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Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa
铜绿​​假单胞菌中细胞内钙浓度测定   

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Abstract

Characterization of the molecular mechanisms of calcium (Ca2+) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca2+in). Here, we describe a protocol optimized to use a recombinantly expressed Ca2+-binding protein, aequorin, for detecting Ca2+in in Pseudomonas aeruginosa. Upon binding to free Ca2+, aequorin undergoes chromophore oxidation and emits light, the log of which intensity linearly correlates with the amount of bound Ca2+, and therefore, can be used to measure the concentration of free Ca2+ available for binding. This protocol involves the introduction of the aequorin gene into P. aeruginosa, induction of apoaequorin production, reconstitution of the holoenzyme with its chromophore, and monitoring its luminescence. This protocol allows continuous measuring of Ca2+in concentration in vivo in response to various stimuli.

Keywords: Intracellular calcium(细胞内钙离子), Regulation(调节), Aequorin(水母发光蛋白), Luminescence(发光), Coelenterazine(腔肠素), Pseudomonas aeruginosa(铜绿假单胞菌)

Background

Ca2+ regulates physiology and virulence of P. aeruginosa (Guragain et al., 2013; Patrauchan et al., 2005; Sarkisova et al., 2014), however, the molecular mechanisms of Ca2+ regulation are not well understood. To characterize these mechanisms, it is critically important to not only measure the concentration of Ca2+in ([Ca2+in]), but to monitor its changes in response to various stimuli. Considering that [Ca2+in] may change in response to even minute alterations in cell physiology (reviewed in [Dominguez et al., 2015]), measuring [Ca2+in] requires a tool specifically recognizing Ca2+ without significantly disturbing cells. One such tool is aequorin, a Ca2+-binding protein, which upon binding to free Ca2+, undergoes chromophore oxidation and emits light. The emitted light can be recorded as a measure of free Ca2+. Aequorin has been successfully used to monitor Ca2+in in eukaryotes (Bonora et al., 2013), as well as several bacterial species (Herbaud et al., 1998; Naseem et al., 2007; Rosch et al., 2008). Sufficient level of aequorin production and its stability within a cell enables continuous monitoring of Ca2+in (Naseem et al., 2007). Use of aequorin offers additional advantages such as targeted intracellular distribution (cytoplasm or periplasm), high dynamic range, high signal-to-noise ratio, and low Ca2+ buffering effect (Bonora et al., 2013). Alternative approaches include application of chemical indicators, such as Fura. However, due to reduced cell membrane permeability in P. aeruginosa, loading cells of this bacterium even with membrane permeable Fura acetoxymethyl (AM, ester form) is challenging and requires additional treatments, which limits physiological relevance of the measurements (not published observations). Therefore, our group pioneered the use of aequorin for measuring [Ca2+in] in P. aeruginosa (Guragain et al., 2013). The original protocol was developed for Escherichia coli (Knight et al., 1991) and further developed in (Jones et al., 1999). Here we present a modified adaptation of the protocol, successfully used to study Ca2+ homeostasis in P. aeruginosa, clinically and environmentally important organism (Guragain et al., 2013).

Materials and Reagents

  1. General supplies
    1. Centrifuge bottles (No specific brand is required)
    2. Microfuge tubes (No specific brand is required)
    3. Lumitrac 96 well white microplates (Greiner Bio One, catalog number: 655075 )
    4. Aluminum foil
    5. Plastic cuvettes (BrandTech Scientific, catalog number: 759086D )

  2. Strains and plasmids
    1. Pseudomonas aeruginosa strain PAO1 carrying pMBB66EH containing aequorin gene
    2. Plasmid pMBB66EH encoding aequorin gene from Aequoria victoria (courtesy: Drs. Delfina Dominguez)

  3. Culture medium
    1. Luria bertani (LB) agar (see Recipes)
    2. Biofilm minimal medium (BMM) (see Recipes)
      1. 10x basal salt solution
      2. Vitamin solution
      3. Trace metal
      4. 1 M MgSO4

  4. Chemicals and buffers
    1. Carbenicillin, disodium (Gold Bio, catalog number: C-103-5 )
    2. IPTG (Gold Bio, catalog number: I2481C )
    3. Calcium chloride dehydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
    4. Live-Dead staining (Molecular Probes)
    5. Yeast extract (BD, Bacto, catalog number: 212750 )
    6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 or VWR, catalog number: E529-500ML )
    7. Tryptone (BD, Bacto, catalog number: 211705 )
    8. Agar (BD, Bacto, catalog number: 214010 )
    9. Nanopure water
    10. Monosodium glutamate (Sigma-Aldrich, catalogue number: 1446600 )
    11. Glycerol (Thermo Fisher Scientific, Fisher Scientific, catalog number: G33 )
    12. Sodium phosphate monobasic dihydrate (NaH2PO4) (Sigma-Aldrich, catalog number: 71500 )
    13. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 17835 )
    14. Biotin (Gold Bio, catalog number: B-950-1 )
    15. Thiamine HCl (Gold Bio, catalog number: T-260-25 )
    16. HCl (Pharmco-Aaper, catalog number: 284000ACS )
    17. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: 209198 ).
    18. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251 )
    19. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: 215422 )
    20. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Avantor Performance Materials, J.T. Baker, catalog number: 2540-04 )
    21. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
    22. HEPES (Sigma-Aldrich, catalog number: H3375 )
    23. MgCl2 (Sigma-Aldrich, catalog number: 230391 )
    24. Tergitol Type NP-40, 70% in H2O (Sigma-Aldrich, catalog number: T1135 )
    25. Coelenterazine (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C2944 )
    26. Ethanol (Pharmco-Aaper, catalog number: AAP-111000190CSGL )
    27. HEPES buffer (see Recipes)
    28. Discharge buffer (see Recipes)
    29. 1 M CaCl2 solution (see Recipes)
    30. 6 mM CaCl2 solution for injection (see Recipes)
    31. Coelenterazine (see Recipes)

