阅读说明：本技术 4-脱氧-n-乙酰氨基葡萄糖作为革兰氏阳性菌抗菌添加剂的应用 (Application of 4-deoxy-N-acetylglucosamine as gram-positive antibacterial additive ) 是由 王佳佳 李霞 马静 王鹏 于 2021-06-24 设计创作，主要内容包括：本发明属于抗菌药物技术领域,尤其涉及4-脱氧-N-乙酰氨基葡萄糖作为革兰氏阳性菌抗菌添加剂的应用。所述的4-脱氧-N-乙酰氨基葡萄糖作为革兰氏阳性菌抗菌添加剂可以与黄芩素联合用药,用于抗金黄色葡萄球菌；另外,4-脱氧-N-乙酰氨基葡萄糖分别与氨苄青霉素、卡那霉素、万古霉素联合应用于抗金黄色葡萄球菌,可降低原有抗生素用药剂量的4倍,几乎达到100％的抑菌效果。(The invention belongs to the technical field of antibacterial drugs, and particularly relates to application of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive. The 4-deoxy-N-acetylglucosamine as a gram-positive bacteria antibacterial additive can be used together with baicalein for resisting staphylococcus aureus; in addition, 4-deoxy-N-acetylglucosamine is respectively combined with ampicillin, kanamycin and vancomycin to be applied to staphylococcus aureus resistance, so that the dosage of the original antibiotic can be reduced by 4 times, and the bacteriostatic effect of 100 percent is almost achieved.)

The application of 4-deoxy-N-acetylglucosamine as antibacterial additive for gram-positive bacteria.

2. Use of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive according to claim 1, wherein the 4-deoxy-N-acetylglucosamine is used as a gram-positive antibacterial additive against staphylococcus aureus, against staphylococcus albus or against ampicillin resistant staphylococcus aureus.

3. Use of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive according to claim 1, wherein the 4-deoxy-N-acetylglucosamine is administered in combination with baicalein for the treatment of staphylococcus aureus.

4. Use of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive according to claim 1, wherein the 4-deoxy-N-acetylglucosamine is used against staphylococcus aureus in combination with ampicillin, kanamycin or vancomycin.

5. Use of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive according to claims 3 to 4, wherein the combination reduces the dose of antibiotic by 4 times to 100% of the antibacterial effect.

Technical Field

The invention belongs to the technical field of antibacterial drugs, and particularly relates to application of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive.

Background

In recent years, bacterial antibiotic resistance has been a major challenge in clinical treatment of infectious diseases. The worldwide review of antibiotic resistance published by Jim O' Neill in UK statistically counts up to 70 ten thousand patients who die of antibiotic resistance every year in the world, and up to 1000 ten thousand patients in 2050. The use of antibiotics reduces human morbidity and mortality caused by infectious diseases. However, this area has been greatly reversed by the emergence of multidrug-resistant pathogenic bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, carbapenem-resistant Acinetobacter baumannii, and carbapenem-resistant Enterobacter. Antibiotic resistance has been listed by the world health organization as one of the most important health hazards for the three public at 21 st century. Therefore, the search for novel antibacterial agents and therapeutic methods that effectively inhibit multiple drug resistance and do not produce a drug resistance mechanism is an urgent problem to be solved by the global public health industry.

Peptidoglycan (PGN) is used as a main structural component of a gram-positive bacterial cell wall, and is a reticular macromolecule formed by alternately connecting N-Acetylglucosamine (NAG) and N-Acetylmuramic acid (NAM) through beta-1 and 4 glycosidic bonds and then mutually crosslinking pentapeptide side chains of NAM residues. The cell wall can maintain the bacterial form, prevent the bacteria from cracking due to the change of the intracellular osmotic pressure, and the integrity of the cell wall structure plays an important role in the normal physiological function of the bacteria. Therefore, biosynthesis of bacterial cell wall peptidoglycan has been a target for many antibacterial drugs and a target of interest for broad-spectrum antibacterial drug development. Penicillin and cephalosporin in beta-lactam antibiotics and vancomycin in glycopeptides inhibit cell wall synthesis to break bacteria and die. However, under long-term drug-target interactions, bacteria either reduce the affinity of vancomycin for the target through alteration of the antibiotic target or develop resistance to the antibiotic through mutants methicillin expressing penicillin binding proteins. In addition, bacteria can also produce inactivated enzymes, and antibiotics are inactivated through enzymatic degradation and modification; the intracellular concentration of the antibiotic is below the inhibitory level by creating efflux pumps and altering the permeability of the cell membrane. The problem of bacterial resistance is becoming more acute due to the development of multiple resistance mechanisms, and in response to this challenge, scientists have proposed combination therapy. The combination therapy mainly combines antibiotics and antibiotics or antibiotics and non-antibiotic compounds, and exerts the antibacterial activity of the existing antibiotics again by interfering the drug resistance mechanism of bacteria and improving the sensitivity of the bacteria to the antibiotics.

