southern Medical University, Guangzhou 510515, China;
4. Department of Gastroenterology, First Affiliated Hospital of
Supported by National Natural Science Foundation of China (81371765)
Abstract: Objective To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7. Methods The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two ppk1 mutant strains. Results At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was Y=1849X+127.5 (R2=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with ppk1 deletion. Conclusion We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.
Keywords: polyP    DAPI staining    Enterohemorrhagic Escherichia coli O157:H7    

无机多聚磷酸盐(polyphosphate, polyP)是由数十至数百个磷酸盐残基通过"高能"磷酸键结合而成的线性多聚体[1], 它广泛存在于生物体中, 不仅影响原核生物稳定生长期的生存、致病性及严谨反应的调控[2-13], 还对真核生物的血栓形成、癌症发生、钙盐沉积、免疫应答等方面具有重要作用[14-18]。大肠杆菌多聚磷酸盐激酶(Polyphosphate kinase, PPK)能催化polyP的合成[19]。为了深入研究polyP在肠出血型大肠埃希菌(Enterohemorrhagic Escherichia coli, EHEC) O157:H7中的生物学功能, 建立一种简便、可靠且特异性高的polyP定性和定量的检测方法十分迫切。

目前, 用于测定polyP方法有:(1)酶和离子交换法, 需要细胞标记32Pi, 分离polyP, 测定时需用酵母提取外切聚磷酸酶(scPPX1), 并将其分解为磷酸盐残基[20];(2)荧光素测定法, 利用polyP合成酶(ppk1), 催化polyP脱磷酸后, 与ADP结合生成ATP, 再用荧光素测定[21]; (3)异染性染色法, 应用甲苯胺蓝染色细胞内polyP [22-24], 蓝色染料将polyP染成紫色, 适用于polyP定性。上述方法步骤繁琐, 仪器设备要求高, 缺乏特异性, 无法测定小于60个磷酸盐残基的polyP, scPPX1不易获得, 难以推广使用。本文拟采用4', 6-二脒基-2-苯基吲哚(DAPI)染色方法直接定量测定大肠杆菌O157:H7内polyP含量。

DAPI广泛应用于双链DNA染色, 在360 nm波长激发下, 最大发射波长460 nm, 在荧光显微镜下呈蓝光[25]。DAPI与DNA结合主要依靠A-T间氢键和脒基间氢键, 还有双螺旋DNA磷酸基团与两个带正电荷的DAPI之间的静电作用[25]

Allan等[26]首先在酿酒酵母菌中报道了用DAPI可以染色浓度比较高的polyP (50 μg/mL), DAPI-polyP在360 nm激发下呈黄绿色荧光。Aschar[27]优化了DAPI定量polyP的激发波长为415 nm、发射波长为550 nm, 应用酶标仪定量polyP。Diaz [28]将这种方法应用于强化的生物除磷过程(EBPR), 但仍采取先提取polyP再用DAPI定量的方式。Gomes等[29]将这种方法应用于寄生虫polyP半定量, 而应用DAPI定量致病性大肠杆菌polyP尚未见报道。

本研究旨在通过应用DAPI结合polyP形成DAPIpolyP复合物并发出黄绿色荧光的特征[26], 通过优化测定条件, 建立DAPI荧光染色法直接定量致病性大肠杆菌polyP的简便、可靠方法, 为深入研究polyP在肠出血型大肠埃希菌O157:H7致病过程中的作用提供技术支撑。

1 材料和方法 1.1 菌株和试剂

EHEC O157:H7 EDL933由中国疾病预防控制中心惠赠; EHEC O157:H7 EDL933 ppk1缺失株(Δppk1)、EHEC O157:H7 EDL933 ppk1回补株(Cppk1)由本实验室构建、保存。4-羟乙基哌嗪乙磺酸(HEPES)、DAPI(nos. D9542)、多聚磷酸钠标准品(PolypType45) (nos.S4379)(Sigma-Aldrich)。其余试剂均为分析纯。

1.2 方法 1.2.1 DAPI-DNA与DAPI-polyP复合物发射光谱的扫描

按照Magen细菌DNA提取试剂盒(Hi Pure Bacterial DNAKit)提取并纯化EHEC O157:H7 EDL933 WT株DNA。取其225 μL与HEPES溶液(20 mmol/L)675 μL混合, 加入DAPI(100 μmol/L) 100 μL, 充分混匀后, 室温避光放置10 min。取polyP标准品polyP45工作液(3 μmol/L) 225 μL与HEPES溶液(20 mmol/L) 675 μL混合, 加入DAPI (100 μmol/L) 100 μL, 充分混匀后, 室温避光孵育7.5 min, 再重复1次, 混匀、孵育。分别应用荧光分光光度计确定360 nm和415 nm激发光的发射光谱。

