CN115950874A

CN115950874A - Method for rapidly and nondestructively detecting ergothioneine in cells

Info

Publication number: CN115950874A
Application number: CN202310057609.1A
Authority: CN
Inventors: 付钰; 王琳淇; 卢维来; 仇昊宁; 谢语嫣
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2023-04-11

Abstract

The invention discloses a method for rapidly and nondestructively detecting ergothioneine in cells. And rapidly detecting the ergothioneine in a single living cell based on Raman spectrum. The specific operation is as follows: obtaining living body cell fluid to be detected, washing, adjusting the concentration of the bacterial suspension, adding the obtained bacterial suspension into a sample detection pool, finding cells in the solution by using a microscope, irradiating the cells by using laser and obtaining a Raman spectrum of the cells, carrying out background removal, smoothing, baseline calibration and normalization on the obtained Raman spectrum data, detecting a characteristic peak of the Raman spectrum, and if the Raman spectrum of the cells has 1210cm ^‑1 And 1507cm ^‑1 And (4) judging that the living body cell to be detected contains ergothioneine by the Raman identification peak, and otherwise, judging that the living body cell to be detected does not contain ergothioneine. The method has the advantages of simple sample preparation, less sample quantity, no cell damage and short detection time, and can realize the detection of the ergothioneine in a single living cell.

Description

Method for rapidly and nondestructively detecting ergothioneine in cells

Technical Field

The invention belongs to the fields of biochemical technology and analytical chemistry, and particularly relates to a method for rapidly and nondestructively detecting ergothioneine in cells.

Background

Ergothioneine (EGT) molecular formula is C ₉ H ₁₅ N ₃ O ₂ S has the molecular weight of 229.3, is easy to dissolve in water and not easy to decompose, and is a rare natural chiral amino acid. Ergothioneine is a safe and nontoxic natural antioxidant, and has multiple physiological functions of relieving inflammation, scavenging free radicals, removing toxic substances, maintaining DNA biosynthesis, normal cell growth, cellular immunity and the like. Has wide application and market prospect in the fields of organ transplantation, cell preservation, food and beverage, cosmetics, animal feed and the like. Natural ergothioneine is known to be produced mainly by a limited microbial population such as actinomycetes, cyanobacteria and mushrooms (hanetal, 2021). Although the intestinal tract of animals and humans has microbial flora, there is currently no clear evidence that intestinal microorganisms are able to synthesize ergothioneine, which is obtained by exogenous uptake and accumulation in tissues such as erythrocytes, bone marrow, liver, kidney, etc. of animals or humans (CheahandHalliwell 2021; eyetal, 2007). It has been reported that ergothioneine can also be synthesized in some pathogenic bacteria, such as Burkholderia (garmagetia) (2018), mycobacterium tuberculosis (mycobacterium tuberculosis) (cummingetial, 2018) and aspergillus fumigatus (aspergillus fumigatus) (sheeridantal, 2016), for use in combating host defenses or enhancing resistance to antibiotics (cheahandhallliwell 2021). Ergothioneine is obtained by two methods of chemical synthesis and biological synthesis. Wherein the biosynthesis has the advantages of short fermentation period, high yield, low cost and the like (Chenjiamin et al, 2022). At present, ergothioneine can be produced by fermentation through recombinant microorganisms of genes such as escherichia coli, saccharomyces cerevisiae, aspergillus and the like.

