Application of citral as virulence protein inhibitor in preparation of gram-positive bacterium virulence resisting medicine

Abstract

The invention discloses an application of citral as a virulence protein inhibitor in preparation of a gram-positive bacterium virulence resistant medicine, and belongs to the technical field of natural medicinal chemistry and medical application. The citral (Citral) is a typical non-cyclic monoterpenoid compound and is a main component of litsea cubeba oil, the CAS number of the citral is 5392-40-5, and the citral targets a dynamic protein SecA1 of a Sec secretion pathway and affects the enzymatic activity of the citral for hydrolyzing ATP, so that the secretion of staphylococcus aureus virulence protein is remarkably inhibited. The inhibitor not only can inhibit the growth of staphylococcus aureus, but also can improve the survival rate of mice infected with the staphylococcus aureus, so that the citral compound can be used for preparing a medicine for resisting the toxicity of gram-positive bacteria.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of natural medicinal chemistry and medical application technology, and relates to application of a new staphylococcus aureus virulence factor inhibitor, in particular to application of a compound citral as a virulence protein inhibitor in preparation of an anti-Gram-positive bacterial virulence drug. BACKGROUND

[0002] Staphylococcus aureus is widely distributed in air, feed, drinking water, ground and object surface, can be obtained from the skin, mucous membrane, intestinal tract, respiratory tract and mammary glands of humans and livestock, and has strong pathogenicity, can cause various pyogenic diseases (wound infection, arthritis, septicemia and septicemia, etc.) and toxin diseases (toxic vomiting, gastroenteritis and shock in food poisoning, etc.). Due to the improper use of clinical antibiotic drugs, drug-resistant bacteria are rampant, especially MRSA, which seriously threatens public health safety.

[0003] The infection of Staphylococcus aureus starts with successful adhesion, and then the bacteria break through the epithelial barrier of the skin and mucous membrane surface of the body or the cell membrane of the wound, and finally multiply in the host cells. Staphylococcus aureus has various virulence factors to promote the adhesion, invasion, spread and immune escape process. According to the signal peptide prediction using various algorithms, researchers believe that most of the outer proteins of Staphylococcus aureus are exported to the cytoplasm outside through the Sec secretion pathway.

[0004] The core component proteins of the Sec secretion pathway are SecA, SecY, SecG and SecE. SecA1 has the functions of ATPase and signal recognition. After the secretory protein precursor with a signal peptide is recognized by SecA, it enters the SecYEG channel for subsequent protein modification and is transported to the corresponding secretion system (such as type I and type III secretion systems, etc.), and finally completes the secretion. In this process, SecA1 consumes ATP to provide energy for the process of protein precursor passing through SecYEG. SecA1 is essential for normal growth of bacteria, and the protein is highly conserved in bacteria. Because there is no corresponding SecA1 in mammalian cells, SecA1 is an ideal target for developing broad-spectrum antibacterial drugs.

[0005] Litsea cubeba is a traditional Chinese medicine, which is the fruit, root and leaf of Litsea cubeba in Lauraceae family, and is a pioneer herb in traditional medicine, widely distributed in southern China. Litsea cubeba essential oil (LCEO) extracted from fresh Litsea cubeba plants is a plant essential oil containing volatile compounds, with strong lemon-like aroma, insoluble in water, and about 60-90% of citral. Citral has been listed in the U.S. Food and Drug Administration (FDA) "Generally Recognized as Safe" (GRAS) list, and is often used as a citrus flavoring in cosmetics and food. Citral can form a chelate with the DNA of MRSA, inhibit the biological activity of bacteria, but the activity of the anti-pathogenic bacteria virulence is reported for the first time. SUMMARY

[0006] The present application first uses citral, the main component of Litsea cubeba essential oil, as a virulence protein inhibitor of gram-positive bacteria. The compound is an inhibitor of SecA1, an important dynamic element of the Sec secretion pathway of Staphylococcus aureus, has good inhibitory effect on the secretion of 26 substrate virulence proteins, and can inhibit the growth of Staphylococcus aureus and reduce the mortality of mice infected with Staphylococcus aureus. Therefore, the present application aims to provide the application of citral in the preparation of anti-gram-positive bacterial virulence drugs.

