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May 2020

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Fluorescence-based Heme Quantitation in Toxoplasma Gondii
基于荧光的弓形虫血红素定量   

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Abstract

Toxoplasma gondii is a highly prevalent protozoan pathogen throughout the world. As a eukaryotic intracellular pathogen, Toxoplasma ingests nutrients from host cells to support its intracellular growth. The parasites also encode full or partial metabolic pathways for the biosynthesis of certain nutrients, such as heme. Heme is an essential nutrient in virtually all living organisms, acting as a co-factor for mitochondrial respiration complexes. Free heme is toxic to cells; therefore, it gets conjugated to proteins or other metabolites to form a “labile heme pool,” which is readily available for the biosynthesis of hemoproteins. Previous literature has shown that Toxoplasma gondii carries a fully functional de novo heme biosynthesis pathway and principally depends on this pathway for intracellular survival. Our recent findings also showed that the parasite’s intracellular replication is proportional to the total abundance of heme within the cells. Moreover, heme abundance is linked to mitochondrial oxygen consumption for ATP production in these parasites; thus, they may need to regulate their cellular heme levels for optimal infection when present in different environments. Therefore, quantitative measurement of heme abundance within Toxoplasma will help us to understand the roles of heme in subcellular activities such as mitochondrial respiration and other events related to energy metabolism.

Keywords: Toxoplasma gondii (弓形虫), Protozoan (原生动物), Heme (亚铁血红素), Fluorescence assay (荧光分析), Biochemical quantitation (生化定量), Protoporphyrin IX (原卟啉IX), Fluorescence plate reader (荧光板读写器)

Background

Heme, an organic molecule, plays a vital role in virtually all living organisms. For example, heme binds to hemoglobin and myoglobin for oxygen transport and serves as a co-factor for several enzymes within the electron transport chain for mitochondrial respiration (Ponka, 1999). Previous literature has shown that Toxoplasma has a fully functional de novo heme biosynthesis pathway (Bergmann et al., 2020). Genetic deletion of Toxoplasma heme biosynthetic enzymes results in decreased replication in vitro (Bergmann et al., 2020; Krishnan et al., 2020; Tjhin et al., 2020) and the loss of acute virulence in a murine model (Bergmann et al., 2020); therefore, inhibition of de novo heme production sheds light on the development of novel therapeutic strategies for managing Toxoplasma infections.


Generally, there are four methods for the measurement of heme abundance in cells: 1) Pyridine hemochrome-based heme quantitation (Sinclair et al., 2001). This strategy replaces the nitrogen ligands of protein-bound heme with pyridine under alkaline conditions. The resulting hemochrome is further reduced and oxidized before spectrophotometric quantitation; 2) Reversed-phase HPLC-based quantitation of heme and its intermediates (Sinclair et al., 2001). An acetone/HCl/water solution is used to extract heme and its biosynthetic intermediates from intact cells or cell homogenates, followed by separation on a C18 HPLC column. The standards of pure heme and its intermediates are run on the column before sample measurement to help recognize their peaks and quantitate their abundances; 3) Protoporphyrin IX (PPIX)-based fluorescence assay (Sinclair et al., 2001). PPIX is produced by the penultimate enzyme within the classic de novo heme biosynthesis pathway (Phillips, 2019). PPIX is further conjugated with a ferrous iron group to form a functional heme molecule. The non-conjugated PPIX molecule displays strong fluorescence, whereas heme is non-fluorescent; therefore, stripping ferrous ion from heme generates fluorescent PPIX, which can be quantitated by a fluorometer and represents heme abundance; 4) Biosensor-based heme quantitation by live-cell imaging or flow cytometry (Song et al., 2015; Hanna et al., 2016; Yuan et al., 2016). Genetically encoded hemoproteins, such as horseradish peroxidase (HRP)- or ascorbate peroxidase (APX)-based biosensors, can be expressed in different organelles to detect their labile heme content (Yuan et al., 2016). Additionally, the heme-binding proteins are genetically fused to heme-sensitive fluorescent proteins to quantitate labile heme within live cells by ratiometric fluorescence quantitation or fluorescence resonance energy transfer (FRET) (Song et al., 2015; Hanna et al., 2016).


The pyridine hemochrome-based strategy is not very sensitive and requires large amounts of parasites as initial material. The HPLC-derived quantitation requires expensive equipment and columns. Additionally, it only can quantitate one sample per run. The biosensor-based method demands the implementation of biosensors in target organisms. Overexpression of biosensors may disturb labile heme pools in the cells and further impair their health status. The PPIX-based fluorescence heme quantitation is economical and sensitive. Moreover, it can be performed in 96-well plate format as a semi high-throughput strategy for multiple samples simultaneously.


In the procedure for the PPIX-based heme quantitation assay, the heme-bound ferrous ion can be chemically removed by oxalic acid via boiling before measurement. During the assay, non-boiled parallel samples must be included to detect background fluorescence signals within parasite strains, which will be subtracted from the corresponding boiled samples. Here, we modify the previously published PPIX-based fluorescence heme quantitation assay (Sinclair et al., 2001). In this protocol, the parasites are mechanically liberated from host cells, filter-purified, and ruptured by sonication prior to heme quantitation. As an example, we measured and compared heme abundances in wildtype parasites and a heme-deficient transgenic Toxoplasma strain. This method employs a plate reader to quantitate heme in a 96-well plate format; it shows high sensitivity and only requires 5-10 × 107Toxoplasma parasites for measurement, which can be harvested from 1-2 T25 flasks. This strategy can be modified for measuring heme abundances in other apicomplexan parasites.