Equipment

  1. Multichannel pipette (Finnipipette F1, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4661030 )
  2. Pipetman classic pipettes (Gilson, catalog numbers: F123600 , F123615 , F123601 , F123602 )
  3. 500 ml glass flasks (No specific brand is required)
  4. SynergyTM Mx multimode microplate reader (Biotek Instruments) with Gen5TM 2.05 PC software (BioTek Instruments)
  5. 37 °C incubator (Bench top incubator) (VWR, catalog number: 89409-314 )
  6. 37 °C shaking incubator (MaxQ 4000 table top shake incubator) (Thermo Fisher Scientific, Thermo ScientificTM, model: SHKA4000 )
  7. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM RC 6 Plus Centrifuge ) with rotor type (Thermo Fisher Scientific, Thermo ScientificTM, model: F13-14x50 cy )
  8. Centrifuge (Eppendorf, model: 5424 )

Procedure

  1. Expression and reconstitution of aequorin
    1. Grow PAO1 strain carrying plasmid pMMB66EH encoding aequorin gene for overnight at 37 °C on LB agar plate with carbenicillin (300 μg/ml).
    2. Inoculate 5 ml of BMM medium with 2-3 single colonies grown on LB agar plate and incubate for 12 h at 37 °C while shaking at 200 rpm. Antibiotic was omitted hereafter to avoid its effect on bacteria.
    3. Transfer 1 ml of 12 h culture of OD600 0.25 (Adjust OD if needed by diluting in fresh BMM medium) to 100 ml of fresh BMM medium in 500 ml flask. Grow culture at 37 °C, 200 rpm until the mid-log phase, as determined in previous growth studies. For example, for PAO1, mid-log phase is reached at OD600 of 0.16 after 12 h of incubation.
    4. Once reached mid-log phase, induce cells with 1 mM IPTG (1 ml of 100 mM IPTG) and incubate for 2 h, at 37 °C, 200 rpm. At the end of induction, measure OD600 of the culture and compare with the previously recorded growth curve, to make sure that cells are still in their logarithmic phase of growth.
    5. Transfer cells into 250 ml ice cold centrifuge bottles. From this step and until the reconstitution step, maintain cells on ice.
    6. Harvest cells by centrifugation at 15,000 x g for 5 min at 4 °C. Discard supernatant and wash the cell pellet with 100 ml of ice-cold HEPES buffer. For this, first carefully resuspend cells by pipetting into of 2 ml HEPES buffer, and then add the remaining 98 ml of HEPES buffer.
    7. Collect cells by centrifuging at 6,000 x g for 5 min at 4 °C. Discard the supernatant.
    8. Resuspend collected cell pellet in 1,250 μl of HEPES buffer by pipetting and transfer 1 ml of cell suspension into a fresh microfuge tube.
    9. Add coelenterazine to a final concentration of 2.5 μM. For this, add 5 μl of 500 μM coelenterazine solution to 1 ml of cell suspension. Since coelenterazine is light sensitive, the procedure from this point onward must be carried out in the dark (see Notes).
    10. After addition of coelenterazine, incubate cell suspension at room temperature without shaking for 30 min.
      Note: This step is referred to as a reconstitution step. Since coelenterazine undergoes slow oxidation in the presence of atmospheric oxygen, shaking should be strictly avoided.
    11. Collect the cell pellet by centrifuging at 6,000 x g for 5 min at 4 °C and wash two times with 1 ml of ice-cold HEPES buffer.
    12. Resuspend the final collected cell pellet into 1 ml HEPES buffer by pipetting.
    13. Measure OD600 of thus prepared sample. Adjust the final OD600 of the cell suspension to 0.4 by adding HEPES buffer if needed.

  2. Measurement of luminescence
    1. Pipette 100 μl of cells suspension with reconstituted aequorin into 96-well luminescence (white) plate and equilibrate at room temperature for 10 min in the dark. When the effect of inhibitors or other compounds on Ca2+in to be tested, add the compounds during this step.
    2. Load the plate with samples into a Synergy Mx plate reader, and record luminescence for 1 min, at 5 sec interval. Use this reading to calculate a basal level of Ca2+in.
    3. To study the response of the intracellular Ca2+ levels to extracellular Ca2+ elevated to the millimolar concentrations commonly present in some environments including a human body, expose the cells prepared as above to the addition of 1 mM CaCl2. For this, inject 20 μl of 6 mM CaCl2 into each well by the plate reader injector. Prior to injection, prime the injector with 5 ml of 6 mM CaCl2. If the immediate effects of other compounds need to be tested, inject the latter at this step (before, after or instead of CaCl2) through a second injector, followed by luminescence measurements.
    4. Immediately after injection, mix the samples for 1 sec, and record luminescence for 20 min at 5 sec interval (Figure 1 A). Mixing and measurements were pre-programmed in the instrument.
    5. In order to estimate the remaining aequorin present in the samples, briefly take out the plate from the reader, and manually add 120 μl of discharge buffer to each sample, mix well, but quickly by pipetting, and the load back the plate into the plate reader. Read luminescence for 10 min at 5 sec interval (Figure 1B). Estimate the total aequorin by summing the luminescence detected during the entire experiment including the discharge. Use this value of total aequorin for data normalization, when calculating the concentration of Ca2+in (Figure 1C).
      Note: it is important to ensure that no bubbles are formed during mixing, as bubbles will interfere with the luminescence reading.

