KstD-like enzyme, engineered bacteria and use thereof in preparing drugs for treating depression

Abstract

The present invention provides a kstD-like enzyme, an engineered bacterium and its use in the preparation of a drug for treating depression, wherein the amino acid sequence of the kstD-like enzyme is shown in SEQ ID NO.1. Through whole genome sequencing, it was found that the kstD-like enzyme expressed by the bacterium plays a role in degrading cortisol, and then the gene sequence of the enzyme was constructed into the probiotic Bacillus subtilis (B. subtilis), and a recombinant probiotic B. subtilis Tongji was obtained. The bacterium is superior to ordinary probiotics B. subtilis, can degrade cortisol in vitro, and protect mice from elevated cortisol and depressive-like behavior caused by stress. Therefore, the probiotic B. subtilis Tongji expressing the kstD-like enzyme can be used as a drug for intervention measures against depression or depression caused by psychological stress.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a kstD-like enzyme, an engineered bacterium and applications thereof in preparing a drug for treating depression. Background Art

[0002] Chronic psychological stress is one of the most important risk factors for depression. About 92% of people with depression have experienced stressful events before the onset of the disease. The body activates the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. Cortisol is a stress hormone and the main effector of the HPA axis stress response system. Long-term exposure to cortisol can lead to depressive symptoms. 20-80% of people with depression show some form of elevated cortisol levels. It is worth thinking about that not everyone who faces stress will develop depression. Among people who have experienced similar traumatic or tragic events, only 10-30% of people develop depression. An individual's sensitivity and ability to cope with stressful events in life is called their susceptibility to stress. Susceptibility to stress makes some people more susceptible to depression. Understanding the heterogeneity of stress susceptibility in depression is crucial for developing prevention and treatment strategies. Recent studies have found that depletion of the microbiota in rodents increases stress hormones. Understanding the regulation of stress hormones or stress susceptibility by the gut microbiota and finding interventions to enhance resistance to stress remains a daunting challenge.

[0003] Therefore, it is necessary to develop a drug for the treatment of depression. Summary of the invention

[0004] The present invention aims to provide a kstD-like enzyme, a carrier and use thereof in the preparation of a medicine for treating depression.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a kstD-like enzyme is provided, and the amino acid sequence of the kstD-like enzyme is shown in SEQ ID NO.1.

[0007] In the second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule is a gene encoding the kstD-like enzyme.

[0008] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2-3.

[0009] In the third aspect of the present invention, a vector containing the encoding gene is provided.

[0010] In the fourth aspect of the present invention, an engineered bacterium containing the vector is provided.

[0011] Furthermore, the host bacteria used by the engineering bacteria include Bacillus subtilis, Escherichia coli (DH5α, Top10, BL21 and Rosetta) or one of other commonly used bacteria. Preferably, the engineering bacteria include Bacillus subtilis expressing the kstD-like enzyme.

[0012] In the fifth aspect of the present invention, a cell line containing the vector is provided.

[0013] In the sixth aspect of the present invention, provided is a use of the kstD-like enzyme, the nucleic acid molecule or the vector or the host bacteria or the cell line in the preparation of a drug for treating depression.

[0014] Furthermore, the depression includes depression or low mood caused by resistance to psychological pressure.

[0015] In the seventh aspect of the present invention, an antiviral drug is provided, the active ingredient of the drug includes the kstD-like enzyme, or the nucleic acid molecule, or the vector, or the engineered bacteria, or the cell line.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0017] The present invention provides a kstD-like enzyme, a vector and an application thereof in the preparation of a drug for treating depression. The present application discovered a kstD-like enzyme expressing the ability to degrade cortisol, and then constructed the gene sequence of the enzyme into the probiotic Bacillus subtilis (B. subtilis) to obtain a recombinant probiotic B. subtilis Tongji. The bacterium is superior to the common probiotic B. subtilis, can degrade cortisol in vitro, and protect mice from elevated cortisol and depressive-like behavior caused by stress. Therefore, the probiotic B. subtilis Tongji expressing the kstD-like enzyme can be used as an intervention measure to resist low mood or depression caused by psychological stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 This is a picture of Pseudomonas aeruginosa isolated in Example 1;

[0020] Figure 2 The LC-MS / MS quantitative analysis results of Pseudomonas aeruginosa degrading cortisol in a time-dependent manner in Example 1;

[0021] Figure 3 The test results of the forced swimming test, tail suspension test, water sprinkling test and novel environment food intake inhibition test of each group of mice in Example 2;

[0022] Figure 4 The results are the test results of serum cortisol levels of mice in each group in Example 2;

[0023] Figure 5 The LC-MS / MS quantitative analysis results of the degradation of cortisol by the probiotic B. subtilis Tongji containing the kstD-like gene in Example 3;

[0024] Figure 6 The test results of the forced swimming test, tail suspension test, water sprinkling test and novel environment food intake inhibition test of each group of mice in Example 3;

[0025] Figure 7 These are the test results of serum cortisol levels of rats in each group in Example 3. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0029] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0030] The present invention successfully isolated a bacterium capable of degrading cortisol from fecal samples of healthy people, and identified it as Pseudomonas aeruginosa by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The bacterium can improve the increase of cortisol and depressive-like behavior caused by stress. Through whole genome sequencing, it was found that the kstD-like enzyme expressed by the bacterium plays a role in degrading cortisol. Sequencing found that the amino acid sequence of the kstD-like enzyme is shown in SEQ ID NO.1.

