参见作者原研究论文

本实验方案简略版
Aug 2019
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Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria
革兰阴性菌中内外膜通透性的同期测定   

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Abstract

The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the membrane-permeabilizing effect and bactericidal efficacy of antibacterial molecules. This study presents an optimized protocol with respect to the classical protocols by incubating bacteria with antibacterial molecules in the culture condition identical to that of antibacterial assays and then detecting the signal of the fluorescent probe in the buffer without broth and antibacterial molecules. This protocol avoids the effect of nutrient deficiency on the physiological status of bacteria and the interference of antibacterial molecules towards the fluorescent probe. Thus, this method can effectively and precisely evaluate the membrane permeability and match the results obtained from other antibacterial assays, such as minimum inhibitory concentration and time–kill curve assays.

Keywords: Gram-negative bacteria (革兰氏阴性菌), Antimicrobial peptides (抗菌肽), Outer and inner membrane permeability (内外膜通透性), N-phenyl-1-naphthylamine (N-苯基-1-萘胺), Propidium iodide (碘化丙啶)

Background

Multidrug resistance in bacteria is a major public health crisis. Gram-negative bacteria currently pose the greatest threat to public health because of the emergence and rapid spread of carbapenem resistance. Consequently, new antibacterial molecules must be identified to combat this urgent problem. The outer membrane (OM) of gram-negative bacteria is not only the target of several traditional antibiotics to exert antibacterial activities but also of novel therapeutic agents, such as antimicrobial peptides (AMPs). These molecules can disrupt the OM integrity and cause bacterial lysis through permeabilization. Thus, detecting the permeability of the outer and inner membranes is a direct and important means of assessing the efficacy of antibacterial molecules. Electron microscopy is usually applied to observe the morphological changes in OM, and the assay of minimal bactericidal concentration is used to examine bacterial viability after treatment. However, these methods are time-consuming and cannot reflect the real-time change in membrane permeability. In this protocol, we provide a convenient and fast approach for evaluating the antibacterial efficacy of agents with potential membrane-permeabilizing effect by using the fluorescent probes N-phenyl-1-naphthylamine (NPN) and propidium iodide (PI).

NPN is a hydrophobic dye that dissolves sparingly in water with very low fluorescent emission. However, the fluorescence intensity increases sharply when NPN binds with nonpolar substances. The intact OM efficiently blocks NPN out of bacteria to ensure that NPN cannot bind to the hydrophobic tail of phospholipids. By contrast, a strong fluorescence emission can be detected when an OM rupture occurs. Thus, the change in fluorescence intensity of NPN can reflect the efficacy of antibacterial molecules on increasing the permeability of OM. PI is a red-fluorescent nucleic acid stain that can bind to DNA and RNA between the bases. Binding to DNA and RNA leads to an enhancement of PI fluorescence by 20- to 30-fold compared with that in aqueous solutions. Given that PI is a membrane-impermeant stain, it can only label bacteria with a compromised inner membrane (IM). Thus, PI is used in this protocol to determine the change in IM permeability after the treatment of antibacterial molecules.