Our earlier work developed a novel small molecule probe: 1,3, 6-triacetyl-4-deoxy-azido-acetamido-Sugar (4deo-Ac3GlcNAz) (A4-deoxy-Analogue of N-acetyl-D-glucosamine inhibition partial phase expression and growth factor binding in vitro; interacting with UDP-GlcNAc Metabolism and heparin surface expression use a Sugar Analogue recovery), which can selectively capture O-GlcNAc glycosylation modified proteins in eukaryotic cells. The deletion of the hydroxyl at the 4-position prevents GlcNAc from extending through beta-1, 4 glycosidic bonds in other sugar chain structures (such as N-/O-glycan), so that O-GlcNAc glycosylation modification of cytoplasmic and nuclear proteins can be efficiently and selectively carried out. The related research also finds that the special structure of 4deo-GlcNAc prevents the extension of the sugar chain structure, can effectively inhibit the synthesis of heparin sulfonate and chitin as the terminal of the sugar chain structure, and has potential development value in the aspects of anti-amyloid, antifungal and the like. UDP-4deo-GlcNAc as glycosyl donor precursor, the study results show that: GlcNAc derivatives such as 4deo-GlcNAc can be efficiently synthesized by protein kinase (NaHK) and pyrophosphatase (GlmU) derived from bacteria. In addition, other studies have reported that 100. mu.L of 4deo-Ac was used₃In the cell lysate of SKVO3 cultured by GlcNAc, a high content of UDP-4deo-GlcNAc could be detected by LC-MS analysis, and the UDP-GlcNAc/UDP-GalNAc level was significantly decreased, to some extent, UDP-4deo-GlcNAc itself or some intermediate could inhibit the production of UDP-GlcNAc or UDP-GalNAc.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of 4-deoxy-N-acetylglucosamine as a gram-positive antibacterial additive.

Specifically, the 4-deoxy-N-acetylglucosamine is used as a gram-positive bacteria antibacterial additive for resisting staphylococcus aureus, staphylococcus albus, ampicillin-resistant staphylococcus aureus and the like.

Furthermore, the 4-deoxy-N-acetylglucosamine as a gram-positive bacteria antibacterial additive can be used in combination with baicalein for resisting staphylococcus aureus.

Furthermore, the 4deo-GlcNAc is respectively combined with ampicillin, kanamycin and vancomycin to be applied to staphylococcus aureus resistance, so that the dosage of the original antibiotic can be reduced by 4 times, and the bacteriostatic effect is almost 100%.

The invention has obvious technical effect.

In the subject, based on the connection mode of gram-positive bacteria cell walls and the framework structure characteristics of the bacteria cell walls, a small molecule compound 4-deoxy-N-acetylglucosamine (4deo-GlcNAc) influencing cell wall synthesis is found, 5-20mg/mL of 4deo-GlcNAc is added into the culture medium of bacteria, the small molecule compound can be converted into corresponding glycosyl donor UDP-4deo-GlcNAc by synthetase in bacteria body, the cell wall framework can continuously take in the 4deo-GlcNAc in the culture medium in the growing process along with the continuous division and proliferation of the bacteria, NAM can not form a compact network structure through beta-1 and 4 connection due to the loss of hydroxyl at the 4 position, and finally the bacteria with damaged cell walls can disintegrate and die under the high osmotic pressure environment, thereby achieving the purpose of bacteriostasis. In the work, the lowest bacteriostatic concentration of 4deo-GlcNAc to normal golden yellow staphylococcus strain N315 is preliminarily determined to be 20mg/mL through an in vitro bacteriostatic experiment, which shows that the 4deo-GlcNAc has good antibacterial activity to normal golden yellow staphylococcus, and the 4deo-GlcNAc is found to be synthesized on bacterial cell walls through bacterial metabolism to promote the breakage of the bacterial cell walls so as to play a bacteriostatic role; the 4deo-GlcNAc and baicalein are used in combination in vitro and in vivo experiments, so that staphylococcus aureus and skin infection caused by staphylococcus aureus can be effectively inhibited, and the 4deo-GlcNAc is suggested to be a potential antibacterial additive, and a new research idea is provided for the development of novel micromolecular antibacterial drugs in the future.

Drawings

FIG. 1 is the MIC values of 4deo-GlcNAc against Staphylococcus aureus in different concentrations of LB medium; wherein, the A picture: MIC of 4deo-GlcNAc and GlcNAc in LB cultures at different fold dilutions (visual panel); and B, drawing: detecting MIC values of 4deo-GlcNAc in LB culture diluted 2.5-fold and 5-fold; panel C is a graph that measures MIC values for GlcNAc in 2.5-fold and 5-fold dilutions in LB culture.

FIG. 2 is a graph showing the effect of 4deo-GlcNAc on the growth curve of Staphylococcus aureus in different concentrations of LB medium; wherein panels A and B are the growth trends of Staphylococcus aureus in LB medium containing different concentrations of 4deo-GlcNAc at 2.5-fold and 5-fold dilutions for 48h, respectively; panels C and D are the growth trends of staphylococcus aureus in LB medium containing 2.5-fold and 5-fold dilutions of GlcNAc at different concentrations, respectively, over 48 h.

FIG. 3 is a fluorescent mapping and analysis of the effect of 4deo-GlcNAc on the bacterial cell wall; wherein, Contrl group is Staphylococcus aureus without any treatment (negative control group); the GlcNAz group is Staphylococcus aureus treated with GlcNAz (10 mg/mL); the 4deo-GlcNAz group was Staphylococcus aureus treated with 4deo-GlcNAz (10 mg/mL).