[0007] In order to achieve the above technical purpose, the technical scheme of the present application is as follows:

[0008] The application of citral as a virulence protein inhibitor in the preparation of anti-gram-positive bacterial virulence drugs, wherein the structure of the citral compound is as follows:

[0009]

[0010] Further, the CAS number of the citral is 5392-40-5.

[0011] Further, the anti-gram-positive bacteria is Staphylococcus aureus.

[0012] Further, the Staphylococcus aureus is a standard strain of Staphylococcus aureus ATCC29213 or a strain of methicillin-resistant Staphylococcus aureus MRSA95.

[0013] Further, the anti-gram-positive bacterial virulence drug comprises citral and a pharmaceutically acceptable excipient.

[0014] Further, the configuration method of the anti-Gram-positive bacterial virulence drug comprises the following steps:

[0015] (1) mixing citral, Tween 80 and water in a certain mass ratio;

[0016] (2) mixing and emulsifying with a vortex oscillator, then storing for standby, and preparing the anti-Gram-positive bacterial virulence drug.

[0017] Further, the mass ratio of citral, Tween 80 and water in step (1) is 1:1:5.

[0018] Further, the storage temperature in step (2) is 4 DEG C.

[0019] Further, the anti-Gram-positive bacterial virulence drug in step (2) is stable for 3 days at room temperature.

[0020] Further, the compound (citral) of the present application can be used as a Sec secretion pathway inhibitor of Gram-positive pathogenic bacteria, inhibits the ATPase activity of SecA1 protein, and can be used for preparing an anti-Staphylococcus aureus virulence drug.

[0021] The present application has the following beneficial effects:

[0022] (1) The present application first reports the active ingredient citral which has the activity of inhibiting the virulence protein transport of Staphylococcus aureus, reduces the secretion of Sec secretion pathway virulence protein, and is a good Sec inhibitor.

[0023] (2) The present application first finds that citral inhibits the Sec secretion pathway transport substrate virulence protein of Staphylococcus aureus, and can significantly reduce the mortality of mice infected with Staphylococcus aureus.

[0024] (3) Since the Sec secretion pathway widely exists in Gram-positive pathogenic bacteria and has high conservation, it is of great significance for discovering a new type of broad-spectrum and efficient anti-bacterial virulence drug with independent intellectual property rights, and the citral compound can be used as an anti-Gram-positive bacterial virulence drug. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 : Citral inhibits the growth of Staphylococcus aureus in a concentration-dependent manner;

[0026] Figure 2 : Citral inhibits the ATPase activity of SecA1 protein of Staphylococcus aureus;

[0027] Figure 3: Citral does not affect the expression of virulence proteins of Staphylococcus aureus Sec secretion pathway substrates;

[0028] Figure 4 : Citral reduces the mortality of mice infected with Staphylococcus aureus. Specific embodiments

[0029] The application will be further described below in conjunction with examples, and the raw materials used are all commercially available if not specifically mentioned.

[0030] Example 1. Effect of Citral on the growth of Staphylococcus aureus

[0031] Test method Dilute the bacterial solution to 1x10 6 CFU / mL with TSB medium, divide into different groups and add different concentrations of Citral, so that the final concentration of Citral is 1 / 2 MIC, 1 / 4 MIC and 1 / 8 MIC, and set up a control group without adding Citral. Incubate at 37℃, 220 rpm for 24h, and take out the bacterial solution at 0, 2, 4, 8, 12 and 24h after incubation, and detect the absorbance value at 600nm wavelength with a spectrophotometer. The experiment is repeated three times.

[0032] Test results According to the results of the test Figure 1 It can be seen that the growth curve of untreated Staphylococcus aureus is "S" shaped, and reaches the logarithmic growth phase at about 3h. After adding 1 / 2 MIC concentration of Citral, the logarithmic growth phase is delayed, and the number of bacteria in the stationary phase is reduced; 1 / 4 MIC concentration of Citral can slightly shift the growth curve of the bacteria downward; the growth curve of 1 / 8 MIC concentration is similar to that of the untreated group; it can be seen that Citral can inhibit the growth of Staphylococcus aureus, and the inhibition is concentration-dependent.

[0033] Example 2. Citral inhibits the ATPase activity of SecA1 protein

[0034] Test principle The work of Sec secretion pathway requires SecA1 protein to consume ATP to provide energy, and increasing or decreasing the concentration of ATP can significantly change the working efficiency of Sec secretion pathway. In this application, Staphylococcus aureus (ATCC29213) is used as the research object, and the inhibitory effect of Citral on the ATPase activity of SecA1 protein is evaluated based on the in vitro enzyme activity detection of malachite green method.