In most of the related studies, the bacterial OM/IM permeability assay is conducted in a 96-well optical-bottom black plate. Bacteria, fluorescent probe, and antibacterial molecules are mixed together and co-cultured throughout the assay, during which the fluorescence intensity is detected at each time point. To avoid the high background fluorescence emitted by the bacterial culture media, the assay buffer (5 mM HEPES, pH 7.2) is used instead. In this case, bacteria grow under an innutritious condition compared with that of the antibacterial assays such as minimum inhibitory concentration and time–kill curve assays, which may affect the physiological status of bacteria. And some antibacterial molecules may also interfere with the function and performance of the fluorescent probe. As a result, the assay may not exactly reflect the change in permeability caused by the antibacterial molecules. The current work presents an optimized procedure with respect to the previously reported protocols (Ma et al., 2016b; Yarlagadda et al., 2016; Krishnamurthy et al., 2019) by culturing bacteria and exposing them to antibacterial molecules in the same medium as those of other antibacterial assays, harvesting bacteria, and detecting the fluorescent yield in the assay buffer at each time point. This methodology can evaluate the membrane permeability more efficiently and precisely and better match the results obtained from the antibacterial assays.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml Eppendorf tube (Eppendorf Safe-Lock, catalog number: 0 22363204 )
  3. Steritop 0.22 μm filter unit (Millipore Millex-GP, catalog number: SLGP033RB )
  4. Flat-bottomed polystyrene 96-well cell culture plates, 0.2 ml well-volume (Corning, Costar®, catalog number: 3599 )
  5. Glass culture tube 20 mm x 150 mm (sterilized by autoclaving)
  6. 50 ml glass conical flask (sterilized by autoclaving) (ShuNiu, China)
  7. E. coli XJ141026 (Isolated from Xijing Hospital)
    Note: Other types of E. coli can also be used.
  8. Luria–Bertani (LB) broth (BD/Difco, catalog number: 244620 )
  9. N-Phenyl-1-naphthylamine (NPN) (Sigma-Aldrich, catalog number: 104043 )
  10. Propidium iodide (PI (Sigma-Aldrich, catalog number: P4170 )
  11. Thanatin (synthesized and purified to over 98%)
    Note: Thanatin was synthesized with the solid-phase method by applying the Fmoc (9-fluorenylmethyloxycarbonyl) active ester chemistry of as described previously (Fehlbaum et al., 1996). Other AMPs of the experimenter’s choice could also be used instead of thanatin.
  12. Glucose (Sigma-Aldrich, catalog number: G8270 )
  13. 1 M HEPES solution (Sigma-Aldrich, catalog number: H0887 )
  14. 10x PBS stock (Life Technologies, Gibco®, catalog number: 70011-044 )
  15. Acetone (Sigma-Aldrich, catalog number: 650501 )
  16. Milli-Q filtered water (ddH2O)
  17. 5 mM HEPES and 5 mM glucose buffer (pH = 7.2) (see Recipes)

Equipment

  1. Pipettes
  2. Shaking incubator (Zhicheng, model: ZHWY-200D )
  3. Static incubators (Taisite Instrument, model: DH4000BII )
  4. Microplate spectrophotometer (BioTek, model: PowerWave HT )
  5. Tabletop centrifuge machine (Hanil Science Industrial, model: Smart R17 )
  6. Fluorescence spectrophotometer (Hitachi, model: F-2500 )
  7. Water purification system (Millipore, model: Milli-Q Advantage A10 )
  8. Quartz cuvettes with 1 cm path length (Starna Cells, catalog number: 3-Q-10 )

Software

  1. Gen 5 (Biotek, USA)
  2. FL Solutions 2.0 (Hitachi, Japan)
  3. Excel 2016 (Microsoft, USA)
  4. Prism 8.0 (GraphPad, USA)

Procedure

  1. Sample preparation for the membrane permeability assay
    1. Inoculate a single colony of E. coli XJ141026 into 5 ml LB broth in a glass culture tube and incubate it overnight in a shaking incubator at 220 rpm at 37 °C.
    2. Take 0.1 ml of stationary growth phase of the E. coli XJ141026 culture and inoculate 10 ml of the LB broth in a 50 ml glass conical flask. Allow the culture to grow for 12 h at 37 °C and 220 rpm.
    3. Add 100 μl of LB broth to 8 wells of the 96-well microtiter plate. Transfer 100 μl of the culture to the first well and make serial twofold dilutions in the LB broth (final volume of 100 μl per well). Determine the OD600 of each well via the microplate spectrophotometer.
    4. Calculate the dilution factor according to the above serial OD600 values. Dilute a portion of the remaining culture to OD600 = 0.1 (determined by using the microplate spectrophotometer with a sample volume of 100 μl) in the LB broth to obtain 40 ml diluted bacterial suspension.
    5. Transfer the diluted bacterial suspension into two 50 ml glass conical flasks, 15 ml per group.
    6. Add 12 μl of 1,000 μM thanatin (dissolved in sterile PBS) to one of the 15 ml bacterial suspensions, in which the final concentration of thanatin is 0.8 μM. Add an equal volume of sterile PBS (pH = 7.2) to the other flask and use as the untreated control. Mix well and grow at 37 °C and 220 rpm.
    7. Collect the samples of thanatin-treated and untreated cultures at the different time intervals of 0, 30, 60, 120, 180, and 240 min. For each group, 2 ml culture was collected and separately added into two 1.5 ml Eppendorf tubes, 1 ml per tube.
    8. Pellet the cells at 8,935 x g for 10 min in 1.5 ml Eppendorf tubes, and remove the supernatant.
    9. Wash the cells through resuspension in 1 ml of 5 mM HEPES and 5 mM glucose buffer (pH = 7.2) and the pellet cells via centrifugation at 8,935 x g for 5 min. Remove the supernatant.
    10. Repeat Step A9 twice.
    11. Resuspend the cells in 1 ml of 5 mM HEPES and 5 mM glucose buffer (pH = 7.2).