FIG. 4 shows the combined bacteriostatic effect of 4deo-GlcNAc and baicalein; FIG. A: measuring the MIC value of the baicalein to the staphylococcus aureus; and B: the growth tendency of bacteria when baicalein with the concentration of 5 mu g/mL and 4deo-GlcNAc with different concentrations are used in combination; and (C) figure: the growth tendency of bacteria when baicalein with the concentration of 10 mu g/mL and 4deo-GlcNAc with different concentrations are used in combination; FIGS. D, E and F are plots of bacterial growth with combinations of 5. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL baicalein and varying concentrations of GlcNAc.

FIG. 5 is a graph of the in vivo efficacy of 4deo-GlcNAc in combination with baicalein on a mouse model of skin infection; FIG. A: an experimental procedure of a model for treating skin infection of mice; and B: colony unit counts of infected skin samples of drug treated and untreated groups; and (C) figure: wound healing results of infected skin of drug-treated and untreated groups.

FIG. 6 shows HE staining of infected skin tissue of treated and untreated mice; FIG. A: HE staining results of back skin tissue samples of uninfected normal mice; and B: HE staining results of dorsal infection skin tissue samples of mice without any treatment; and (C) figure: HE staining of dorsal infection skin tissue samples of mice treated with 20mg/mL of 4 deo-GlcNAc; FIG. D: HE staining results of dorsal infection skin tissue samples of mice treated by 80 mu g/mL of baicalein; FIG. E: staining results of HE (high intensity staining) of dorsal infection skin tissue samples of mice treated with 20mg/mL of 4deo-GlcNAc in combination with 40. mu.g/mL of baicalein.

FIG. 7 shows the results of immunofluorescent staining of neutrophils in infected skin tissues of treated and untreated mice; normal skin tissue in Normal group; the Untreated group is infected skin tissue that has not been treated with an antibiotic; baicalein (80. mu.g/mL) group is infected skin tissue treated with Baicalein (80. mu.g/mL); the 4deo-GlcNAc (20mg/mL) group was 4deo-GlcNAc (20mg/mL) treated infected skin tissue; baicalein (40. mu.g/mL) +4deo-GlcNAc (20mg/mL) group is the infected skin tissue treated with a combination of Baicalein (40. mu.g/mL) and 4deo-GlcNAc (20 mg/mL).

FIG. 8 shows 4deo-GlcNAc and Amp⁺MIC for staphylococcus aureus standard and drug resistant strains; FIG. A: amp⁺For SaN315 and SaN315-Amp⁺A minimum inhibitory concentration measurement value; and B: 4deo-GlcNAc pairs SaN315 and SaN315-Amp⁺The minimum inhibitory concentration of (c).

Detailed description of the invention

The technical solutions of the present invention are further described in detail with reference to the accompanying drawings and the detailed description, and unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and all the raw materials are commercially available.

Example 1 recovery of bacterial strains and plate preparation.

(1) Preparation of diluted LB medium of different concentrations and normal LB agar plates: dissolving 2.5-fold diluted LB culture medium (weighing 1.6g of Tryptone, 0.8g of Yeast Extract and 2g of NaCl) and 5-fold diluted LB culture medium (weighing 0.8g of Tryptone, 0.4g of Yeast Extract and 2g of NaCl) in 400mL of double distilled water respectively, performing high-pressure sterilization at 120 ℃ for 20min to prepare LB liquid culture medium, cooling and storing in a refrigerator at 4 ℃; weighing 4g of Tryptone, 2g of Yeast Extract, 2g of NaCl and 6g of agar powder, adding 400mL of double distilled water for dissolving, carrying out autoclaving treatment at 120 ℃ for 20min to obtain LB culture solution, pouring the solution into a disposable culture dish by 15mL in an aseptic super clean bench, cooling and solidifying to obtain an LB solid agar plate, and sealing and storing the LB solid agar plate in a refrigerator at 4 ℃ in a sealing film manner after ultraviolet disinfection.

(2) Preparation of staphylococcus aureus N315 flat plate: taking out the frozen standard strain N315 at-80 ℃ for thawing at room temperature, taking a little bacterial liquid in a sterile super clean bench by using a sterile inoculating loop, carrying out three-zone lineation on an LB (Luria Bertani) nonreactive solid culture plate, putting the plate in a constant-temperature bacterial incubator at 37 ℃ for culturing for 18h, taking out, making golden colony with a regular shape on the plate by naked eyes, sealing by a sealing membrane, and storing at 4 ℃ in a refrigerator.

The antibacterial activity of 4deo-GlcNc on staphylococcus aureus is preliminarily evaluated by an in vitro experiment and a mouse skin infection model.

EXAMPLE 2 determination of the minimum inhibitory concentration of an antibacterial agent against Staphylococcus aureus

Because the infection caused by bacteria wound on the skin surface is a microenvironment with relatively deficient nutrient components, LB culture medium with dilution multiple of 2.5 times and LB culture medium with dilution multiple of 5 times are prepared, staphylococcus aureus is respectively cultured in two culture media containing 4deo-GlcNAc with final concentration of 0, 1.25, 2.5, 5, 10 and 20mg/mL for 24 hours, and then the OD of bacteria liquid growing under different drug concentrations is respectively determined₆₀₀Exploring the effect of Staphylococcus aureus on its growth by ingesting large amounts of 4deo-GlcNAc in nutrient-deficient culture environments.

