Pathogenic fusobacterium nucleatum adapted to acid environment and culture method and application thereof

Abstract

The invention provides a pathogenic fusobacterium nucleatum adapted to an acid environment and a culture method and application thereof, a new strain is obtained through culture of acid adaptive evolution F.nucleatum, and the new strain is named fusobacterium nucleatum LA57 and F.nucleatum LA57 and preserved in Guangdong Microbial Culture Collection Center with the preservation number of GDMCC No.64989. According to the invention, the new strain is named F.nucleatum LA57 and preserved in Guangdong Microbial Culture Collection Center with the preservation number of GDMCC No.64989; the bacterial strain can improve the proliferation capacity of colorectal cancer cells and improve the erosion capacity of colorectal cancer; the method can be applied to a real process experiment of reducing bacterial infected tumor cells in tumor tissues, and has important significance on further revealing the microbial pathogenic effect in the CRC generation and development process and improving the tumor prevention and treatment effect.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a pathogenic Fusobacterium nucleatum adaptable to an acidic environment and a culture method and application thereof. Background Art

[0002] Colorectal cancer (CRC) is one of the common digestive tract malignancies. According to the latest global cancer statistics, CRC is the third most common cancer in terms of incidence and the second most common cancer in terms of mortality. It has been confirmed that intratumoral bacteria colonized in tumor tissues are one of the important components of the tumor microenvironment (TME), which is closely related to the occurrence and progression of CRC.

[0003] There are significant differences in the structure and function of the microbiota between CRC tumor tissues and adjacent normal tissues. The abundance of Fusobacterium nucleatum (F.nucleatum) in tumor tissues is significantly increased, and it is a confirmed intratumoral pathogen of CRC. Studies have found that the adhesion of F.nucleatum to tumor cells is the key to its colonization in tumor tissues and induction of carcinogenesis, but the mechanism that causes F.nucleatum to tend to colonize in tumor tissues is not fully understood.

[0004] Anaerobic metabolism leading to tissue acidification is one of the characteristics of the tumor microenvironment, which is closely related to tumor invasion and metastasis. It is the instinct of bacteria to respond to the external environment and adjust their own adaptive evolution to achieve reproduction. Therefore, the acidic environment may be conducive to the adhesion of F. nucleatum to CRC cells and play a role in promoting cancer. F. nucleatum that has been cultured in an acidic environment and adaptively evolved can restore the true process of bacterial infection of tumor cells in tumor tissues better than commercial strains such as standard strains ATCC 23726 and ATCC 25586, which is more convincing for exploring its role in adhesion and promoting the proliferation, invasion and migration of colorectal cancer.

[0005] The acid-adaptive evolved F. nucleatum LA57 cultivated in this patent is of great significance for further revealing the pathogenic role of microorganisms in the occurrence and development of CRC and improving the effect of tumor prevention and treatment in this field of research. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a pathogenic Fusobacterium nucleatum that can adapt to an acidic environment and a culture method and application thereof. The new strain of the present invention can improve the proliferation ability of colorectal cancer cells and the erosion ability of colorectal cancer, and can be used in experiments to restore the real process of bacteria infecting tumor cells in tumor tissues.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A pathogenic Fusobacterium nucleatum that adapts to an acidic environment is named Fusobacterium nucleatum LA57, Fusobacterium nucleatum LA57, and is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC No. 64989.

[0009] The aforementioned pathogenic Fusobacterium nucleatum that adapts to an acidic environment, Fusobacterium nucleatum LA57, can enhance the proliferation ability of colorectal cancer cells.

[0010] The aforementioned pathogenic Fusobacterium nucleatum that adapts to an acidic environment, Fusobacterium nucleatum LA57, can enhance the invasive ability of colorectal cancer cells.

