阅读说明：本技术 水杨酸甲酯在制备沙门氏菌ⅲ型分泌系统抑制剂中的应用 (Application of methyl salicylate in preparation of salmonella III type secretion system inhibitor ) 是由 吕强华 王建锋 邓旭明 李淑芳 韩伟 张蕊 冯海华 王琳 于 2021-01-14 设计创作，主要内容包括：本发明涉及水杨酸甲酯在制备沙门氏菌Ⅲ型分泌系统抑制剂中的应用,以人宫颈癌细胞(HeLa)为感染载体,证实水杨酸甲酯通过抑制Ⅲ型分泌系统的功能,从而对沙门氏菌感染具有显著的抑制作用。相比传统抗生素在直接杀死沙门氏菌的作用机制,中药化合物水杨酸甲酯以T3SS为靶标的治疗策略可以解除病原细菌的武装,但不直接杀死它们,给予细菌的选择压力小,从而降低了产生耐药性的风险。此外,天然的水杨酸甲酯最初发现于自然界的柳树皮、白珠树叶及甜桦树中；之后实现了可以人工制备,具有来源广泛、成本低廉和治愈率高的特点。(The invention relates to an application of methyl salicylate in preparation of a salmonella III type secretion system inhibitor, which takes human cervical carcinoma cells (HeLa) as an infection carrier to prove that the methyl salicylate has a remarkable inhibiting effect on salmonella infection by inhibiting the function of a III type secretion system. Compared with the action mechanism of the traditional antibiotics for directly killing salmonella, the treatment strategy of the traditional Chinese medicine compound methyl salicylate taking T3SS as the target can relieve the armed state of pathogenic bacteria, but does not directly kill the pathogenic bacteria, and the selection pressure given to the bacteria is small, so that the risk of drug resistance is reduced. In addition, natural methyl salicylate was originally found in willow bark, beautyberry leaves and betula in nature; the preparation method realizes the artificial preparation, and has the characteristics of wide sources, low cost and high cure rate.)

1. Application of methyl salicylate in preparing salmonella III type secretion system inhibitor is provided.

2. The use of claim 1, wherein the methyl salicylate is for inhibiting a biological function of a salmonella type III secretion system.

3. The use of claim 1, wherein the methyl salicylate inhibits Salmonella type III secretory system function and reduces Salmonella virulence.

4. The use of claim 1, wherein the methyl salicylate is used in the manufacture of a medicament for the treatment of salmonella infection.

5. The use of claim 1, wherein the Salmonella infection is a human or animal infection caused by Salmonella, including typhoid fever, paratyphoid fever, gastroenteritis, pullorum disease, and the like.

Technical Field

The invention relates to an application of methyl salicylate in preparation of salmonella III type secretion system inhibitor, belonging to the technical field of medical pharmacy.

Background

Antibiotic-resistant bacteria are threatening global public health and are threatening our ability to treat common infectious diseases. With the decline in antibiotic efficacy, treatment of infections (such as pneumonia, tuberculosis, sepsis, gonorrhea and food-borne diseases) is becoming increasingly challenging. There is an urgent need for novel anti-infective therapies to prevent or treat bacterial pathogens, either alone or in combination with traditional antibiotics. The type III secretion system (T3SS) transports effector proteins into eukaryotic host cells to induce infection, which is critical for the virulence of certain bacterial pathogens. Therapeutic strategies targeting T3SS may disarm pathogenic bacteria, but do not kill them directly, thereby reducing the risk of developing drug resistance. Therefore, T3SS appears to be a new target for the development of drugs against bacterial infections.