Materials and Reagents

  1. Nucleopore track-etched hydrophilic membrane filter, 3.0-µm pore size (VWR, catalog number:28158-624) with filter holder (Fisher Scientific, catalog number: SX0002500)

  2. 15 ml polystyrene conical tubes (VWR, catalog number: 10026-084)

  3. 1.5 ml Eppendorf microcentrifuge tubes (VWR, catalog number: 89000-028)

  4. Black 1.5 ml microcentrifuge tubes (VWR, catalog number: 47751-688)

  5. 10 ml syringes (VWR, catalog number: BD309646)

  6. Blunt-end needles (McMaster-Carr, catalog numbers: 75165A761 [20G]; 76165A759 [25G])

  7. Cellstar® black 96-well plates without lids (VWR, catalog number: 82050-732)

  8. 1,000 ml glass beaker (VWR, catalog number: 10754-960)

  9. 5 ml and 10 ml serological pipettes (VWR, catalog numbers: 89130-896, 89130-898)

  10. 20 µl, 200 µl, and 1,000 µl micropipette tips (VWR, catalog numbers: 76322-134, 76322-150, 76322-138)

  11. Toxoplasma gondii RH∆ku80hxg strain lacking the ku80 and hypoxanthine-xanthine-guanine phosphoribosyl transferase genes. This strain is provided by the Carruthers lab at the University of Michigan Medical School. Lack of the ku80 gene boosts homology-dependent recombination in the parasites (Huynh and Carruthers, 2009). Loss of hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXG) allows this strain to be genetically modified by exogenous DNA vectors carrying the HXG gene. This strain is widely used in the Toxoplasma research field as a wildtype parental strain.

  12. RH∆ku80hxgcpox strain (abbreviated to ∆cpox). A heme-deficient parasite strain was generated in a previous study (Bergmann et al., 2020). This strain lacks coproporphyrinogen-III oxidase (TgCPOX) that catalyzes the antepenultimate reaction within the parasite’s heme biosynthesis pathway.

  13. RH∆ku80hxgcpoxCPOX strain (abbreviated to ∆cpoxCPOX). A TgCPOX complementation strain was generated in a previous study (Bergmann et al., 2020). Heme deficit is restored in this strain.

  14. Human Foreskin Fibroblasts (HFFs) (American Type Culture Collection (ATCC), catalog number: SCRC-1041)

  15. Corning® Dulbecco’s Modified Eagle Medium (DMEM) (VWR, catalog number: 45000-304)

  16. 100× Penicillin-Streptomycin (VWR, catalog number: 45000-652)

  17. 200 mM L-glutamine (VWR, catalog number: 45000-676)

  18. 1 M HEPES Free Acid (VWR, catalog number: 45000-696)

  19. Hyclone® Cosmic Calf Serum (Cytiva, catalog number: SH30087.03)

  20. HyClone® Dulbecco’s Phosphate-Buffered Saline (DPBS) powder, without calcium and magnesium (Powder) (Cytiva, catalog number: SH30013.04)

  21. Pig hemin powder (VWR, catalog number: BT138155-1G)

  22. Oxalic acid dihydrate (VWR, catalog number: 97064-978)

  23. Isopropanol (VWR, catalog number: BDH11744)

  24. DPBS (see Recipes)

  25. D10 medium (see Recipes)

  26. 1 mM heme solution (see Recipes)

  27. 2 M oxalic acid solution (see Recipes)

Equipment

  1. Eppendorf CellXpert® C170 cell culture incubator (Eppendorf)

  2. Eppendorf refrigerated centrifuge (Eppendorf, model: 5810R)

  3. Branson Analog Sonifier ultrasonic cell disrupter S-250A (VWR, catalog number: 33995-309) with a tapered 1/8-inch microtip (VWR, catalog number: 33996-163)

  4. Biotek Synergy H1 Hybrid Multi-Mode microplate reader (Biotek Instruments)

  5. Micropipette set (P-1000, P-200, and P-20) (VWR, catalog number: 75788-456)

  6. Hemocytometer (VWR, catalog number: 15170-168). Instructions can be found in http://www.hausserscientific.com/products/reichert_bright_line.html

  7. Hot plate (VWR, Advanced Hot Plate, catalog number: 97042-642)

Software

  1. BioTek® Gen5 Data Analysis Software

  2. Microsoft Excel

  3. GraphPad Prism (Version 9)

Procedure

Please see Figure 1 for a schematic description of the entire procedure.



Figure 1. A schematic outline of the fluorescence-based heme quantitation in Toxoplasma gondii. Please refer to the text for more details.


