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May 2019
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A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells
基于Fura-2-AM探针的荧光光度法测定丁香假单胞菌复合菌胞内Ca2+水平   

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Abstract

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.

Keywords: Fura 2-AM (Fura2-乙酰氧基甲酯), Cytosolic calcium concentration (胞质钙离子浓度), Spectrophotometer (分光光度计), Pseudomonad (假单胞菌), Live cell signaling (活细胞信号)

Background

Ca2+ is an emerging intracellular messenger of bacteria that impacts a wide array of cellular processes such as the maintenance of cell integrity, cell division (Dominguez et al., 2015), motility (Tisa and Alder, 1995; Gode-Potratz et al., 2010; Cruz et al., 2012; Guragain et al., 2013; Parker et al., 2015; Fishman et al., 2018), type III secretion (DeBord et al., 2003; Dasgupta et al., 2006; Gode-Potratz et al., 2010; Fishman et al., 2018), gene expression (Dominguez et al., 2015), quorum sensing (Werthén and Lundgren, 2001), biofilm formation (Patrauchan et al., 2005; Sarkisova et al., 2005; Rinaudi et al., 2006; Cruz et al., 2012; Das et al., 2014; Zhou et al., 2014; Parker et al., 2016) or biofilm suppression (Bilecen and Yildiz, 2009; Shukla and Rao, 2013). Recently, it was demonstrated that the intracellular Ca2+ concentration controls virulence of Pseudomonas savastanoi pv. savastanoi (Psav) (Moretti et al., 2019). Furthermore, several known virulence genes were upregulated in the presence of increasing Ca2+ concentrations in Pseudomonas syringae pv. tomato (Pto) DC3000 (Fishman et al., 2018), and Xylella fastidiosa (Parker et al., 2016). Measurement of the intracellular free-calcium concentration, [Ca2+]i, in prokaryotes has, therefore, become of great interest to study its role as intracellular messenger of bacteria in response to environmental cues. Monitoring the [Ca2+]i within bacterial cells, which is indispensable for understanding the correlations between the transport of Ca2+ across the plasma membrane and cellular processes was thus far difficult, as it was hampered by the small size of the bacterial cells, the semi-selective nature of the bacterial cell wall, the low membrane permeability, and the toxicity of many Ca2+ chelators used (Gangola and Rosen, 1987; Knight et al., 1991; Futsaether and Johnsson, 1994; Norris et al., 1996; Herbaud et al., 1998; Jones et al.,1999; Torrecilla et al., 2001). A well-established method to determine changes in the [Ca2+]i in prokaryotes is based on the heterologous expression of Aequorin (a calcium-activated photoprotein) bacterial cells (Watkins et al., 1995). This method employs the expression of recombinant Aequorin (from a plasmid or integrated in the bacterial genome), which emits light upon Ca2+ binding. This method is rather time-consuming, requires the availability of molecular biology tools, and is technically challenging. In fact, the method is better suited for eukaryotic cells even if it was successfully used to monitor [Ca2+]i in several bacterial species (Naseem et al., 2007; Guragain et al., 2016). To overcome these limitations of Aequorin, we developed an alternative and complementary spectrofluorometric method based on the chemical probe Fura 2-AM {1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2’-amino-5’-methylphenoxy) ethan-N,N,N’,N’-tetraacetic acid}. Importantly, both the assay solution used and the Fura 2-AM do not compromise cell viability at the concentrations here used (Gangola and Rosen, 1987; Futsaether and Johnsson, 1994; Tisa and Alder, 1995; Norris et al., 1996; Jones et al., 1999), meaning that this method allows quantification of [Ca2+]i in response to external cues and different conditions without the need of advanced equipment (Moretti et al., 2019). It must be pointed out that Fura 2-AM is a probe that diffuses across the cell membrane of viable bacterial cells and its subsequent rapid de-esterification by cellular esterases yields Fura 2, which retains the ability to bind the cytosolic Ca2+ while it losses the ability to diffuse across the cell membrane (Figure 1). When Fura 2 forms a complex with Ca2+, the intensity of the fluorescence at λ=510 nm increases with increasing Ca2+ concentration (Grynkiewicz et al., 1985) (Figure 2). In addition, Fura 2 is unable to permeate bacterial cells itself due to the selective permeability of the cell walls and membranes (Grynkiewicz et al., 1985). Of notice, since the measurements make use of a fluorescence signal that only becomes apparent inside cells (when Fura 2-AM is converted to Fura 2), it is not necessary to use unloaded viable cells as negative control. In fact, if the cells are not viable, the cell membrane loses its integrity and the probe would not be trapped in the cells but rather remain dispersed in the incubation medium.



Figure 1. The fate of Fura 2-AM in cells. Fura 2-AM is de-esterified by cellular esterases and transformed in Fura 2, which is able to form a complex with cytosolic calcium (Ca2+) and cannot passively cross the cell membrane.