Salmonellosis is one of zoonosis having important significance in public health, the pathogenic salmonella enteric bacteriaceae of the salmonellosis can infect various animals including human beings, the animals can be in an asymptomatic germ-carrying state after infection, and can also be shown as a lethal disease with clinical symptoms, the salmonella can aggravate morbidity or mortality, or reduce the reproductive productivity of the animals, thereby not only bringing heavy burden to public health, but also causing economic loss to society. The salmonella serotype salmonella typhimurium induces inflammatory diarrhea and invades non-phagocytic epithelial cells using T3SS encoded on salmonella pathogenetic island 1(SPI-1), the T3SS device is a needle-like structure that can inject bacterial effector proteins into host cells to establish infection, and the mechanism is widely distributed among gram-negative bacteria, so salmonella T3SS can be used as a new strategy for developing anti-virulence drugs.

Traditionally, anti-infective medicinal plants are used in treasury for drug discovery. Some flavonoids, such as baicalein and quercetin, inhibit invasion by blocking the T3SS effect and translocation of salmonella typhimurium to epithelial cells. Thymol, a natural monoterpene phenol derivative of cymene, inhibits the translocation of SipA from Salmonella typhi to HeLa cells. At present, no report that methyl salicylate resists salmonella infection by inhibiting the function of salmonella III type secretion system is seen at home and abroad.

Disclosure of Invention

The CAS number of the methyl salicylate is 119-36-8, and the molecular formula is C₈H₈O₃And the molecular weight is 152.15.

The chemical structural formula of methyl salicylate is as follows:

according to the research, the natural compound methyl salicylate obtained by screening by taking the salmonella T3SS effector protein as a drug target is a salmonella pathogenic island 1(SPI-1) inhibitor, and the inhibitor can block secretion of several SPI-1 related effector proteins without influencing bacterial growth, so that the inhibitor has a strong inhibition effect on SPI-1 mediated HeLa cell invasion. Further studies have shown that methyl salicylate significantly reduces transcription of certain SPI-1 genes, such as SipA, SipB and HilA. In animal infection models, methyl salicylate is effective in protecting mice from death and pathological damage caused by Salmonella typhimurium infection. In summary, this study proposed a potent SPI-1 inhibitor, salicylic acid, which reduces the expression of the SPI-1 effector protein by regulating transcription of major regulatory genes.

Drawings

FIG. 1: methyl salicylate inhibits translocation of effector protein SipA and thus inhibits function of T3SS (blue indicates normal transport function of Salmonella T3SS effector protein SipA; Salmonella viridans T3SS effector protein SipA cannot normally transport)

FIG. 2: methyl salicylate did not affect the growth of Salmonella typhimurium (time on the horizontal axis and OD on the vertical axis)_600nmMeasured value)

FIG. 3 shows that methyl salicylate is not cytotoxic to HeLa cells (the horizontal axis represents the concentration of methyl salicylate and the vertical axis represents the LDH release amount from HeLa cells)

FIG. 4: protective Effect of methyl Salicylate on Salmonella-mediated HeLa cell injury (lateral axis shows methyl Salicylate treatment groups at different concentrations and vertical axis shows LDH Release amount)

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the invention in any way, and any modifications or changes that can be easily made by a person skilled in the art without departing from the technical solution of the invention will fall within the scope of the claims of the invention.

Example 1

The methyl salicylate can be used as a salmonella T3SS inhibitor and used for preparing a medicament for treating salmonella infection, and can be used in any pharmaceutically acceptable carrier.

Example 2

Methyl salicylate is used as salmonella T3SS inhibitor for preparing medicine for treating infectious diseases.

Example 3

Methyl salicylate is used as salmonella T3SS inhibitor for treating infectious diseases caused by bacteria, especially human and animal infections caused by salmonella, including typhoid, paratyphoid, gastroenteritis, pullorum disease, etc.

1. Screening for inhibitors of the Salmonella type III secretory system

The research enables a target protein SipA to be expressed in a mode of fusing TEM by constructing a TEM reporter gene plasmid, the constructed plasmid is electrically transferred into salmonella typhimurium, a Hela cell is infected for 2h after a natural compound and the salmonella typhimurium are incubated together in advance, a CCF4-AM substrate is added, when the SipA protein fused with beta-lactamase enters a host cell, the substrate CCF2 is decomposed under the action of the beta-lactamase to interfere the FRET phenomenon, and emits 447nm blue light under the excitation of light with the wavelength of 409nm, if the natural compound inhibits the SipA protein from translocating to the host cell, the FRET phenomenon is generated under the excitation of the light with the wavelength of 409nm to emit 520nm green light, so that inhibitor screening is carried out through fluorescent signals with different colors under a laser confocal microscope.