(1) Preparing bacterial liquid: picking single colonies from a staphylococcus aureus N315 flat plate by using an aseptic gun tip to a test tube with 1mL of LB culture medium, placing the test tube in a shaking table with 37 ℃ and 220rpm, and culturing for 16 h; standardizing and diluting the suspension liquid, taking LB blank culture medium as a reference, obtaining the relation between the plate clone count and the optical density value of the suspension liquid, and measuring OD₆₀₀When the concentration is 0.5, the bacterial liquid concentration is 2 × 10⁹CFU/mL, diluting the adjusted suspension to 1X 10⁶CFU/mL is ready for use.

(2) Preparing an antibacterial agent: dissolving baicalein powder into solution with final concentration of 20mg/mL by DMSO, and diluting with double dilution method to 640, 320, 160, 80, 40, 20 μ g/mL for use; dissolving the compound 4deo-GlcNAc in autoclaved double distilled water to obtain a solution with the final concentration of 200mg/mL, and diluting the solution to 40, 20, 10, 5 and 2.5mg/mL by a double dilution method for later use; prepared AMP with a storage concentration of 100mg/mL⁺Diluting into concentration gradient of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 μ g/mL by double dilution method for use; the negative control compound GlcNAc was dissolved in autoclaved double distilled water to a final concentration of 200mg/mL and diluted to 40, 20, 10, 5, 2.5mg/mL by two-fold dilution for use. All the prepared antibacterial agents are sterilized by filtration through a 0.22 μm filter membrane.

(3) Sample adding: 1mL of the quantified inoculum was pipetted into each of the tubes containing 1mL of the diluted antimicrobial agent and the positive control tubes containing no antimicrobial agent and mixed. This resulted in a dilution of 1:2, the dilution of the inoculum is 1:2, the final concentration of the baicalein in each test tube is respectively 320, 160, 80, 40, 20 and 10 mu g/mL, and the final concentration of the 4deo-GlcNAc in each test tube is respectively 20, 10, 5, 2.5 and 1.25 mg/mL; the final GlcNAc concentration in each tube was made 20, 10, 5, 2.5, 1.25mg/mL, respectively; for AMP⁺Using broth microdilution, 50. mu.L of each of the quantified bacterial solutions and drug concentrations were placed in each well of a 96-well plate at a ratio of 1:1, and AMP was allowed to flow through each sample well⁺To a final concentration of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64. mu.g/mL, respectively, in the absence of AMP⁺Wells containing 100 μ L LB medium alone served as positive controls and wells containing 100 μ L LB medium alone served as negative controls.

(4) Determining MIC results: and putting a sample to be tested in a shaking table at 37 ℃, rotating speed of 220rpm and a 96-hole plate in a constant-temperature incubator for incubation culture for 24h, wherein when a result is read, the negative control is clear and transparent, the positive hole is suspension bacteria liquid, and the lowest bacteriostatic concentration is obtained when no obvious bacterial colony can be seen by eyes in the sample to be tested. The results are shown in FIG. 1: in LB medium diluted 2.5 times, the growth of bacteria was inhibited as the concentration of the drug increased, and the minimum inhibitory concentration of 4deo-GlcNAc to Staphylococcus aureus N315 was 20mg/mL (FIG. 1-A); the effective antimicrobial concentration of 4deo-GlcNAc was 10mg/mL in the 5-fold dilution medium (FIG. 1-B); in contrast, in the negative control group, GlcNAc at different concentrations had substantially no inhibitory effect on the growth of Staphylococcus aureus in LB medium at different dilution times (FIG. 1-C).

Example 34 Deo-GlcNAc Effect on the growth tendency of Staphylococcus aureus

To better illustrate the in vitro bacteriostatic effect of 4deo-GlcNAc on Staphylococcus aureus, we monitored the effect of 4deo-GlcNAc on the growth curve of Staphylococcus aureus. By taking GlcNAc as a control group, 4deo-GlcNc and GlcNAc with different concentrations and staphylococcus aureus were cultured for 48h respectively, and OD values of the bacteria liquid at different time points at the absorbance of 600nm were measured.

(1) Preparing bacterial liquid: picking single colonies from a staphylococcus aureus N315 flat plate by using an aseptic gun tip to a test tube of 1ml LB culture medium, placing the test tube in a shaking table at 37 ℃ and 220rpm, and culturing for 16 h; standardizing and diluting the suspension liquid, taking LB blank culture medium as a reference, obtaining the relation between the plate clone count and the optical density value of the suspension liquid, and measuring OD₆₀₀When the concentration is 0.5, the bacterial liquid concentration is 2 × 10⁹CFU/mL。

(2) Preparing an antibacterial agent: the compound 4deo-GlcNAc was dissolved in autoclaved double distilled water to a final concentration of 200mg/mL and diluted to 20, 10, 5, 2.5, 1.25, 0mg/mL by two-fold dilution method for use. Sample preparation was carried out in the same manner with GlcNAc as a negative control. All the prepared antibacterial agents are sterilized by filtration through a 0.22 μm filter membrane.

