Application of combination of zinc and vitamin K3 in inhibition of bacteria

Abstract

The invention discloses application of zinc combined with vitamin K3 in inhibiting bacteria, and belongs to the technical field of medical biology. According to the invention, zinc with a safe concentration and vitamin K3 with a low concentration are used in a combined manner, so that an efficient antibacterial effect on bacteria such as escherichia coli, klebsiella pneumoniae and the like is realized. Zinc specifically inhibits iron-sulfur cluster assembly of SoxR protein in bacteria, so that expression of soxS is reduced, the superoxide dismutase level is reduced, the resistance of the bacteria to superoxide anions is weakened, and the antibacterial effect is enhanced. Therefore, a novel antibacterial strategy is provided on the basis of combination of zinc and vitamin K3, the concentration is low, certain clinical application value is achieved, the zinc-vitamin K3 can be used for improving the antibacterial effect and overcoming the defects in an existing antibacterial method, and a new thought and theoretical basis are provided for development of an efficient and safe antibacterial method.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and particularly to the application of zinc combined with vitamin K3 in inhibiting bacteria. Background Art

[0002] At present, in the field of anti-bacterial, zinc, as an important trace element, has been widely studied and applied. Zinc has a variety of biological functions, including promoting the normal operation of the immune system, participating in protein synthesis and cell division, etc. In recent years, it has been found that high concentrations of zinc (usually in the concentration range of several millimoles per liter) have significant bactericidal effects in animal husbandry, can effectively reduce the number of pathogenic bacteria, and improve the health level and production performance of animals. In addition, vitamins are also widely used in the anti-bacterial field, and some vitamins are found to have direct or indirect anti-bacterial activities, and can play an anti-bacterial role by regulating the immune response of the host or affecting the metabolic process of bacteria.

[0003] Although certain achievements have been made in anti-bacterial with zinc and vitamins, there are still some problems and defects in the prior art. For the application of high-concentration zinc, although it can effectively kill bacteria to a certain extent, too high zinc concentration may be toxic to host cells, affecting the normal physiological functions of animals. And in practical applications, how to precisely control the zinc concentration to achieve the best anti-bacterial effect while avoiding adverse effects on the host is still a problem to be solved. Moreover, people have less understanding of bacterial zinc homeostasis and are not sufficient to utilize its understanding in the application of bacterial drug resistance. On the other hand, although vitamins also have certain applications in the anti-bacterial field, for specific types of vitamins such as vitamin K3, its research on anti-bacterial effects is relatively less, and its potential anti-bacterial value has not been fully exerted. In addition, the applications of zinc and vitamins in the prior art are mostly used alone, lacking in-depth research and effective utilization of their synergistic effects, unable to fully exert their advantages of combined anti-bacterial to achieve a more ideal anti-bacterial effect. And the separate anti-bacterial in the prior art requires a relatively high concentration and has no clinical application value. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of zinc combined with vitamin K3 in inhibiting bacteria, so as to solve the problems existing in the above-mentioned prior art. By combining safe concentrations of zinc (≤1.2 mM) and low concentrations of vitamin K3 (0.1 mM - 2 mM), high-efficiency anti-bacterial is achieved, overcoming the potential toxicity of high-concentration zinc and the limitations of vitamin K3 in the application of the anti-bacterial field, and providing new ideas and theoretical bases for the development of efficient and safe anti-bacterial methods.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides the use of zinc combined with vitamin K3 in any of the following:

[0007] (1) Use in inhibiting bacteria for non-therapeutic purposes;

[0008] (2) Use in preparing a drug for inhibiting bacteria;

[0009] (3) Use in preparing a drug for treating diseases caused by bacteria.

[0010] Preferably, the concentration of zinc ≤ 1.2 mM; the concentration of vitamin K3 is 0.1 mM - 2 mM. The combined antibacterial mechanism within the above range can optimize and adjust the specific concentrations of zinc and vitamin K3 according to different experimental conditions and application scenarios to meet different antibacterial requirements.

[0011] Preferably, the bacteria include Escherichia coli and / or Klebsiella pneumoniae.

[0012] The present invention also provides a drug for inhibiting bacteria, and the drug uses zinc and vitamin K3 as active ingredients.

[0013] Preferably, the concentration of zinc ≤ 1.2 mM; the concentration of vitamin K3 is 0.1 mM - 2 mM.