  2. Outer membrane permeability
    1. To determine the outer membrane permeability of thanatin, add 8 μl of 500 μM NPN (dissolved in acetone) to 1 ml resuspended cells (A11) and vortex well.
    2. Incubate in darkness for 30 min at room temperature.
    3. Turn on the fluorescence spectrometer, and allow the lamp to warm up for 30 min.
    4. Define the operating parameters of the spectrometer as follows: photomultiplier tube (PMT) voltage: 700 V, excitation wavelength: 350 nm, and emission wavelength: 420 nm.
      Note: The PMT voltage settings are machine- and sample-dependent. To prevent the fluorescence signal values from exceeding the measurement range, the appropriate PMT voltage for the experiments must be established before a series of experiments is started. To set the appropriate PMT voltage, collect and stain the antimicrobial agent-treated bacteria at the last time point of the experiment, detect the fluorescence signal using different PMT voltages, and choose the optimal PMT voltage that can maximally amplify the signal in the detector range.
    5. Carefully clean a fluorescence cuvette with water and ethanol.
    6. Transfer 1 ml of the incubated culture to the cuvette and place it in the spectrometer.
    7. Start the data acquisition and record.

  3. Inner membrane permeability
    1. To determine the inner membrane permeability of thanatin, add 5 μl of 1 mM PI (dissolved in sterile ddH2O) to 1 ml of resuspended cells (A11).
    2. Incubate in darkness for 30 min at room temperature.
    3. Define the operating parameters of the spectrometer as follows: PMT voltage: 700 V, excitation wavelength: 535 nm, emission wavelength: 617 nm.
    4. Carefully clean the fluorescence cuvette with water and ethanol.
    5. Transfer 1 ml of the incubated culture to the cuvette and place it in the spectrometer.
    6. Start the data acquisition and record.

Data analysis

  1. Repeat the experiment three times independently.
  2. Import all the detected values into the GraphPad Prism 8.0 to visualize them graphically. As shown in Figure 1, time and relative fluorescence intensity are depicted in the x- and y-axes, and grouped comparison of untreated control vs. thanatin-treated.
  3. Use a two-way ANOVA to analyze the data between the different groups.
  4. OM integrity is damaged by Thanatin in a time-dependent manner, and a distinct increase in the fluorescence intensity of NPN is observed 1 h post-incubation (Figure 1A).
  5. The increase in fluorescence intensity of PI approximately 2 h post-incubation indicates the increase in IM permeability of the bacteria (Figure 1B).
  6. The basal fluorescence (Control group in Figure 1) also increases with time, which may due to the increasing number of aging and dead bacteria along with culture time. Thus, the final fluorescence data in the thanatin-treated group are normalized by subtracting the fluorescence values of the corresponding control group at each time point (Figure 2).


    Figure 1. Outer and inner membrane permeabilization of thanatin (Tha) is measured by detecting the fluorescence intensity of NPN (A) and PI (B). All the data are shown as the mean ± SEM of the three independent experiments. P-values were determined via two-way ANOVA; ***P < 0.001 vs. untreated control (Ma et al., 2019).


    Figure 2. Outer and inner membrane permeabilization of thanatin is measured by detecting the fluorescence intensity of NPN (A) and PI (B) after normalization. All the data are shown as the mean ± SEM of the three independent experiments. P-values were determined via one-way ANOVA; ***P < 0.001.

Recipes

  1. 5 mM HEPES and 5 mM glucose buffer (pH = 7.2)
    1 M HEPES solution is diluted to 5 mM HEPES with ddH2O (pH = 7.2)
    Dissolve 90.08 mg glucose in 100 ml of 5 mM HEPES (pH = 7.2)
    Sterilize using a 0.22 μm filter

Acknowledgments

This protocol was adapted from previous work (Ma et al., 2016a; Ma et al., 2019). We thank Xiuli Xu and Shan Zhou for providing us with clinical strain from the Department of Clinical Laboratory Medicine of Xijing hospital. This work was supported by grants from the National Natural Science Foundation of China (no. 81673477, 81903671, 81471997 and 81001460).