[0031] The gene sequence of the enzyme (sequence as shown in SEQ ID NO: 2) was constructed into the probiotic Bacillus subtilis (B. subtilis) to obtain the recombinant probiotic B. subtilis Tongji. This bacterium is superior to the common probiotic B. subtilis, can degrade cortisol in vitro, and protect mice from stress-induced cortisol elevation and depressive-like behavior. Therefore, the probiotic B. subtilis Tongji expressing the kstD-like enzyme can be used as an intervention measure to resist low mood or depression caused by psychological stress.

[0032] The following will describe in detail a kstD-like enzyme, a vector and its application in the preparation of a drug for treating depression in combination with examples and experimental data.

[0033] Example 1 Acquisition of Pseudomonas aeruginosa and its ability to reduce serum cortisol and improve depression-like behavior in rats

[0034] 1. Screening and acquisition of Pseudomonas aeruginosa capable of degrading cortisol

[0035] Based on the hypothesis that intestinal bacteria can reduce serum cortisol levels by degrading cortisol, the inventors mixed 0.5 g of fresh fecal samples from healthy people with selective culture medium (cortisol as the only carbon source). The culture was incubated in the dark at 37°C and 250 rpm on a rotary shaker. The culture medium was changed after three to four days of culture. After five repetitions, the culture suspension was inoculated on blood agar plates to isolate strains, such as Figure 1 shown.

[0036] Matrix-assisted laser desorption ionization-time of flight mass spectrometry identified it as Pseudomonas aeruginosa. The ability of Pseudomonas aeruginosa to degrade testosterone was verified by incubating the bacteria with cortisol. Figure 2 As shown, LC-MS / MS detection showed that cortisol gradually decreased with the culture time.

[0037] Pseudomonas aeruginosa reduces serum cortisol and improves depressive-like behavior in rats

[0038] In order to investigate whether Pseudomonas aeruginosa in the intestine can cause depressive-like behavior in rats, we administered Pseudomonas aeruginosa to mice by oral gavage:

[0039] Antibiotic group: Mice were given antibiotics consisting of 0.5 mg / mL vancomycin, 1 mg / mL neomycin, 1 mg / mL metronidazole, and 1 mg / mL ampicillin in their drinking water for 1 week to deplete intestinal microorganisms.

[0040] Aeruginosa gavage group: The mice were given antibiotics consisting of 0.5 mg / mL vancomycin, 1 mg / mL neomycin, 1 mg / mL metronidazole, and 1 mg / mL ampicillin in their drinking water for 1 week to deplete intestinal microorganisms. After another 24 hours, the mice were gavaged with about 1 mL of overnight Pseudomonas aeruginosa bacterial culture (about 1×10 8 cfu), with the gavage frequency being once a day, 3 consecutive days a week, for 4 weeks.

[0041] Control group: mice not gavaged;

[0042] Depression group: mice were directly gavaged with cortisol;

[0043] Behavioral experiments were then used to verify whether the mice in the above groups showed depressive-like behaviors.

[0044] like Figure 3 As shown in the results, the immobility time of the Pseudomonas aeruginosa gavage group in the forced swimming test and tail suspension test was similar to that of the control group; in the novel environment feeding inhibition test and water sprinkling test, the latency time of the Pseudomonas aeruginosa gavage group was similar to that of the control mice. After the Pseudomonas aeruginosa was eliminated with antibiotics, the mice showed depressive-like behavior.

[0045] like Figure 4 As shown, LC-MS / MS detection of cortisol found that the cortisol of mice in the Pseudomonas aeruginosa gavage group did not increase significantly after the depression model was established. After the Pseudomonas aeruginosa was eliminated with antibiotics, the depression model caused the cortisol of mice to increase.

[0046] These data suggest that oral administration of P. aeruginosa reduces serum cortisol and improves depressive-like behavior in rats.

[0047] Example 2 Obtaining a gene encoding a kstD-like enzyme

[0048] We speculate that Pseudomonas aeruginosa causes depressive-like behavior because it has the ability to degrade cortisol. We tried to find the gene sequence of the cortisol-degrading enzyme in Pseudomonas aeruginosa. By sequencing the whole genome of the isolated Pseudomonas aeruginosa, we obtained the whole genome sequence information. The gene encoding the kstD-like enzyme was found in the whole genome of our Pseudomonas aeruginosa chromosome. The amino acid sequence of the kstD-like enzyme is shown in SEQ ID NO.1, and the gene encoding the kstD-like enzyme described in claim 1 is shown in SEQ ID NO:2.