  1. In this protocol, wildtype (RH∆ku80hxg), ∆cpox (a heme-deficient strain), and ∆cpoxCPOX (a TgCPOX complementation strain) strains are included as examples. If parasite strains are stored at -80°C, they must be thawed at 37°C and immediately passed to a T25 tissue culture flask containing fully confluent human foreskin fibroblast (HFF) cells. After the parasites lyse the host cells, ~300 µl fully lysed wildtype and ∆cpoxCPOX parasites were passed to one T25 tissue culture flask coated with HFF cells for a 2-day passage. For the ∆cpox strain, the inoculum was increased to 2 ml to compensate for its severe growth defects. 1-2 T25 flasks of parasites per strain are needed depending on their growth phenotype. Since the ∆cpox showed severe growth defects, two flasks of ∆cpox parasites were used in this assay. The wildtype and ∆cpoxCPOX strains were passed into one T25 flask.

  2. Pre-cool DPBS on ice and keep the infected cells on ice. Scrape the infected HFFs and syringe them three times using a 20 G needle, followed by three times using a 25 G needle. The lysates were passed through a 3.0-µm filter to remove intact host cells and large host cell debris. Rinse the flasks with 5 ml ice-cold DPBS and pass through the filter to wash.

  3. Fill a 1,000-ml glass beaker with water and keep it boiling on a hot plate for sample heating. The boiling water will be used in Step 16 to heat the samples.

  4. Centrifuge parasites at 1,000 × g for 10 min at 4°C. After centrifugation, a parasite pellet is expected to be seen at the bottom of the centrifuge tube.

  5. Carefully aspirate the supernatant and resuspend the parasite pellet in 10 ml ice-cold DPBS. Repeat Step 4. Check for the pellet before proceeding.

  6. Aspirate the supernatant carefully and resuspend the pellet in 10 ml ice-cold DPBS.

  7. Quantitate the yield of purified parasites using a hemocytometer following the vendor’s instructions.

  8. Pellet the parasites at 1,000 × g for 10 min at 4°C.

  9. Aspirate the supernatant and resuspend the parasites in 1 ml ice-cold DPBS and transfer to a 1.5-ml microcentrifuge tube.

  10. Spin down the parasites at 5,000 × g at 4°C for 5 min.

  11. Aspirate the supernatant and resuspend the remaining pellet in 400 µl ice-cold DPBS.

  12. Sonicate each purified parasite strain inside a biosafety cabinet using a Branson Analog Sonifier ultrasonic cell disrupter S-250A with a tapered 1/8-inch microtip, using the following settings: output intensity = 3 and Duty% = 20%. Sonicate each purified parasite strain for 10 s and repeat 4 times. Wait for 30 s between each repeat and keep samples on ice to avoid overheating.

  13. A hemin standard curve is needed for the measurement of absolute heme abundance per parasite. The hemin stock is initially diluted in DPBS to 1,200 nM, followed by a 3-fold serial dilution to generate 5 additional concentrations at 400, 133.3, 44.4, 14.8, and 4.9 nM. DPBS alone is also included as a blank for 0 nM hemin.

  14. Add 100 µl each sample or hemin standard to black 1.5-ml centrifuge tubes. Two sets for each strain or hemin standard are prepared; one will be boiled and another one will remain at room temperature.

  15. Add 900 µl 2 M oxalic acid to each sample and vortex for complete mixing.

  16. Keep one set of samples and hemin dilutions in a rack at room temperature and place another set in a tube holder to boil for 30 min.

  17. Place the boiled samples on ice for 5 min and then leave them at room temperature for 10 min. Mix all samples by vortexing.

  18. Pipette 200 µl each parasite stain or hemin standard into each well of a black 96-well plate in triplicate.

  19. Read the samples using a BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader with the following settings: Excitation: 400 nm, Emission: 608 nm, Optics: Top, Gain: 135, Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10, and Read Height: 7 mm. Export the acquired data to an Excel spreadsheet.

  20. Repeat the assay in at least three biological replicates for statistical significance comparison.

Data analysis

  1. Calculation of the normalized heme abundance in Toxoplasma parasites (Figure 2):



    Figure 2. Example data and analysis for PPIX-based heme quantitation in Toxoplasma parasites. A. Excel table of the raw data from one biological replicate of heme quantitation in wildtype, ∆cpox, and ∆cpoxCPOX parasites. B. Data analysis of normalized heme abundances in Toxoplasma parasites. The heme abundance in wildtype parasites was set as 100% for normalization of heme amounts in other strains.


    1. Calculate the average readings for each boiled and non-boiled sample.

    2. Subtract the fluorescence of every non-boiled sample from the signal of the corresponding boiled sample.

    3. Subtract the blank value (0 nM hemin sample) from each parasite strain.

    4. Divide the mean values for the individual samples by the number of parasites calculated by the hemocytometer counts.

      Note: One-twentieth of the purified parasites are included in each well for quantitation of heme abundance (A quarter of the total purified parasites are mixed with oxalic acid before boiling, and one-fifth of the boiled mixture is pipetted into each well of a 96-well plate for fluorescence measurement).

    5. Divide the average fluorescence value per parasite for each sample by that from the wildtype strain for normalization. The normalized value for wildtype parasites is set as 100% for comparison with other strains.