Data analysis

  1. Calculation of free intracellular calcium concentration ([Ca2+in])
    [Ca2+in] (Figure 1C) was calculated from the luminescence (Figure 1A) values using the formula: pCa = 0.612 (-log10k) + 3.745
    Where,
    k is a rate constant for luminescence decay (sec-1) as described in Jones et al., 1999.
    [Ca2+in] at each time point was calculated as an average of at least three independent biological replicates. Even slight inconsistencies during harvesting and preparing cells may cause fluctuations in the [Ca2+in] profile. Therefore, in case of inconsistent [Ca2+in] profile, it is strongly recommended to repeat the experiment with three independent biological replicates, making sure that cells are synchronized and harvested at exactly the same point of growth. Certain mutants are particularly sensitive and produce more fluctuations in their [Ca2+in] levels. In these cases, additional replicates were added, and a [Ca2+in] profile, shared by at least 70% of biological replicates, was considered for further calculations. Replicates significantly deviating from this profile were excluded. Statistical significance was calculated by standard deviations among biological replicates (Figure 1D).


    Figure 1. Measuring [Ca2+]in in PAO1 by using aequorin. A. Representative luminescence profile. Arrow indicates addition of 1 mM CaCl2. B. Representative discharge luminescence profile. Arrow indicates addition of discharge buffer (see Recipes). C. Representative calculated [Ca2+]in. Arrow indicates addition of 1 mM CaCl2. D. Averaged [Ca2+]in calculated from three independent biological replicates. Arrow indicates addition of 1 mM CaCl2.

Notes

  1. Two types of controls were included in the study
    1. Control 1 was to verify that addition of HEPES buffer alone does not affect the [Ca2+in]. For this, HEPES buffer alone was injected instead of 1 mM CaCl2 challenge, and the entire procedure was followed as described. No buffer effect was detected.
    2. Control 2 was to ensure that aequorin was not leaking through cell membranes. For this, 100 μl of cell suspension prepared for the measurements was incubated for 30 min in the dark at room temperature. Cells were removed by centrifugation 15,000 x g for 5 min, and the supernatant was collected, and mixed with 1 mM CaCl2. Luminescence was monitored for 1 min. We did not observe any increase in luminescence during this experiment, thus confirming that there was no aequorin leakage from the cells into the supernatant. We also verified cells viability after the procedure by Live-Dead staining (Molecular Probes).
  2. Luminescence profiles may vary between replicates due to possible differences in the levels of apoaequorin production. However, the calculated concentrations of Ca2+in normalized by the total available aequorin should be consistent.
  3. To ensure consistent [Ca2+]in measurements, it is important to induce and harvest cells at the same point of growth. During our study, we followed both OD and incubation time to determine when to harvest. Only 30 min window was allowed for cells to reach the expected density, which was estimated based on prior growth analysis.
  4. Aequorin is light-sensitive, and therefore all the solutions with aequorin should be kept and handled in dark tubes or tubes covered with aluminum foil. We also recommend turning off the lights in the room, only allowing either dimmed daylight or indirect light from the neighboring room. Although not tested, since aequorin absorbance maximum is at 350 nm (Shimomura and Johnson, 1969), using blue light could be safe.
  5. Although aequorin is reported to be stable in solution (Prendergast, 2000), we do not recommend storing cell samples with reconstituted aequorin (even on ice) for extended period of time, as this may cause fluctuations in the luminescence profiles. For this reason, only three samples were monitored at a time.