[0035] E. coli DH5a competent cells, 5x in-fusion Cloning mix were purchased from Hangzhou Baosai Biotechnology Co., Ltd. IPTG (Isopropyl β-D-Thiogalactoside) was purchased from Invitrogen, USA. Adenosine triphosphate solution (ATP, 10 mmol / L), malachite green and ammonium molybdate were purchased from Beijing Solaybao Technology Co., Ltd. Rose Bengal (95%) was purchased from Shanghai Mokang Biotechnology Co., Ltd.

[0036] The secA1 gene sequence of Staphylococcus aureus ATCC 29213 strain was obtained from the NCBI database: ACCESSION NZ_CP094857, REGION: 752408..754939, and the constructed recombinant plasmid pCZN1-secA1 was transformed into E. coli Arctic Express competent cells. IPTG was added to a final concentration of 0.2 mM to induce expression, and the His-tag fusion protein was captured by a nickel column chromatography system. The eluate was collected and used later. The effect of citral on the ATPase activity of SecA1 was determined by the malachite green method. The buffer was prepared from 0.5 M Tris-HCl (pH = 7.6), 0.2 M KCl, 0.2 M NH4Cl, 10 mM DTT, and 20 mM Mg(OAc)2. 4.2% ammonium molybdate prepared by mixing 0.45% malachite green and 4N HCl at a ratio of 3:1 was filtered and 0.1% Triton X-100 was added to obtain the final color developing agent. 3 μL of buffer, 3 μg of ATPase (SecA1 protein), 2 μL of ATP, 5 μL of citral (with Rose Bengal as a positive control and distilled water as a negative control) were mixed and incubated at 37°C for 30 min. After completion, 160 μL of color developing agent was added and incubated for 1 min, 20 μL of 34% citrate was added, and OD660 was measured to calculate the ATPase activity.

[0037] The test results are shown in Table 1. Figure 2 As shown in Table 1, moderate and high concentrations of citral can significantly inhibit the ATPase activity of SecA1 (p < 0.05), and show a dose-dependent relationship, with Rose Bengal as a positive control. The above results prove that citral can inhibit the ATPase activity of SecA1, thereby inhibiting the transport function of the substrate protein.

[0038] Example 3. Effect of citral on the SecA secretion system of Staphylococcus aureus

[0039] The Sec secretion pathway is a multi-protein complex, mainly consisting of the dynamogen SecA1, responsible for substrate validation and ATP hydrolysis, and the SecYEG channel, which enables substrate co-translation or post-translational secretion. Under normal circumstances, nearly one-third of bacterial proteins are transported to the extracellular space via the Sec secretion pathway after synthesis to exert their function. This invention uses Staphylococcus aureus ATCC29213 as the research object, and uses RT-qPCR and secretory proteomics to detect the intracellular expression and extracellular transport of Sec secretion pathway substrates, evaluating the inhibitory effect of citral on the virulence proteins of the SecA secretion system substrates.

[0040] The test method involves picking a single colony of ATCC29213 and adding it to 3.5 mL of TSB. The colony is incubated at 37°C and 250 rpm for 8 hours. Then, 1 mL of the bacterial culture is added to 200 mL of TSB and incubated with shaking at 37°C and 250 rpm. Simultaneously, 1 / 4 MIC of citral is added, and the mixture is incubated until OD reaches [value missing]. 600 When the concentration is 2.0, centrifuge at 5000 rpm for 20 min, collect the precipitate for RT-qPCR detection, and collect the supernatant through a 0.22 μm filter to remove bacterial cells before using it for secretory proteomics detection.

[0041] The test results, obtained using secretory proteomics, examined whether citral could inhibit substrate transport in the Sec secretion pathway. As shown in Table 1, compared to the control group, the transport of substrate proteins from 26 Sec secretion pathways in the extracellular supernatant of *Staphylococcus aureus* strain ATCC 29213 decreased after treatment with 1 / 4 MIC of citral. To explore whether citral significantly reduces Sec substrate secretion by downregulating the expression of bacterial Sec substrates, the expression of several selected Sec substrates was detected by RT-qPCR. Figure 3 As shown, different concentrations of citral were co-incubated with Staphylococcus aureus ATCC 29213 until OD500. 600 At a concentration of 2.0, citral did not decrease the expression of Sec substrates; in fact, it may have increased the intracellular expression of Sec substrates. In conclusion, citral does not affect the expression of virulence proteins in the Staphylococcus aureus Sec secretion pathway, but it reduces the content of virulence proteins in the culture supernatant and inhibits the transport of virulence proteins.

