Method for improving vibrio splendidus infection resistance of stichopus japonicus

Abstract

The invention provides a method for improving vibrio splendidus infection resistance of stichopus japonicus, which is a method for preventing and treating stichopus japonicus diseases caused by vibrio splendidus by inhibiting pathogenesis related to vibrio splendidus outer membrane vesicles. The composition capable of improving the vibrio splendidus infection resistance of the stichopus japonicus comprises chlorpromazine and nystatin. The composition provided by the invention has no obvious toxicity to coelomic cells of the stichopus japonicus, and can inhibit endocytosis of OMVs to the maximum extent, so that the optimal prevention and control effect is realized. After the OMVs are treated by chlorpromazine and nystatin, the endocytosis efficiency of the OMVs is reduced by more than 50%.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture disease prevention and control technology, specifically relating to a method for improving the resistance of sea cucumbers to Vibrio splendidus infection. Background Technology

[0002] Sea cucumbers are an important marine aquaculture species in my country, possessing extremely high nutritional and economic value. However, in recent years, with the continuous expansion of sea cucumber farming scale and the widespread implementation of intensive farming models, farming density has been constantly increasing. Coupled with increasingly prominent problems such as water pollution and eutrophication in farming areas, the living environment of sea cucumbers has been severely damaged, leading to frequent outbreaks of various diseases. Among these, Vibrio brevicornu (… Vibrio splendidus The "skin rot syndrome" caused by septic tank rot has become one of the most serious diseases threatening the sea cucumber farming industry. "Skin rot syndrome" is characterized by its rapid onset, rapid spread, and high mortality rate. Infected sea cucumbers typically exhibit symptoms such as skin ulceration, skin peeling, and damage to internal organs. Once it breaks out in aquaculture ponds, it can cause large-scale sea cucumber deaths in a short period, resulting in devastating economic losses to the farming industry and severely hindering the healthy and sustainable development of my country's sea cucumber farming industry.

[0003] Studies have shown that the pathogenic mechanism of Vibrio splenium is closely related to its secreted outer membrane vesicles (OMVs). OMVs are nanoscale vesicle structures formed by bacteria during growth and reproduction through cell membrane budding and shedding, and are essentially important carriers for information exchange and material transfer between bacteria and host cells. The OMVs produced by Vibrio splenium encapsulate various pathogenic factors, among which a key virulence small RNA (sRNA063) plays a central role in the pathogenic process. When Vibrio splenium OMVs enter the coelomic cells of sea cucumbers, the sRNA063 they carry is released and directly binds specifically to the autophagy-related protein AjATG2 in the sea cucumber. This binding triggers a series of molecular reactions, accelerating the degradation of AjATG2 through the ubiquitin-proteasome system, leading to a decrease in AjATG2 protein levels, thereby disrupting the sea cucumber's autophagy defense function and promoting the survival and proliferation of the pathogen within the host cell.

[0004] Autophagy is an important innate immune mechanism for sea cucumbers to resist pathogenic microbial infection. AjATG2, as a key positive regulator of the autophagy pathway, is crucial for maintaining the sea cucumber's resistance to infection. sRNA063 disrupts the autophagy process through its interaction with AjATG2, and is one of the core mechanisms by which Vibrio splenium achieves immune evasion. Therefore, blocking the delivery of sRNA063 to sea cucumber coelomic cells is a key approach to preventing this disease.