Recipes

  1. Luria Bertani (LB) agar
    5 g yeast extract
    5 g NaCl
    10 g tryptone
    15 g agar
    Combine the ingredients in 1 L of nanopure water and autoclave
  2. Biofilm minimal media (BMM) (Patrauchan et al., 2005)
    Mix 100 ml of sterile 10x basal salt solution (Recipe 2a) to 900 ml of sterile nanopure water. Add 1 ml of vitamin solution (Recipe 2b), 200 μl of trace metals solution (Recipe 2c), and 20 μl of MgSO4 (Recipe 2d). Mix properly.
    1. 10x basal salt solution (9.0 mM sodium glutamate, 50 mM glycerol, 0.15 mM NaH2PO4, 0.34 mM K2HPO4, 145 mM NaCl):
      15 g monosodium glutamate
      46 g glycerol
      0.18 g sodium phosphate monobasic dehydrate (NaH2PO4)
      0.78 g potassium phosphate dibasic (K2HPO4)
      84.7 g sodium chloride (NaCl)
      Combine the ingredients and dissolve completely in 850 ml nanopure water. Adjust the pH to 7.0. Adjust the final volume to 1,000 ml with nanopure water. Sterilize by autoclaving.
    2. Vitamin solution
      Dissolve 1 mg of biotin in 10 ml of nanopure water. Aliquot 1 ml of biotin stock solution in fresh tube and add 50 mg thiamine HCl to it and mix properly. Adjust the final volume to 100 ml with nanopure water. Filter sterilize and store at 4 °C.
    3. Trace metal
      Dilute 10 ml concentrated HCl into 70 ml of nanopure water. Add the following ingredients:
      0.5 g CuSO4·5H2O
      0.5 g ZnSO4·7 H2O
      0.5 g FeSO4·7H2O
      0.2 g MnCl2·4H2O
      Dissolve completely and adjust the final volume to 100 ml with nanopure water. Filter-sterilize or autoclave.
    4. 1 M MgSO4
      24.64 g of MgSO4·7H2O was dissolved in final volume of 100 ml of nanopure water. The solution was sterilized by autoclaving.
  3. HEPES buffer (25 mM HEPES, 125 mM NaCl, 1 mM MgCl2, pH7.5)
    5.96 g HEPES
    7.3 g NaCl
    0.0952 MgCl2
    Dissolve the ingredients in 900 ml nanopure water. Adjust the pH to 7.5 and titrate to the final volume of 1,000 ml with nanopure water.
  4. Discharge buffer (12.5 mM CaCl2, 2% NP-40 in HEPES buffer)
    To 5 ml of HEPES buffer, add 62.5 μl of 1 M CaCl2 solution and 143 μl of Tergitol. Gently mix by stirring on a magnetic stir plate to avoid foaming of NP-40 detergent
  5. 1 M CaCl2 solution
    Dissolve 36.75 g of CaCl2 dihydrate in 250 ml nanopure water. Sterilize by autoclaving
  6. 6 mM CaCl2 solution for injection
    240 μl of 1 M CaCl2 solution was added to 39.76 ml of HEPES buffer and mixed properly
  7. Coelenterazine
    Pulse-centrifuge the tube to collect the entire quantity of the reagent (250 μg coelenterazine) at the bottom. Add 1,136 μl of 95% ethanol to the tube and mix by pipetting thoroughly but quickly (to avoid light and ethanol evaporation). Resting the tube (closed) on bench for a few minutes helps dissolving. After dissolving, aliquot 50 μl of the coelenterazine solution into 0.5 ml microfuge tubes. Cover the microfuge tubes with aluminum foil and store at -20 °C
    Note: Coelenterazine is highly light sensitive and should be handled in the dark.

Acknowledgments

We thank Dr. Delfina Dominguez from The University of Texas at El Paso for sharing E. coli strain carrying pMMB66EH. We thank Ian Reutlinger for transformation of P. aeruginosa PAO1 strains with pMMB66EH plasmid containing aequorin gene. This work was supported by the Grant-in-Aid from American Heart Association (Award 09BGIA2330036) and the Research Grant from OCAST (Award HR12-167).

References

  1. Bonora, M., Giorgi, C., Bononi, A., Marchi, S., Patergnani, S., Rimessi, A., Rizzuto, R. and Pinton, P. (2013). Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes. Nat Protoc 8(11): 2105-2118.
  2. Dominguez, D. C., Guragain, M. and Patrauchan, M. (2015). Calcium binding proteins and calcium signaling in prokaryotes. Cell Calcium 57(3): 151-165.
  3. Guragain, M., Lenaburg, D. L., Moore, F. S., Reutlinger, I. and Patrauchan, M. A. (2013). Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility. Cell Calcium 54(5): 350-361.
  4. Herbaud, M. L., Guiseppi, A., Denizot, F., Haiech, J. and Kilhoffer, M. C. (1998). Calcium signalling in Bacillus subtilis. Biochim Biophys Acta 1448(2): 212-226.
  5. Jones, H. E., Holland, I. B., Baker, H. L. and Campbell, A. K. (1999). Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium. Cell Calcium 25(3): 265-274.
  6. Knight, M. R., Campbell, A. K., Smith, S. M. and Trewavas, A. J. (1991). Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli. FEBS Lett 282(2): 405-408.
  7. Naseem, R., Davies, S. R., Jones, H., Wann, K. T., Holland, I. B. and Campbell, A. K. (2007). Cytosolic Ca2+ regulates protein expression in E. coli through release from inclusion bodies. Biochem Biophys Res Commun 360(1): 33-39.
  8. Patrauchan, M. A., Sarkisova, S., Sauer, K. and Franklin, M. J. (2005). Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp. Microbiology 151(Pt 9): 2885-2897.
  9. Prendergast, F. G. (2000). Structural biology: Bioluminescence illuminated. Nature 405(6784): 291-293.
  10. Rosch, J. W., Sublett, J., Gao, G., Wang, Y. D. and Tuomanen, E. I. (2008). Calcium efflux is essential for bacterial survival in the eukaryotic host. Mol microbiol 70(2): 435-444.
  11. Sarkisova, S. A., Lotlikar, S. R., Guragain, M., Kubat, R., Cloud, J., Franklin, M. J. and Patrauchan, M. A. (2014). A Pseudomonas aeruginosa EF-hand protein, EfhP (PA4107), modulates stress responses and virulence at high calcium concentration. PLoS One 9(2): e98985.
  12. Shimomura, O. and Johnson, F. H. (1969). Properties of the bioluminescent protein aequorin. Biochemistry 8(10): 3991-3997.