Competing interests

The authors declare no conflict of interest.

References

  1. Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hetru, C. and Hoffmann, J. A. (1996). Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides. Proc Natl Acad Sci U S A 93(3): 1221-1225.
  2. Krishnamurthy, M., Lemmon, M. M., Falcinelli, E. M., Sandy, R. A., Dootz, J. N., Mott, T. M., Rajamani, S., Schaecher, K. E., Duplantier, A. J. and Panchal, R. G. (2019). Enhancing the antibacterial activity of polymyxins using a nonantibiotic drug. Infect Drug Resist 12: 1393-1405.
  3. Ma, B., Fang, C., Lu, L., Wang, M., Xue, X., Zhou, Y., Li, M., Hu, Y., Luo, X. and Hou, Z. (2019). The antimicrobial peptide thanatin disrupts the bacterial outer membrane and inactivates the NDM-1 metallo-beta-lactamase. Nat Commun 10(1): 3517.
  4. Ma, B., Niu, C., Zhou, Y., Xue, X., Meng, J., Luo, X. and Hou, Z. (2016a). The disulfide bond of the peptide thanatin is dispensible for its antimicrobial activity in vivo and in vitro. Antimicrob Agents Chemother 60(7): 4283-4289.
  5. Ma, L., Wang, Y., Wang, M., Tian, Y., Kang, W., Liu, H., Wang, H., Dou, J. and Zhou, C. (2016b). Effective antimicrobial activity of Cbf-14, derived from a cathelin-like domain, against penicillin-resistant bacteria. Biomaterials 87: 32-45.
  6. Yarlagadda, V., Manjunath, G. B., Sarkar, P., Akkapeddi, P., Paramanandham, K., Shome, B. R., Ravikumar, R. and Haldar, J. (2016). Glycopeptide Antibiotic To Overcome the Intrinsic Resistance of Gram-Negative Bacteria. ACS Infect Dis 2(2): 132-139.

简介

[摘要 ] 细菌中多药耐药性的出现和迅速扩散导致对新型抗菌剂的迫切需求。膜通透性是许多针对革兰氏阴性细菌的抗菌分子的机制。因此,为了确定潜在的抗菌分子的功效,重要的是评估治疗后细菌膜通透性的变化。这项研究描述了使用荧光探针N-苯基-1-萘胺和碘化丙啶测定外膜和内膜通透性的方案。与其他实验(例如电子显微镜和最小杀菌浓度的测定)相比,该方法提供了一种更简单,更快速且经济高效的方法来评估抗菌分子的膜通透性和杀菌功效。这项研究提出了一种相对于传统方案的优化方案,该方案是在与抗菌测定相同的培养条件下,将细菌与抗菌分子一起孵育,然后在不含肉汤和抗菌分子的缓冲液中检测荧光探针的信号。此协议可避免的效果养分缺乏对细菌的生理状态和抗菌molecul的干扰ES 朝向的F luorescent探针。因此,该方法可以有效,准确地评估膜的渗透性,并与从其他抗菌测定法(如最小抑菌浓度和时间杀灭曲线测定法)获得的结果相匹配。

[背景 ] 在细菌中多药耐药性是一个重大的公共卫生危机。革兰氏阴性细菌由于对碳青霉烯类药物的耐药性的出现和迅速传播,目前对公共卫生构成最大威胁。因此,必须确定新的抗菌分子来解决这一紧急问题。革兰氏阴性细菌的外膜(OM)不仅是几种传统抗生素发挥抗菌活性的目标,而且还是新型治疗剂(例如抗菌肽(AMPs))的目标。这些分子可破坏OM完??整性并通过通透性引起细菌裂解。因此,检测外膜和内膜的渗透性是评估抗菌分子功效的直接和重要手段。通常使用电子显微镜观察OM的形态变化,并使用最小杀菌浓度的分析方法来检查处理后的细菌生存力。但是,这些方法很耗时,不能反映出膜渗透率的实时变化。在该协议中,我们通过使用荧光探针N-苯基-1-萘胺(NPN)和碘化丙啶(PI),提供了一种方便,快速的方法来评估具有潜在膜通透作用的药物的抗菌功效。

