Method for super-resolution imaging of bacterial extracellular newborn protein

Abstract

The invention relates to the field of biological analysis and detection, in particular to a method for super-resolution imaging of bacterial extracellular newborn protein. On the basis of biological orthogonal amino acid and alkyne fluorescent dye, super-resolution imaging in the in-situ visualized space-time deposition process of the extracellular newborn protein is realized by utilizing a super-resolution microscope. Compared with a traditional research method, the method has the advantages of being easy to operate, rapid, efficient, high in universality and low in biological disturbance, the imaging challenge of space-time deposition of the extracellular matrix is solved, key information related to the extracellular matrix is provided, understanding of the biological membrane forming process is deepened, and good application prospects are achieved.

Description

Technical Field

[0001] The invention relates to the field of biological analysis and detection, and in particular to a method for super-resolution imaging of bacterial extracellular nascent proteins. Background Art

[0002] Biofilm is a membrane tissue formed by extracellular polymers (EPS) surrounding microorganisms. The formation of biofilm is a dynamic process, which mainly includes the microbial colonization stage, aggregation stage, maturity stage, and microbial shedding and recolonization stage. As a protective survival method preferred by prokaryotes, biofilm is extremely widely distributed, causing persistent and stubborn biofilm pollution problems in industrial production, human health and other aspects. In this process, EPS produced by microorganisms plays an important role.

[0003] EPS is composed of high molecular polymers secreted by microorganisms outside the body, and its main components include proteins, polysaccharides, etc. Usually, EPS is attached to the surface of bacteria or surrounds bacteria, hindering the contact between cells and the external environment. Some functional macromolecules in it determine the structure and function of biofilms in terms of biofilm adhesion and chemical functional group adsorption. At present, traditional research methods for EPS cover in vitro analysis such as molecular sequencing and chemical colorimetry, as well as in vivo studies such as molecular imaging. The in vitro analysis process is overly dependent on the extraction of EPS components. Since it is impossible to accurately identify intracellular and extracellular substances, this method is still controversial. In vivo imaging studies on key components of EPS provide a more direct visual basis for revealing EPS identity information. For example, extracellular proteins are one of the main components of EPS and are also participants in key functions. Methods such as radiolabeled isotopes and immunofluorescence staining can achieve in vivo labeling and imaging of a specific target molecule in extracellular proteins. However, these methods lack spatiotemporal resolution and universality, and have problems such as long time and high cost, and can provide very limited identity information about EPS. Bioorthogonal amino acids can be incorporated by cells and participate in protein synthesis. By combining cells with specific chemical molecules with fluorescent groups, fluorescent labeling of extracellular nascent proteins can be achieved. This labeling method is fast, simple, and efficient, and is widely used in the study of in situ activity of microorganisms. Microscopic imaging technology combined with bioorthogonal amino acid fluorescent staining has become a powerful tool for studying EPS structure, composition, and dynamic processes.

[0004] In recent years, super-resolution microscopy (SRM) technology has been continuously developed, increasing the imaging resolution to tens or even a few nanometers. Among them, the most widely used SRM technologies include structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). Among them, STED has the advantages of real-time imaging and rapid acquisition. Its resolution is five times higher than that of traditional living cell microscopy. It is the only pure optical super-resolution imaging technology that does not require post-image reconstruction. STED has great application potential in the imaging of fluorescent molecules in complex systems. Therefore, it is of great practical significance to provide a STED-based method for imaging bacterial extracellular nascent proteins. Summary of the invention

[0005] In view of this, the present invention provides a method for super-resolution imaging of bacterial extracellular nascent proteins, the purpose of which is to achieve super-resolution imaging of the spatiotemporal deposition process of extracellular nascent proteins in situ visualization. The method of the present invention is simple to operate, fast and efficient, has strong universality, and has low biological disturbance. It solves the imaging challenge of spatiotemporal deposition of extracellular matrix, provides key information about extracellular matrix, deepens the understanding of the biofilm formation process, and has good application prospects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for imaging a nascent protein, comprising: incorporating bioorthogonal amino acids into a culture medium, culturing cells with the culture medium to obtain a culture, and staining the culture multiple times to obtain an image;

[0008] The bioorthogonal amino acid is: (i) an amino acid having an azide group; or (ii) an amino acid derivative having an azide group;

[0009] The multiple staining is to stain the culture multiple times with an alkyne dye labeled with a fluorescent group, wherein the fluorescent group is different in each staining;

[0010] The multiple times are two, three, four, five, six, seven, eight, nine, ten or more times.