And (4) conclusion: the blue color of a HeLa cell infected by beta-lactamase-SipA indicates that T3SS effector protein SipA can be normally transported into the cell, the normal function of the cell is lost after a key gene invA for transporting T3SS system effector protein is knocked out, the infected HeLa cell presents green color, and the blue color of the infected HeLa cell presents partial blue color after the methyl salicylate and the beta-lactamase-SipA are co-incubated, which indicates that the salicylic acid inhibits the translocation of salmonella T3SS effector protein SipA to influence the function of salmonella T3SS (see figure 1).

2. Effect of methyl Salicylate on Salmonella growth

Inoculating Salmonella to LB liquid medium (0.3M NaCl) for overnight culture (37 deg.C, 200rpm), and expanding culture at a ratio of 1:100 to OD the next day_600nmAbout 0.3, and the mixture is subpackaged into 5 conical flasks (20 ml/flask), and a drug-free group and methyl salicylate treatment groups with different concentrations are set. The culture was continued at 37 ℃ and 200rpm, and the OD of each sample was measured and recorded every 0.5h_600nmUntil the bacteria grow to plateau.

And (4) conclusion: in the culture time of 7h, compared with the group without the drug, the methyl salicylate treatment group (0-32 mu g/ml) has the bacteria growing to the plateau OD_600nmThere was no significant difference in the values, indicating that methyl salicylate did not affect normal bacterial growth in the effective concentration range (see figure 2).

Effect of methyl 3-Salicylate on Salmonella invasion of host cells

3.1 methyl Salicylate is not cytotoxic to host cells

The HeLa cell line was maintained in DMEM high-glucose medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin, and the cell concentration was adjusted to 2X 10 cells per ml⁴Individual cells, seeded in 96-well plates (2X 10)⁴One/well) was incubated overnight. Adding methyl salicylate, DMEM and 0.1% TritonX-100 with different concentrations in the next day, setting three repeats for each group, culturing at 37 deg.C for 8h, collecting cell culture supernatant, determining absorbance at 492nm according to the instructions of Lactate Dehydrogenase (LDH) kit, and calculating L of each groupThe DH release rate.

The formula is as follows: LDH release rate (%) ═ (test group-DMEM control group)/(0.1% TritonX group-DMEM control group)

And (4) conclusion: compared with a group without adding methyl salicylate, the methyl salicylate hardly has toxicity to HeLa cells within the concentration range of 2-64 mu g/ml (see figure 3).

3.2 protective Effect of methyl Salicylate on Salmonella-mediated HeLa cell injury

HeLa cells were suspended in DMEM supplemented with 10% fetal bovine serum in 96-well plates at 2X 10⁴Density culture overnight per well. Wild type strains SL1344 and Δ invA-SL1344 were diluted 20-fold in LB broth containing 0.3M NaCl, after pretreatment with methyl salicylate at various concentrations for 4h, MOI was 100 infected cells, each group was replicated three by three, after infection for 5h, cell culture supernatants were collected by centrifugation at 1000rpm for 10min in new 96-well plates, and the LDH release rate was calculated for each group by measuring the absorbance at 492nm according to the instructions of the Lactate Dehydrogenase (LDH) kit.

And (4) conclusion: LDH release in culture supernatants was an indicator of cell death, and LDH release was significantly reduced in the methyl salicylate-treated group at 16 μ g/ml compared to the control group infected with SL1344 but not treated with methyl salicylate, indicating that methyl salicylate was effective in alleviating salmonella-mediated HeLa cell damage (see fig. 4).