The detection of the ergothioneine in the cells is important for the research of germplasm resource excavation, breeding of the ergothioneine-producing microorganisms, high-yield cell screening in recombinant strains and the like, industrial application and the like. The current methods for detecting ergothioneine mainly comprise the following methods: (1) Spectrophotometric methods, i.e., thiol oxidation mediated by copper ions in alkaline medium followed by rapid reaction with 2,2 '-dithiodipyridine (2, 2' -dipyridylisillide) at around pH1 (carlsonetal, 1974); (2) Thin layer chromatography, i.e., the extracted sample is placed in a glass plate filled with an adsorbent system, and the difference in the degree of adsorption of the compound to the adsorbent is used to distinguish the target compound (kanekoetal, 1980); (3) A high-efficiency capillary electrophoresis method, wherein a sample to be detected is added into a capillary, different components are migrated at a certain speed under the action of an electric field due to the difference of the sizes, the charged properties, the amounts and the like of different chemical molecules, and the different components are detected by laser-induced fluorescence in the migration process of the different chemical components (Sotgiaetal, 2013); (4) High Performance Liquid Chromatography (HPLC) (sotgiaetal, 2013, vanderhoeketal, 2022), the current HPLC method is the predominant method for detecting ergothioneine. Firstly, damaged cells or fermentation liquor are required to be extracted, crude ergothioneine in the cells or in the fermentation liquor is extracted through a reflux extraction method, an enzymolysis extraction method, an ultrasonic microwave combined extraction method and the like, finally, a sample to be detected is obtained through concentration, methanol or acetonitrile-water and the like are used as mobile phases, and whether the sample has the ergothioneine and the content thereof is detected through a hydrophilic column and a UV detector (with the wavelength of 254-257 nm). However, the detection method requires a complex preparation process of a sample to be detected, damages cells, has high requirement on the purity of a required reagent in the detection process, and is not suitable for detecting the change of ergothioneine in a single cell; (5) An inductively coupled plasma tandem mass spectrometry (HPLC-ICP-QQQ-MS) combined with HPLC is used, firstly, cell lysate is collected, cell fragments, proteins and the like are removed, the lysate is frozen and thawed by liquid nitrogen, TCEP and IAM solutions are added according to a certain proportion, after incubation for 2 hours at room temperature, the proteins in the lysate are removed through a molecular sieve and the like, the filtrate is added with HPLC/ICP-QQ-MS, and analysis is carried out by utilizing a 8800-ICP-QQQ-MS system, for example, the conditions for detecting ergothioneine in red blood cells are set to have the gas temperature of 500 ℃, the flow rate of 65, the spray voltage of 3500V, the capillary temperature of 300 ℃ and the scanning range of m/z of nuclear mass ratio of 170-600 (Kroepfuelet, 2019); (6) Also used in conjunction with HPLC was LC-MS/MS triple quadrupole tandem mass spectrometry, the sample was sparged with nitrogen, introduced into the detection chamber at 520 degrees, analyzed for optimal collision energy of ergothioneine by positive ion sparging, precursor and product ions were monitored using MRM as they passed through the mass spectrometer, and the data finally analyzed with analytical software (wangtal, 2013). The detection technologies all need complicated sample preparation work in the early stage, damage of cells, and need a large amount of samples, and cannot be applied to detection of the ergothioneine of single cells.

Disclosure of Invention

The invention aims to provide a method for rapidly and nondestructively detecting ergothioneine in cells. The method has the advantages of simple sample preparation, less sample amount, no cell damage and short detection time, and can realize the detection of the ergothioneine in a single living cell.

The method for rapidly and nondestructively detecting the ergothioneine in the cells is used for rapidly detecting the ergothioneine in the cells based on Raman spectrum.

The cells are single or multiple cells within the laser irradiation range.

In one embodiment of the invention, the cell is a cryptococcus neoformans cell of wild type WT, EGT1 gene mutant EGT1 Δ and EGT1 gene complementation strain PEGT1-EGT1 in Q phase (resting phase).

In the research of virulence and infection mechanism of conditional pathogenic fungi, the cryptococcus neoformans can form a dormant state, and cells in the dormant state can well resist the elimination of the immune system of a host. Metabolome research discovers that the resting cells can efficiently express ergothioneine for the first time. The detection of the ergothioneine in the cell is usually detected by an HPLC method, the rapid nondestructive detection of the single-cell ergothioneine is extremely challenging, and no rapid nondestructive single-cell ergothioneine detection technology is reported at present. In order to detect the ergothioneine in the cells more efficiently, quickly and nondestructively, a vibration spectrum (Raman spectrum) detection technology capable of detecting at a single cell level is adopted.

The method for rapidly and nondestructively detecting the ergothioneine in the cells comprises the following steps:

obtaining living body cell fluid to be detected, washing, adjusting the concentration of the bacterial suspension, adding the obtained bacterial suspension into a sample detection pool, finding cells in the solution by using a microscope, irradiating the cells by using laser, and obtaining a Raman spectrum of the cells, wherein if the Raman spectrum of the cells exists 1210cm ^-1 And 1507cm ^-1 If the identification peak is not the same, determining that the living body cell to be detected contains ergothioneine, otherwise, determining that the living body cell to be detected contains the ergothioneineDoes not contain ergothioneine.