[0014] Preferably, the bacteria include Escherichia coli and / or Klebsiella pneumoniae.

[0015] The present invention also provides a method for inhibiting bacteria for non-therapeutic purposes, including the step of simultaneously applying zinc and vitamin K3 to a test sample containing the bacteria.

[0016] Preferably, the concentration of zinc ≤ 1.2 mM; the concentration of vitamin K3 is 0.1 mM - 2 mM.

[0017] Preferably, the bacteria include Escherichia coli and / or Klebsiella pneumoniae.

[0018] The technical solution of the present invention has at least the following beneficial effects:

[0019] The present invention realizes a novel antibacterial strategy by using a safe concentration of zinc (less than or equal to 1.2 mM) combined with a low concentration (within 100 μM to 2 mM) of vitamin K3 in an LB medium. This strategy has the following technical effects:

[0020] (1) Synergistic antibacterial effect: By jointly using zinc and vitamin K3, and taking advantage of the impact of zinc on the zinc homeostasis mechanism of Escherichia coli, the iron-sulfur cluster assembly of SoxR protein is specifically inhibited, thereby reducing the expression of downstream soxS, and further decreasing the level of superoxide dismutase (SOD). This synergistic effect renders Escherichia coli unable to effectively resist the attack of superoxide anions under the induction of low-concentration vitamin K3, thus achieving the antibacterial effect.

[0021] (2) Inhibition of bacterial growth: Experimental results show that the joint use of zinc and vitamin K3 can effectively inhibit the growth of Escherichia coli. In the LB medium containing 1.2 mM zinc and 0.6 mM vitamin K3, the growth of Escherichia coli is significantly inhibited, while the same antibacterial effect cannot be achieved by using zinc or vitamin K3 alone.

[0022] (3) Regulation of zinc homeostasis: This invention reveals the zinc homeostasis regulation mechanism of Escherichia coli in a high-zinc environment. By affecting the iron-sulfur cluster assembly of SoxR protein, zinc can inhibit the expression of soxS, thereby reducing the uptake of zinc ions and maintaining zinc homeostasis within the cell. This regulation mechanism provides a theoretical basis for the development of novel antibacterial strategies.

[0023] (4) Enhancement of oxidative stress sensitivity: Due to the inhibitory effect of zinc on SoxR protein, the expression of SOD is reduced, leading to a decline in the resistance of Escherichia coli to superoxide anions. Under the induction of vitamin K3, Escherichia coli generates reactive oxygen species (ROS), and the reduction of SOD renders the bacteria unable to effectively scavenge ROS, thus enhancing their sensitivity to oxidative stress.

[0024] (5) Safety and practicality: The concentrations of zinc and vitamin K3 adopted in this invention are within the safe range, with low toxicity to host cells, and possess high safety and practicality.

[0025] (6) Universality: The joint antibacterial strategy of zinc and vitamin K3 provided by this invention is applicable not only to Escherichia coli but also to other bacteria such as Klebsiella pneumoniae, etc., and has universality. Brief Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is a diagram of the antibacterial mechanism of zinc combined with vitamin K3;

[0028] Figure 2Determination results of the sensitivity of Escherichia coli to different concentrations of vitamin K3 in the presence of a relatively high concentration (1 mM) of zinc; 1, wildtype E.coli cells of MC4100: wild-type Escherichia coli MC4100; 2, soxR - mutant: Escherichia coli MC4100 with the soxR - gene knocked out, Escherichia coli MC4100 with the soxS - gene knocked out, Escherichia coli MC4100 with the iscU - , iscA - and fdx - genes knocked out;

[0029] Figure 3 Determination results of the sensitivity of Escherichia coli to different concentrations of vitamin K3 in the presence of a relatively low concentration (0.6 mM) of zinc; 1, pBAD+MC4100: empty plasmid pBAD transferred into Escherichia coli MC4100; 2, pBSOSX+MC4100: pBAD plasmid overexpressing the soxS gene transferred into Escherichia coli MC4100; 3, pBAD+soxS - : empty plasmid pBAD transferred into Escherichia coli MC4100 lacking the soxS gene; 4, pBSOXS+soxS - : pBAD plasmid overexpressing the soxS gene transferred into Escherichia coli MC4100 lacking the soxS gene;