Figure 2. Excitation spectra of Fura 2. Excitation spectra of Fura 2 in solution containing 0 to 39.8 µM of free calcium (Ca2+). Modified from Grynkiewicz et al. (1985).


We find that the fluorescent probe Fura 2-AM is highly sensitive allowing us to determine changes in cytosolic Ca2+ levels in Pseudomonas savastanoi pv. savastanoi DAPP-PG 722 (Moretti et al., 2019) and Pseudomonas syringae pv. tomato DC3000 (Trabalza et al., in preparation) cells by using a spectrofluorometer equipped with a stirred semi-micro cuvette.

Materials and Reagents

  1. Inoculation loops 10 µl (Laboindustria S.P.A., catalog number: 21131 )

  2. Petri dish Ø 90 (Laboindustria S.P.A., catalog number: 21050 )

  3. Pipette tips (Mettler Toledo, catalog numbers: 17007956, 17007952 )

  4. High clarity polypropylene (PP) conical centrifuge tube 50 ml (Falcon, catalog number: 352070 )

  5. NIR Quartz SUPRASIL 300 Rectangular Macro Cell with Lid, volume 3.5 ml (PerkinElmer, catalog number: B0631015 )

  6. PIREX Media bottles, graduated, Corning, 500 ml (VWR, PIREX, catalog number: 1395-500 )

  7. Pseudomonas savastanoi pv. savastanoi (Psav) DAPP-PG 722 strain (Moretti et al., 2014), stored at -80 °C in 15% glycerol

  8. Pseudomonas syringae pv. tomato (Pto) DC3000 strain (Gizjen, 2008), stored at -80 °C in 15% glycerol

  9. MilliQ double distilled water

  10. Tris base (Sigma-Aldrich, catalog number: T1503 ); prepare 0.12 M aqueous solution, adjust pH to 8.0 with 1 M HCl, autoclave and store at room temperature

  11. Fura 2-AM (Sigma-Aldrich, catalog number: F0888 ); prepare 2 mM solution in DMSO in aliquots of 50 µl and freeze at -20 °C. Store at -20 °C, wrap it in aluminum foil to avoid photodegradation. Avoid repeated freezing and thawing of the aliquots

  12. EGTA (Sigma-Aldrich, catalog number: E3889 ); prepare a 0.5 M aqueous stock solution, adjust pH to 8.0 with 0.5 M NaOH, autoclave and store at room temperature. Prepare a 2 mM aqueous solution from the stock solution

  13. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )

  14. Tryptone (Sigma-Aldrich, Millipore, catalog number: T7293 )

  15. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )

  16. Calcium chloride (Sigma-Aldrich, catalog number: 449709 ); prepare a 50 mM aqueous solution and autoclave at 121 °C for 20 min

  17. Hank’s Buffered Salt Solution (HBSS) buffer; prepare 1 L aqueous solution with 8.18 g/L NaCl, 0.4 g/L KCl (Sigma-Aldrich, catalog number: P9541 ), 5.96 g/L HEPES (Sigma-Aldrich, catalog number: H3375 ), adjust pH to 7.4 and autoclave at 121 °C for 20 min

  18. EDTA (Sigma-Aldrich, catalog number: E9884 ); prepare a 0.5 M aqueous stock solution, adjust pH to 8.0 with NaOH, autoclave at 121 °C for 20 min and store at room temperature. Prepare a 0.1 mM aqueous solution from the stock solution

  19. Triton X-100 (Sigma-Aldrich, catalog number: X100 ); prepare a 1% aqueous solution and autoclave at 121 °C for 20 min

  20. Luria Bertani (LB) medium (see Recipes)

Equipment

  1. 1 L measuring cylinder (DWK Life Sciences, catalog number: 21 390 54 08 )

  2. BRAND magnetic stirring bar (Sigma-Aldrich, Aldrich, catalog number: BR137630 )

  3. Magnetic stirrer (Heidolph MR 2000, catalog number: 200-505-20000-00 )

  4. Eppendorf Research Plus G pippetes (Sigma-Aldrich, Eppendorf, catalog number: EP3123000918 )

  5. Autoclave (EXAPro, Lequeux, catalog number: P80602001 )

  6. Shaking incubator SI500 (Stuart, catalog number: FV-79520-00 )

  7. Eppendorf® Centrifuge 5804R (Sigma-Aldrich, Sigma, catalog number: EP022628146 )

  8. HerathermTM Incubator (Thermo Scientific, catalog number: 51028112 )

  9. LS-50B Luminescence Spectrometer (PerkinElmer, catalog number: 17931 )

  10. Laminar flow cabinet Gelaire BSB 6A (Gelaire)

Software

  1. FL WinLab Software, version 3 (PerkinElmer)

  2. Prism 8 (GraphPad, https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Bacterial growth (Day 1: ~15 min followed by 16 h incubation)

    Note: Do not use any antibiotics in the growth medium, because it can interfere with the experiments. Do not use more than 10% of maximum tube volume to ensure growth of the bacterium. Conduct all steps under sterile conditions.