  2. Calculation of the absolute heme abundance in Toxoplasma parasites (Figure 3):



    Figure 3. Calculation of the absolute heme abundance in Toxoplasma parasites. A. Table of fluorescence (A.U.) signals under each concentration of hemin standard. B. Graph of a linear regression of the plotted hemin standard curve. C. Linear regression analysis to generate the equation for calculating heme concentrations in lysates of purified parasites. D. Step-by-step data analysis for calculating heme amounts per parasite.


    If the absolute heme abundance per parasite is desired, the standard heme curve is required to determine the heme concentration in sonicated parasite lysates by substituting fluorescence values from each strain in the heme standard curve equation. The total amount of heme in each parasite lysate is determined by multiplying the heme concentration of each sample by the volume (400 µl) and dividing by the total number of parasites to yield the absolute heme abundance per parasite.

    To generate the hemin standard curve:

    1. Average the fluorescence intensities of boiled and non-boiled samples for each concentration of hemin standard.

    2. Subtract the average fluorescence values for the individual non-boiled samples from the corresponding boiled samples.

    3. Subtract the blank value (0 nM hemin) from the individual hemin concentrations.

    4. Plot the subtracted fluorescence values against the individual hemin concentrations at 4.9, 14.8, 44.4, 133.3, 400, and 1,200 nM.

    5. Perform linear regression on the plotted data points for curve fitting to produce the heme standard curve equation, which will be used to calculate heme abundances in the tested parasite lysates.

Recipes

  1. DPBS

    Add 9.6 g HyClone Dulbecco's phosphate-buffered saline powder in 1 L deionized water. The solution is autoclaved before use.

  2. D10 medium

    Dulbecco’s Modified Eagle Medium (DMEM) is supplemented with 10 mM HEPES, 10% (v/v) Cosmic calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

  3. 1 mM heme solution

    Dissolve 32.6 mg hemin in 50 ml 20 mM NaOH.

  4. 2 M oxalic acid solution

    Add 126.07 g oxalic acid dihydrate to 500 ml deionized water. Heat water to ~50°C for solution preparation. The solution is oversaturated, and the solute will precipitate when the solution cools to room temperature. Use the supernatant in the assay.

Acknowledgments

The authors would like to thank Dr. Carruthers for sharing the RH∆ku80hxg Toxoplasma strain for this study. This work was supported by the Clemson Startup fund (to Z.D.), Knights Templar Eye Foundation Pediatric Ophthalmology Career-Starter Research Grant (to Z.D.), a pilot grant of an NIH COBRE grant P20GM109094 (to Z.D.), and NIH R01AI143707 (to Z.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This protocol was modified from the previously published PPIX-based heme quantitation assay (Sinclair et al., 2001) used for heme quantification in mammalian cells.

Competing interests

The authors declare no conflicts or competing interests.

References

  1. Bergmann, A., Floyd, K., Key, M., Dameron, C., Rees, K. C., Thornton, L. B., Whitehead, D. C., Hamza, I. and Dou, Z. (2020). Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection. PLoS Pathog 16(5): e1008499.
  2. Hanna, D. A., Harvey, R. M., Martinez-Guzman, O., Yuan, X., Chandrasekharan, B., Raju, G., Outten, F. W., Hamza, I. and Reddi, A. R. (2016). Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc Natl Acad Sci U S A 113(27): 7539-7544.
  3. Huynh, M. H. and Carruthers, V. B. (2009). Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8(4): 530-539.
  4. Krishnan, A., Kloehn, J., Lunghi, M., Chiappino-Pepe, A., Waldman, B. S., Nicolas, D., Varesio, E., Hehl, A., Lourido, S., Hatzimanikatis, V. and Soldati-Favre, D. (2020). Functional and Computational Genomics Reveal Unprecedented Flexibility in Stage-Specific Toxoplasma Metabolism. Cell Host Microbe 27(2): 290-306 e211.
  5. Phillips, J. D. (2019). Heme biosynthesis and the porphyrias. Mol Genet Metab 128(3): 164-177.
  6. Ponka, P. (1999). Cell biology of heme. Am J Med Sci 318(4): 241-256.
  7. Sinclair, P. R., Gorman, N. and Jacobs, J. M. (2001). Measurement of heme concentration. Curr Protoc Toxicol Chapter 8: Unit 8 3.
  8. Song, Y., Yang, M., Wegner, S. V., Zhao, J., Zhu, R., Wu, Y., He, C. and Chen, P. R. (2015). A Genetically Encoded FRET Sensor for Intracellular Heme. ACS Chem Biol 10(7): 1610-1615.
  9. Tjhin, E. T., Hayward, J. A., McFadden, G. I. and van Dooren, G. G. (2020). Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis. J Biol Chem 295(6): 1539-1550.
  10. Yuan, X., Rietzschel, N., Kwon, H., Walter Nuno, A. B., Hanna, D. A., Phillips, J. D., Raven, E. L., Reddi, A. R. and Hamza, I. (2016). Regulation of intracellular heme trafficking revealed by subcellular reporters. Proc Natl Acad Sci U S A 113(35): E5144-5152.

