A method of 5-iodoindole inhibiting vibrio splendidus virulence factors

Abstract

The present application provides a method and application of 5-iodoindole inhibiting virulence factors of Vibrio splendidus, and it is found that different concentrations of 5-iodoindole can significantly reduce the expression of virulence factors such as hemolytic activity and protease activity of Vibrio splendidus under the premise of basically not affecting the growth of Vibrio splendidus. Through the method, the infection toxicity of Vibrio splendidus can be weakened, so as to reduce the infection and disease probability of host sea cucumber and alleviate the infection symptoms. Meanwhile, the present application also provides the application of 5-iodoindole in reducing the expression of Vibrio splendidus related virulence genes and the application of 5-iodoindole in improving the survival rate of host sea cucumber after Vibrio splendidus attack, which proves that 5-iodoindole with a concentration of 20 muM has guiding significance for actual sea cucumber production and culture. And through the research on virulence factors of Vibrio splendidus, the present application is helpful to reduce the infection and disease of sea cucumber caused by Vibrio splendidus.

Description

Technical Field

[0001] This invention belongs to the field of biological products, specifically relating to a method and application of 5-iodoindole to inhibit the virulence factor of Vibrio splenium. Background Technology

[0002] *Vibrio splendidus* is a Gram-negative, facultative anaerobic marine bacterium widely distributed in estuaries, coastal waters, and aquaculture environments. As an important member of the Vibrio family, its cells are short, curved, and arc-shaped, possessing unilateral flagella and motility. In aquaculture, *Vibrio splendidus* is significantly pathogenic to various commercially important marine fish species (such as yellow croaker, pomfret, and turbot), often spreading through water or the feed chain, causing acute infections. Simultaneously, this bacterium is a major pathogenic factor in sea cucumber skin rot syndrome, causing skin ulceration, internal organ damage, and large-scale mortality in the host, resulting in annual economic losses of nearly one million yuan for the sea cucumber aquaculture industry. Furthermore, contaminated seafood may become a source of human infection, as its secreted hemolysins and extracellular proteins pose a potential threat to food safety. Therefore, controlling *Vibrio splendidus* infection is of great significance for sea cucumber research and human health.

[0003] Bacteria breach host defenses and establish infection by secreting virulence factors. Virulence factors are a series of substances or mechanisms specific to pathogens (including bacteria, viruses, etc.) to enhance their pathogenicity, causing disease by damaging host tissues, suppressing immune responses, or interfering with cellular function. These factors are diverse, mainly including exotoxins and endotoxins, adhesion and invasion factors, and secretion systems. The virulence factors in *Vibrio splenida* mainly include hemolysins, proteases, biofilms, and fluidity. Hemolysins can directly destroy red blood cells, leading to anemia; extracellular proteases can decompose the tissue matrix, promoting diffusion; and fluidity helps bacteria migrate, enabling rapid spread.

[0004] Currently, antibiotics are the most commonly used antibacterial method in aquaculture. However, the extensive use of antibiotics has led to the emergence of drug-resistant bacteria, disrupting water balance and environmental stability. Furthermore, sea cucumber products treated with antibiotics often contain drug residues, affecting product quality and discouraging consumer purchases of sea cucumber-related products. The virulence factors of Vibrio splenida are crucial for its ability to infect hosts and enhance its competitive survival. Inhibiting these virulence factors reduces bacterial toxicity, thereby decreasing host invasion and increasing host survival rates. Compared to antibiotics, this method involves less environmental selection pressure and reduces the emergence of drug-resistant bacteria. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for inhibiting the virulence factor of Vibrio splenium with 5-iodoindole, thereby overcoming the shortcomings of the prior art.

[0006] This invention first provides an application of 5-iodoindole in inhibiting Vibrio virulence factors;

[0007] The present invention also provides another use of the 5-iodoindole, which is its use in the preparation of articles that inhibit Vibrio virulence;

[0008] As a specific example, the Vibrio species described is Vibrio Brilliantii.