    1. Prepare a fresh culture of Psav DAPP-PG 722 or Pto DC3000 strain by inoculating bacterial cells, scraped with a inoculation loop from a 15% glycerol stock at -80 °C, on Petri dish containing LB agar medium and incubate for 16 h at 27 °C.


  2. Bacterial growth (Day 2: ~15 min followed by 16 h incubation)

    Note: Do not use any antibiotics in the growth medium, because it can interfere with the experiments. Do not use more than 10% of maximum tube volume to ensure growth of the bacterium. Conduct all steps under sterile conditions.

    1. Inoculate a loop of bacterial cells into a 50 ml tube containing 5 ml LB broth. Incubate the tubes in the shaking incubator for 16 h at 27 °C and 200 rpm (until the OD660 = 0.8).


  3. Cell preparation with Fura 2-AM (Day 3: ~3.5 h)

    Note: Do not centrifuge the bacterial suspension at >16,000 × g to avoid any cell damage. Wrap the tube containing the Fura 2-AM with aluminum foil or work in the dark to avoid photodegradation of the probe.

    1. Collect the bacterial cells from the 5 ml cell culture by centrifugation (15,585 × g, 3 min) at room temperature (RT).

    2. Discard the supernatant and resuspend the cells in 5 ml of sterile 0.12 mM Tris HCl (pH 8.0).

    3. Adjust the bacterial suspension to 1 × 108 CFU ml-1 by bringing the OD660 at 0.06.

    4. Collect again 10 ml of this bacterial suspension by centrifugation (15,585 × g, 3 min) at RT.

    5. Discard the supernatant and resuspend the cell pellet in 5 ml 0.12 mM Tris HCl (pH 8.0) (= 5 × 109 CFU), add 0.5 ml of a 2 mM EGTA solution (pH 8.0), and incubate at 25 °C for 5 min in an air incubator to render the bacteria receptive to the Ca2+ probe.

    6. Add 20 µl of 2 mM CaCl2 solution to quench the EGTA.

    7. Pellet the cells by centrifuging them at 15,585 × g for 3 min at RT.

    8. Discard the supernatant and resuspend the cells in 5 ml HBSS supplemented with 2 µl of 2 mM Fura 2-AM stock solution. Note, add the Fura 2-AM freshly to the HBSS prior to usage from a fresh stock solution.

    9. Incubate this cell suspension in HBSS + Fura2-AM in the incubator without shaking at 25 °C for 2 h.

    10. Pellet the cells by centrifuging them at 15,585 × g for 3 min at RT.

    11. Add 5 ml HBSS (without the dye), resuspend the cells and incubate for 1 min at RT.


  4. Measurement of the Ca2+ levels (Day 3: ~1.5 h)

    Note: Set the LS-50B luminescence spectrometer with FL WinLab Software using the User’s Guide.

    1. Pour 1 ml of the cell suspension in the NIR Quartz SUPRASIL 300 Rectangular Macro Cell containing the BRAND magnetic stirring bar which reduces the swirling and guarantees the correct reading of the samples.

    2. Place the Rectangular Macro Cell in the LS-50B luminescence spectrometer, wait until the Traffic Light bottom becomes green to initialize the spectrometer (Figure 3A) and click on it to start the measurement. Similar results could be obtained with analogous spectrometer.

    3. Wait about 50 s (a time useful for the signal stabilization) and add 30-240 µl of a 50 mM CaCl2 solution to reach a final concentration ranging from 0.5 to 4 mM (Figure 4).

    4. Add 1 ml of 1% Triton X-100 to disrupt the cells, record the time and wait until the signal stabilizes. Triton X-100 is added at the end of the measurement because it allows one to determine the maximum Ca2+ level in the system.

    5. Add 1 ml of 0.1 mM EDTA (pH 8.0) to chelate the Ca2+ ions in solution and record the exact time appearing in the display as it is important for the final Rmin determination, wait until the signal stabilizes and press the Traffic Light buttom (Figure 3A), make sure it becomes red to acquire the signals and save the value. By adding EDTA the Ca2+ ions are chelated allowing one to determine the minimum Ca2+ level in the system.

      Note: Triton X-100 and EGTA are added at the end of the determination so the measurement is performed with intact cells. If the cells were not intact, the minimum and maximum Ca2+ value could not be determined.