简介

[摘要]弓形虫是世界范围内高度流行的原生动物病原体。作为一种真核细胞内病原体,弓形虫从宿主细胞中摄取营养以支持其细胞内生长。寄生虫还编码某些营养物质(例如血红素)的生物合成的全部或部分代谢途径。血红素是在几乎所有生物体必需的营养素,作为一个辅助因子的线粒体呼吸复合物。游离血红素对细胞有毒;因此,它与蛋白质或其他代谢物结合形成“不稳定的血红素池” , ”,可用于血红素蛋白的生物合成。先前的文献表明,弓形虫具有功能齐全的从头血红素生物合成途径,并且主要依赖该途径进行细胞内存活。我们最近的研究结果还表明,寄生虫的细胞内复制与细胞内血红素的总丰度成正比。此外,H EME丰度与线粒体氧消耗ATP产量本身寄生虫; 因此,当存在于不同环境中时,y可能需要调节其细胞血红素水平以获得最佳感染。因此,弓形虫内血红素丰度的定量测量将有助于我们了解血红素在亚细胞活动中的作用,如线粒体呼吸和其他与能量代谢相关的事件。


[背景]血红素是一种有机分子,几乎在所有生物体中都起着至关重要的作用。例如,血红素结合血红蛋白和肌红蛋白对氧传输,并作为一个辅因子为电子传递链为线粒体呼吸内几种酶(Ponka,1999) 。以前的文献有显示Ñ该弓形虫具有全功能的从头血红素生物合成途径(贝格曼等人,2020) 。弓形虫血红素生物合成酶的遗传缺失导致体外复制减少(Bergmann等人,2020 年;Krishnan等人,2020 年;Tjhin等人,2020 年)和小鼠模型中急性毒力的丧失(Bergmann等人,2020 年)。, 2020) ; 吨herefore,抑制从头血红素生产阐明了新的治疗策略的开发,用于管理弓形虫感染。

一般地,有四种方法用于血红素丰度在细胞中的测定:1)将吡啶血色素基血红素孔定量吨通货膨胀(辛克莱。等人,2001) 。该策略在碱性条件下用吡啶代替蛋白质结合的血红素的氮配体。将得到的血色素被进一步还原和氧化之前光度法孔定量吨通货膨胀; 2)反相HPLC为基础的孔定量吨血红素和其中间体的通货膨胀(辛克莱等人,2001) 。甲N A cetone / HCl /水溶液用于提取血红素和从完整细胞或细胞匀浆,接着分离其生物合成中间体上一个C18 HPLC柱。纯血红素及其中间体的标准品在样品测量前在色谱柱上运行,以帮助识别ir峰并对其丰度进行定量;3) 基于原卟啉 IX (PPIX) 的荧光测定(Sinclair等,2001)。PPIX 由经典的从头血红素生物合成途径中的倒数第二个酶产生(Phillips, 2019) 。PPIX 进一步与亚铁基团结合形成功能性血红素分子。未结合的 PPIX 分子显示出强烈的荧光,而血红素则没有n-荧光;吨herefore,从汽提血红素亚铁离子产生的荧光PPIX,其可以是孔定量达ED由一荧光计,并且表示血红素丰度; 4)生物传感器为基础的血红素孔定量吨由活细胞成像通货膨胀或流式细胞术(宋等人,2015 ;汉纳。等人,2016;元。等人,2016) 。基因编码的血红素蛋白,例如基于辣根过氧化物酶 (HRP) 或抗坏血酸过氧化物酶 (APX) 的生物传感器,可以在不同的细胞器中表达以检测其不稳定的血红素含量(Yuan等,2016)。另外,所述血红素结合蛋白基因融合于血红素敏感的荧光蛋白以孔定量泰特由不稳定的血红素活细胞内比率荧光孔定量吨通货膨胀或荧光共振能量转移(FRET) (宋等人,2015;汉纳等人。, 2016) 。

基于吡啶血色素的策略不是很敏感,需要大量的寄生虫作为初始材料。HPLC的衍生孔定量吨通货膨胀需要昂贵的设备和列。此外,它每次运行只能定量一个样品。基于生物传感器的方法需要在目标生物体中实施生物传感器。生物传感器的过度表达可能会扰乱细胞中不稳定的血红素池,并进一步损害其健康状况。基于PPIX荧光血红素孔定量吨通货膨胀是经济的和敏感的。此外,它可以在 96 孔板格式中进行,作为同时处理多个样品的半高通量策略。

在该过程中为所述基于PPIX血红素孔定量吨通货膨胀测定中,血红素结合的亚铁离子可化学通过草酸经由测量之前煮沸除去。在测定过程中,必须包括未煮沸的平行样本,以检测寄生虫菌株内的背景荧光信号,这些信号将从相应的煮沸样本中减去。在这里,我们修改先前公布的基于PPIX荧光血红素孔定量吨通货膨胀测定(辛克莱等人,2001) 。在这个协议中,这些寄生虫在机械上的宿主细胞,过滤和纯化的释放,并通过超声处理之前血红素孔定量破裂吨通货膨胀。作为一个例子,我们测量和比较在野生型寄生虫和血红素丰度一个血红素缺陷型的转基因弓形虫菌株。该方法采用一板读出器孔定量泰特血红素一个96孔板格式; 它显示出高灵敏度,只需要5 - 10 × 10 7弓形虫进行测量,可以从1 - 2个T25烧瓶中收获。可以修改此策略以测量其他顶端复合体寄生虫中的血红素丰度。