简介

钙的分子机制的表征(Ca 2+ 2+)调节细菌生理学和毒力需要能够测量和监测游离钙的细胞内水平的工具(Ca 2+ 2+ in )。在这里,我们描述了优化使用重组表达的Ca 2+ 2+结合蛋白水母发光蛋白的方案,用于检测 em>铜绿假单胞菌 。在结合游离Ca 2+ 2+时,水母发光蛋白经历发色团氧化并发射光,其强度与结合的Ca 2+ 2+的量线性相关,因此,可以可用于测量可用于结合的游离Ca 2+的浓度。该方案包括将水母发光蛋白基因导入em。铜绿假单胞菌,诱导脱辅基水母发光蛋白产生,用其发色团重建全酶并监测其发光。该方案允许在体内响应于各种刺激连续测量Ca <2> 浓度。
关键词:/strong>细胞内钙,调节,水母发光,发光,腔肠素,绿脓杆菌

[背景] Ca 2 + 调节P的生理学和毒力。铜绿假单胞菌(Guragain等人,2013; Patrauchan等人,2005; Sarkisova等人,2014),然而,Ca 2 + 调节的分子机制尚不清楚。为了表征这些机制,非常重要的是不仅测量Ca 2+中的Ca 2+的浓度([Ca 2+] + ]),但监测其响应各种刺激的变化。考虑到细胞生理学中的甚至微小的改变,[Ca 2 + ]可能改变(综述于[Dominguez等人, ]),测量[Ca 2+ 2+]需要特异性识别Ca 2+的工具而不显着干扰细胞。一种这样的工具是水母发光蛋白,Ca 2+ - 结合蛋白,其在结合游离Ca 2+上时经历发色团氧化并发光。发射的光可以被记录为游离Ca 2+的测量。水母发光蛋白已经成功地用于在真核生物(Bonora等人,2013)中监测Ca 2+ 2+,并且几种细菌物种Herbaud等人,1998; Naseem等人,2007; Rosch等人,2008)。足够水平的水母发光蛋白产生及其在细胞内的稳定性使得能够连续监测Ca 2+ 2 +(Naseem等人,2007)。水母发光蛋白的使用提供了另外的优点,例如靶向的细胞内分布(细胞质或周质),高动态范围,高信噪比和低Ca 2+缓冲效应(Bonora等人。,2013)。替代方法包括应用化学指示剂,例如Fura。然而,由于细胞膜通透性降低。即使用膜渗透性Fura乙酰氧基甲基(AM,酯形式)加载细胞也是挑战性的,并且需要另外的处理,这限制了测量的生理相关性(未公开的观察)。因此,我们的组首先使用水母发光蛋白测量 P中的[Ca 2 + in ]。铜绿假单胞菌(Guragain 。,2013)。原始方案是为大肠杆菌开发的(Knight等人,1991),并在(Jones等人,1999)中进一步开发。在这里我们提出一个修改的协议适应,成功地用于研究Ca 2 + 体内平衡。铜绿假单胞菌,临床和环境重要的生物体(Guragain等人,2013)。

关键字:细胞内钙离子, 调节, 水母发光蛋白, 发光, 腔肠素, 铜绿假单胞菌

材料和试剂

  1. 一般用品
    1. 离心瓶(无需特定品牌)
    2. Microfuge管(不需要特定品牌)
    3. Lumitrac 96孔白色微孔板(Greiner Bio One,目录号:655075)
    4. 铝箔
    5. 塑料比色杯(BrandTech Scientific,目录号:759086D)

  2. 菌株和质粒
    1. 携带含有水母发光蛋白基因的pMBB66EH的铜绿假单胞菌菌株PAO1
    2. 编码来自Aequoria victoria的水母发光蛋白基因的质粒pMBB66EH (礼貌:Drs.Delfina Dominguez)

  3. 培养基
    1. Luria bertani(LB)琼脂(见Recipes)
    2. 生物膜基本培养基(BMM)(参见配方)
      1. 10x基础盐溶液
      2. 维生素溶液
      3. 痕量金属
      4. 1 M MgSO 4/v/v
  4. 化学品和缓冲液
    1. 羧苄青霉素,二钠(Gold Bio,目录号:C-103-5)
    2. IPTG(Gold Bio,目录号:I2481C)
    3. 氯化钙脱水物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
    4. 活死染色(Molecular Probes)
    5. 酵母提取物(BD,Bacto,目录号:212750)
    6. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653或VWR,目录号:E529-500ML)
    7. 胰蛋白胨(BD,Bacto,目录号:211705)
    8. 琼脂(BD,Bacto,目录号:214010)
    9. 纳米水
    10. 谷氨酸钠(Sigma-Aldrich,目录号:1446600)
    11. 甘油(Thermo Fisher Scientific,Fisher Scientific,目录号:G33)
    12. 磷酸二氢钠二水合物(NaH 2 PO 4)(Sigma-Aldrich,目录号:71500)
    13. 磷酸氢二钾(K 2 HPO 4)(Sigma-Aldrich,目录号:17835)
    14. 生物素(Gold Bio,目录号:B-950-1)
    15. 硫胺HCl(Gold Bio,目录号:T-260-25)
    16. HCl(Pharmco-Aaper,目录号:284000ACS)
    17. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Sigma-Aldrich,目录号:209198)。
    18. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z0251)
    19. 硫酸铁(II)七水合物(FeSO 4·7H 2 O)(Sigma-Aldrich,目录号:215422)
    20. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(Avantor Performance Materials,J.T.Baker,目录号:2540-04)
    21. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:230391)
    22. HEPES(Sigma-Aldrich,目录号:H3375)
    23. MgCl 2(Sigma-Aldrich,目录号:230391)
    24. Tergitol型NP-40,70%,在H 2 O中(Sigma-Aldrich,目录号:T1135)
    25. Coelenterazine(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C2944)
    26. 乙醇(Pharmco-Aaper,目录号:AAP-111000190CSGL)
    27. HEPES缓冲区(参见配方)
    28. 放电缓冲区(参见配方)
    29. 1 M CaCl 2溶液(参见配方)
    30. 6mM CaCl 2注射用溶液(参见配方)
    31. 腔肠素(见配方)