    6. To automatically determine the Ca2+ concentration according to the formula of Grynkiewicz et al. (1985) [see also Data analysis], set the calibration values Rmin and Rmax (Figure 3B) in the Calibration Tab layout of the FL WinLab Software, as reported in the User’s Guide.

      Note: Without this software it is necessary to determine the Rmin and Rmax for each sample as indicated in point 1 of Data analysis.

    7. Press the Convert to [ion] button (Figure 3B) to convert the raw dataset of the signals into Ca2+ ion concentration dataset using the calibration values set before and save the generated file. Without FL WinLab Software Ca2+ ion concentration has to be calculated using the formula of Grynkiewicz et al. (1985).



      Figure 3. FL WinLab Software. A. The Traffic light button allows to start and stop the analysis and describes the status of the instrument. B. The Calibration Tab layout permits to manage the raw data and the Convert to [ion] button to visualize the data (from the FL WinLab User’s Guide, PerkinElmer, Inc., UK).


    8. Calculate the difference between the Ca2+ ions present before and after CaCl2 addition (Figure 4).



      Figure 4. Example of a plot generated by FL WinLab Software used to calculate Δ[Ca2+]c before and after CaCl2 addition. The presence of the peaks in the trace is due to the change of the wavelength that LS-50B Luminescence Spectrometer shifts every two seconds. Therefore, the calculation of the Ca2+ concentration has to be performed considering the baseline (dotted line). After about 50 s, when the signal is stabilized, the CaCl2 is added (red arrow) and the Δ[Ca2+]c is calculated between the highest Ca2+ concentration value reached in the baseline (blue arrow) and the value when the CaCl2 is added (red arrow).


    9. Incubate the cells in HBSS buffer supplemented with Fura 2-AM (basal condition) or in HBSS buffer supplemented with 2 mM Fura 2-AM and different carbon sources (e.g., 5 mM glucose, fructose or sucrose), 50 μM ATP, or other conditions of interest. Of note, one should optimally add these compounds only after 50s from the start of the measurement (Figure 5).

Data analysis

  1. The free intracellular Ca2+ concentration was calculated using the formula of Grynkiewicz et al. (1985):



    Kd: Dissociation constant for the Ca-Fura-2 complex

    β: Fluorescence intensity ratio with an excitation wavelength of 380 nm, with and without Ca2+

    R: Ratio of fluorescence intensities obtained

    Rmin and Rmax: Fluorescence intensity ratio excited at 340 and 380 nm, in the absence (min) and in the presence (max) of Ca2+, respectively

  2. To validate the results, multiple biological replicates are necessary. Here, ten independent experiments were carried out and the data points and error bars (Figure 5) represent the mean and standard error, respectively. In Psav DAPP-PG 722 cells it has been observed that under basal conditions (i.e., HBSS buffer) an increase in external Ca2+ results in an increase in the cytosolic Ca2+ concentrations. The cytosolic Ca2+ concentrations rapidly increase in response to external Ca2+ concentration in the medium (Figure 5). This trend was suppressed when different carbon sources (glucose, fructose or sucrose) or ATP were added in a combination with Ca2+ (Figure 5).



    Figure 5. An increase of cytosolic Ca2+ levels in Pseudomonas savastanoi pv. savastanoi (Psav) DAPP-PG 722. Psav bacterial cells incubated in HBSS medium alone (basal condition, closed squares) or in the presence of glucose, fructose, sucrose, indole 3 acetic acid (IAA) or tryptophan (open circles) over a concentration range of extracellular calcium chloride. Each point represents the mean of 10 independent experiments ± SE. From Moretti et al. (2019).

Recipe

  1. Luria Bertani (LB) medium (1 L)

    1. Add 800 ml of MilliQ water into a 1 L measuring cylinder

    2. Put the measuring cylinder on magnetic stirrer with a magnetic rod

    3. Add 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl

    4. Add MilliQ water to 1 L

    5. Pour 250 ml medium into four 500 ml PYREX® glass bottles

    6. Autoclave at 121 °C for 20 min

Acknowledgments

This work was financially supported by DSA3 research funds “Fondo di base” to the co-authors CM, RB and CAP. This method has been used in Moretti et al. (2019).

Competing interests

The authors declare no conflict-of-interest and have no competing financial interests.

Informed consent was obtained from all individual participants included in the study.