[0011] A method for culturing pathogenic Fusobacterium nucleatum adapted to an acidic environment comprises the following steps:

[0012] Acid adjustment was used to simulate an acidic environment in vitro, and the growth of the strains at various concentrations was compared. The concentration point at which the specific growth rate (μ) was 20-30% of the normal growth condition at pH 7.0 was selected as the initial adaptation concentration;

[0013] When the culture μ reaches the same concentration as the previous culture, increase the acid concentration by 0.1-0.2 g·L from the current value. -1 , continuously improve the strain tolerance adaptability; after culturing at a given concentration for 18-24 hours, if the strain growth rate is observed to be higher than the critical value by 0.15 hours -1 , then the strain is transferred to a higher lactic acid concentration for further cultivation;

[0014] According to the degree of TME pH acidification, the adaptive evolution process terminated at pH 5.7;

[0015] In OD 600 The growth of the strains was monitored by spectrophotometer, and 16S sequencing was performed regularly during the acid adaptation evolution process;

[0016] The acid-evolved strain LA57 was isolated and named Fusobacterium nucleatum LA57, which was deposited in Guangdong Microbial Culture Collection Center with the deposit number GDMCC No.64989.

[0017] In the aforementioned method for culturing pathogenic Fusobacterium nucleatum that is adapted to an acidic environment, the acid is lactic acid.

[0018] The aforementioned application of the pathogenic Fusobacterium nucleatum that adapts to an acidic environment is used in experiments to restore the actual process of bacteria infecting tumor cells in tumor tissues.

[0019] The present invention is beneficial in that:

[0020] The present invention obtains acid-resistant pathogenic Fusobacterium nucleatum through acid-adaptive evolution of F. nucleatum, which can be applied to the experiment of reducing the real process of bacteria infecting tumor cells in tumor tissues;

[0021] The new strain obtained by the present invention has been verified by experiments to be able to improve the proliferation ability of colorectal cancer cells and the erosion ability of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a simplified diagram of the adaptive evolution process of an embodiment of the novel strain of the present invention;

[0023] Figure 2 is the growth curve of the new strain of the present invention;

[0024] Figure 3 It is the intracellular NADP of the new strain of the present invention. + / NADPH results;

[0025] Figure 4 This is the scanning electron microscopy result of Fusobacterium nucleatum in Example 3 of the present invention;

[0026] Figure 5 The absorbance (A) value of each well measured at a wavelength of 450 nm by the microplate reader in Example 5 of the present invention;

[0027] Figure 6 This is an inverted microscope photograph of the new strain in Example 6 of the present invention after acting on colorectal cancer cells;

[0028] Figure 7 The new strain in Example 7 of the present invention has an enhanced level of adhesion to colorectal cancer cells;

[0029] Figure 8 This is the result of the new strain in Example 8 of the present invention causing colorectal cancer proliferation in mice. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] The pathogenic Fusobacterium nucleatum of the present invention is named Fusobacterium nucleatum LA57, Fusobacterium nucleatum LA57, deposited in Guangdong Provincial Microbiological Culture Collection Center, with a taxonomic name: Fusobacterium nucleatum, a deposit number of GDMCC No. 64989, a deposit date of August 7, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0032] Example 1: Adaptive evolution of Fusobacterium nucleatum

[0033] like Figure 1 As shown in the figure, the reference genome standard strain F. nucleatum ATCC 25586 was used as the starting strain for adaptive evolution. The acidic environment was simulated in vitro (using lactic acid for regulation), and the growth of the strains under different concentrations was compared. The concentration point with a specific growth rate (μ) of 20-30% under normal growth conditions (pH 7.0) was selected as the initial adaptation concentration. The "gradient increase" was used to control the time for the lactic acid concentration to increase. Once the culture reached the increase standard (μ reached the same concentration node as the previous culture), the acid concentration was increased by 0.1-0.2 g·L from the current value. -1 , continuously improving the strain tolerance adaptability. After culturing at a given concentration for a period of time (such as 18-24 hours), if the strain growth rate is observed to be higher than the critical value (such as 0.15 hours -1 ), the strain was transferred to a higher lactic acid concentration for further cultivation. According to the degree of TME pH acidification, the adaptive evolution process was terminated at pH 5.7. 600 The growth of the strain was monitored by spectrophotometer. 16S sequencing was performed regularly during the acid adaptation evolution process to ensure the purity of Fusobacterium nucleatum and avoid contamination by foreign bacteria affecting the evolution process. Finally, the acid evolution strain LA57 was isolated; the growth curve of the strain is shown in Figure 2 shown.