设备

  1. 多通道移液管(Finnipipette F1,Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4661030)
  2. Pipetman经典移液管(Gilson,目录号:F123600,F123615,F123601,F123602)
  3. 500 ml玻璃烧瓶(无需特定品牌)
  4. Synergy TM sup/TM Mx多模微板读数器(Biotek Instruments)和Gen5 TM sup 2.05 PC软件(BioTek Instruments)
  5. 37℃培养箱(台式培养箱)(VWR,目录号:89409-314)
  6. 37℃摇动培养箱(MaxQ 4000台式摇动培养箱)(Thermo Fisher Scientific,Thermo Scientific TM ,型号:SHKA4000)
  7. 使用转子型(Thermo Fisher Scientific,Thermo Scientific TM )离心(Thermo Fisher Scientific,Thermo Scientific ,型号:Sorvall >,型号:F13-14x50 cy)
  8. 离心机(Eppendorf,型号:5424)

程序

  1. 水母发光蛋白的表达和重建
    1. 将携带有编码水母发光蛋白基因的质粒pMMB66EH的PAO1菌株在含有羧苄青霉素(300μg/ml)的LB琼脂平板上在37℃下生长过夜。
    2. 接种5ml生长在LB琼脂板上的2-3个单菌落的BMM培养基,并在37℃下以200rpm振荡孵育12小时。此后省略抗生素以避免其对细菌的影响
    3. 转移1ml的12小时OD 600 600(如果需要通过稀释在新鲜的BMM培养基中调整OD)培养物至100ml新鲜的BMM培养基的500ml烧瓶中。在37℃,200rpm生长培养物直至中期对数期,如在先前的生长研究中确定的。例如,对于PAO1,在孵育12小时后OD OD 600为0.16时达到对数中期。
    4. 一旦达到对数中期,用1mM IPTG(1ml的100mM IPTG)诱导细胞并在37℃,200rpm温育2小时。在诱导结束时,测量培养物的OD 600,并与先前记录的生长曲线进行比较,以确保细胞仍处于其生长的对数生长期。
    5. 转移细胞到250毫升冰冷离心瓶中。从该步骤直到重构步骤,将细胞保持在冰上
    6. 通过在4℃下以15,000×g离心5分钟收获细胞。弃去上清液,用100ml冰冷的HEPES缓冲液洗涤细胞沉淀。为此,首先通过移液管将细胞重新悬浮于2ml HEPES缓冲液中,然后加入剩余的98ml HEPES缓冲液。
    7. 通过在4℃下以6000xg离心5分钟收集细胞。弃去上清液。
    8. 通过吸取并将1ml细胞悬浮液转移到新鲜的离心管中,将悬浮液收集在1250μlHEPES缓冲液中。
    9. 加入腔肠素至终浓度为2.5μM。为此,加入5μl的500μM腔肠素溶液到1 ml的细胞悬液。因为腔肠素对光敏感,所以从这一点开始的程序必须在黑暗中进行(参见注释)。
    10. 加入腔肠素后,在室温下孵育细胞悬浮液,不摇动30分钟。
      注意:此步骤称为重构步骤。由于腔肠素在大气氧存在下进行缓慢氧化,因此应严格避免摇晃。
    11. 通过在4℃下以6000xg离心5分钟收集细胞沉淀,并用1ml冰冷的HEPES缓冲液洗涤两次。
    12. 通过移液将最终收集的细胞沉淀重悬在1ml HEPES缓冲液中
    13. 测量这样制备的样品的OD 600。如果需要,通过加入HEPES缓冲液将细胞悬浮液的最终OD 600调节至0.4。

  2. 发光测量
    1. 吸取100微升的细胞悬浮液与重建水母发光蛋白进入96孔发光(白色)板,并在室温下在黑暗中平衡10分钟。当抑制剂或其他化合物对待测试的Ca 2 + 的影响时,在此步骤中添加化合物。
    2. 将样品板装入Synergy Mx板读数器,并以5秒的间隔记录1分钟的发光。使用此读数计算中的Ca 2 + 的基础水平。
    3. 为了研究细胞内Ca 2+ 2+水平对提高到在包括人体的一些环境中通常存在的毫摩尔浓度的细胞外Ca 2+ 2 +的响应,将制备的细胞暴露于以上加入1mM CaCl 2。为此,通过板读取器注射器将20μl的6mM CaCl 2注入每个孔中。在注射之前,用5ml的6mM CaCl 2引发注射器。如果需要测试其他化合物的即时效应,则通过第二注射器在该步骤(在CaCl 2之前,之后或代替CaCl 2)注入后者,然后进行发光测量。
    4. 注射后立即将样品混合1秒,并以5秒的间隔记录20分钟的发光(图1A)。混合和测量在仪器中预编程
    5. 为了估计样品中存在的剩余水母发光蛋白,简要地从读取器中取出板,并且向每个样品中手动添加120μl放电缓冲液,混合好,但是通过移液快速,并且将板负载回板中读者。以5秒间隔读取发光10分钟(图1B)。通过对在包括放电的整个实验期间检测到的发光求和来估计总水母发光蛋白。当计算Ca 2 + 的浓度时,使用总水母发光蛋白的这个值进行数据归一化(图1C)。
      注意:重要的是确保混合过程中不会形成气泡,因为气泡会干扰发光读数。