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简介

[摘要]钙信号传导是细菌对环境线索作出反应的一种新兴机制。为了测量细菌细胞中细胞内游离钙的浓度,在此使用假单胞菌细菌细胞,提出一种基于化学探针Fura 2-乙酰氧基甲基酯(Fura 2-AM)的简单分光荧光法[Ca 2+ ] i 。这是一种可替代的定量方法,可在短时间内以低成本完成,并且不需要诱导异源表达的基于蛋白质的探针(如水母发光蛋白)。此外,有可能验证参与Ca 2+从细胞外基质进入的膜通道的特性。该方法对于在像原核生物一样的小细胞中测量[Ca 2+ ] i在0.1-39.8 µM范围内特别有价值。

[背景] Ca 2+是一种新兴的细菌细胞内信使,会影响多种细胞过程,例如维持细胞完整性,细胞分裂(Dominguez等人,2015),运动性(Tisa和Alder,1995;Gode-Potratz等人,2010;Cruz等人,2012;Guragain等人,2013; Parker等人,2015;Fishman等人,2018 ),III型分泌物(DeBord等人,2003;Dasgupta等人,2003)。 ,2006; Gode-Potratz等,2010; Fi shman等,2018),基因表达(Dominguez等,2015),群体感应(Werthén和Lundgren,2001),生物膜形成(Patrauchan等,, 2001)。 2005年; Sarkisova等人,2005年; Rinaudi等人,2006年; Cruz等人,2012年; Das等人,2014年; Zhou等人,2014年; Parker等人,2016年)或生物膜抑制作用(Bilecen和Yildiz ,2009; Shukla和Rao,2013)。最近,证实了细胞内Ca 2+浓度控制毒力假单胞菌savastanoi PV 。savastanoi (Psav )(Moretti等,2019)。此外,一些已知的毒力基因在提高Ca的存在被上调2+浓度Pse时udomonas丁香PV 。番茄(Pto )DC3000(Fishman等人,2018)和Xylella fastidiosa (Parker等人,2016)。在测量细胞内游离钙浓度的[Ca 2+ ]我在原核生物中都有,因此,成为极大的兴趣来研究其作为细胞内我的角色在应对环境线索细菌ssenger。监测细菌细胞内的[Ca 2+ ] i ,这对于理解Ca 2+跨质膜的运输与细胞过程之间的相互关系是必不可少的,因为它受细菌细胞尺寸的限制,细菌细胞壁的半选择性,低的膜通透性以及使用的许多Ca 2+螯合剂的毒性(Gangola和Rosen,1987; Knight等,1991;Futsaether和Johnsson ,1994; Norris等。等人,1996;Herbaud等,1998; Jones等,1999;Torrecilla等,2001)。确定原核生物中[Ca 2+ ] i变化的一种公认的方法是基于Aequorin (钙激活的光蛋白)细菌细胞的异源表达(Watkins等,1995)。该方法利用重组Aequorin的表达(来自质粒或整合到细菌基因组中),该结合蛋白在Ca 2+结合后发光。该方法相当耗时,需要分子生物学工具的可用性,并且在技术上具有挑战性。实际上,即使该方法已成功用于监测多种细菌中的[Ca 2+ ] i (Naseem等,2007;Guragain等,2016),该方法也更适合真核细胞。为了克服Aequorin的这些局限性,我们基于化学探针Fura 2-AM {1- [2-(5-(羧甲基恶唑-2-基)-6-氨基-苯并呋喃-5-氧基] -2-(2'-氨基-5'-甲基苯氧基)乙烷- ñ ,ñ ,N ' N' - 四乙酸}。重要的是,所使用的测定溶液和Fura 2-AM都不会在此处使用的浓度下损害细胞活力(Gangola和Rosen,1987;Futsaether和Johnsson ,1994; Tisa和Alder,1995; Norris等,1996; Jones等人,1999年),这意味着该方法无需外部先进设备即可根据外部提示和不同条件对[Ca 2+ ] i进行定量(Moretti等人,2019)。必须指出的是的Fura 2-AM是探针跨细胞膜活的细菌细胞和其随后的快速去的扩散通过蜂窝酯化酯酶产生的Fura 2,其保留结合在细胞内的Ca的能力2+而它失去了在细胞膜上扩散的能力(图1)。当Fura 2与Ca 2+形成络合物时,λ= 510 nm处的荧光强度随Ca 2+浓度的增加而增加(Grynkiewicz等,1985)(图2)。另外,由于细胞壁和细胞膜的选择性渗透性,Fura 2不能渗透细菌细胞本身(Grynkiewicz等,1985)。值得注意的是,由于测量利用的荧光信号仅在细胞内部变得明显(当Fura 2-AM转化为Fura 2时),因此无需使用未加载的活细胞作为阴性对照。事实上,如果细胞是不可行的,该电池隔膜失去其完整性和探头不会被困在细胞而是保持分散在孵育液。


图1. Fura 2-AM在细胞中的命运。Fura 2-AM被细胞酯酶去酯化并在Fura 2中转化,Fura 2能够与胞质钙(Ca 2+ )形成复合物,并且不能被动地穿过细胞膜。