关键字:弓形虫, 原生动物, 亚铁血红素, 荧光分析, 生化定量, 原卟啉IX, 荧光板读写器



材料和试剂


核孔径迹蚀刻亲水膜过滤器,3.0 µm 孔径(VWR,目录号:28158-624)带过滤器支架(Fisher Scientific,目录号:SX0002500)
15毫升聚苯乙烯锥形管(VWR,目录号:10026-084)
1.5 ml Eppendorf 微量离心管(VWR,目录号:89000-028)
黑1.5 ml离心管小号(VWR,目录号:47751-688)
10毫升注射器小号(VWR,目录号:BD309646)
钝-端针(麦克马斯特-卡尔,目录号小号:75165A761 [ 20G ] ; 76165A759 [ 25G ] )
蜂星®黑色96孔板无盖小号(VWR,目录号:82050-732)
1,000 ml玻璃烧杯(VWR,目录号:10754-960 )
5毫升和10ml血清移液管(VWR,目录号小号:89130-896,89130-898 )
20微升,200微升,和1 ,000微升微量提示(VWR,目录号小号:76322-134,76322-150,76322-138 )
弓形虫RHΔ Ku80蛋白Δ HXG应变缺乏荷兰国际集团的ķ U80和次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶基因。该菌株由密歇根大学医学院的 Carruthers 实验室提供。大号的ACK的Ku80蛋白在寄生虫基因提升同源-依赖性重组(黄长发和Carruthers的,2009年)。大号次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶(HXG)的OSS允许该菌株进行基因修饰,通过携带外源DNA的载体的HXG基因。该菌株作为野生型亲本菌株广泛用于弓形虫研究领域。
RH∆ ku80 ∆ hxg ∆ cpox应变(缩写为 ∆ cpox )。在已生成血红素缺陷寄生虫株一个先前的研究(贝格曼等人,2020) 。该菌株缺乏可催化寄生虫血红素生物合成途径中倒数第二次反应的粪卟啉原-III 氧化酶 ( TgCPOX ) 。
RH∆ ku80 ∆ hxg ∆ cpoxCPOX应变(缩写为 ∆ cpoxCPOX )。甲TgCPOX在产生互补应变一个先前的研究(贝格曼等人。,2020) 。在该菌株中血红素缺乏得以恢复。
人包皮成纤维细胞(HFF)(美国典型培养物保藏中心(ATCC),目录号:SCRC-1041)
康宁® Dulbecco改良的Eagle培养基(DMEM)(VWR,目录号:45000-304)
100×青霉素-链霉素(VWR,目录号:45000-652)
200 mM L-谷氨酰胺(VWR,目录号:45000-676)
1 M HEPES游离酸(VWR,目录号:45000-696)
Hyclone ® Cosmic Calf Serum(Cytiva ,目录号:SH30087.03)
HyClone ® Dulbecco 磷酸盐-缓冲盐水(DPBS)粉末,不含钙和镁(粉末)(Cytiva ,目录号:SH30013.04)
猪血红素粉(VWR,目录号:BT138155-1G)
草酸一个CID二水合物(VWR,目录号:97064-978)
异丙醇(VWR,目录号:BDH11744)
DPBS(小号EE食谱)
D10培养基(小号EE食谱)
1mM的血红素溶液(小号EE食谱)
2 M草酸溶液(见配方)


设备


的Eppendorf CellXpert ® C170 Ç ELL Ç ulture我ncubator(Eppendorf)中
Eppendorf 冷冻离心机(Eppendorf ,型号:5810R )
Branson Analog Sonifier超声波细胞破碎仪 S-250A(VWR,目录号:33995-309),带有锥形 1/8 英寸微尖端(VWR,目录号:33996-163)
Biotek的协同H1混合多模式米icroplate ř EADER(Biotek的仪器)
微量移液器套装(P-1000、P-200 和 P-20)(VWR,目录号:75788-456)
血细胞计数器(VWR,目录号:15170-168)。说明可以在http://www.hausserscientific.com/products/reichert_bright_line.html中找到
热板(VWR,Advanced Hot Plate,目录号:97042-642)


软件


BioTek ® Gen5 数据分析软件
微软Excel
的GraphPad Prism(V版为9)