(3) Sample adding: the activated bacteria were again inoculated in 1:1000 tubes containing 1mL of fresh 2.5-fold and 5-fold dilutions of LB medium, respectively, and the drugs were added to the tubes in order according to the designed concentration gradient.

(4) Measuring a sample value: placing the sample in a shaking table for incubation culture at 37 ℃ and 220rpm, and taking 100 mu L of bacterial liquid at fixed time point in a 96-well plate for OD₆₀₀And (4) detecting, and evaluating the bacteriostatic effect of the 4deo-GlcNAc through the OD value. The results are shown in FIG. 2: in LB medium at 2.5-fold dilution and at 5-fold dilution with constant NaCl concentration, phases4deo-GlcNAc at the concentration required was effective in inhibiting the growth of Staphylococcus aureus within 48h (FIG. 2-A, B). In the negative control group, different concentrations of GlcNAc in LB culture medium with different dilution times have little influence on the growth of bacteria; GlcNAc can be used as a nutrient to promote bacterial growth in nutrient deficient culture environments compared to the blank (Con) (fig. 2-C, D).

Example 4 molecular Probe Fluorescently labeled assay to assess the effect of 4deo-GlcNAc on the bacterial cell wall.

To explore the effect of 4deo-GlcNAc on the cell wall of Staphylococcus aureus, we used the compounds GlcNAz and 4deo-GlcNAz as small molecule probes against Staphylococcus aureus. Through a fluorescence labeling experiment, a membrane dye Alk488 and a nuclear dye PI are used for processing staphylococcus aureus, and a labeling effect is observed in a confocal super-resolution microscope.

(1) Strain activation: picking single colonies from a staphylococcus aureus N315 flat plate by using an aseptic gun tip to a test tube with 1mL of LB culture medium, placing the test tube in a shaking table with 37 ℃ and 220rpm, and culturing for 16 h; and (5) turbid activation of the bacterial liquid.

(2) Preparing a sample: the overnight activated Staphylococcus aureus was diluted at 1:1000 and inoculated in 1mL of LB medium diluted 2.5 times, and 10mg of the corresponding molecular probes GlcNAz and 4deo-GlcNAz were added to give a final concentration of 10mg/mL, respectively, after culturing at 37 ℃ with a shaker at 220rpm for 6 hours, OD was then measured₆₀₀The value is about 0.53.

(3) Collecting 100 μ L of the bacterial solution to 1.5mL centrifuge tube, centrifuging at 6000g for 5min, removing supernatant, washing with 200 μ L of 1 × PBS for three times, suspending the bacterial solution in 100 μ L of PBS solution, and adding 1mM CuSO to the final concentration₄The reaction mixture was mixed with 100. mu.M TBTA (100mM stored in DMSO), 1mM TCEP (ready prepared) and 20. mu.M Alk488 and incubated at room temperature in the dark for 30 min.

(4) The reacted sample was centrifuged at 6000g for 5min, the supernatant was discarded, and washed twice with 200. mu.L PBS and then fixed to 100. mu.L PBS solution, 30. mu.M Propidium Iodide (PI) was added to the solution, and after incubation for 15min at room temperature in the dark, the sample was centrifuged at 6000g for 5min to discard the excess dye and washed three times with PBS.

(5) The fluorescence-labeled bacteria are fixed in 100 mu L PBS solution, 20 mu L bacterial solution is respectively dripped in the center of an adhesive glass slide, and the adhesive glass slide is covered by a cover glass and sealed by nail polish. The fluorescently labeled bacteria were observed under a super-resolution confocal microscope at 488nm and 594 nm. The results are shown in FIG. 3, where both GlcNAz and 4deo-GlcNAz can be metabolised to the cell wall, and the blank panel is free of green fluorescence due to the absence of azide-labeled sugar structures (Contrl panel); GlcNAz does not affect the integrity of the cell wall backbone, so PI dyes cannot enter the bacteria through the cell wall to stain the nuclear region red, so no red fluorescence is observed at the emission wavelength of 594nm, and no red fluorescently labeled nuclear region is seen after fusion (GlcNAz group); based on the previous theoretical hypothesis that 4deo-GlcNAz can be metabolically labeled onto the cell wall through the biosynthetic pathway of bacteria and will interfere with the linkage between cell wall NAM and NAG, causing the cell wall structure to break, while PI dyes can penetrate the structurally incomplete cell wall, causing the nuclear region of the bacteria to appear red; after fusion, the green fluorescently labeled cell wall was seen to be wrapped with a nuclear region colored with PI red dye (4deo-GlcNAz panel). The above experimental results indicate that the 4deo-GlcNAc treated S.aureus cell wall is disrupted.

Example 54 deo-GlcNAc and baicalein combination in vitro bacteriostasis assay

According to the action mode of a compound 4deo-GlcNAc on bacterial cell walls and the action mechanism of an antibacterial drug, a flavonoid compound baicalein with broad-spectrum antibacterial activity is selected, and the action mechanism of the flavonoid compound baicalein on methicillin-resistant staphylococcus aureus exerts antibacterial action by influencing bacterial membrane permeability, inhibiting protein synthesis and influencing SDH and DNA topoisomerase activity. Firstly preparing 4deo-GlcNAc with a series of concentration gradients, then respectively adding equivalent baicalein, testing a sample OD600 at different time points, and evaluating the antibacterial effect of the 4deo-GlcNAc and baicalein combined drug.