[0042] Table 1. Substrate virulence proteins that were significantly downregulated in Staphylococcus aureus before and after citral treatment (CITvs CON).

[0043]

[0044]

[0045] Example 4. Citral improves the survival rate of mice infected with Staphylococcus aureus.

[0046] The test method involved dividing mice into three groups: a healthy control group (Control), a model group (MRSA 95), and a citral group (MRSA 95 + Citral, 0.5 g / kg), with 30 mice in each group, half male and half female. For three days prior to challenge, the experimental groups received the drug orally once daily via gavage, while the healthy control and model groups received the same volume of physiological saline via gavage. On the third day, challenge was performed 3 hours after gavage (6 × 10⁻⁶). 10 (CFU / mL, 0.1mL / 10g). Mice were continued to be administered the drug once daily after challenge until the end of the experiment. The survival status of mice in each group was observed and recorded every 12 hours after challenge, until no mice in any group died after 24 hours.

[0047] Test results are as follows Figure 4 As shown, mice began to die 24 hours after challenge, and all mice in the model group died after 48 hours. In contrast, mice in the citral group remained alive after 48 hours and did not die. Ultimately, the survival rate of the citral group was 50.75%. These results indicate that citral can improve the survival rate of mice infected with Staphylococcus aureus.

[0048] The above content should not be construed as limiting the specific implementation of this invention to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of this invention, and all such deductions or substitutions should be considered as belonging to this invention as determined by the submitted claims.

Claims

1. Use of citral as a virulence protein inhibitor for the preparation of a drug against virulence of Gram-positive bacteria, characterized in that, The structure of the citral compound is shown as follows:

2. Use of citral as a virulence protein inhibitor in the manufacture of a medicament for the treatment of Gram-positive bacteria according to claim 1, characterized in that, The CAS number of the citral is 5392-40-5.

3. Use of citral as a virulence protein inhibitor in the manufacture of a medicament for the treatment of Gram-positive bacteria according to claim 2, characterized in that, The citral is used as a SecA1 inhibitor of gram-positive pathogenic bacteria to prepare a drug for resisting staphylococcus aureus infection.

4. The use of citral as a virulence protein inhibitor in the manufacture of a drug for the treatment of Gram-positive bacteria according to claim 1, characterized in that, The gram-positive bacteria is staphylococcus aureus.

5. The use of citral as a virulence protein inhibitor in the manufacture of a drug for the treatment of Gram-positive bacteria according to claim 4, characterized in that, The staphylococcus aureus is a standard strain ATCC29213 or a methicillin-resistant staphylococcus aureus MRSA95.

6. Use of citral as a virulence protein inhibitor in the manufacture of a medicament against virulence of Gram-positive bacteria according to any one of claims 1 to 5, characterized in that, The anti-gram-positive bacteria virulence drug comprises the citral and a pharmaceutically acceptable excipient.

7. The use of citral as a virulence protein inhibitor in the manufacture of a drug for the treatment of Gram-positive bacteria according to claim 6, characterized in that, The configuration method of the anti-gram-positive bacteria virulence drug comprises the following steps: (1) mixing the citral, Tween 80 and water according to a certain mass ratio; (2) mixing and emulsifying by using a vortex oscillator, then storing for standby, and preparing the anti-gram-positive bacteria virulence drug.

8. Use of citral as a virulence protein inhibitor in the manufacture of a medicament for the treatment of Gram-positive bacteria according to claim 7, characterized in that, The mass ratio of the citral, Tween 80 and water in step (1) is 1:1:

5.

9. The use of citral as a virulence protein inhibitor in the manufacture of a drug for the treatment of Gram-positive bacteria according to claim 7, characterized in that, The storage temperature in step (2) is 4℃.

10. The use of citral as a virulence protein inhibitor in the manufacture of a drug for the treatment of Gram-positive bacteria according to claim 7, characterized in that, The anti-gram-positive bacteria virulence drug in step (2) is stable for 3 days at room temperature.

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