NPN是一种疏水性染料,在荧光发射极低的情况下可微溶于水。但是,当NPN与非极性物质结合时,荧光强度会急剧增加。完整的OM可有效阻止NPN进入细菌,以确保NPN不能与磷脂的疏水尾结合。相反,当发生OM破裂时,可以检测到强烈的荧光发射。因此,NPN荧光强度的变化可以反映抗菌分子对增加OM渗透性的功效。PI是一种红色荧光核酸染色剂,可以与碱基之间的DNA和RNA结合。与水溶液相比,与DNA和RNA的结合导致PI荧光增强20到30倍。鉴于PI是一种不渗透膜的污渍,它只能用受损的内膜(IM)来处理细菌。因此,在该协议中使用PI来确定抗菌分子处理后IM渗透性的变化。

在大多数相关研究中,细菌OM / IM渗透性测定是在96孔光学底黑色板上进行的。在整个测定过程中,将细菌,荧光探针和抗菌分子混合在一起并共培养,其间在每个时间点检测荧光强度。为避免细菌培养基发出的高背景荧光,可使用测定缓冲液(5 mM HEPES,pH 7.2)代替。在这种情况下,与抗菌测定(例如最小抑菌浓度和时间杀灭曲线测定)相比,细菌在营养条件下生长,这可能会影响细菌的生理状态。并且某些抗菌分子也可能干扰荧光探针的功能和性能。结果,该测定法可能不能准确反映出由抗菌分子引起的通透性变化。通过对细菌进行培养并将其暴露于与细菌相同的培养基中的抗菌分子,目前的工作针对先前报道的方案(Ma 等,2016b ; Yarlagadda 等,2016 ; Krishnamurthy 等,2019)提出了一种优化程序。那些其它的抗菌试验,公顷的rvesting细菌,并检测在每个时间点测定缓冲液荧光产率。这种方法可以更有效,更准确地评估膜的通透性,从而更好地匹配抗菌测定的结果。

关键字:革兰氏阴性菌, 抗菌肽, 内外膜通透性, N-苯基-1-萘胺, 碘化丙啶

材料和试剂


 


P ipette提示
1.5毫升Eppendorf管(Eppendorf Safe-Lock,货号:022363204)
Steritop 0.22 微米过滤装置(Millipore 的Millex -GP,目录号:SLGP033 RB )
平底聚苯乙烯96 - 孔细胞培养板,0.2毫升公体积(康宁,Costar公司? ,目录号:3599)
玻璃培养管20 mm x 150 mm(通过高压灭菌器灭菌)
50 ml玻璃锥形烧瓶(高压灭菌)(中国牛市)
大肠杆菌XJ141026 (西京医院分离)
注意:也可以使用其他类型的大肠杆菌。


Luria–Bertani(LB)汤汁(BD / Difco ,目录号:244620)
N-苯基-1-萘胺(NPN)(Sigma-Aldrich,目录号:104043)
碘化丙啶(PI (Sigma-Aldrich,目录号:P4170)
Thanatin(合成和纯化至98%以上)
注意:通过应用如前所述的Fmoc (9-芴基甲氧羰基)活性酯化学方法,通过固相法合成了他纳汀(Fehlbaum et al。,1996)。也可以使用实验者选择的其他AMP代替thanatin。


葡萄糖(Sigma-Aldrich,目录号:G8270 )
1 M HEPES溶液(Sigma-Aldrich,目录号:H0887)
10X PBS股票(Life Technologies公司,GIBCO ? ,目录号:70011-044)
甲cetone (Sigma-Aldrich公司,目录号:650501 )
的Milli-Q 过滤器ED 水(DDH 2 O)
5 mM HEPES和5 mM葡萄糖缓冲液(pH = 7.2)(请参阅食谱)
 


设备


 


移液器
小号克竞保温箱(志成,型号:ZHWY-200D)
小号tatic孵化器(泰斯特仪器,型号:DH4000BII)
中号icroplate分光光度计(BioTek的,型号:PowerWave的HT)
台式离心机(Hanil Science Industrial,型号:Smart R17)
?F luorescence分光光度计(日立,型号:F-2500)
水净化系统(Millipore,型号:Milli-Q Advantage A10)
路径长度为1厘米的Q uartz比色皿(Starna Cells,目录号:3-Q-10)
 