(1) Preparing bacterial liquid: picking single colonies from a staphylococcus aureus N315 flat plate by using an aseptic gun tip to a test tube with 1mL of LB culture medium, placing the test tube in a shaking table with 37 ℃ and 220rpm, and culturing for 16 h; standardized dilution of the suspension, comparison with LB blank medium, passingThe relationship between the plate clone count and the optical density of the bacteria liquid can be obtained, and OD is measured₆₀₀When the concentration is 0.5, the bacterial liquid concentration is 2 × 10⁹CFU/mL, diluting the adjusted suspension to 1X 10⁶CFU/mL is ready for use.

(2) Preparing an antibacterial agent: the compound 4deo-GlcNAc was dissolved in autoclaved double distilled water to a final concentration of 200mg/mL, and diluted in a test tube at a final concentration of 20, 10, 5, 2.5, 1.25, 0mg/mL in 2.5-fold diluted LB medium by a double dilution method for use. Sample preparation was performed in the same manner with GlcNAc as a negative control. Baicalein powder was dissolved in DMSO to give a solution having a final concentration of 20mg/mL, and the same amount of baicalein was added to test tubes containing 4deo-GlcNAc or GlcNAc at different concentrations to give final concentrations of 5. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL, respectively, for use. All the prepared antibacterial agents are sterilized by filtration through a 0.22 μm filter membrane.

(3) Sample adding: and respectively adding the standardized bacterial liquid into prepared drug concentration gradient test tubes.

(4) Measuring a sample value: placing the sample in a shaking table for incubation culture at 37 ℃ and 220rpm, and taking 100 mu L of bacterial liquid at fixed time point in a 96-well plate for OD₆₀₀And (4) detecting, and evaluating the combined bacteriostasis effect of the 4deo-GlcNAc and the baicalein through the OD value. The results are shown in FIG. 4: when the baicalein acts on staphylococcus aureus independently, the minimum inhibitory concentration is 160 mug/mL, which is equivalent to the value reported in the literature (figure 4A); the results show that the combination of 4deo-GlcNAc and baicalein can significantly improve the activity of baicalein targeting cells when the baicalein with the same concentration is used alone, the growth of bacteria can be significantly inhibited within 8h (figure 4B) by 5 mug/mL of baicalein and 10mg/mL of 4deo-GlcNAc, the inhibition time can be prolonged to 12h (figure 4C) when the concentration of the baicalein is 10 mug/mL, and the OD600 value of the bacteria can reach about 0.3 (figure 4A). The GlcNAc negative control group showed no bacteriostatic activity under the combined action of baicalein at different concentrations (FIGS. 4D-F).

Example 64 deo-GlcNAc and Baicalein combination in vivo antibacterial assay

The experimental results show that the 4deo-GlcNAc and the baicalein have good in-vitro antibacterial effect on the staphylococcus aureus. Subsequently, in order to evaluate the antibacterial efficacy of 4deo-GlcNAc and baicalein in vivo. Randomly dividing a successfully constructed mouse skin infection model into 5 groups, wherein each group comprises 5 mice, namely a GlcNAc group, a blank group, a baicalein group, a 4 deo-GlcNAc/baicalein combined drug administration group for drug treatment, and subcutaneously administering at a wound part for 3 multiplied by 20 mu L/day; after 40h, the healing degree of the wound surface of the mouse is observed by photographing and the wound skin of the mouse is taken for CFU statistics, so that the antibacterial effect of each group of medicines is evaluated.

6.1 mouse skin infection model construction and treatment experiment

(1) In the experiment, 25 male BALB/C mice with the age of 8 weeks are used for carrying out back depilation one day in advance, and the mice are randomly divided into 5 groups, each group is 5, and the mice are divided into a blank group, a GlcNAc group, a baicalein group, 4deo-GlcNAc, 4 deo-GlcNAc/baicalein combined drug group.

(2) Picking single colonies on a staphylococcus aureus N315 flat plate by using a sterile gun tip to a test tube of 1mL LB culture medium, placing the test tube in a shaking table at 37 ℃ and 220rpm, and culturing for 16 h; standardizing and diluting the suspension liquid, taking LB blank culture medium as a reference, obtaining the relation between the plate clone count and the optical density value of the suspension liquid, and measuring OD₆₀₀When the concentration is 0.5, the bacterial liquid concentration is 2 × 10⁹CFU/mL, diluting the adjusted suspension to 1X 10⁷CFU/mL。

(3) Cultured Staphylococcus aureus (Staphylococcus aureus) 20. mu.L (1X 10)⁷CFU/mL) was inoculated on the dorsal skin of mice with a 0.3 × 13RWLB disposable sterile syringe needle, and the mice were observed for molding after 16 h. Then, the mice of each group are respectively treated by corresponding medication, namely subcutaneous administration at the wound part, 3 multiplied by 20 mu L/day, photographing is carried out after 40h, the model of the mouse model and the conditions before and after treatment are observed, and the antibacterial effect of the drugs of each group is evaluated. As a result, as shown in FIG. 5, the combination of 4deo-GlcNAc (20mg/mL) and baicalein (40. mu.g/mL) was effective in antibacterial action and promoted wound scab healing, as compared with the other groups (FIG. 5C). CFU statistics is carried out on the skin of the mouse, and the results show that the 4deo-GlcNAc and baicalein combined treatment has obvious colony order reduction compared with a blank group and also has obvious bacteriostatic effect compared with single-drug treatment (figure 5B), which shows that the 4deo-GlcNAc and baicalein combined treatment has good effect on treating the staphylococcus aureus infected wound skinHas curative effect.