[0011] In some specific embodiments of the present invention, the different in the above imaging method includes that the fluorescent colors emitted by the fluorescent groups are different.

[0012] In some specific embodiments of the present invention, the nascent protein in the above imaging method may be a nascent protein derived from a biological membrane.

[0013] In some specific embodiments of the present invention, the nascent protein of the above imaging method may be a nascent protein derived from a biofilm produced by at least one bacterium.

[0014] In some specific embodiments of the present invention, the final concentration of the bioorthogonal amino acid in the culture medium of the above imaging method is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.55 mM, 0.6 mM, 0.65 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1.0 mM.

[0015] In some specific embodiments of the present invention, the bioorthogonal amino acid of the above imaging method can be L-azidohomoalanine.

[0016] In some specific embodiments of the present invention, the cells in the above imaging method may be model organisms for studying biofilms.

[0017] In some specific embodiments of the present invention, the cell in the above imaging method may be Pseudomonas aeruginosa PAO1.

[0018] In some specific embodiments of the present invention, the acquired images in the above imaging method are acquired based on a super-resolution microscope and / or a laser confocal microscopy imaging system.

[0019] In some specific embodiments of the present invention, the acquired image of the above imaging method can be acquired based on a laser confocal microscopy imaging system, and its excitation wavelength can be 405 nm and / or 488 nm.

[0020] In some specific embodiments of the present invention, the final concentration of the alkyne dye labeled with a fluorescent group in the above imaging method is 30 µM, 35 µM, 40 µM, 45 µM, 50 µM, 55 µM, 60 µM, 65 µM or 70 µM.

[0021] In some specific embodiments of the present invention, the alkyne dye labeled with a fluorescent group in the above imaging method includes a cyclooctyne dye labeled with a fluorescent group.

[0022] In some specific embodiments of the present invention, the culture carrier in the above imaging method is at least one of a cell slide, a glass-bottomed culture dish, and a glass slide.

[0023] In some specific embodiments of the present invention, the above imaging method further comprises the step of fixing the culture after multiple stainings and before acquiring images.

[0024] In some specific embodiments of the present invention, the imaging method further comprises the step of staining the culture with 4',6-diamidino-2-phenylindole.

[0025] In some specific embodiments of the present invention, the above imaging method comprises the following steps:

[0026] Step (1): culturing Pseudomonas aeruginosa PAO1 in a culture medium containing L-azidohomoalanine to obtain a biofilm;

[0027] Step (2): immersing the biofilm with a cyclooctyne dye labeled with a fluorescent group 1, reacting, and obtaining a first stained biofilm;

[0028] Step (3): immersing the biofilm with a cyclooctyne dye labeled with a fluorescent group 2, reacting to obtain a second stained biofilm;

[0029] Step (4): Obtain an image of the second stained biofilm by acquiring laser confocal microscopy imaging.

[0030] In some specific embodiments of the present invention, the imaging method further includes a step of fixing the biofilm stained for the second time after step (3) and before step (4), wherein fixation refers to a process of using any method to make the morphology, structure and position of intracellular substances as close as possible to that of their living state.

[0031] In some specific embodiments of the present invention, the imaging method further comprises a step of staining the biofilm with 4',6-diamidino-2-phenylindole after step (1) and before step (2).

[0032] In some specific embodiments of the present invention, the imaging method further comprises a step of staining the first biofilm with 4',6-diamidino-2-phenylindole after step (2) and before step (3).

[0033] In some specific embodiments of the present invention, the imaging method further comprises a step of staining the second biofilm with 4',6-diamidino-2-phenylindole after step (3) and before step (4).

[0034] The method of the present invention has the following effects:

[0035] The present invention provides a method for super-resolution imaging of extracellular nascent proteins in the process of bacterial film formation, which provides key information of extracellular proteins in the film formation process. The experimental operation is simple and efficient, has low biological disturbance, and has strong universality, and has good promotion prospects in biofilm application research.