图2. Fura 2的激发光谱。Fura2在含有0至39.8 µM游离钙(Ca 2+ )的溶液中的激发光谱。修改自Grynkiewicz等。(1985)。

我们发现荧光探针Fura 2-AM高度敏感,这使我们能够确定假单胞菌(Pseudomonas savastanoi pv)中胞质Ca 2+水平的变化。savastanoi DAPP-PG 722 (Moretti等,2019)和丁香假单胞菌pv 。番茄DC3000细胞(Trabalza等,正在制备),使用配备了搅拌半微量比色杯的分光荧光计。

关键字:Fura2-乙酰氧基甲酯, 胞质钙离子浓度, 分光光度计, 假单胞菌, 活细胞信号



材料和试剂

1.接种环10 µl(Laboindustria SPA,目录号:21131)     

2.培养皿Ø 90 (Laboindustria SPA,目录号:21 050 )     

3.移液管头(梅特勒托莱多,目录号š :1 7007956,17007952)     

4. 50毫升高清晰度聚丙烯(PP)锥形离心管(Falcon,目录号:352070)     

5.带有盖子的NIR石英SUPRASIL 300矩形宏单元,容积3.5毫升(PerkinElmer,目录号:B0631015)     

6. PIREX介质瓶,刻度,Corning,500毫升(VWR,PIREX,目录号:1395-500)     

7.假单胞菌savastanoi PV 。savastanoi (Psav )DAPP-PG 722菌株(Moretti等人,2014),于-80 °C的15%甘油中保存     

8.丁香假单胞菌PV 。番茄(Pto )DC3000菌株(Gizjen ,2008),在-80 °C下用15%甘油存储     

9. MilliQ双蒸馏水     

10.的Tris碱(Sigma-Aldrich公司,目录号:T1503); 制备0.12 M水溶液,用1 M HCl调节pH到8.0 ,高压灭菌并在室温下保存 

11.的Fura 2-AM(Sigma-Aldrich公司,目录号:F0888); 在DMSO中以50 µl等分试样制备2 mM溶液,并在-20 °C冷冻。存放在-20 °C,用铝箔包裹,以免发生光降解。避免反复冷冻和解冻等份试样 

12. EGTA(Sigma-Aldrich,目录号:E3889);制备0.5 M的储备水溶液,用0.5 M NaOH调节pH到8.0 ,高压灭菌并在室温下保存。从储备溶液中制备2 mM水溶液 

13.氯化钠(西格玛奥德里奇,目录号:S7653) 

14.胰蛋白((西格玛奥德里奇,密理博,目录号:T7293) 

15.酵母提取物(Sigma-Aldrich,目录号:Y1625) 

16.氯化钙(Sigma-Aldrich,目录号:449709);制备50 mM水溶液并在121 °C下高压灭菌20分钟 

17. Hank的缓冲盐溶液(HBSS)缓冲液;用8.18 g / L NaCl ,0.4 g / L KCl (Sigma-Aldrich,目录号:P9541),5.96 g / L HEPES(Sigma-Aldrich,目录号:H3375)制备1 L水溶液,将pH调节至7.4并高压灭菌在121 °C下20分钟 

18. EDTA(Sigma-Aldrich公司,目录号:E9884); 制备0.5 M的原液,用NaOH调节pH到8.0 ,在121 °C高压灭菌20分钟,然后在室温下保存。从储备溶液中制备0.1 mM水溶液 

19. Triton X-100(Sigma-Aldrich,目录号:X100);准备1%的水溶液并在121 °C下高压灭菌20分钟 

20. Luria Bertani (LB)培养基(请参阅食谱) 



设备

1 L量筒(DWK Life Sciences,目录号:21 390 54 08)
BRAND磁力搅拌棒(Sigma-Aldrich,Aldrich,目录号:BR137630)
磁力搅拌器(Heidolph MR 2000,目录号:200- 505-20000-00)
Eppendorf Research Plus G木偶(Sigma-Aldrich,Eppendorf,目录号:EP3123000918 )
高压灭菌器(EXAPro ,Lequeux ,目录号:P80602001)
摇床SI500 (Stuart,目录号:FV-79520-00)
的Eppendorf ®离心机5804R(Sigma-Aldrich公司,Sigma,目录号:EP022628146)
Heratherm TM培养箱(该RMO科学,目录号:51028112)
LS-50B发光光谱仪(PerkinElmer,目录号:17931)
层流柜Gelaire BSB 6A(Gelaire )


软件

FL WinLab软件版本3(PerkinElmer)
棱镜8(GraphPad ,https : //www.graphpad.com/scientific-software/prism/ )




程序

细菌生长(第1天:〜15分钟,然后孵育16小时)
注意:请勿在生长培养基中使用任何抗生素,因为它会干扰实验。请勿使用超过最大试管体积10%的量,以确保细菌生长。在无菌条件下进行所有步骤。