1.2.2 细菌前处理

EHEC O157:H7 WT株常规培养后, 测定WT株A600值, 用不含抗生素的LB液体培养基稀释为A600=1.0。取1.5 mL细菌悬液, 4 ℃ 10 000 r/min离心15 min, 弃上清。加入20 mmol/L HEPES(pH= 7.0)1 mL涡旋重悬细菌, 4 ℃ 10 000 r/min离心15 min, 弃上清。加入20 mmol/L HEPES (pH=7.0)1 mL涡旋重悬细菌。各取上述重悬菌液1 mL, 分别进行6种方法处理, 即液氮速冻后室温下自然溶解、-80 ℃速冻溶解、-20 ℃速冻溶解、60 ℃加热10 min、Triton x-100作用及室温下不作处理, 以确定最佳的细菌前处理方法(表 1)。将6种方法处理后的细菌取100 μL进行倍比稀释到106, 涂LB平板, 确定不同处理方法对细菌生存的影响。6种方法处理后的菌液在原管中充分涡旋混匀, 取300 μL加入1.5 mL EP管; 再加入600 μL 20 mmol/LHEPES溶液; 实验组再加入100 μL DAPI溶液, 对照组均加100 μL ddH2O代替DAPI。溶液充分涡旋混匀, 室温避光孵育10 min。96孔板上样, 每孔200 μL。避光, 酶标仪荧光模块测量荧光值。激发波长为415 nm, 发射波长为550 nm。根据荧光值确定最佳的细菌前处理方法。

表 1 细菌前处理方法改善细胞膜通透性 Tab.1 Six pretreatment methods for improving the permeability of cells
1.2.3 EHEC O157:H7 WT株polyP的共聚焦定性观察及发射光谱扫描

EHEC O157:H7 WT株常规培养后, 根据上一步确定的最佳细菌前处理方法处理细菌, 步骤同上。上述溶液充分涡旋混匀, 室温避光孵育10 min。离心, 4 ℃ 10 000 r/min离心3 min, 弃上清。HEPES洗1次后, 加入1 mL HEPES重悬。制作玻片, 取3~5 μL重悬液制作玻片, 置于激光共聚焦显微镜下, 60×油镜和10×目镜, 以405 nm和488 nm激发光激发, 用Nikon NIS Elements v4.5.观察荧光。余液应用荧光分光光度计确定360 nm和415 nm激发光的发射光谱。

1.2.4 polyP含量的直接测定

按前述步骤配制3 μmol/LpolyP45工作液。按照表 2比例配制polyP标准系列。其中ru指900 μL (HEPES液+polyP标准品)混合液中标准品的体积比。混匀后, 室温避光孵育7.5 min, 再重复1次。96孔板每孔上样200 μL, 每个浓度至少做2个平行孔。测定polyP荧光值, 激发波长415 nm, 发射波长550 nm。EHEC O157:H7 WT、△ppk1Cppk1均采用常规培养。

表 2 polyP标准系列配制 Tab.2 Dilution series of standard polyP45 for constructing the standard calibration curve

前处理后的菌液充分混匀, 取300 μL, 置1.5 mL EP管中, 再加入600 μL 20 mmol/L HEPES溶液; 实验组再加入100 μL DAPI溶液, 对照组均加100 μL ddH2O代替DAPI。溶液充分涡旋混匀, 室温避光孵育5~7 min。96孔板上样, 每孔上样200 μL。避光, 测量荧光值, 激发波长415 nm, 发射波长550 nm。根据标准曲线推算各株细胞内polyP含量。

1.2.5 统计学处理

计量数据用均数或均数±标准差描述, 6种处理方法及3株细菌polyP含量差异的比较应用单因素方差分析, 多重比较采用SNK and Bonferroni tests, 检验水准α=0.05。

2 结果 2.1 测定出DAPI-DNA与DAPI-polyP不同的最大发射波长

EHEC O157:H7 WT株、纯化WT株DNA分别与DAPI作用后, 应用360 nm激发波长, 获得DAPI-DNA的发射光谱(图 1AC)。再通过标准品polyP45、细菌WT株分别与DAPI作用后, 应用415 nm激发波长, 测定DAPI-polyP的发射光谱(图 1BD), DAPI-polyP的最大发射波长是550 nm, 而DAPI-DNA的最大发射波长是460 nm。