程序


请参见图 1了解整个过程的示意图。




图1.基于荧光的血红素孔定量的示意性轮廓TA在灰弓形虫。请参阅正文以获取更多详细信息。


在这个协议中,野生型(RH Δku80Δhxg ),Δ CPOX (一个血红素缺陷型菌株),和Δ cpoxCPOX (一个TgCPOX互补株)菌株一个包括作为例子重。如果寄生虫菌株储存在-80 ° C,它们必须在 37 °C下解冻,并立即传递到含有完全融合的人类包皮成纤维细胞 (HFF) 细胞的 T25 组织培养瓶中。后的寄生虫裂解的宿主细胞中,约300μ升充分裂解野生型和Δ cpoxCPOX寄生虫传递到涂有HFF细胞用于一个T25组织培养瓶一个2天的通年龄。对于所述Δ CPOX株,接种物增加至2μm升,以补偿其严重的生长缺陷。1 - 2 T25每株寄生虫的烧瓶需要根据它们的生长表型。由于 Δ cpox表现出严重的生长缺陷,因此在该测定中使用了两个 Δ cpox寄生虫烧瓶。将野生型和ΔcpoxCPOX菌株传递到一个 T25 烧瓶中。
在冰上预冷 DPBS并将感染的细胞保持在冰上。刮的感染HFFS和注射器他们三次使用一20号针,随后三次使用一个25号针。将裂解物穿过一个3.0微米的过滤器以除去完整的宿主细胞和宿主大细胞碎片。用 5 ml 冰冷的DPBS冲洗烧瓶并通过过滤器进行洗涤。
将水装满 1,000 毫升玻璃烧杯,并在加热板上保持沸腾以加热样品。沸水将在步骤16中使用,以加热所述样品。
在 4 °C 下以1,000 × g离心寄生虫10 分钟。离心后,寄生虫沉淀在预期的底部待观察的离心管中。
小心吸上清,重悬寄生虫沉淀在10毫升冰冷DPBS。重复小号TEP 4.检查在继续之前沉淀。
小心吸出上清液,将沉淀重悬在10 ml 冰冷的 DPBS 中。
孔定量泰特纯化寄生虫的产量使用下列供应商的说明血球。
在 4 °C 下以 1,000 × g 的速度将寄生虫颗粒化10 分钟。
吸出上清液,将寄生虫重悬在1 ml 冰冷的 DPBS 中,然后转移到 1.5 ml 微量离心管中。
在 4 °C下以5,000 × g 的速度旋转寄生虫5 分钟。
吸出上清液并将剩余的沉淀重悬在400 µl 冰冷的 DPBS 中。
使用带有锥形 1/8 英寸微尖端的Branson Analog Sonifier超声波细胞破坏器 S-250A ,使用以下设置对生物安全柜内的每个纯化寄生虫菌株进行超声处理:输出强度 = 3 和占空比 = 20%。对每个纯化的寄生虫菌株进行超声处理 10 秒并重复 4 次。等待的每个重复之间30秒,并保持在冰样本,以避免过热。
需要用于甲氯化血红素标准曲线的每寄生虫绝对血红素丰度的测量。氯高铁血红素的库存最初稀释在DPBS至1200纳米,接着一个3倍连续稀释,以产生5个在附加浓度400,133.3,44.4,14.8,和4.9 nM的。DPBS单独也作为一个空白为0纳米血红素。
将100 µl每种样品或氯化血红素标准品加入黑色 1.5 毫升离心管中。每个菌株或氯化血红素标准品准备两套;一个将被煮沸,另一个将保持在室温。
向每个样品中加入900 µl 2 M 草酸并涡旋以完全混合。
在室温下将一组样品和氯化血红素稀释液放在架子上,并将另一组样品放在管架中煮沸30 分钟。
放置在冰上煮沸的样品5分钟,然后让它们在室温下搅拌10分钟。通过涡旋混合所有样品。
移液管将200μl各寄生虫污渍或氯高铁血红素标准到每个孔中的一个黑色96孔平板中,一式三份。
读取使用样品一个BioTek的协同H1混合多模式酶标仪使用以下设置:激发:400纳米,发射:608纳米,光学:顶部,增益:135,读取速度:正常,延时:100毫秒,测量/数据点:10,读取高度:7 毫米。导出的采集的数据的Excel电子表格。
在至少三个生物学重复中重复该测定以进行统计显着性比较。


数据一nalysis


的计算的归一化血红素丰度弓形虫寄生虫(图2 ):




图2.实施例的数据和分析基于PPIX血红素孔定量吨通货膨胀在弓形虫寄生虫。一个。从血红素的一种生物复制的原始数据的Excel表格孔定量吨通货膨胀在野生型,Δ CPOX ,和Δ cpoxCPOX寄生虫。乙。数据分析的标准化血红素丰度弓形虫的寄生虫。野生型寄生虫的血红素丰度设置为 100%,用于其他菌株血红素量的标准化。


计算每个煮沸和未煮沸的样品的平均读数。
从相应煮沸样品的信号中减去每个非煮沸样品的荧光。
减去该空白值(0 nM的从每个寄生虫株氯高铁血红素样品)。
除以平均值为在通过计算寄生虫数量个别样品通过了血球计数。
注:二十分之一的纯化寄生虫被包括在每个孔中孔定量吨血红素丰度(四分之一的通货膨胀的总纯化寄生虫沸腾之前与草酸混合,五分之一的煮沸混合物移液到每个孔用于荧光测量的96 孔板)。


分割的每寄生虫平均荧光值用于从每个样品由与野生型菌株进行归一化。野生型寄生虫的归一化值设置为 100%,以便与其他菌株进行比较。


的计算的绝对丰度血红素在弓形体寄生虫(图3 ):




图 3. 计算弓形虫寄生虫的绝对血红素丰度。一个。Ť能够荧光的(AU)信号氯高铁血红素标准的每个浓度下。乙。ģ绘制的氯高铁血红素的标准曲线的线性回归的拍摄和。Ç 。线性回归分析,以发电机密封e为方程为CALCULAT荷兰国际集团中纯化寄生虫溶胞产物血红素浓度。d 。用于计算每个寄生虫的血红素量的分步数据分析。