              制备的新鲜培养物PSAV DAPP-PG 722或PTO通过接种细菌细胞,用刮DC3000菌株一个在-80从15%的甘油原液接种环℃,在含有LB琼脂培养基并孵育16小时,在27陪替氏培养皿℃。


细菌生长(第2天:〜15分钟,然后孵育16小时)
注意:请勿在生长培养基中使用任何抗生素,因为它会干扰实验。请勿使用超过最大试管体积10%的量,以确保细菌生长。在无菌条件下进行所有步骤。


              将细菌细胞环接种到装有5 ml LB肉汤的50 ml管中。将试管在振荡培养箱中于27 °C和200 rpm孵育16 h (直到OD 660 = 0.8)。


用Fura 2-AM进行细胞制备(第3天:〜3.5小时)
注意:请勿以> 16,000 × g的速度离心细菌悬液,以免损坏细胞。用铝箔纸包裹装有Fura 2-AM的试管,或在黑暗中工作,以免探头发生光降解。


通过在室温下(RT)离心(15,585 × g ,3分钟)从5 ml细胞培养物中收集细菌细胞。
              弃去上清液,将细胞重悬于5 ml无菌0.12 mM Tris HCl (pH 8 .0)中。
调整的细菌悬浮液以1 × 10 8 CFU毫升-1通过使OD 660为0.06。             
通过在室温下离心(15,585 × g,3分钟)再次收集10 ml这种细菌悬液。
弃去上清液并将细胞沉淀重悬于5 ml 0.12 mM Tris HCl (pH 8 .0)(= 5 × 10 9 CFU)中,加入0.5 ml 2 mM EGTA溶液(pH 8 .0),并于25孵育在空气培养箱中保持5分钟,使细菌易于吸收Ca 2+探针。
              加入20 µl 2 mM CaCl 2溶液淬灭EGTA。
              通过在室温下以15585 × g离心3分钟来沉淀细胞。
              弃去上清液,将细胞重悬于5 ml HBSS中,并加入2 µl 2 mM Fura 2-AM储备液。请注意,在使用新鲜储备溶液使用之前,应将Fura 2-AM新鲜添加到HBSS中。
              孵育在培养箱中在HBSS + Fura2-AM该细胞悬浮液,而不在25摇动℃下进行2小时。
              通过在室温下以15585 × g离心3分钟来沉淀细胞。
加入5 ml HBSS(不含染料),重悬细胞,在室温下孵育1分钟。


Ca 2+含量的测量(日期3:〜1.5小时)
注意:使用《用户指南》,使用FL WinLab软件设置LS-50B发光光谱仪。


              将1 ml的细胞悬液倒入装有BRAND磁力搅拌棒的NIR Quartz SUPRASIL 300矩形宏样品池中,以减少涡旋并确保正确读取样品。             
              放置矩形宏切尔升在LS-50B荧光分光光度计,等到至i红绿灯底部变成绿色nitialize分光计(图3A),并点击它来开始测量。用类似的光谱仪可以得到类似的结果。
等待约50 s(对于信号稳定有用的时间),然后添加30-240 µl的50 mM CaCl 2溶液以达到0.5至4 mM的最终浓度(图4)。
              加入1 ml的1%Triton X-100破坏细胞,记录时间并等待信号稳定。在测量结束时添加Triton X-100,因为它可以确定系统中的最大Ca 2+水平。
              1毫升0.1添加毫EDTA (pH为8 .0)到CHEL吃了的Ca 2+离子在溶液中,并记录精确的时间在出现DISPLA ÿ,因为它是最后的重要Rmin的判定,等到信号稳定,然后按在“交通信号灯”按钮(图3A)中,确保其变为红色以获取信号并保存值。通过添加EDTA,Ca 2+离子被螯合,从而可以确定系统中的最低Ca 2+水平。
注意:在测定结束时添加Triton X-100和EGTA,以便使用完整的细胞进行测量。如果细胞不完整,则无法确定最小和最大Ca 2+值。


根据Grynkiewicz等人的公式自动确定Ca 2+的浓度。(1985)[也参见d ATA分析],设定校准值Rmin的和RMAX (图3B)在FL的校准标签布局WINLAB软件,据报道在用户指南中。
注意:如果没有此软件,则必须按照数据分析第1点中的说明确定每个样品的Rmin和Rmax 。


按转换为[离子]按钮(图3B),使用之前设置的校准值将信号的原始数据集转换为Ca 2+离子浓度数据集,并保存生成的文件。如果没有FL WinLab软件,则必须使用Grynkiewicz等人的公式计算Ca 2+离子浓度。(1985)。






图3. FL WinLab软件。A.牛逼他红绿灯按钮,可以启动和停止的分析和介绍了仪器的状态。乙。Ť他校准选项卡布局允许以管理原始数据和转换为[离子]按钮以可视化的数据(从FL WINLAB用户指南,PerkinElmer公司,英国)。