Further, if the Raman spectrum of the cell is 1210cm ^-1 And 1507cm ^-1 The ergothioneine content in the living cells is judged to be higher if the Raman characteristic peak intensity is high.

1210cm in Raman spectrum of the cell ^-1 And 1507cm ^-1 The Raman identification peak is limited by the resolution of the instrument and equipment, and the identification peak can be 1-2cm ^-1 Of (3) is detected.

The number of cells irradiated by the laser is a single cell or a plurality of cells within the laser irradiation range.

In the method, the washing is to wash the cells in the obtained living cell fluid to be detected with sodium chloride solution or cell isotonic solution, and the washing is performed for a plurality of times, specifically 2-3 times;

wherein the sodium chloride solution is 0.8-0.9% NaCl or a NaCl solution suitable for the physiological concentration of the cell;

adjusting the concentration of the bacterial suspension to 10 ⁴ -10 ⁷ CFU/mL；

The sample detection pool can be a liquid sample pool which is disclosed in Chinese patent ZL2020101989793 and is used for detecting aerobic or facultative anaerobic pathogenic microorganisms by using micro-Raman spectrum; or a sample detection cell with a quartz or calcium fluoride substrate; or a substrate sample detection cell without Raman scattering background interference;

the Raman spectrum system used for cell Raman spectrum detection is an upright or inverted micro-Raman spectrum system, wherein the wavelength of a laser of the Raman spectrum system is any one or combination of 532nm, 633nm or 785nm and the like.

Before cell Raman spectrum detection, firstly, a characteristic peak of a material special for Raman spectrometer calibration is adopted to correct a Raman spectrum system;

the material for calibrating the Raman spectrometer is polystyrene microspheres or silicon wafers and the like;

when the cell Raman spectrum is detected, the total integration time is more than 3 seconds, and the power of the lasers with different wavelengths is based on no damage to the cells;

in one embodiment of the invention, a 785nm continuous laser is used as the light source for exciting raman scattering of the cells, for a total integration time of 20 seconds or more.

The method further comprises performing background removal, smoothing, baseline calibration and normalization on the obtained Raman spectrum data,

the smoothing method can adopt a convolution smoothing method, a moving average method, gaussian filtering, bilateral filtering or mean filtering and the like;

the baseline calibration method can adopt a polynomial fitting method, a BEADS algorithm, a wavelet algorithm, empirical Mode Decomposition (EMD) and the like;

the normalization method may employ a min-max method, area normalization, vector normalization, or the like.

The method has the advantages of simple sample preparation, less sample amount, no cell damage and short detection time, and can realize the detection of the ergothioneine in a single living cell.

Drawings

FIG. 1 is a graph of the Raman spectra of ergothioneine at different concentration gradients, with the concentration of ergothioneine being in mg/mL.

FIG. 2 is a graph showing the results of HPLC detection of ergothioneine in different strains of Cryptococcus neoformans. WT: wild type, egt1 Δ ergothioneine synthesis gene-deleted strain, Q: rest period, P: reproductive phase, standard: ergothioneine standard spectrum.

FIG. 3 is a Raman spectrum of L.neoformans at Q-phase for detection of ergothioneine by different strains of Cryptococcus neoformans. A. Raman spectrum overall pattern of different strains. B. Characteristic peaks of ergothioneine in cells are enlarged. C-D, ergothioneine Raman peak intensity statistical chart, t-test double-tail detection, ^** P<0.01， ^**** P<0.0001,ns means no significant difference.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Examples