数据分析

  1. 计算游离细胞内钙浓度([Ca 2 + ])
    使用以下公式从发光(图1A)值计算[Ca 2+] +(图1C):pCa = 0.612(-log 10 < sub> k)+ 3.745
    在哪里,
    k是如Jones等人在1999年所描述的发光衰减速率常数(sec <-1>)。
    在每个时间点计算作为至少三个独立生物学重复的平均值的[Ca 2+ 2 + in ]。即使在收获和制备细胞期间的微小不一致也可能引起[Ca 2+]分布中的波动。因此,在不一致的情况下,强烈推荐用三个独立的生物重复重复该实验,确保细胞同步和收获在完全相同的增长点。某些突变体是特别敏感的,并且在它们的水平上产生更多的波动。在这些情况下,添加额外的重复,并且考虑至少70%的生物学重复所共享的[Ca 2+] + [+]/- >图谱用于进一步计算。排除显着偏离此轮廓的重复。通过生物学重复之间的标准偏差计算统计学显着性(图1D)

    图1.通过使用水母发光蛋白在PAO1中测量[Ca 2 + ] 。 A.代表性发光曲线。箭头表示添加1mM CaCl 2。 B.代表性放电发光分布。箭头表示添加放电缓冲液(参见配方)。 C.代表计算的[Ca 2 + ] in 。箭头表示添加1mM CaCl 2。 D.从三个独立的生物学重复计算的平均值[Ca 2 + ] 。箭头表示添加1mM CaCl 2。

笔记

  1. 两种类型的对照包括在研究中
    1. 对照1验证单独添加HEPES缓冲液不影响[Ca 2+] + [/sup] 。为此,注射单独的HEPES缓冲液代替1mM CaCl 2攻击,并且如所述遵循整个程序。未检测到缓冲效果。
    2. 对照2是为了确保水母发光蛋白不通过细胞膜渗漏。为此,将制备用于测量的100μl细胞悬浮液在黑暗中在室温下温育30分钟。通过15,000×g离心15分钟除去细胞,收集上清液,并与1mM CaCl 2混合。监测发光1分钟。在该实验期间,我们没有观察到发光的任何增加,因此证实没有水母发光蛋白从细胞渗漏到上清液中。我们还通过Live-Dead染色(Molecular Probes)验证了程序后的细胞活力
  2. 由于脱辅基水母发生蛋白的产生水平可能存在差异,所以发光曲线可能在重复之间变化。然而,由总的可用水母发光蛋白标准化的Ca 2+ 2+的计算浓度应该是一致的。
  3. 为了确保测量中的一致性,在相同的生长点诱导和收获细胞是重要的。在我们的研究期间,我们遵循OD和孵育时间以确定何时收获。仅允许细胞达到预期密度的30分钟窗口,其是基于先前生长分析估计的
  4. 水母发光蛋白是光敏感的,因此所有具有水母发光蛋白的溶液应该在黑色的管或覆盖有铝箔的管中保存和处理。我们还建议关闭房间里的灯,只允许暗淡的日光或来自相邻房间的间接光。虽然没有测试,因为水母发光蛋白的最大吸光度在350nm(Shimomura和Johnson,1969),使用蓝光可以是安全的。
  5. 尽管水母发光蛋白据报道在溶液中是稳定的(Prendergast,2000),我们不建议将细胞样品与重构水母发光蛋白(甚至在冰上)一起长时间储存​​,因为这可能引起发光分布的波动。因此,每次只监测三个样品。