[0036] Specifically, the method of the present invention has the following outstanding advantages over the prior art:

[0037] (1) The operation steps of the present invention have low bioturbation and can achieve in situ imaging of extracellular nascent proteins in a complex biofilm system without the need for any component extraction;

[0038] (2) The present invention is universal and applicable to the labeling of extracellular proteins of all microorganisms that can utilize bioorthogonal amino acids, rather than just labeling a specific protein of a specific microorganism;

[0039] (3) The present invention solves the challenge of distinguishing new and old extracellular proteins. Dyes with different fluorescent groups are used to perform sequential extracellular protein staining, and the order of fluorescence appearance of the sequential staining is used to identify which are the original extracellular proteins and which are the new extracellular proteins; the excitation and emission wavelengths of the fluorescent groups are far apart, eliminating the phenomenon of fluorescence cross-coloring;

[0040] (4) The present invention utilizes super-resolution microscopy STED imaging technology, which does not require subsequent image reconstruction and is simple and efficient to operate, thus achieving super-resolution imaging of extracellular nascent proteins in complex biomembrane systems;

[0041] (5) The operation steps of the present invention are simple, rapid and efficient, and the imaging of extracellular proteins can be obtained within three hours after obtaining the biofilm sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0043] Figure 1 shows the cell distribution map after whole-cell staining;

[0044] Figure 2 Figure 2 shows the visualization of extracellular newly generated proteins, where (a) is a schematic diagram of extracellular newly generated protein staining, (b) is a super-resolution imaging of extracellular newly generated proteins and cells in the biofilm incubated for 16 h, and (c) is the fluorescence distribution curve of extracellular newly generated proteins and cells corresponding to line 1;

[0045] Figure 3 The super-resolution images of extracellular newly formed proteins and cells at different biofilm incubation periods (5 h, 19 h, 3 d) are shown, where (a) shows the labeling of extracellular newly formed proteins in the biofilm incubated for 5 h; (b) shows the labeling of extracellular newly formed proteins in the biofilm incubated for 19 h; (c) shows the labeling of extracellular newly formed proteins in the biofilm incubated for 3 d;

[0046] Figure 4Figure 4 shows the time-series labeling imaging of extracellular newly generated proteins in the biofilm, where (a) is the overlay of two fluorescence channels, (b) is the imaging of the extracellular original proteins under the 488 channel, and (c) is the imaging of the extracellular newly generated proteins under the 405 channel. DETAILED DESCRIPTION

[0047] The present invention discloses a method for super-resolution imaging of bacterial extracellular nascent proteins. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] The present invention uses bioorthogonal amino acids to incubate Pseudomonas aeruginosa (a model strain for biofilm research). The method uses a cyclooctyne dye with a fluorescent group to mark and image the extracellular protein incorporated with the amino acid at different time points in the film formation process; by changing the fluorescent group of the cyclooctyne dye, the extracellular nascent protein incorporated with the amino acid is sequentially fluorescently labeled; 4',6-diamidino-2-phenylindole is used to label the whole cell to distinguish the intracellular and extracellular structures; and the labeled sample is subjected to STED super-resolution imaging. Under a super-resolution microscope, the method can clearly and intuitively observe the distribution morphology and spatiotemporal evolution process of extracellular proteins at different biofilm formation periods. It can distinguish the nascent proteins from the original extracellular proteins and capture the generation of extracellular nascent proteins in situ.

[0049] The technical solution of the present invention is as follows:

[0050] (1) Activate the strain by streaking, pick a single clone and place it in LB medium, and culture at 37°C with shaking until the OD 600 =0.2~0.8. The cultured bacteria were collected by centrifugation, resuspended in an equal volume of mineral salt medium and then incubated in biofilm;

[0051] (2) Transfer the above bacterial solution to a 24-well plate containing cell slides or a laser confocal cell culture dish with a glass bottom or a slide at a ratio of 1:1000. Add bioorthogonal amino acids at a final concentration of 0.1~1.0 mM and incubate the biofilm at 37°C for 2 h~5 d;

[0052] (3) Add cyclooctyne dye to PBS solution to prepare staining solution with a final concentration of 10-50 µM.