[0009] The present invention also provides an article for inhibiting the virulence of Vibrio, the article containing a pharmacologically effective concentration of 5-iodoindole;

[0010] The preferred pharmacologically effective concentration is not less than 5 μM;

[0011] Furthermore, the pharmacologically effective concentration is not less than 10 μM;

[0012] Preferably, the pharmacologically effective concentration is 20 μM.

[0013] The present invention also provides a method for inhibiting the virulence factor of Vibrio splenium, which is to use 5-iodoindole to reduce the virulence of Vibrio splenium.

[0014] This invention provides a method for inhibiting virulence factors of Vibrio splenium using 5-iodine-indole. Vibrio splenium is inoculated twice into 2216E liquid medium at the same inoculation ratio and cultured for a certain period to obtain different concentrations of Vibrio splenium bacterial suspensions. These suspensions are then cultured in vitro with different concentrations of 5-iodine-indole. The results show that different concentrations of 5-iodine-indole can significantly reduce the expression of virulence factors such as hemolytic activity and protease activity in Vibrio splenium without significantly affecting its growth. This method can weaken the infectious virulence of Vibrio splenium, thereby reducing the probability of infection and disease in sea cucumbers and alleviating infection symptoms. Furthermore, this invention also provides the application of 5-iodine-indole in reducing the expression of Vibrio splenium-related virulence genes and in improving the survival rate of sea cucumbers after Vibrio splenium challenge, demonstrating that a 20 μM concentration of 5-iodine-indole has guiding significance for practical sea cucumber production and cultivation. Moreover, this invention's research on Vibrio splenium virulence factors helps reduce infection and disease in sea cucumbers caused by Vibrio splenium. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the method for inhibiting the virulence factor of Vibrio splenida with 5-iodoindole in an embodiment of the present invention.

[0016] Figure 2 : This is a diagram showing the effect of 5-iodoindole on the expression of the virulence factor Vsh gene of Vibrio brilliance in the embodiments of the present invention;

[0017] Figure 3: This is a diagram showing the effect of 5-iodoindole on the expression of the virulence factor Vsm gene of Vibrio brilliance in the embodiments of the present invention;

[0018] Figure 4 : This is a diagram showing the effect of 5-iodoindole on the expression of the virulence factor Flic gene of Vibrio brilliance in the embodiments of the present invention;

[0019] Figure 5 : This is a diagram showing the effect of 5-iodoindole on the growth of Vibrio brilliance in an embodiment of the present invention;

[0020] Figure 6 : This is a diagram showing the effect of 5-iodoindole on the motility of Vibrio brilliance in an embodiment of the present invention;

[0021] Figure 7 : This is a graph showing the effect of 5-iodoindole on the relative protease activity of Vibrio splenium in the embodiments of the present invention;

[0022] Figure 8 : This is a graph showing the effect of 5-iodoindole on the hemolytic activity of Vibrio splenti in the embodiments of the present invention;

[0023] Figure 9 Example 5 of this invention is a flowchart illustrating the specific operation of iodoindole in challenging Vibrio brilliance.

[0024] Figure 10 : This is a graph showing the effect of 5-iodoindole on the survival rate of sea cucumbers after being challenged with Vibrio brilliance in the embodiments of the present invention. Detailed Implementation

[0025] This invention provides a method for inhibiting the virulence factors of Vibrio splenaki, achieving high efficacy with low concentrations. It significantly inhibits the virulence expression of Vibrio splenaki without affecting bacterial growth and increases the protection rate of sea cucumbers by over 90%. Experiments using this method have shown that 20 μM 5-iodoindole can significantly reduce various indicators of Vibrio splenaki, including protease activity, motility, and hemolytic activity. This invention combines in vitro inhibition of Vibrio splenaki virulence factors with animal experiments, providing a detailed description of the significant effects of 5-iodoindole on the virulence expression of Vibrio splenaki and its infection process. This offers valuable insights for reducing Vibrio splenaki infection and disease incidence, and for the development of the aquaculture industry.