食谱

  1. Luria Bertani(LB)琼脂
    5g酵母提取物
    5克NaCl
    10g胰蛋白胨
    15克琼脂
    将成分在1L超纯水和高压釜中混合
  2. 生物膜基本培养基(BMM)(Patrauchan等人,2005)
    混合100毫升无菌10x基础盐溶液(食谱2a)到900毫升无菌纳米纯水。加入1ml维生素溶液(配方2b),200μl痕量金属溶液(配方2c)和20μlMgSO 4(配方2d)。正确混合。
    1. 将10×基础盐溶液(9.0mM谷氨酸钠,50mM甘油,0.15mM NaH 2 PO 4,0.34mM K 2 HPO 4, 4mM,145mM NaCl):
      15g谷氨酸单钠 46克甘油 0.18g磷酸二氢钠一水合物(NaH 2 PO 4)
      0.78g磷酸氢二钾(K 2 HPO 4)
      84.7g氯化钠(NaCl)
      结合成分,并完全溶解在850毫升超纯水中。调节pH至7.0。使用纳米水将最终体积调节至1,000ml。通过高压灭菌消毒。
    2. 维生素溶液
      将1mg生物素溶解在10ml的超纯水中。等分1毫升生物素储备溶液在新管,并加入50毫克盐酸硫胺素,并适当混合。使用纳米纯水将最终体积调节至100 ml。过滤灭菌并储存在4°C。
    3. 痕量金属
      将10 ml浓盐酸稀释至70 ml超纯水中。添加以下成分:
      0.5g CuSO 4·5H 2 O 2 / 0.5g ZnSO 4·7H 2 O·h/v 0.5g FeSO 4·7H 2 O·h/v 0.2g MnCl 2·4H 2 O·m/2 完全溶解,使用纳米纯水将最终体积调节至100 ml。过滤灭菌或高压灭菌。
    4. 1 M MgSO 4/v/v 将24.64g MgSO 4·7H 2 O溶解在100ml的纳米纯水的最终体积中。该溶液通过高压灭菌消毒。
  3. HEPES缓冲液(25mM HEPES,125mM NaCl,1mM MgCl 2,pH7.5)
    5.96g HEPES
    7.3克NaCl
    0.0952 MgCl 2
    将成分溶解在900毫升超纯水中。将pH调节至7.5,用超纯水滴定至最终体积为1,000ml
  4. 放电缓冲液(12.5mM CaCl 2,2%NP-40的HEPES缓冲液)
    向5ml HEPES缓冲液中加入62.5μl1M CaCl 2溶液和143μlTergitol。通过在磁力搅拌板上搅拌轻轻混合以避免NP-40洗涤剂发泡
  5. 1 M CaCl 2溶液
    将36.75g的CaCl 2·2水合物溶解在250ml纳米纯水中。高压灭菌
    灭菌
  6. 6mM CaCl 2注射用溶液
    将240μl1M CaCl 2溶液加入到39.76ml HEPES缓冲液中并适当混合
  7. 腔肠素
    脉冲离心管收集全部数量的试剂(250μg腔肠素)在底部。加入1,136微升95%乙醇的试管,并通过移液充分但快速混合(避免光和乙醇蒸发)。将试管(封闭)在台上静置几分钟有助于溶解。溶解后,将50μl腔肠素溶液等分到0.5ml微量离心管中。用铝箔盖住微量离心管,-20℃保存 注意:colenterazine对光敏感度高,应在黑暗中处理。

致谢

我们感谢来自德克萨斯大学埃尔帕索的Delfina Dominguez博士分享了 E。携带pMMB66EH的大肠杆菌菌株。我们感谢Ian Reutlinger转换 p。铜绿假单胞菌PAO1菌株与含有水母发光蛋白基因的pMMB66EH质粒。这项工作得到了美国心脏协会(Award 09BGIA2330036)的授予助学金和OCAST的研究资助(奖HR12-167)的支持。

参考文献

  1. Bonora,M.,Giorgi,C.,Bononi,A.,Marchi,S.,Patergnani,S.,Rimessi,A.,Rizzuto,R. and Pinton,P.(2013)。  使用水母光蛋白水母发光蛋白探针的哺乳动物细胞中的亚细胞钙测量。 em> Nat Protoc 8(11):2105-2118。
  2. Dominguez,DC,Guragain,M。和Patrauchan,M。(2015)。  钙结合蛋白和钙信号在原核生物中。细胞钙 57(3):151-165。
  3. Guragain,M.,Lenaburg,DL,Moore,FS,Reutlinger,I。和Patrauchan,MA(2013)。  钙稳态在绿脓杆菌中需要多种转运蛋白和调节群体运动性。细胞钙 :350-361。
  4. Herbaud,ML,Guiseppi,A.,Denizot,F.,Haiech,J.and Kilhoffer,MC(1998)。  在枯草芽孢杆菌中的钙信号。 Biochim Biophys Acta 1448(2):212-226。
  5. Jones,HE,Holland,IB,Baker,HL和Campbell,AK(1999)。  在大肠杆菌中胞质游离Ca 2+缓慢变化突出两种推定的流入机制以响应细胞外钙的变化。 em> Cell Calcium 25(3):265-274。
  6. Knight,MR,Campbell,AK,Smith,SM和Trewavas,AJ(1991)。 
  7. Naseem,R.,Davies,SR,Jones,H.,Wann,KT,Holland,IB和Campbell,AK(2007)。  细胞溶质Ca 2+在 E中调节蛋白质表达。大肠杆菌通过从包涵体中释放。 Biochem Biophys Res Commun 360(1):33-39。
  8. Patrauchan,MA,Sarkisova,S.,Sauer,K.and Franklin,MJ(2005)。  钙影响海洋假单胞菌属生物膜相关生长期间的细胞和细胞外产物形成。微生物学151(Pt 9 ):2885-2897。
  9. Prendergast,FG(2000)。  结构生物学:生物发光照明。 自然 405(6784):291-293。
  10. Rosch,JW,Sublett,J.,Gao,G.,Wang,YD and Tuomanen,EI(2008)。  钙流出对于真核宿主中的细菌存活是必不可少的。 Mol microbiol 70 :435-444。
  11. Sarkisova,SA,Lotlikar,SR,Guragain,M.,Kubat,R.,Cloud,J.,Franklin,MJ and Patrauchan,MA(2014)。  A绿脓假单胞菌EF手蛋白EfhP(PA4107)调节高钙胁迫应答和毒力 PLoS One 9(2):e98985。
  12. Shimomura,O.和Johnson,FH(1969)。  生物发光蛋白水母发光蛋白的性质。生物化学 8(10):3991-3997。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Guragain, M., Campbell, A. K. and Patrauchan, M. A. (2016). Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa. Bio-protocol 6(23): e2041. DOI: 10.21769/BioProtoc.2041.
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