6.2 mice infected wound skin CFU count

(1) The mice are killed by dislocation of cervical vertebrae, the skin of the infected part of the mice is taken down, the skin is cut into pieces and then fully ground in 3mL sterile LB culture medium, the pieces are diluted into different gradients, and 100 mu L of each is uniformly smeared on an LB solid culture plate.

(2) And (3) putting the solid culture plate into a constant-temperature incubator at 37 ℃ for incubation for 18h, and taking out the solid culture plate for single colony counting.

6.3 HE staining of infected wounded skin in mice

Subsequently, we took the infected skin area of the back of the mice for pathological analysis by histological HE staining.

(1) Mouse infected skin tissue fixation: the mice were sacrificed by dislocation of cervical vertebrae, the skin was taken out, washed clean with PBS repeatedly, and fixed in 4% paraformaldehyde for 48 h.

(2) Preparing a paraffin section:

(a) skin tissue was laid flat in a white embedding frame and then pencil marked to clarify the different grouped samples.

(b) The tissue was rinsed with fine running water for 30min, then placed in an embedding frame, then placed in an automatic dehydrator for dehydration, transparency, and wax immersion. The procedure is as follows: 1h of 75% ethanol, 1h of 85% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1h of 100% ethanol I, 1h of 100% ethanol II, 45min of xylene I, 45min of xylene II, 1h of paraffin I, 1h of paraffin II and 45min of paraffin III.

(c) After the wax dipping is finished, the tissue is taken out and immediately placed into the wax melted in the embedding machine. Pouring a certain amount of wax liquid, erecting the tissue in an embedding mould, and filling the wax liquid when the wax liquid is in a semi-solidified state. The mixture is cooled at room temperature and then placed on a freezing table for rapid solidification.

(d) Cutting wax block tissue with a microtome to a thickness of 5 μm, flattening in a 37 deg.C water bath, lightly sticking on a glass slide, and baking for 4 hr.

(3) HE staining of tissue

(a) Dewaxing: xylene I, II, III each for 10min, anhydrous ethanol I, II, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol each for 5 min;

(b) rehydration: slowly washing with running water for 10 min;

(c) and (3) carrying out hematoxylin nuclear staining: dripping hematoxylin on the tissue with pipette, staining for 1min, and flushing with running water for 10 min;

(d) and (3) differentiation is transparent: differentiating with 1% hydrochloric acid alcohol for 1s, washing with running water, returning blue for 10min, and observing stained nucleus under the microscope;

(e) eosin staining of cytoplasm: dripping eosin on the tissue with pipette, staining for 3min, flushing with flowing water for 1min,

observing cytoplasmic staining under a microscope;

(f) sealing: sealing a sheet by using neutral resin adhesive;

(g) and scanning the section by a full-automatic microscope to carry out under-mirror observation.

The results are shown in FIG. 6: in normal (healthy, uninfected) skin tissue samples, the skin tissue stratification can be clearly seen (fig. 6A). Staphylococcus aureus infected skin showed severe inflammatory response and extensive inflammatory cell infiltration of dermis and subcutaneous tissue was seen compared to control (fig. 6B). In contrast, the section of the combination treatment of 4deo-GlcNAc and baicalein showed the lightest inflammatory response and minimal inflammatory cell infiltration, and a large number of adipocytes were accumulated at the injured skin site (fig. 6E). The inflammatory response in 4deo-GlcNAc and baicalein treated infected skin alone was less in the untreated infected skin (FIGS. 6C-D). The data show that in the mouse model, the combined administration of 4deo-GlcNAc and baicalein can more effectively reduce the infection of staphylococcus aureus and show good curative effect.

6.4 immunofluorescence staining of infected wound skin of mice

Neutrophils are the first line of defense against bacterial pathogens that breach the epithelial barrier. Within the short time of bacterial invasion, neutrophils respond to soluble factors including chemokines and cytokines and recruit to the site of infection and begin to phagocytose the pathogen. To further determine the changes in inflammatory cells following drug treatment of infected skin on the back of mice, we performed immunofluorescent staining analysis of wounded skin in mice and evaluated the therapeutic effect of 4deo-GlcNAc and baicalein by the neutrophil-specific fluorescent antibody Ly6 g.