[0034] Example 2 Intracellular NADP of Fusobacterium nucleatum + / NADPH detection experiment:

[0035] NADP + Extraction: Take 100μL bacterial solution, add 0.9mL acidic extract solution, ultrasonically disrupt for 1min, boil for 5min, cool in an ice bath, centrifuge at 10000g at 4℃ for 10min, take the supernatant, add an equal volume of alkaline extract solution to neutralize, mix well, and store on ice for testing. NADH extraction: Take 100μL bacterial solution, add 0.9mL alkaline extract solution, ultrasonically disrupt for 1min, boil for 5min, cool in an ice bath, centrifuge at 10000g at 4℃ for 10min, take the supernatant, add an equal volume of acidic extract solution to neutralize, mix well, and store on ice for testing. Add samples in sequence according to the steps in the instruction manual, and measure the absorbance at 570nm. The test results are as follows. Figure 3 shown.

[0036] Example 3 Scanning electron microscope observation of morphological changes of Fusobacterium nucleatum

[0037] Freshly cultured Fusobacterium nucleatum that has reached the stable period is gently rinsed with PBS and quickly placed in electron microscopy fixative at room temperature for 2 hours, then transferred to 4°C for storage. The fixed sample is rinsed three times with 0.1M phosphate buffer PB (PH7.4), each time for 15 minutes. After 1% osmium acid is prepared in 0.1M phosphate buffer PBS (pH 7.4), it is fixed at room temperature in the dark for 1-2 hours and then rinsed again with PBS of the same concentration three times, each time for 15 minutes. The bacteria are sequentially immersed in 30%-50%-70%-80%-90%-95%-100%-100% alcohol for 15 minutes each time, and isoamyl acetate for 15 minutes. The sample is placed tightly on the conductive carbon film double-sided tape and placed on the sample stage of the ion sputtering instrument for gold spraying for about 30 seconds. Observe the elemental analysis of the area to be tested under a scanning electron microscope. The electron microscopy results are as follows Figure 4 shown.

[0038] Example 4 Cell culture

[0039] HCT116 and HT29 were inoculated in 5A medium and placed in culture bottles to grow as monolayer adherent growth. The medium contained 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. The cells were kept at 5% CO. 2 , 37°C in a cell culture incubator. When the cell confluence reaches about 80%-90%, that is, 2-3 days, digest with 0.25% trypsin and pass at a ratio of 1:2. When the cells are in good condition and in the logarithmic growth phase, they are used for subsequent experiments.

[0040] Example 5 CCK-8 cell proliferation assay

[0041] Logarithmic phase colorectal cancer cells in good condition were seeded in a 96-well plate at a number of 10,000 cells / well and placed in a 5% CO 2 and 37°C. After 24 hours of cell culture, the culture medium was discarded; the experimental group was added with Fusobacterium nucleatum LA57 at an MOI of 100:1, the control group 1 was not added with bacteria, and the control group 2 was added with the original Fusobacterium nucleatum at the same ratio. After 24 hours of co-culture, the culture medium was discarded, and CCK-8 reagent and 5A pure culture medium were mixed at 1:9 per well and added to the 96-well plate, and the cells were incubated at 37°C for another 2 hours. The absorbance (A) value of each well was then measured at a wavelength of 450nm on an ELISA reader. Figure 5 LA57 enhances the proliferation ability of colorectal cancer cells.

[0042] Example 6 Scratch Test

[0043] HCT116 and HT29 cells in the logarithmic growth phase were cultured at 2 × 10 cells per well. 6 The cells were seeded in six-well plates and incubated in 5% CO 2, cultured in a constant temperature incubator at 37°C until the cell mass reached 100%, washed twice with PBS, streaked with a 200 μL pipette tip and replaced with serum-free culture medium, added with original Fusobacterium nucleatum and acid-evolved Fusobacterium nucleatum LA57 at an MOI of 100:1, and photographed with an inverted microscope. Figure 6 It can be seen that LA57 promotes the migration ability of colorectal cancer cells.