[0053] (4) Remove excess culture medium from the incubated biofilm sample and wash it three times with PBS solution; add the pre-prepared staining solution and react in the dark for 15-60 min. Wash it three times with PBS solution; for the time-sequential labeling of extracellular newly generated proteins, the labeled biofilm is continued to be cultured at 37°C in fresh mineral salt culture medium containing 0.1-1.0 mM bioorthogonal amino acids. After the incubation is completed, the extracellular newly generated proteins are labeled with cyclooctyne dyes with different fluorescent groups;

[0054] (5) The labeled biofilm samples were fixed in 4% paraformaldehyde and washed three times with PBS;

[0055] (6) Prepare whole-cell staining solution in PBS solution. Stain the fixed biofilm with 4',6-diamidino-2-phenylindole to a final concentration of 5-10 µg mL -1 ; Temporal labeling of extracellular nascent proteins skips this step;

[0056] (7) Using STED and confocal laser microscopy systems, images of extracellular nascent proteins and whole cells were obtained at the excitation wavelengths of various fluorescence signals (405 nm and 488 nm).

[0057] The model strain Pseudomonas aeruginosa in the present invention is purchased from a bacterial strain bank.

[0058] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0059] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0060] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0061] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0062] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0063] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0064] The present invention will be further described below in conjunction with the embodiments.

[0065] Example 1: Bacterial culture

[0066] a. Inoculate the Pseudomonas aeruginosa PAO1 (WT) stored in a glycerol tube into liquid LB medium at a volume ratio of 1%, and culture at 37°C with shaking. After overnight culture, use the plate streak method to obtain a monoclonal strain. Pick a monoclonal strain and culture it in LB medium at 37°C with shaking, and grow it to OD 600 The value is around 0.2~0.8.

[0067] b. Collect the bacterial liquid in the LB tube, centrifuge to obtain the bacterial pellet, resuspend it in an equal volume of mineral salt medium (35.9 mM K2HPO4, 14.2 mM KH2PO4, 9.3 mM NH4Cl, 42.8 mM NaCl, 1.0 mM MgSO4, 7.5μM FeSO4·7H2O, 0.8 μM CoCl2·6H2O, 0.5 μM MnCl2·4H2O, 0.5 μM ZnCl2, 0.2 μM Na2MoO4·2H2O, 0.1 μMNiCl2·6H2O, 0.1 μM H3BO3, 0.01 μM CuSO4) and incubate the biofilm.

[0068] Example 2: Biofilm incubation

[0069] There are three optional culture methods according to the culture carrier:

[0070] i. Cell culture system: Add cell culture slides and 1.8 mL of mineral salt medium into a 24-well plate;

[0071] ii. Glass-bottomed culture dish: Add 2 mL of mineral salts medium to a glass-bottomed culture dish suitable for the imaging system.

[0072] iii. Slide system: Place the slide in a bacterial culture dish and add 0.8 mL of mineral salt medium on the slide surface;

[0073] The bacterial solution in Example 1 was transferred to the mineral salt culture medium in the above-mentioned different culture methods at a ratio of 1:1000, and 3.6 µL of L-azidohomoalanine (click chemistry tools, product number: CCT:1066) was added to a final concentration of 0.6 mM; the biofilm was incubated at 37°C for 5 h to 3 d.

[0074] Example 3: Whole cell staining

[0075] a. The biofilm sample obtained in Example 2 was stained with 4',6-diamidino-2-phenylindole for whole-cell staining, with a final staining concentration of 10 µg mL -1 The reaction was allowed to proceed in the dark for 15 min and then the cells were immediately washed three times with PBS solution.

[0076] b. Fluorescence imaging of the 405 excitation channel using confocal laser microscopy.

[0077] like Figure 1 As shown, the results show that when cells are labeled with 4',6-diamidino-2-phenylindole, the blue fluorescence occupies the entire volume of the cell, allowing the cell outline to be clearly described.