[0030] Figure 4 Determination results of the sensitivity of Klebsiella pneumoniae to different concentrations of vitamin K3 in the presence of a safe concentration (1 mM) of zinc; 1, wild type E.coli cells of MC4100: wild-type Escherichia coli MC4100; 2, Klebsiella pneumoniae: Klebsiella pneumoniae. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0036] This invention has deeply studied the zinc homeostasis regulation mechanism of Escherichia coli in a high-zinc environment and found that high concentrations of zinc can specifically affect the iron-sulfur cluster assembly process of the SoxR protein. The iron-sulfur cluster assembly of the SoxR protein is blocked, thereby inhibiting the expression of the downstream SoxS protein and resulting in a decrease in the expression level of superoxide dismutase (SOD). The decrease in the SOD expression level significantly weakens the resistance of Escherichia coli to superoxide anions induced by a superoxide inducer (Vitamin K3, abbreviated as VitK3), so that an effective antibacterial effect can be achieved under the action of a low concentration of Vitamin K3. More specifically, the basic components of the antibacterial strategy are as follows:

[0037] (1) Zinc concentration used: Escherichia coli uses a zinc concentration greater than 0.6 mM and less than 1.2 mM, which is lower than the threshold at which zinc has a significant toxic effect on Escherichia coli and can ensure less impact on the normal physiological functions of Escherichia coli during the combined antibacterial process.

[0038] (2) Concentration of superoxide inducer (Vitamin K3) used: A Vitamin K3 concentration within 100 micromoles to 2 millimoles is used. Vitamin K3 within this concentration range can effectively induce the production of superoxide anions while avoiding toxicity to host cells.

[0039] The above antibacterial (against Escherichia coli, Klebsiella pneumoniae, etc.) strategy of zinc combined with vitamin K3 is as Figure 1 shown.

[0040] The above antibacterial mechanism will be further illustrated by examples below.

[0041] Example 1: Antibacterial effect of zinc combined with vitamin K3 at a higher concentration (bacterial safe concentration) against Escherichia coli

[0042] 1. Experimental materials

[0043] Strains: Escherichia coli: MC4100, soxR - , soxS - , iscU - / iscA - / fdx - Four strains (- indicates gene knockout). soxR - and soxS - strains have been disclosed in the literature "Lu, J., Yang, J., Tan, G., and Ding, H. (2008) Complementary roles of SufA and IscA in the biogenesis of iron-sulfur clusters in Escherichia coli. The Biochemical journal 409, 535-543"; iscU - / iscA - / fdx - strains have been disclosed in the literature "Li, J., Ren, X., Fan, B., Huang, Z., Wang, W., Zhou, H., Lou, Z., Ding, H., Lyu, J., and Tan, G. (2019) Zinc Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli. Applied and environmental microbiology". The construction methods of the strains are detailed in the corresponding literature.

[0044] Culture medium: LB solid medium.

[0045] Reagents: Zinc salts (such as ZnSO4), vitamin K3.

[0046] 2. Experimental methods

[0047] Experimental grouping: Set up an experimental group with the addition of a higher concentration of zinc ions (1 mM) and a control group without the addition of zinc, and add different concentrations of VitK3: 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM respectively.

[0048] Experimental procedure: Add 1.0 mM zinc (ZnSO4) and different concentrations of vitamin K3 to LB solid medium respectively. Adjust the absorbance of overnight resuscitated Escherichia coli at 600 nm to 0.02, and inoculate it into the above medium. Set three replicates for each treatment. Incubate at 37 °C for 24 hours, and observe and record the colony growth situation.

[0049] 3. Experimental results

[0050] As Figure 2 shown, the results show that in the medium supplemented with 1.0 mM zinc, Escherichia coli was effectively inhibited at a lower concentration of vitamin K3 (such as 0.6 mM), manifested as significant inhibition or complete non-growth of colony growth.

[0051] The above results indicate that by comparing the colony growth of different treatment groups, it is confirmed that zinc combined with low-concentration vitamin K3 has a significant antibacterial effect on Escherichia coli.

[0052] Example 2: Antibacterial effect of lower concentration zinc ions combined with vitamin K3 on Escherichia coli

[0053] 1. Experimental materials

[0054] Strains: Escherichia coli: pBAD+MC4100, pBSOXS+MC4100, pBAD+soxS - , pBSOXS+soxS - Four strains (pBSOXS refers to the pBAD plasmid transfected with the overexpressed soxS gene in the strain; pBAD was purchased from Invitrogen (Thermo Fisher) company, product number V430-01).