[0049] Example 3 Obtaining an engineered bacterium (Bacillus subtilis Tongji) expressing kstD-like enzyme

[0050] 1. In order to verify whether the protein expressed by the sequence has the ability to degrade cortisol, we introduced the kstD-like enzyme into the probiotic B. subtilis to obtain B. subtilis Tongji that can constitutively express the kstD-like enzyme. The construction method of the engineered bacteria is as follows:

[0051] (1) The sequence of the gene for synthesizing the kstD-like enzyme is shown in SEQ ID NO: 2;

[0052] (2) Optimizing the gene of kstD-like enzyme, as shown in SEQ ID NO: 3;

[0053] (3) The gene of the optimized kstD-like enzyme was cloned into the extracellular protease nprE site of B. substilis 168 (plasmid vector network, product number: zl-059601), and P43 was selected as the promoter to obtain recombinant B. subtilis Tongji.

[0054] 2. Detection of the ability to degrade testosterone

[0055] The ability of Pseudomonas aeruginosa to degrade testosterone was verified by incubating cortisol with the B. subtilis Tongji bacteria expressing the kstD-like enzyme. Figure 5 As shown, LC-MS / MS detection found that B. subtilis Tongji could degrade cortisol in LB medium containing cortisol, while B. subtilis that could not express kstD-like enzyme had no ability to degrade cortisol.

[0056] Example 4 The engineered bacteria expressing kstD-like enzyme can improve the depressive-like behavior of mice caused by stress

[0057] To investigate whether B. subtilis Tongji could improve the depressive-like behavior of mice induced by stress, we gavaged the mice in groups.

[0058] Control group: no gavage;

[0059] Depression group: mice were directly gavaged with cortisol;

[0060] B. subtilis Tongji gavage group: mice were given antibiotics consisting of 0.5 mg / mL vancomycin, 1 mg / mL neomycin, 1 mg / mL metronidazole, and 1 mg / mL ampicillin in their drinking water for 1 week to deplete intestinal microorganisms. After another 24 hours, mice were gavaged with about 1 mL of overnight B. subtilis Tongji culture (about 1×10 8 cfu), with the gavage frequency being once a day, 3 consecutive days a week, for 4 weeks.

[0061] B. Subtilis: The mice were given antibiotics consisting of 0.5 mg / mL vancomycin, 1 mg / mL neomycin, 1 mg / mL metronidazole, and 1 mg / mL ampicillin in their drinking water for 1 week to deplete intestinal microorganisms. After another 24 hours, the mice were gavaged with approximately 1 mL of an overnight culture of B. Subtilis bacteria that did not express the kstD-like enzyme (approximately 1×10 8 cfu), with the gavage frequency being once a day, 3 consecutive days a week, for 4 weeks.

[0062] Behavioral experiments were performed and the procedures were as follows: Behavioral assays were performed within a week with at least 24 h between each assessment. The analyses were performed by an experimenter who was blind to the experimental conditions. In the forced swimming test, mice were transferred individually into a glass cylinder. The cumulative duration of immobility within 5 min was noted. In the tail suspension test, mice were suspended above the floor. The cumulative duration of immobility within 5 min was noted. In the novel environment feeding inhibition test, food pellets were placed on a white paper in the center of the container. The mouse was placed in a corner of the container, and the time required to start eating was recorded within 10 min. In the water sprinkling test, a 10% sucrose solution was applied to the back of the mouse, and the grooming time was recorded for 6 min.

[0063] The results are as follows Figure 6 As shown in the behavioral experiment, compared with the control group mice, the immobility time of the mice in the B. subtilis Tongji gavage group after depression modeling was not prolonged in the forced swimming test and the tail suspension test, and the latency time in the novel environment food inhibition test and the water sprinkling test was not prolonged. These behavioral experimental data indicate that the depressive-like behavior of mice in the B. subtilis Tongji gavage group has been improved.

[0064] like Figure 7 As shown, the detection of serum testosterone by LC-MS / MS revealed that after the B. subtilis Tongji gavage group mice were subjected to depression modeling, the serum cortisol level was not significantly different from that of the control group, indicating that B. subtilis Tongji resisted the increase in cortisol caused by stress.

[0065] In summary, the research results of the present application prove that the probiotic B. subtilis Tongji expressing kstD-like can be used to prevent depressive-like behavior caused by stress.

[0066] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0067] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0068] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a kstD-like enzyme having an amino acid sequence as shown in SEQ ID NO.1, a nucleic acid molecule encoding the kstD-like enzyme, a vector containing the nucleic acid molecule, or an engineered bacterium or cell line containing the vector in the preparation of a drug for treating depression.

2. The use according to claim 1, characterized in that: The depression involves low mood caused by resistance to psychological stress.

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