(1) Baking the prepared paraffin section in a 65 ℃ oven for 40min until the section is dried;

(2) slice dewaxing and rehydration: xylene I, II was added each for 15min and then dried in a fume hood. The slices were then processed further as follows: anhydrous ethanol I, II, 90% ethanol, 85% ethanol, 70% ethanol each for 5min, and then adding ddH₂Flushing for 3 times (5 min each time) with O;

(3) antigen retrieval: soaking the slices in citrate buffer solution (pH 6.0), boiling in microwave oven, cutting off power, heating with middle and high fire for 20s every 2min, and ending the process after the duration of 15 min;

(4) PBS washing for 3 times, 5min each time;

(5) blocking for 2h at room temperature by using 2% BSA blocking buffer;

(6) a first antibody: the primary antibody was diluted with PBS containing 2% BSA, 0.2% TritonX-100 at the ratio recommended by the specification and incubated overnight at 4 ℃;

(7) washing with PBST for 3 times, 5min each time;

(8) secondary antibody: diluting the secondary antibody with PBS containing 2% BSA and 0.2% TritonX-100, and incubating for 60min at room temperature;

(9) washing with PBST for 3 times, 5min each time;

(10) DAPI staining of nuclei: diluting DAPI with PBS at a ratio of 1:200, and incubating at room temperature for 7 min;

(11) washing with PBST for 3 times, 5min each time, and ddH₂Washing with water twice for 5 min;

(12) mounting and observing and analyzing under a fluorescence microscope.

The results are shown in FIG. 7, and the intense green fluorescence in the skin of untreated infected mice indicates a high neutrophil accumulation (Normal group); in the drug-treated group, the 4deo-GlcNAc and baicalein-treated group alone had a gradual decrease in neutrophils (Untreated); neutropenia trended towards normal tissue in the combination treatment group of baicalein and 4deo-GlcNAc (FIG. 7 m); it is demonstrated again that the combination of baicalein and 4deo-GlcNAc can effectively reduce the inflammatory reaction of the infected skin on the back of the mouse.

Example 7 Induction experiments and MIC value determination of AMP + Staphylococcus aureus resistant strains

The above experimental results have shown that 4deo-GlcNAc has good bacteriostatic action on normal Staphylococcus aureus and can effectively treat skin infection caused by Staphylococcus aureus in combination with baicalein. To better illustrate whether 4deo-GlcNAc has the same effect on drug-resistant strains, we first induced AMP resistance⁺Staphylococcus aureus, i.e., Staphylococcus aureus in sub-MIC-AMP⁺Is continuously cultured in the growth environment of the strain, and finally, the AMP capable of being induced⁺The MIC of a drug-resistant strain grown in an environment with a final concentration of 28. mu.g/mL was determined by broth microdilution.

(1) AMP + strain-resistant induction: picking single colonies on a staphylococcus aureus N315 flat plate by using a sterile gun tip to a test tube of 1mL LB culture medium, placing the test tube in a shaking table at 37 ℃ and 220rpm, and culturing for 16 h; and (5) activating the bacterial liquid. Mixing bacterial liquid in a ratio of 1:100 in proportion, inoculating the mixture in LB culture medium containing AMP + with sub-inhibitory concentration (0.5 × MIC), culturing at 37 deg.C and 220rpm for 16 h; the method is used for continuously culturing 11 generations to be used as a final drug-resistant detection strain. Taking 500 mu L of bacterial liquid and 50% of glycerol, mixing the mixture in a ratio of 1:1, and freezing and storing at-80 ℃ for later use.

(2) Minimum Inhibitory Concentration (MIC) determination

(a) And (3) standardizing and diluting the drug-resistant bacteria suspension, taking an LB blank culture medium as a reference, obtaining the drug-resistant bacteria suspension through the relation between plate clone counting and the optical density value of the bacteria liquid, measuring OD600 to be 0.48, wherein the concentration of the bacteria liquid is about 2 × 109CFU/mL, and diluting the adjusted bacteria liquid suspension to be 1 × 106CFU/mL for later use.

(b) Diluting prepared AMP + with preservation concentration of 100mg/mL into concentration gradient of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 and 256 mug/mL by a double dilution method for later use; diluting 4deo-GlcNAc with final concentration of 200mg/mL into 40, 20, 10, 5, 2.5mg/mL by a double dilution method for later use;

(c) the adjusted inoculum was added to a 96-well plate at a 1:1 ratio and mixed gently with 50 μ L of the diluted series of antimicrobials. The final concentrations of 4deo-GlcNAc in the wells were 20, 10, 5, 2.5, 1.25mg/mL, respectively, and the final concentrations of AMP + in the wells were 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128. mu.g/mL, respectively.

(d) And (3) covering a plate cover, putting the 96-well plate in an electric heating constant-temperature incubator for culturing for 18h at 37 ℃, and observing the concentration of the bacteria liquid which is not turbid by naked eyes, namely the minimum inhibitory concentration, which is shown in table 1. The OD at a wavelength of 600nm was then measured by a full-wavelength microplate reader.

TABLE 14 determination of minimum inhibitory concentration of deo-GlcNAc against AMP + Staphylococcus aureus.

TABLE 14 determination of minimum inhibitory concentration of deo-GlcNAc on Amp + Staphylococcus aureus

The results are shown in FIG. 8: 4deo-GlcNAc AMP tolerance⁺The minimal inhibitory concentration of Staphylococcus aureus was 20mg/mL (FIG. 8B), while AMP was⁺The minimum inhibitory concentration against the drug-resistant strain was 64. mu.g/mL (FIG. 8A). The primary result shows that 4deo-GlcNAc with the same MIC still has obvious bacteriostatic effect on the drug-resistant strain, and a new idea is provided for the selection of candidate drugs for clinically treating the drug-resistant strain.