如果需要每个寄生虫的绝对血红素丰度,则需要标准血红素曲线,通过替换血红素标准曲线方程中每个菌株的荧光值来确定超声处理的寄生虫裂解物中的血红素浓度。血红素的各寄生虫溶胞产物的总量是由含多处确定颖通过每个样品的浓度血红素的体积(400 μ升)和DIVID荷兰国际集团由寄生虫的总数,得到每寄生虫的绝对丰度血红素。


要生成氯化血红素标准曲线:


平均每个浓度的血红素标准的煮沸和非煮沸样品的荧光强度。
减去的平均荧光值用于所述从相应的煮沸的样品个别非煮沸样品。
减去空白值(0 nM的从氯高铁血红素)的个体氯高铁血红素浓度。
绘制针对减去荧光值的个别氯高铁血红素浓度4.9,14.8,44.4,133.3,400,和1 ,200 nM的。
上进行线性回归的绘制的数据点进行曲线拟合,以产生血红素标准曲线方程,这将被用于计算丰度血红素在所述测试寄生虫溶胞产物。


食谱


DPBS
添加9.6克HyClone公司Dulbecco氏p hosphate -b uffered小号在1次艾琳粉末大号去离子水。该溶液在使用前进行高压灭菌。


D10 中号
Dulbecco改良的Eagle培养基(DMEM)中补充有10mM HEPES,10%(V / V)宇宙Ç ALF小号erum,2mM的L-谷氨酰胺,100IU / ml青霉素,和100μg/ ml链霉素。


1 mM 血红素溶液
将 32.6 mg 氯化血红素溶解在 50 ml 20 mM NaOH 中。


2M草酸溶液
将 126.07 克二水草酸加入 500 毫升去离子水中。将水加热至~ 50 °C以进行溶液制备。溶液过饱和,当溶液冷却至室温时溶质会沉淀。在测定中使用上清液。


致谢


作者想感谢卡拉瑟斯博士分享了RHΔ的Ku80 Δ HXG弓形虫株这项研究。这项工作得到了克莱姆森启动基金(对 ZD)、圣殿骑士眼科基金会儿科眼科职业启动研究补助金(对 ZD)、NIH COBRE 赠款 P20GM109094(对 ZD)和 NIH R01AI143707(对 ZD)的试点资助)。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。该协议是从以前出版的基于PPIX血红素孔定量改性吨通货膨胀阿萨ý (辛克莱等人,2001)用于在哺乳动物细胞中血红素定量。


利益争夺


作者声明没有冲突或竞争利益。


参考


Bergmann, A., Floyd, K., Key, M., Dameron , C., Rees, KC, Thornton, LB, Whitehead, DC, Hamza, I. 和 Dou, Z. (2020)。弓形虫需要其类似植物的血红素生物合成途径进行感染。PLoS病原体16(5):e1008499。              
Hanna, DA, Harvey, RM, Martinez-Guzman, O., Yuan, X., Chandrasekharan, B., Raju, G., Outten , FW, Hamza, I. 和Reddi , AR (2016)。基因编码的荧光血红素传感器揭示的血红素动力学和贩运因素。Proc Natl Acad Sci USA 113(27): 7539-7544。
Huynh, MH 和 Carruthers, VB (2009)。标记在一个内源基因的弓形虫弓形虫应变缺乏Ku80蛋白。 真核细胞8(4):530-539。
Krishnan, A., Kloehn , J., Lunghi , M., Chiappino -Pepe, A., Waldman, BS, Nicolas, D., Varesio , E., Hehl , A., Lourido , S., Hatzimanikatis , V.和 Soldati-Favre, D. (2020)。功能和计算基因组学揭示了阶段特异性弓形虫代谢前所未有的灵活性。细胞宿主微生物27(2):290-306 e211。              
菲利普斯,JD(2019 年)。血红素生物合成和卟啉症。Mol Genet Metab 128(3): 164-177。
Ponka , P. (1999)。血红素细胞生物学。Am J Med Sci 318(4): 241-256。
Sinclair, PR, Gorman, N. 和 Jacobs, JM (2001)。血红素浓度的测量。Curr Protoc Toxicol第 8 章:第 8 单元 3.              
Song, Y., Yang, M., Wegner, SV, Zhao, J., Zhu, R., Wu, Y., He, C. 和 Chen, PR (2015)。用于细胞内血红素的基因编码 FRET 传感器。ACS 化学生物学10(7): 1610-1615。
Tjhin , ET, Hayward, JA, McFadden, GI 和 van Dooren , GG (2020)。弓形虫中顶质体定位酶 TgUroD 的表征揭示了顶质体在血红素生物合成中的关键作用。J Biol Chem 295(6): 1539-1550。              
Yuan, X., Rietzschel , N., Kwon, H., Walter Nuno, AB, Hanna, DA, Phillips, JD, Raven, EL, Reddi , AR 和 Hamza, I. (2016)。亚细胞记者揭示的细胞内血红素运输的调节。 Proc Natl Acad Sci USA 113(35):E5144-5152。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bergmann, A. and Dou, Z. (2021). Fluorescence-based Heme Quantitation in Toxoplasma Gondii. Bio-protocol 11(12): e4063. DOI: 10.21769/BioProtoc.4063.
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