[0026] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0027] Example 1: Verification of the effect of 5-iodoindole on inhibiting the virulence factor of Vibrio splenita.

[0028] See Figure 1 This embodiment provides a method for inhibiting the virulence factor of Vibrio splenida with 5-iodoindole. The Vibrio splenida strain used is the standard strain (CGMCC1.1586). The method includes the following steps:

[0029] 1. Co-culturing 5-iodoindole with Vibrio splendidus

[0030] (1) Prepare 2216E liquid culture medium

[0031] Weigh 5g of Tryptone powder and 1g of Yeast extract powder, stir and dissolve them in 1000mL of filtered seawater, and autoclave at 121℃ for 20min.

[0032] (2) Co-culture of 5-iodoindole and Vibrio splenium

[0033] 1) Take out Vibrio Brilliantus glycerol culture from a -80℃ freezer and inoculate it into 2216E liquid medium (culture: 2216E medium = 1:1000). Incubate at 180 rpm for 12 h in a constant temperature shaker at 28℃ to obtain the first bacterial solution.

[0034] 2) Inoculate the first bacterial culture into a new 2216E liquid medium (first bacterial culture: 2216E medium = 1:1000) and incubate it in a constant temperature shaker at 28℃ and 180 rpm for 12 h to obtain the second bacterial culture.

[0035] 3) Dispense the second bacterial solution into prepared sterile centrifuge tubes, and add pre-diluted 5-iodine indole solution to each centrifuge tube to make the final concentration of 5-iodine indole 0, 5, 10, 20, 40, 50, 80, 100 μmol / L, so as to obtain a third bacterial solution containing different concentrations of 5-iodine indole.

[0036] 2. Effects of 5-iodoindole on the growth of Vibrio splendidus

[0037] After mixing the third bacterial suspension at concentrations of 0, 5, 10, 20, 40, 80, and 100 μmol / L, 200 μL of each suspension was transferred to a 96-well plate. A negative control group, a *Vibrio splendidus* group, and a 5-iodoindole + *Vibrio splendidus* group were also set up, with three replicates for each sample. The plates were incubated at 28°C in a shaker at 180 rpm, and the absorbance (OD600) was measured every 1 hour using a microplate reader.

[0038] 3. Effects of 5-Iodoindole on the expression of virulence genes in Vibrio splenium

[0039] (1) Primer design

[0040] Primers used for detecting the virulence gene expression of Vibrio splenium were designed and synthesized based on the whole genome sequence of Vibrio splenium CGMCC1.1586 and the virulence factor gene sequences in the VFDB database, as shown in Table 1.

[0041] Table 1 Primer list for Vibrio brilliance gene virulence.

[0042]

[0043] (2) RNA extraction

[0044] The second bacterial culture was incubated at 28°C and 180 rpm in a shaker until OD600 = 0.3-0.4. Different concentrations of 5-iodine-indole solution were then added to achieve working concentrations of 10, 50, and 100 μM. A control group was also included. The cultures were incubated again at 28°C for 1 hour. The bacterial culture was centrifuged at 6000×g for 10 minutes to collect the bacterial cell pellet. The pellet was washed three times with PBS and centrifuged again at the same speed for 3 minutes. Trizol solution was added, and the mixture was mixed thoroughly and lysed at -80°C for 12 hours. The sample was thawed on ice and centrifuged at 12000×g at 4°C for 5 minutes. The supernatant was transferred to a new centrifuge tube, and 1 / 5 of the supernatant volume of chloroform solution was added to exchange the RNA lysed in Trizol into the chloroform solution. After inverting and mixing, the mixture was allowed to stand for 5 minutes. Centrifuged at 12000×g, 4℃ for 15 minutes, resulting in layering. The supernatant in the upper layer was a chloroform solution containing dissolved RNA. The supernatant was transferred to new centrifuge tubes, and an equal volume of isopropanol was added to precipitate the nucleic acid. After inverting and mixing, the mixture was allowed to stand on ice for 15 minutes. Subsequently, the mixture was centrifuged at 12000×g, 4℃ for 10 minutes, and the supernatant was discarded. 1 mL of 75% ethanol solution was added to the precipitate and mixed well. Finally, the mixture was centrifuged at 12000×g, 4℃ for 5 minutes, and the supernatant was discarded. DEPC-treated water was added to dissolve the RNA. The mixture was repeatedly tapped with a pipette to fully dissolve the RNA, and the RNA concentration was measured.