软件


 


第五代(美国Biotek )
FL Solutions 2.0 (日本日立)
Excel 2016(Microsoft,美国)
棱镜8 .0(美国GraphPad )




程序


 


膜渗透性测定的样品制备
在玻璃培养管中将大肠杆菌XJ141026 的单个菌落接种到5 ml LB肉汤中,并将其在振荡培养箱中以2 2 0 rpm在37°C 下孵育过夜。
取0.1 ml 大肠杆菌XJ141026培养物的固定生长期,并在50 ml玻璃锥形烧瓶中接种10 ml LB肉汤。使培养物在37°C 和2 2 0 rpm下生长12 h 。
100添加微升的LB肉汤,以8个孔的96孔的微量滴定板。传送1 0 0 微升的培养物以第一井,并在LB进行连续的两倍稀释液(100的最终体积微升每孔)。确定OD 6 0 0 的经由微孔板分光光度计以及每个。
根据上述系列OD 6 0 0 值计算稀释倍数。稀释剩余的培养物的一部分,以OD 6 0 0 = 0.1 (通过使用与样品体积微孔板分光光度计测定1 0 0 微升)在LB肉汤以获得40毫升稀释的细菌悬浮液。
转移稀释细菌悬浮液中,以2个50ml的玻璃锥形瓶中,每组15毫升。
加入12 微升的1 ,000 μM 的泛素(溶解于无菌PBS中)至15毫升细菌悬浮液,其中的最终浓度之一死亡素是0.8 μM 。将等体积的无菌PBS (pH = 7.2)加入另一个烧瓶中,并用作未处理的对照。充分混合并在37 °C 和220 rpm下生长。
分别在0、30、60、120、180和240分钟的不同时间间隔收集经坦那坦处理和未经处理的培养物的样品。对于每个组,2毫升培养物收集和分开添加在到2支1.5毫升的微量试管,加入1ml每管。
P ellet 吨他细胞在8935 X 克1 0 在1.5ml Eppendorf管分钟,并除去上清液。
通过重悬于1 ml 5 mM HEPES和5 mM葡萄糖缓冲液(pH = 7.2)中洗涤细胞,并通过以8,935 xg离心5分钟离心沉淀细胞。除去上清液。
重复步骤A9两次。
在1 ml 5 mM HEPES和5 mM葡萄糖缓冲液(pH = 7.2)中重悬细胞。
 


外膜通透性
为了确定出的ER膜渗透性死亡素,添加8 微升500 μM NPN(溶解在一cetone )至1 ml的[R esuspend 编细胞(A11)和涡流井。
在室温下于黑暗中孵育30分钟。
打开荧光光谱仪,让灯预热30分钟。
如下定义光谱仪的工作参数:光电倍增管(PMT)电压:700 V,激发波长:350 nm,发射波长:420 nm。
注意:PMT电压设置取决于机器和样品。为了防止荧光信号值超出测量范围,必须在开始一系列实验之前为实验确定适当的PMT电压。要设置适当的PMT 电压,在实验的最后一个时间点收集和染色经抗菌剂处理的细菌,使用不同的PMT电压s 检测荧光信号,并选择最佳的PMT电压以最大程度地放大检测器中的信号范围。


用水和乙醇小心清洁荧光比色杯。
将1 ml培养的培养物转移到比色杯中,并将其放入光谱仪中。
开始数据采集和记录。
 


内膜通透性
要确定的内膜渗透性死亡素,添加5 微升1mM的的PI (溶解于无菌DDH 2 O) ,以1 ml的为r的esuspend ED 细胞(A11)。
我在黑暗中于室温下孵育30分钟。
如下定义光谱仪的操作参数:PMT电压:700 V,激发波长:535 nm,发射波长:617 nm。
用水和乙醇小心清洁荧光比色杯。
将1 ml培养的培养物转移到比色杯中,并将其放入光谱仪中。
开始数据采集和记录。
 


数据分析


 