计算添加CaCl 2之前和之后存在的Ca 2+离子之间的差异(图4)。






图4. FL WinLab软件生成的用于在添加CaCl 2之前和之后计算Δ[ Ca 2+ ] c的图的示例。迹线中存在峰是由于LS-50B发光光谱仪每两秒钟移动一次波长的变化而引起的。因此,必须考虑基线(虚线)来进行Ca 2+浓度的计算。大约50 s后,当信号稳定后,添加CaCl 2 (红色箭头),并在基线达到的最高Ca 2+浓度值(蓝色箭头)和该值之间计算Δ[Ca 2+ ] c添加CaCl 2时(红色箭头)。

孵育在HBSS将细胞缓冲液补充有的Fura 2-AM(基础条件)或HBSS缓冲液补充有2毫米的Fura 2-AM和不同碳源(例如。,5 mM的葡萄糖,果糖或蔗糖),50 μM ATP,或其他感兴趣的条件。值得注意的是,应该从测量开始仅在50秒后最佳地添加这些化合物(图5)。


数据分析

1.使用Grynkiewicz等人的公式计算游离细胞内Ca 2+的浓度。(1985年):     




Ca-Fura-2配合物的K d :D缔合常数


β :˚F luorescence强度比为380nm的激发波长,并用不含Ca 2+


R:- [R得到的荧光强度的ATIO


ř分钟和- [R最大:˚F的Ca luorescence强度比激发在340nm和380nm处,在不存在(分钟),并在存在(最大)2+ ,分别


2.为了验证结果,必须进行多次生物学复制。在这里,进行了十次独立的实验,数据点和误差线(图5)分别代表平均值和标准误差。在PSAV DAPP-PG 722个细胞中,这已经观察到,在基础条件下(我。ê 。,HBSS缓冲液)增加外部的Ca 2+的增加的细胞内的Ca结果2+浓度。响应于培养基中外部Ca 2+浓度,胞质Ca 2+浓度迅速增加(图5)。当不同的碳源(葡萄糖,果糖或蔗糖)或ATP与Ca 2+结合使用时,这种趋势得到了抑制(图5)。     







图5.一种细胞内的Ca的增加2+水平假单胞菌savastanoi PV 。萨瓦斯坦(Psav )DAPP-PG 722 。在HBSS培养基中(基础条件,实心正方形)或在葡萄糖,果糖,蔗糖,吲哚3乙酸(IAA)或色氨酸(空心圆)存在下,在细胞外氯化钙的浓度范围内孵育的Psav细菌细胞。每个点代表10个独立实验的平均值±SE。从Moretti等人。(2019 )。

食谱

Luria Bertani(LB)培养基(1升)
将800毫升MilliQ水加到1升量筒中
用磁棒将量筒放在磁力搅拌器上
加入10克胰蛋白,、 5克酵母提取物和5克氯化钠
加入MilliQ水至1升
倾250毫升介质分为四个500毫升PYREX ®玻璃瓶
在121 °C下高压灭菌20分钟


致谢

这项工作得到了DSA3研究基金“ Fondo di base”的财政支持,并由他们共同撰写了CM,RB和CAP。此方法已在Moretti等人中使用。(2019)。

利益争夺

作者宣称没有利益冲突,也没有竞争的经济利益。


从研究中包括的所有个体参与者获得知情同意。

参考

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克鲁斯(LF),宾夕法尼亚州科宾(Cobine)和德拉富恩特(L.La La Fuente)(2012)。钙会增加木糖衣藻的表面附着,生物膜形成和抽搐运动力。应用环境微生物学78(5):1321-1331。
Das,T.,Sehar ,S.,Koop,L.,Wong,YK,Ahmed,S.,Siddiqui,KS和Manefield ,M.(2014)。钙对细胞外DNA介导的细菌聚集和生物膜形成的影响。PLoS One 9(3):e91935。
Dasgupta ,N.,Ashare ,A.,Hunninghake ,GW和Yahr ,TL(2006)。低Ca 2+和宿主细胞的接触导致铜绿假单胞菌(Pseudomonas aeruginosa)III型分泌系统的转录诱导通过两个不同的信号途径进行。感染免疫74(6):3334-3341。
DeBord ,KL,Galanopoulos,NS和Schneewind ,O。(2003)。所述TTSA基因需要烨蛋白和毒力的低钙诱导的III型分泌小肠结肠炎耶尔森菌W22703。Ĵ细菌学185(12):3499-3507。
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引用:Trabalza, S., Buonaurio, R., Del Pino, A. M., Palmerini, C. A., van den burg, H. A. and Moretti, C. (2021). A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells. Bio-protocol 11(6): e3949. DOI: 10.21769/BioProtoc.3949.
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