[0005] Currently, disease control methods commonly used in my country's sea cucumber farming industry mainly rely on traditional chemical agents such as antibiotics and chemical disinfectants. While these agents can achieve certain bactericidal effects in the short term, long-term use leads to numerous serious problems. On the one hand, long-term overuse of antibiotics can cause pathogens such as Vibrio splenida to develop drug resistance, gradually reducing the effectiveness of the agents or even rendering them ineffective. On the other hand, chemical agents leave residues in the farming environment, polluting water and sediment, disrupting the balance of the farming ecosystem, and affecting the normal growth and development of sea cucumbers. Furthermore, drug residues also pose a potential threat to consumer health. Therefore, developing a novel, environmentally friendly, and side-effect-free disease control technology that targets the pathogenic mechanisms of pathogens is an urgent need for the sustainable development of the current sea cucumber farming industry and is the important background for the proposal of this invention. Summary of the Invention

[0006] The purpose of this invention is to provide a method for improving the resistance of sea cucumbers to Vibrio splendid infection. This method prevents and treats diseases caused by Vibrio splendid by inhibiting the pathogenic mechanism related to the outer membrane vesicles of Vibrio splendid.

[0007] The present invention first provides a product that can improve the resistance of sea cucumber to Vibrio scintillans infection, and the product can inhibit sea cucumber diseases caused by the outer membrane vesicles of Vibrio scintillans; The disease described, as a specific example, is skin rot syndrome; Furthermore, the product contains chlorpromazine (CPZ) and nystatin. In another aspect, the present invention also provides an article for improving the resistance of sea cucumber to Vibrio splenzi infection, wherein chlorpromazine (CPZ) and nystatin are used as core inhibitors.

[0008] Furthermore, the mass ratio of chlorpromazine (CPZ) to nystatin is 1:4-7.

[0009] Preferably, the mass ratio of chlorpromazine to nystatin is 1:5.

[0010] The product described herein contains a carrier acceptable to aquaculture animals; As described in the embodiments, the carrier is physiological saline, phosphate buffered saline (PBS), or seawater-adapted buffer. Furthermore, the article provided by the present invention may also contain other components for enhancing or maintaining the effects of chlorpromazine and nystatin.

[0011] The product composition provided by this invention has no significant toxicity to sea cucumber coelomic cells and can maximize the inhibition of OMV endocytosis, achieving the best control effect. After treatment with chlorpromazine and nystatin, the endocytosis efficiency of OMVs decreased by more than 50%. Attached Figure Description

[0012] Figure 1 : Inhibitory effects of different endocytosis pathway inhibitors on the entry of Vibrio splenti OMVs into sea cucumber coelomic cells; Figure 2 : The inhibitory effect of the composition inhibitor on the entry of Vibrio splendidiomycosis OMVs into the coelomic cells of sea cucumber; Figure 3 : The effect of the composition of the present invention on the mortality rate and skin rot of sea cucumber infected with Vibrio splenium; Figure 4 : Changes in the binding levels of sRNA063 and AjATG2 after treatment with the composition inhibitor; Figure 5 : Effect of composition inhibitor treatment on AjATG2 protein expression and autophagosome formation in coelomic cells; Figure 6 : Effect of composition inhibitor treatment on intracellular bacterial count. Detailed Implementation

[0013] The core idea of ​​this invention is to target the pathogenic pathway of Vibrio brilliance OMVs carrying sRNA063 into the coelomic cells of sea cucumbers. By inhibiting related endocytosis pathways and blocking the delivery of virulence factors, the autophagy defense function of sea cucumbers is maintained, thereby preventing "skin rot syndrome". The specific details are as follows: 1. Identification of the endocytic pathway of Vibrio brilliance OMVs entering the coelomic cells of sea cucumber To clarify the specific pathway by which Vibrio splenium OMVs enter sea cucumber coelomic cells, we treated sea cucumber coelomic cells with different endocytosis pathway inhibitors and quantitatively analyzed the endocytosis efficiency of OMVs using fluorescent labeling and flow cytometry. The results showed that the entry of Vibrio splenium OMVs into sea cucumber coelomic cells depends on multiple endocytosis pathways, with clathrin-dependent endocytosis and caverin-dependent endocytosis being the main pathways. To further verify the roles of these two pathways, we used the specific inhibitor chlorpromazine (CPZ) to inhibit the clathrin-dependent endocytosis pathway and nystatin to inhibit the caverin-dependent endocytosis pathway. The results showed that treatment with chlorpromazine and nystatin reduced the endocytosis efficiency of OMVs by more than 50%. This result fully demonstrates the crucial role of the clathrin-dependent and caverin-dependent endocytosis pathways in the entry of Vibrio splenium OMVs into sea cucumber coelomic cells, providing a clear target for subsequent screening of targeted inhibitors.