图 1 DAPI-DNA和DAPI-polyP的发射光谱 Fig.1 Emission spectra of DAPI-DNA and DAPI-polyP complexes. A: Emission spectra of DNA from the living EHEC WT with 360 nm excitation; B: Emission spectra of 0.25 relative unit (ru) of polyP 45 (equals 0.75 μmol/L P) excited at 415 nm; C: Emission spectra of living EHEC WT with 360 nm excitation; D: Emission spectra of living EHEC WT with 415 nm excitation.
2.2 比较发现-80 ℃速冻室温下自然溶解是最佳细菌前处理方法

6种处理方法作用WT株后, 倍比稀释涂平板, 观察对细菌存活的影响。结果显示(表 3), 6种处理方法对细菌存活具有显着性差异(F=37.91, P=0.000);多重比较结果显示, 常温、-20 ℃、-80 ℃、0.5%Triton x-100等4种方法的菌落数无显着性差异。而与常温组相比, 60℃作用10 min及液氮速冻后自然溶解这2种方法直接导致细菌数量减少(细菌存活平均菌落数分别是0和15.25×106 cfu), 尤其60℃细菌作用10 min后细菌全部死亡(表 3)。

表 3 6种细菌前处理方法对细菌WT株存活影响的比较 Tab.3 Comparison of wild-type strain survival after 6 pretreatments

对细菌存活无显着影响的4种处理方法与DAPI作用后, 测定并比较荧光值。结果显示, 4种处理方法的荧光值有显着性差异(F=78.67, P=0.000);多重比较, 结果发现最佳处理方法为-80 ℃速冻后室温下自然融化; 最差方法为室温下放置不作任何处理(表 4)。

表 4 4种细菌前处理方法与DAPI作用后荧光值比较(WT株菌) Tab.4 Comparison of fluorescence after 4 pretreatments and DAPI staining
2.3 细胞内polyP的定性观察

共聚焦显微镜观察, 结果显示, 当应用405 nm激发光, 在425~475 nm范围内收集到蓝色荧光, 为DAPIDNA。而当应用488 nm激发光时, 在500~560 nm范围收集到绿色荧光, 为DAPI-polyP (图 2)。

图 2 共聚焦显微镜观察DAPI-DNA和DAPI-polyP Fig.2 Confocal images of EHEC obtained with a Nikon A1R confocal microscope under the 60 × oil immersion objective with 10 × ocuLars. A: 405 nm laser line for detecting DNA (blue); B: 488 nm laser line for detecting polyP (green); C: No laser line for the Figure of cells; D: Merged images ofA, B and C. Scale bars: 2 μm
2.4 建立标准曲线

利用标准品polyP45建立荧光值与其浓度的标准曲线。结果显示, 反应体系中, 10 μmol/L DAPI与polyP45结合的最大量是0.25 ru。因此polyP45在0~ 0.25 ru范围内, 荧光值与其成线性关系, 而随着polyP45量的增加, 荧光值不再增加(图 3A)。由此, 得出荧光值与polyP45相对单位(ru)的标准曲线Y=5546X+127.5, R2=0.991 (图 3B)。进一步根据表 2的换算获得荧光值与polyP45浓度(μmol/L)的标准曲线为Y=1849X + 127.5, R2=0.991 (图 3C)。

图 3 polyP45标准曲线 Fig.3 The relationship and standard calibration curve between polyP45 and fluorescence (measured emission at 550 nm when excited at 415 nm) with a final DAPI concentration of 10 μmol/L in each sample. A: The fluorescence increased gradually at polyP45 0-0.25 relative unit (ru), while it remained relatively stable at 0.25 to 1.0 ru; B: Standard curve of polyP45 ru and fluorescence; C: Standard curve of polyP45 concentration (based on B) and fluorescence. Both B and C had the same intercept β0 and R2 value of the regression line.
2.5 各株菌polyP含量测定

各株细菌-80 ℃速冻, 室温下自然融化, 与DAPI作用, 测荧光值, 比较结果显示, 各株细菌polyP荧光值有显着性差异; 多重比较结果显示, Δppk1株的荧光值显着低于WT株和Cppk1株(表 5)。

表 5 各株细菌polyP荧光值比较 Tab.5 Comparison of fluorescence among different bacteria strains (EHEC WT, Δppk1 and Cppk1)

根据标准曲线, 推算1 mLA600=1.0的各株菌所含polyP量(表 6)。

表 6 各株菌polyP浓度 Tab.6 polyP concentration (μmol/L Pi) deduced through standard calibration curve
3 讨论