Genome analysis of cryptococcus neoformans and knockout experiments of key synthetic genes of ergothioneine prove that the key gene for synthesizing the ergothioneine is EGT1 gene, and the mutant strain EGT1 delta of the gene causes cryptococcus neoformans cells to be incapable of forming the ergothioneine. We examined in vitro culture of Cryptococcus neoformans for 6 hours (propagation, labeled P) and 72 hours (stationary phase Quiescience, labeled Q) wild type and egt 1. DELTA. By HPLC. It was found that only wild-type cells cultured to the resting stage produced ergothioneine, and the EGT1 gene was a key gene for the synthesis of intracellular ergothioneine. In order to further realize the rapid and nondestructive detection of the ergothioneine in the cells, the Raman spectroscopy technology is utilized to detect the unicellular Raman spectrum of the cryptococcus neoformans. Detecting whether the tested strain is Wild Type (WT), gene mutant strain egt1 delta for synthesizing ergothioneine, and gene mutation anaplerotic strain P for synthesizing ergothioneine _EGT1 Raman spectrum of EGT 1. Meanwhile, the Raman spectrum of the ergothioneine compound in the aqueous solution is detected by using the Raman spectrum.

The specific steps for detecting ergothioneine in cryptococcus neoformans by HPLC are as follows:

the cryptococcus neoformans ergothioneine synthesis related gene EGT1 is knocked out by using a CRISPR/Cas9 gene editing system. The cell concentration was 2.5X 10 ⁸ CFU/mL cells in a 6-hour propagation period and 72-hour resting period are cultured, the culture medium supernatant is removed by centrifugation, ceramic beads are added to shatter the thalli, and the whole process is kept at a low temperature. Then adding 50% methanol to constant volume, shaking up, placing on ice to cool for 20min, and centrifuging at 1000rpm for 2min. Transferring the supernatant to a 3kDa ultrafiltration tube, ultrafiltering at low temperature (5000 rpm, 60min), collecting the filtrate as sample solution, and refrigerating in refrigerator. The samples were injected into a High Performance Liquid Chromatography (HPLC) system. Mobile phase: 0.1% of threeFluoroacetic acid-acetonitrile (99, v/v); flow rate: 0.7mL/min; detection wavelength: 260nm.

WT, egt 1. Delta. And P of Cryptococcus neoformans _EGT1 Three strains of-EGT 1 were simultaneously transferred to YPD medium and cultured at 37 ℃ for 6 hours and 72 hours. Collecting the thallus of the three strains. Collecting cell thallus by centrifuging at 8000rpm for 5min to obtain living cell liquid, and washing cells in the living cell liquid with 0.85% saline or cell isotonic solution for 2-3 times. Adjusting the concentration of the bacterial suspension to 10 ⁴ -10 ⁷ CFU/mL. The bacterial suspension was pipetted using a 20. Mu.L pipette and added to the sample detection cell (patent No. ZL 2020101989793). The Raman spectrum system is a micro Raman spectrum system, and the excitation wavelength of the Raman spectrum system is 785nm. The characteristic peak of the polystyrene microsphere is used for correcting the Raman spectrum system before the cell Raman spectrum is detected. And finding cells in the solution under a microscope, and irradiating the single cells by using laser, wherein the total integration time is more than 20 seconds, and further acquiring the Raman spectrum of the single cells. The number of the measured Raman spectra of each strain is not less than 50. The raman spectra obtained for all cells were subjected to background removal, savitzky _ Golay smoothing, 6 th order polynomial fit baseline calibration and min-max normalization. Raman spectrum analysis of ergothioneine solutions with different concentrations shows that the ergothioneine concentration is 1210cm ^-1 And 1507cm ^-1 The most prominent Raman characteristic peak (figure 1 is a Raman spectrum peak diagram of ergothioneine with different concentration gradients, the concentration unit of the ergothioneine is mg/mL, the operation is as follows, the concentration of the ergothioneine solution is prepared to be 1.0, 1.4, 1.6, 1.8 and 2.0mg/mL, an inverted microscopic Raman spectrometer is adopted to detect the Raman spectra of the ergothioneine with different concentrations, the wavelength of a laser of the Raman spectrometer is 785nm, the integral time of laser irradiation on the ergothioneine with different concentrations is more than 3 seconds, the average number of the spectra of the collected solutions under each gradient solution is not less than 3, the obtained Raman spectra are subjected to background removal, savitzky _ Golay smoothing treatment, 6-order polynomial fitting baseline calibration and minimum-maximum normalization treatment), and can be used as a candidate peak for detecting the identification peak of the ergothioneine in the cell.