[0014] 2. Preparation of the control composition Core Component Screening and Concentration Optimization: Based on the identification results of the aforementioned endocytosis pathway, this invention screened chlorpromazine (CPZ) and nystatin as core inhibitors. Chlorpromazine is a classic clathrin-dependent endocytosis pathway inhibitor, which can inhibit OMV endocytosis by interfering with clathrin assembly and preventing the formation of endocytic vesicles. Nystatin specifically disrupts the lipid raft structure required for the caveolin-dependent endocytosis pathway, thereby blocking the entry of OMVs into cells mediated by this pathway. To determine the optimal concentrations of the two inhibitors, numerous concentration gradient experiments were conducted. By testing the toxicity of different concentrations of inhibitors to sea cucumber coelomic cells and their inhibitory effect on OMV endocytosis, the effective concentration of chlorpromazine was finally determined to be 10 μM, and the effective concentration of nystatin was determined to be 50 μM. This concentration combination not only showed no significant toxicity to sea cucumber coelomic cells but also maximized the inhibition of OMV endocytosis, achieving the best control effect.

[0015] Carrier Selection and Composition Preparation: To ensure the inhibitor remains stable in the aquaculture environment and effectively acts on the coelomic cells of sea cucumbers, this invention uses carriers acceptable for aquaculture to mix with the inhibitor to prepare the control composition. Selected carriers include physiological saline, phosphate-buffered saline (PBS), and seawater-adapted buffer. These carriers have good biocompatibility, will not adversely affect sea cucumbers or the aquaculture environment, and can provide a suitable dissolution environment for the inhibitor, ensuring its activity. During preparation, chlorpromazine powder and nystatin powder are first dissolved separately with the selected carriers to prepare high-concentration stock solutions (10 mM chlorpromazine stock solution and 50 mM nystatin stock solution), which are then stored at -20 ℃ in the dark to prevent inhibitor degradation and inactivation. Before use, according to actual aquaculture needs, the stock solutions are diluted with aquaculture seawater to the above-mentioned optimal working concentration. After thorough mixing, the control composition can be directly used in the aquaculture environment or for in vitro treatment of sea cucumbers.

[0016] 3. Implementation of prevention methods Scope of Application and Timing of Implementation: The prevention method of this invention is mainly applicable to the prevention of "skin rot syndrome" in sea cucumbers, especially during the peak disease season (water temperature 16-22 ℃), when it can achieve the best control effect. This is because within this water temperature range, Vibrio splenti multiplies rapidly, and the secretion of OMVs increases, significantly increasing the risk of sea cucumbers contracting "skin rot syndrome." Using the method of this invention at this time can effectively block the pathogenic process of the pathogen.

[0017] This invention employs an in vitro immersion method to treat sea cucumbers. This method is simple to operate, easy to scale up, and allows the inhibitor to act evenly on the sea cucumber's surface and fully contact the coelomic cells. Specific operating parameters are as follows: The prepared control composition is diluted in the aquaculture water. The immersion temperature is strictly controlled at 15-18℃, consistent with the sea cucumber aquaculture environment temperature, to avoid stress reactions caused by temperature changes. Each immersion lasts 24 hours, repeated every 4 days, with 2-3 consecutive treatments constituting a complete control cycle.