[0044] Example 7 Immunofluorescence

[0045] Prepare slides in advance, inoculate HT-29 and HCT-116 cells (30% of the cell mass) in six-well slides, and co-culture with F. nucleatum for 4 hours after the cells adhere to the wall. Fix the cells with 4% paraformaldehyde for 15 minutes and wash 3 times with PBS. Permeabilize the cells with 0.5% TritonX-100 in PBS for 15 minutes and wash 3 times with PBS. Block with 5% goat serum at 37°C for 30 minutes and incubate with primary antibody overnight. Wash 5 times with 0.1% PBST, incubate with secondary antibody at room temperature for 1 hour, and wash 3 times with PBST, each time for 5 minutes. DAPI staining for 4 minutes, wash 5 times with PBST, and then seal with anti-fluorescence quencher, and photograph with a confocal microscope (Zeiss LSM 710NLO / 7MP).

[0046] Example 8 In vivo experiment

[0047] Male BALB / c mice were fed 1×10 8 Colorectal cancer HCT116 cells were subcutaneously inoculated. One week after the tumor was formed, the tumor-bearing mice were randomly divided into three groups, each with 5 mice. One group was injected with multiple sites of the original Fusobacterium nucleatum, and the other group was injected with multiple sites of Fusobacterium nucleatum LA57. The intervention was once every other day for two consecutive weeks. The PBS intervention group was used as a control. 24 hours after the last injection, the mice were anesthetized and killed, and the subcutaneous tumor tissue was collected. All animal studies followed the animal procedures approved by the Experimental Animal Welfare and Ethics Committee of the National Science Ningbo Life and Health Industry Research Institute, and followed the "Guidelines for the Welfare and Ethics Review of Experimental Animals" (GB / T35892-2018).

[0048] like Figure 8 As shown, the tumor volume was observed, and the results showed that the tumors of mice treated with LA57 were larger than those of the original Fusobacterium nucleatum and the control group, indicating that LA57 does promote tumor proliferation. After weighing the peeled tumor tissue, it was found that the weight of the tumor tissue of mice treated with LA57 also increased significantly. Figure 8 Multi-site injection of LA57 promotes the growth of colorectal cancer tumors.

[0049] The present invention obtains a new strain through acid adaptive evolution F. nucleatum culture. The strain is verified by experiments to be able to improve the proliferation ability of colorectal cancer cells and the erosion ability of colorectal cancer. It is of great significance to further reveal the pathogenic role of microorganisms in the occurrence and development of CRC and to improve the tumor prevention and treatment effect.

[0050] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A pathogenic Fusobacterium nucleatum adapted to an acidic environment, characterized in that: The pathogenic Fusobacterium nucleatum is named Fusobacterium nucleatum LA57, and is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC No.64989.

2. The pathogenic Fusobacterium nucleatum adapted to an acidic environment according to claim 1, characterized in that: The Fusobacterium nucleatum LA57 can enhance the proliferation ability of colorectal cancer cells.

3. The pathogenic Fusobacterium nucleatum adapted to an acidic environment according to claim 1, characterized in that: The Fusobacterium nucleatum LA57 can enhance the invasive ability of colorectal cancer cells.

4. A method for culturing pathogenic Fusobacterium nucleatum adapted to an acidic environment, characterized in that: The steps include: Acid adjustment was used to simulate an acidic environment in vitro, and the growth of the strains at various concentrations was compared. The concentration point at which the specific growth rate (μ) was 20-30% of the normal growth condition at pH 7.0 was selected as the initial adaptation concentration; When the culture μ reaches the same concentration as the previous culture, increase the acid concentration by 0.1-0.2 g·L from the current value. -1 , continuously improve the strain tolerance adaptability; after culturing at a given concentration for 18-24 hours, if the strain growth rate is observed to be higher than the critical value by 0.15 hours -1 , then the strain is transferred to a higher lactic acid concentration for further cultivation; According to the degree of TME pH acidification, the adaptive evolution process terminated at pH 5.7; In OD 600 The growth of the strains was monitored by spectrophotometer, and 16S sequencing was performed regularly during the acid adaptation evolution process; The acid-evolved strain LA57 was isolated and named Fusobacterium nucleatum LA57, which was deposited in Guangdong Microbial Culture Collection Center with the deposit number GDMCC No.64989.

5. The method for culturing pathogenic Fusobacterium nucleatum adapted to an acidic environment according to claim 4, characterized in that: The acid is lactic acid.

6. The use of a pathogenic Fusobacterium nucleatum adapted to an acidic environment as claimed in claim 1, characterized in that: It is used to restore the real process experiment of bacteria infecting tumor cells in tumor tissue.

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