[0078] Example 4: Staining and imaging of extracellular proteins at different biofilm stages

[0079] a. Prepare staining solution: Prepare cyclooctyne dye (Click Chemistry Tools, Catalog No.: CCT-1278:1278-1, with green fluorescent group) stock solution at a concentration of 5 mM; dilute the stock solution in PBS solution to a final concentration of 50 µM;

[0080] b. The biofilm obtained by incubation in Example 3 was removed from the culture medium and washed three times with PBS solution;

[0081] c. Add an appropriate volume of staining solution to the biofilm sample, requiring the staining solution to immerse the biofilm; add 400 µL of staining solution to the cell slide system; add 800 µL of staining solution to the glass slide system and the slide system; react in the dark for 60 min, and then immediately wash three times with PBS solution;

[0082] d. The labeled biofilm samples were fixed with 4% paraformaldehyde solution;

[0083] e. STED microscope imaging: The cell slide system keeps the cell slide moist, and the cell slide is taken out and placed on a glass slide for microscopic imaging; the glass bottom culture dish keeps the inside of the glass bottom culture dish moist, and the glass bottom culture dish is placed on the stage for microscopic imaging; the glass slide system keeps the surface of the glass slide moist, covers it with a coverslip, presses it and ensures that no bubbles are generated; use STED microscope imaging.

[0084] like Figure 2 As shown, the results show that this method can realize the visualization of extracellular nascent proteins, namely Figure 2 The green fluorescence in (b) and the green curve in (c) show that the green fluorescence of the extracellular newly formed protein is strong around the edges and weak in the middle, which means that the extracellular newly formed protein is attached to the bacterial surface or surrounds the cells. Figure 3 The temporal and spatial deposition process of extracellular nascent proteins was presented in the figure. The results showed that as the biofilm incubation time increased, the extracellular proteins initially adhered to or wrapped around the cells, and then slowly expanded into the matrix around the cells, showing irregular fluorescence distribution forms such as flakes.

[0085] Example 5: Time-sequential labeling imaging of extracellular nascent proteins

[0086] a. The biofilm sample was stained with a cyclooctyne dye with a green fluorescent group for the first time according to the operating steps in Example 4, and then the same volume of mineral salt medium and L-azidohomoalanine in Example 2 were added and incubated at 37°C for 16 hours; and then the biofilm sample was stained with a cyclooctyne dye with a blue fluorescent group (ClickChemistry Tools, Catalog No.: CCT-1310) according to the operating steps in Example 4 for the second time;

[0087] b. Channels 488 and 405 were imaged using confocal laser microscopy.

[0088] Figure 4 The results showed that the newly generated proteins in the biofilm system gradually occupied the vacancies in the original matrix carrier and tended to continue to be generated on the surface of the original extracellular proteins.

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Imaging methods for nascent proteins, including: Incorporating bioorthogonal amino acids into a culture medium, culturing cells with the culture medium to obtain a culture, and acquiring an image after staining the culture multiple times; The bioorthogonal amino acid is: (i) an amino acid having an azide group; or (ii) an amino acid derivative having an azide group; The multiple staining is to stain the culture multiple times with an alkyne dye labeled with a fluorescent group, wherein the fluorescent group is different in each staining; The multiple times are two or more times.

2. The imaging method according to claim 1, characterized in that The final concentration of the bioorthogonal amino acid in the culture medium was 0.6 mM.

3. The imaging method according to claim 1 or 2, characterized in that: The bioorthogonal amino acids include L-azidohomoalanine.

4. The imaging method according to any one of claims 1 to 3, characterized in that: The cell is Pseudomonas aeruginosa PAO1.

5. The imaging method according to any one of claims 1 to 4, characterized in that: The acquired images are acquired based on a super-resolution microscope and / or a laser confocal microscopy imaging system.

6. The imaging method according to any one of claims 1 to 5, characterized in that: The final concentration of the fluorophore-labeled alkyne dye is 50 µM.

7. The imaging method according to any one of claims 1 to 6, characterized in that: The alkyne dye labeled with a fluorescent group includes a cyclooctyne dye labeled with a fluorescent group.

8. The imaging method according to any one of claims 1 to 7, characterized in that: The culture carrier is at least one of a cell slide, a glass-bottomed culture dish, and a glass slide.

9. The imaging method according to any one of claims 1 to 8, characterized in that: After multiple stainings and before acquiring images, the method further includes a step of fixing the culture.

10. The imaging method according to any one of claims 1 to 9, characterized in that: Also included is the step of staining the culture with 4',6-diamidino-2-phenylindole.

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