[0018] Control Mechanism and Effects: Through the above treatment, chlorpromazine and nystatin in the control composition can act on the clathrin-dependent endocytosis and caverin-dependent endocytosis pathways of sea cucumber coelomic cells, respectively, effectively blocking the entry of Vibrio spleniculitis OMVs into sea cucumber coelomic cells. This prevents the delivery of the virulence factor sRNA063 carried by OMVs into sea cucumber coelomic cells, thereby reducing the binding of sRNA063 to AjATG2 protein. The degradation of AjATG2 protein is inhibited, and its expression level is maintained within the normal range, ensuring the integrity and normal function of the sea cucumber autophagy pathway. Normal autophagy function can help sea cucumbers effectively clear invading Vibrio spleniculitis, significantly enhancing the sea cucumber's resistance to this pathogen and achieving the goal of preventing "skin rot syndrome".

[0019] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0020] Example 1: Preparation of the control composition 1. Preparation of experimental materials: chlorpromazine powder, nystatin powder, physiological saline, volumetric flask, pipette, refrigerator (-20 ℃).

[0021] 2. Preparation of mother liquor: Accurately weigh appropriate amounts of chlorpromazine and nystatin powder, add sterile physiological saline, stir thoroughly to dissolve, and prepare chlorpromazine mother liquor with a concentration of 10 mM and nystatin mother liquor with a concentration of 50 mM.

[0022] 3. Preservation of mother liquor: The two prepared mother liquors are placed into sterile reagent bottles, sealed, and stored at -20 ℃ in a light-protected environment for later use.

[0023] 4. Preparation of working solution: Before use, remove the stock solution from the refrigerator and allow it to thaw at room temperature. Based on the volume of the aquaculture water, dilute the stock solution to the working concentration using aquaculture seawater, following the requirements of a final concentration of 10 μM for chlorpromazine and 50 μM for nystatin. Stir continuously during dilution to ensure the inhibitor is evenly dispersed in the seawater, ultimately obtaining the control composition.

[0024] Example 2: Effect of Inhibitors on OMV Internalization 1. Experimental Materials and Grouping: Vibrio spleniculina OMVs were extracted and purified, and labeled with PKH67 fluorescent dye for later use. Cultured sea cucumbers were divided into two groups: experimental group (treated with a combination of chlorpromazine and nystatin inhibitors, with final concentrations of 10 μM and 50 μM, respectively) and control group (treated with an equal volume of physiological saline).

[0025] 2. OMVs endocytosis efficiency assay: PKH67-labeled Vibrio brilliance OMVs were injected into two groups of cultured sea cucumbers for 12 hours. Flow cytometry was used to detect intracellular fluorescence intensity, which directly reflects the endocytosis efficiency of the OMVs.

[0026] Depend on Figure 1 It is evident that the entry of Vibrio splenita OMVs into the coelomic cells of sea cucumbers relies on multiple endocytic pathways, with clathrin-dependent endocytosis and caveolin-dependent endocytosis being the primary pathways. Figure 2 It is evident that the fluorescence intensity of the experimental group cells was significantly lower than that of the control group, indicating that the combined use of chlorpromazine and nystatin can effectively block OMVs from entering the coelomic cells of sea cucumbers.

[0027] Example 3: In vitro immersion treatment experiment of sea cucumber 1. Selection of experimental subjects: Forty healthy sea cucumbers with uniform weight (100 ± 10 g) were selected and confirmed to be free from Vibrio splenita infection and other diseases after quarantine. The sea cucumbers were randomly divided into an experimental group and a control group, with 20 sea cucumbers in each group. They were placed in two 50 L culture tanks, with the water temperature controlled at 16 ℃ and the salinity at 28‰ to simulate the actual culture environment.

[0028] 2. Experimental Treatment: The prepared control composition was added to the aquaculture tanks of the experimental group and stirred thoroughly to ensure that the final concentrations of chlorpromazine and nystatin reached 10 μM and 50 μM, respectively. An equal volume of sterile saline was added to the aquaculture tanks of the control group as a blank control. Both groups underwent immersion treatment for 24 hours. After immersion, all seawater in both aquaculture tanks was replaced with fresh seawater. Thereafter, the treatment was repeated every 4 days for a total of 3 treatments, completing one control cycle.