独立地重复实验三遍。
将所有检测到的值导入到GraphPad Prism 8.0中以图形方式对其进行可视化。如图1所示,时间和相对荧光强度分别在x轴和y轴上进行了描述,并对未处理的对照组与经唐宁处理的组进行了比较。
ü 知的双因素ANOVA 吨?分析不同组之间的数据。
OM完整性通过Thanatin基因以时间依赖的方式损坏,并且在荧光强度明显增加的NPN 观察到1个小时后的培养(图URE 1A)。
孵育后约2小时,PI的荧光强度增加表明细菌的IM渗透性增加(图1B )。
基础荧光(图1中的对照组)也随时间增加,这可能是由于衰老和死亡细菌的数量随培养时间的增加而增加。因此,通过在每个时间点减去相应对照组的荧光值,将唐那汀处理组的最终荧光数据归一化(图2)。
 


D:\ Reformatting \ 2020-1-6 \ 1902939--1307郑厚821825 \ Figs jpg \图1.jpg


图1. 通过检测NPN(A)和PI(B)的荧光强度来测量thanatin (Tha )的内外膜通透性。所有数据均显示为三个独立实验的平均值±SEM 。通过双向方差分析确定P 值;*** 与未处理的对照相比,P <0.001 (Ma 等,2019)。


 


D:\ Reformatting \ 2020-1-6 \ 1902939--1307郑厚821825 \ Figs jpg \图2.jpg


图2 。通过在归一化后检测NPN(A)和PI(B)的荧光强度来测量坦那汀的外膜和内膜通透性。所有数据均显示为三个独立实验的平均值±SEM 。通过单向方差分析确定P 值;*** P <0.001 。


 


菜谱


 


5 mM HEPES和5 mM葡萄糖缓冲液(pH = 7.2)
用ddH 2 O (pH = 7.2)将 1 M HEPES溶液稀释至5 mM HEPES


将90.08 mg葡萄糖溶于100 ml 5 mM HEPES(pH = 7.2)


消毒使用0.22 微米过滤器


 


致谢


 


该协议改编自以前的工作(Ma 等,2016a;Ma 等,2019)。感谢徐秀丽和周珊为西京医院临床检验医学科提供的临床菌株。这项工作得到了中国国家自然科学基金(No. 81673477、81903671、81471997和81001460)的资助。


 


利益争夺


 


作者宣称没有利益冲突。


 


参考文献


 


Fehlbaum ,P.,Bulet ,P.,Chernysh ,S.,Briand,JP,Roussel,JP,Letellier ,L.,Hetru ,C.和Hoffmann,JA(1996)。thanatin的结构活性分析,thanatin是一种21残基可诱导的昆虫防御肽,与青蛙皮肤抗菌肽具有序列同源性。PROC国家科科学院科学USA 93(3):1221至1225年。
克里希纳穆尔蒂,M.,Lemmon的,MM,Falcinelli的,EM,桑迪,RA,Dootz ,JN,莫特,TM,Rajamani ,S.,Schaecher ,KE,Duplantier ,AJ和潘卡尔,RG(2019)。使用非抗生素药物增强多粘菌素的抗菌活性。感染抗药性12:1393-1405。
马斌,方成,陆琳,王明,薛雪,周艳。,李敏,胡彦,罗雪,侯正( 2019)。抗菌肽thanatin破坏细菌外膜并使NDM-1金属β-内酰胺酶失活。Nat Commun 10(1):3517。
Ma B.,Niu,C.,Zhou,Y.,Xue,X.,Meng,J.,Luo X. and Hou,Z.(2016a)。肽thanatin的二硫键由于其在体内和体外的抗菌活性是必需的。抗微生物剂Chemother 60(7):4283-4289。
Ma L.,Wang,Y.,Wang,M.,Tian,Y.,Kang,W.,Liu,H.,Wang,H.,Dou,J. and Zhou,C.(2016b)。源自cathelin样域的Cbf-14对耐青霉素的细菌的有效抗菌活性。生物材料87:32-45。
Yarlagadda,V.,Manjunath,GB,Sarkar,P.,Akkapeddi,P.,Paramanandham,K.,Shome,BR,Ravikumar,R.和Haldar,J.(2016)。糖肽抗生素可以克服革兰氏阴性细菌的内在抗性。ACS 传染病2(2):132-139。

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引用:Ma, B., Fang, C., Zhang, J., Wang, M., Luo, X. and Hou, Z. (2020). Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria. Bio-protocol 10(5): e3548. DOI: 10.21769/BioProtoc.3548.
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