[0055] Medium: LB solid medium.

[0056] Reagents: Zinc salts (such as ZnSO4).

[0057] 2. Experimental methods

[0058] Experimental grouping: Set an experimental group supplemented with lower concentration zinc ions (0.6 mM) and a control group without zinc, and add different concentrations of VitK3 (0.4 mM, 0.8 mM, 1.2 mM) respectively, and add 20% L-Arab (L-arabinose, as an inducer to overexpress SoxS) at a ratio of 1:100.

[0059] Experimental procedure: Add 0.6 mM of zinc (ZnSO4), different concentration gradients of VitK3, and 20% L-Arab as the pBAD plasmid inducer at a ratio of 1:100 to the LB solid medium. Adjust the absorbance of the overnight-revived Escherichia coli at 600 nm to 0.02, and inoculate it into the above medium. Set three replicates for each treatment. Incubate at 37 °C for 24 hours, and observe and record the colony growth.

[0060] 3. Experimental results

[0061] As Figure 3 shown, the results show that in the medium supplemented with 0.6 mM of zinc, the growth of Escherichia coli is inhibited to a certain extent, manifested as a decrease in the number of colonies or slow growth.

[0062] The above results indicate that the combination of low-concentration zinc ions and vitamin K3 has a certain improvement in the antibacterial effect on Escherichia coli, proving that under the combination of very low-concentration zinc ions, the antibacterial effect still exists and has clinical significance.

[0063] Example 3: Antibacterial effect of zinc combined with vitamin K3 on Klebsiella pneumoniae

[0064] 1. Experimental materials

[0065] Strains: Klebsiella pneumoniae.

[0066] Medium: LB solid medium.

[0067] Reagents: Zinc salts (such as ZnSO4), vitamin K3.

[0068] 2. Experimental methods

[0069] Experimental grouping: Set an experimental group supplemented with a safe concentration of zinc ions (1.0 mM) and a control group without zinc, and add different concentrations of VitK3 (0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, and 1.2 mM) respectively.

[0070] Experimental procedure: Add 1.0 mM of zinc (ZnSO4) and different concentrations of VitK3 to the LB solid medium respectively. Inoculate Klebsiella pneumoniae into the above medium. Set three replicates for each treatment. Incubate at 37 °C for 24 hours, and observe and record the colony growth.

[0071] 3. Experimental results

[0072] As Figure 4As shown, the results show that in the medium supplemented with 1.0 mM zinc, Klebsiella pneumoniae is effectively inhibited at a lower concentration of vitamin K3 (such as 0.8 mM), manifested as significant inhibition of colony growth or complete non-growth.

[0073] The above results indicate that the combination of zinc and low-concentration vitamin K3 also has a significant antibacterial effect on the common pathogenic bacterium Klebsiella pneumoniae, indicating that this strategy has a certain universality.

[0074] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of zinc combined with vitamin K3 in any of the following: (1) Application in the inhibition of bacteria for non-therapeutic purposes; (2) Application in the preparation of drugs for inhibiting bacteria; (3) Application in the preparation of drugs for treating diseases caused by bacteria.

2. The use according to claim 1, characterized in that The concentration of zinc is ≤1.2mM; the concentration of vitamin K3 is 0.1mM-2mM.

3. The use according to claim 1, characterized in that The bacteria include Escherichia coli and / or Klebsiella pneumoniae.

4. A drug for inhibiting bacteria, characterized in that: The medicine has zinc and vitamin K3 as active ingredients.

5. The drug according to claim 4, characterized in that The concentration of zinc is ≤1.2mM; the concentration of vitamin K3 is 0.1mM-2mM.

6. The drug according to claim 4, characterized in that The bacteria include Escherichia coli and / or Klebsiella pneumoniae.

7. A method for inhibiting bacteria for non-therapeutic purposes, characterized in that: The method comprises the step of simultaneously applying zinc and vitamin K3 to a test sample containing the bacteria.

8. The method according to claim 7, characterized in that The concentration of zinc is ≤1.2mM; the concentration of vitamin K3 is 0.1mM-2mM.

9. The method according to claim 7, characterized in that The bacteria include Escherichia coli and / or Klebsiella pneumoniae.

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