The HPLC detection of different strains of cryptococcus neoformans is only foundWild-type WT and cells containing the egt1 gene in Q phase were able to synthesize ergothioneine (FIG. 2). The Raman spectrum analysis of the cells of the corresponding strains revealed that two characteristic peaks of ergothioneine could be observed in the Raman spectrum of cryptococcus neoformans (FIGS. 3A-B), thus 1210cm ^-1 And 1507cm ^-1 Can be determined as the peak of the marker for detecting the ergothioneine in the cell. Wild type WT at 1210cm ^-1 And 1507cm ^-1 The Raman peak intensity is obvious, while the characteristic peak of the egt1 delta deletion strain (cells can not synthesize ergothioneine) at the position is obviously reduced or disappeared, and P _EGT1 The marked peak intensity of ergothioneine in cells of strains with-EGT 1 complementing EGT1 genes (capable of synthesizing ergothioneine) was significantly higher than that of the EGT 1. Delta. Deleted strain and similar to wild type (FIGS. 3C-D). The analysis results of HPLC and cell Raman spectrum prove that the Raman spectrum can be applied to the ergothioneine detection of single cells and only needs to pass through the identification peak 1210cm ^-1 And 1507cm ^-1 And determining whether the cells contain ergothioneine. The Raman spectrum is used as a technique for detecting ergothioneine in living cells, and has the advantages of less sample amount, no need of breakage, rapidness, single cell detection and the like.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

1. A method for rapidly and nondestructively detecting ergothioneine in cells is used for rapidly detecting the ergothioneine in the cells based on Raman spectrum.

2. The method of claim 1, wherein: the method for rapidly and nondestructively detecting ergothioneine in cells comprises the following steps:

obtaining living body cell fluid to be detected, washing, adjusting the concentration of the bacterial suspension, adding the obtained bacterial suspension into a sample detection pool, finding cells in the solution by using a microscope, irradiating the cells by using laser, and obtaining a Raman spectrum of the cells, wherein if the Raman spectrum of the cells exists 1210cm ^-1 And 1507cm ^-1 If the identification peak is positive, the ergothioneine in the living body cell to be detected is judged to be contained, otherwise, the ergothioneine in the living body cell to be detected is judged not to be contained.

3. The method of claim 2, wherein: if the Raman spectrum of the cell is 1210cm ^-1 And 1507cm ^-1 And if the Raman identification peak intensity is high, the ergothioneine content in the living body cell to be detected is judged to be higher.

4. A method according to claim 2 or 3, characterized in that: 1210cm in Raman spectrum of cell ^-1 And 1507cm ^-1 The Raman identification peak is limited by the resolution of the instrument and equipment, and the identification peak can be 1-2cm ^-1 Of (3) is detected.

5. The method according to any one of claims 2-4, wherein: the number of cells irradiated by the laser is a single cell or a plurality of cells within the laser irradiation range.

6. The method according to any one of claims 2-5, wherein: the washing is to wash the cells in the obtained living body cell sap to be detected with sodium chloride solution or cell isotonic solution, and the washing is carried out for a plurality of times, specifically for 2-3 times;

adjusting the concentration of the bacterial suspension to 10 ⁴ -10 ⁷ CFU/mL。

7. The method according to any one of claims 2-6, wherein: the Raman spectrum system used for cell Raman spectrum detection is an upright or inverted micro-Raman spectrum system, wherein the excitation wavelength of the Raman spectrum system is any one or combination of 532nm, 633nm and 785 nm;

when the cell Raman spectrum is detected, the total integration time is more than 3 seconds.

8. The method of claim 7, wherein: a785 nm continuous laser is used as a light source for exciting Raman scattering of cells, and the total integration time is more than 20 seconds.

9. The method according to any one of claims 2-8, wherein: the method further comprises performing background removal, smoothing, baseline calibration, and normalization on the obtained raman spectral data.

10. The method of claim 9, wherein: the smoothing method adopts a convolution smoothing method, a moving average method, gaussian filtering, bilateral filtering or mean filtering;

the baseline calibration method adopts a polynomial fitting method, a BEADS algorithm, a wavelet algorithm and empirical mode decomposition;

the normalization method adopts a minimum-maximum value method, area normalization and vector normalization.