DAPI通常被用来检测细胞内DNA, 是否可应用于定性和定量检测大肠杆菌O157:H7内polyP尚不清楚。本研究通过荧光分光光度计测定固定激发波长时, DAPI-DNA及DAPI-polyP复合物的最大发射波长, 发现DAPI分别与polyP标准品polyP45及WT结合, 用415 nm波长激发时, 发射光谱最大波长是550 nm (图 1BD); 而DAPI-DNA复合物用360 nm激发, 最大发射波长是460 nm (图 1AC), 二者并无交叉。激光共聚焦显微镜观察发现, DAPI-DNA呈蓝色荧光, 而DAPI-polyP呈绿色荧光(图 2)。因此, 上述结果从定性和定量两方面说明DAPI可有效定量检测大肠杆菌polyP, 并可避免DNA的干扰。

应用DAPI直接定量polyP, DAPI可直接渗透入细菌内结合polyP, 而不需要将细菌杀死后提取polyP。polyP的DAPI荧光定量法和polyP提取法进行对比, 结果显示, polyP提取法比DAPI定量法低估了28~55% polyP含量[28, 30]

DAPI与polyP的作用机制可能为[25]: DAPI与带负电荷的离子相互作用, 尤其DAPI通过N-H基团产生静电吸引力。polyP是带负电荷的磷酰基团, 它作为阴离子吸引大量DAPI分子结合, 并引起染料间的相互作用。

Zink等[31]认为DAPI穿透活细胞的效率比较低。为了获得DAPI渗透入细胞最佳的细菌前处理方法, 改善细胞膜通透性, 使DAPI易于进入活细胞, 我们进行了6种细菌前处理方法的比较(表 1)。细菌经前处理后, 取等量菌液倍比稀释后涂LB平板, 观察前处理方法对细菌生存的影响。结果显示, 6种处理方法对细菌的存活具有显着性差异, 细菌存活菌落数显着不同。常温、-20 ℃、-80 ℃、0.5% Triton x-100四种前处理方法未明显影响细菌的存活; 而液氮速冻后再自然溶解的方法导致部分细菌死亡, 60 ℃细菌作用10 min后出现细菌全部死亡, 分解的细胞将影响DAPI结合polyP。此外, 加热导致长链polyP裂解(长度 < 5个Pi), DAPI也无法结合[28]

再将对细菌生存无显着影响的4种处理方法与DAPI作用后, 应用荧光分光光度计测定并比较荧光值。结果显示, 细菌前处理最好的方法是-80 ℃速冻后在室温下自然融化。另外, -20 ℃速冻后室温自然融化与-80 ℃的处理无显着性差异, 说明这些物理方法只是改变了细胞膜的通透性而未改变其形态; Triton x-100方法对于细菌渗透效果远没有真核细胞明显; 效果最差的处理方法是室温下不作任何处理。

DAPI荧光法定量检测方法的建立为polyP相关研究打下坚实基础。在大肠杆菌内, 多聚磷酸盐激酶(ppk1)催化polyP的合成, 为了验证本方法的适用性, 在EHEC O157:H7野生株(WT)基础上, 我们构建了ppk1基因缺失株(Δppk1)和回补株(Cppk1)。将3株细菌应用-80 ℃速冻自然融化, 进行DAPI染色, 测定荧光值进行比较。结果显示, 3株细菌荧光值有显着性差异; 多重比较显示, Δppk1株的荧光值显着低于WT株与Cppk1株, 说明ppk1基因缺失突变株细胞内显polyP的合成显着减少, 与理论预设相符, 也与致脑膜炎大肠杆菌K1 (RS218) [32]结果一致。

应用标准品polyP45建立的荧光值与标准品相对浓度的标准曲线, 即Y=1849X+127.5, R2=0.991说明自变量Y (荧光值)与因变量X (polyP标准品浓度)呈极线性相关。标准曲线的斜率和截距会随着不同实验室的实验条件而变化[28]。因此, 建议在不同实验室或应用不同的polyP标准品时构建新的标准曲线。

对于体外培养细菌, 我们建议用A600=1.0时1 mL细菌内polyP的浓度μmol/L Pi/mL为单位更为简单合理。将三株菌荧光值带入标准曲线, 换算成A600=1.0时1 mL菌液中polyP的浓度, 结果显示WT为4.25 μmol/L Pi/mL, Δppk1Cppk1分别为1.06和4.64 μmol/L Pi/mL。细菌浓度无差异的情况下, 3株的polyP浓度具有显着性差异。WT和Cppk1显着高于Δppk1株, 与预期结果一致。

鉴于此, 本研究应用DAPI与polyP结合发出不同于DAPI-DNA荧光的特点, 建立了肠出血型大肠埃希菌polyP的DAPI直接定量荧光检测方法, 为进一步研究polyP及其催化酶在该菌生存、致病、进化过程中的作用提供技术支持。

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