[0029] 3. Infection Experiment and Result Observation: After the control period, *Vibrio splendidus* infection experiments were conducted on sea cucumbers in both the experimental and control groups. The pathogen was inoculated by immersion, and *Vibrio splendidus* was cultured to the logarithmic growth phase and diluted with sterile seawater to a concentration of 10. 7 CFU / mL. For 15 days post-infection, the health status, morbidity, and mortality of the two groups of sea cucumbers were observed and recorded daily, and the mortality rate was calculated.

[0030] Depend on Figure 3It is evident that the mortality rate of sea cucumbers in the experimental group was only 50% within 15 days of infection, while the mortality rate of sea cucumbers in the control group was as high as 90%. The onset of disease in the experimental group sea cucumbers was significantly delayed, and the symptoms of infected individuals were relatively mild; some infected sea cucumbers were even able to recover spontaneously. This result fully demonstrates that the control composition and prevention method of the present invention can significantly improve the resistance of sea cucumbers to Vibrio splenida infection and effectively prevent the occurrence of "skin rot syndrome".

[0031] Example 4: Effect of the inhibitor on sRNA063-AjATG2 interaction 1. Detection of sRNA063 and AjATG2 binding levels: The binding levels of sRNA063 and AjATG2 were detected using RNA immunoprecipitation (RIP). Cells from both groups were lysed, and AjATG2-specific antibodies were added for immunoprecipitation. After washing and elution of the precipitate complex, RNA was extracted. The content of sRNA063 in the precipitate complex was detected using real-time quantitative PCR to reflect the binding amount of sRNA063 and AjATG2.

[0032] Depend on Figure 4 It is evident that the content of sRNA063 in the experimental group was significantly lower than that in the control group, indicating that the inhibitor effectively inhibited the interaction between sRNA063 and AjATG2 by blocking the endocytosis of OMVs and reducing the amount of sRNA063 entering the coelomic cells of sea cucumber.

[0033] 2. Autophagy-related indicators and intracellular bacterial count: Western blotting was used to detect the AjATG2 protein and autophagosome count in both groups of cells. Simultaneously, intracellular bacterial counts were performed on both groups of cells to determine the number of viable Vibrio splendens bacteria.

[0034] Depend on Figure 5 It was evident that the expression level of AjATG2 protein in the experimental group was significantly higher than that in the control group; the number of autophagosomes in the experimental group was also significantly higher than that in the control group. Figure 6 It is evident that the number of Vibrio brilliance in the cells of the experimental group was significantly lower than that in the control group.

[0035] These results demonstrate that the inhibitor combination of the present invention can effectively maintain the autophagy function of sea cucumber coelomic cells and enhance the cells' ability to clear Vibrio splenita, thereby helping sea cucumbers resist pathogen infection.

Claims

1. A product capable of enhancing the resistance of sea cucumber to Vibrio splenida infection, characterized in that, The product described above can inhibit sea cucumber diseases caused by the outer membrane vesicles of Vibrio splenium.

2. The article of claim 1, characterized in that, The disease described is skin rot syndrome.

3. The article of claim 1, characterized in that, The product contains chlorpromazine and nystatin.

4. The article of claim 3, characterized in that, The mass ratio of chlorpromazine to nystatin is 1:4-7.

5. The article of claim 3, characterized in that, The mass ratio of chlorpromazine to nystatin is 1:

5.

6. The article of claim 3, characterized in that, The product contains a carrier acceptable to aquaculture animals.

7. The article of claim 6, characterized in that, The carrier is physiological saline, phosphate buffer, or seawater-adapted buffer.

8. The article of claim 6, characterized in that, The carrier also contains components that can enhance or maintain the efficacy of the composition against Vibrio splenti.

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