[0045] (3) RNA reverse transcription

[0046] 1) DNAase (without RNAase) treatment

[0047] The configuration system is as follows:

[0048]

[0049] The PCR instrument was set to 42℃ for 2 minutes and then stored at 4℃.

[0050] 2) Configure the reverse transcription reaction system

[0051]

[0052]

[0053] 3) Reverse transcription reaction

[0054]

[0055] (3) mRNA expression level detection

[0056] 1) The cDNA obtained in (2) was subjected to mRNA detection. The reaction system is as follows:

[0057]

[0058] 2) qPCR reaction

[0059]

[0060] 4. Effect of 5-iodoindole on the motility of Vibrio splenium

[0061] Take 5 μL of Vibrio Brilliantii bacterial suspension containing different concentrations of 5-iodoindole and drop it onto 2216E solid medium with an agar concentration of 0.2%, keeping it horizontal and not deviating. Use sealing film to wrap the petri dishes to avoid contamination and incubate them in a constant temperature shaker at 28℃ and 180 rpm for 48 h. Perform 6 replicates for each group and observe and record the motility diameter of Vibrio Brilliantii colonies.

[0062] 5. Effect of 5-Iodoindole on Vibrio splenium protease activity

[0063] Different concentrations of 5-iodoindole were added to *Vibrio splenicus* bacterial suspensions and incubated at 28°C with a shaker at 180 rpm. Samples were collected at 6, 12, 24, and 48 h and transferred to 96-well plates. Each group was repeated three times, and a blank control was included. The OD600 absorbance was measured using a microplate reader. The samples were then centrifuged at 10,000 rpm at 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter to obtain sterile supernatant. 50 μL of the sterile supernatant was added to 450 μL of azocasein solution and incubated at 37°C for 2 h. 500 μL of 10% trichloroacetic acid was added to the reaction mixture, and the mixture was incubated at 4°C for 15 min. After centrifugation at 5000 rpm for 5 min, the supernatant was collected and transferred to 96-well plates. The OD350 was measured using a microplate reader. Each group was repeated three times. The relative protease activity of *Vibrio splenicus* was expressed as OD350 / OD600.

[0064] 6. Effect of 5-Iodoindole on the hemolytic activity of Vibrio splenium

[0065] Different concentrations of 5-iodoindole were added to *Vibrio splenium* cultured in 2216E medium and incubated at 28°C with a shaker at 180 rpm until OD600 = 1.0. The resulting samples were then transferred to 96-well plates, and the absorbance at OD600 was measured using a microplate reader. A 1% sheep blood erythrocyte suspension was then added. The plates were incubated at 28°C for 5 hours, gently mixed, and then transferred to 96 wells to measure the absorbance at OD450. The hemolytic activity of *Vibrio splenium* was expressed as OD450 / OD600.

[0066] qPCR results showed that... Figures 2-4 As shown, 10, 50, and 100 μM 5-iodoindole all reduced the expression of Vibrio splenium-related virulence genes Vsh, Vsm, and Flic (p < 0.05). These results demonstrate that only a small amount of 5-iodoindole is needed to exert an inhibitory effect on the virulence of Vibrio splenium.

[0067] Depend on Figure 5 It is evident that concentrations of 5-iodoindole at 5, 10, 20, 40, and 80 μM have virtually no effect on the growth of Vibrio splenium, meaning that 5-iodoindole can exert virulence-inhibiting capabilities without affecting the growth of Vibrio splenium.

[0068] Depend on Figure 6 It is evident that 10, 20, and 50 μM 5-iodoindole can all significantly inhibit the motility of Vibrio splentospira, meaning that 5-iodoindole can effectively inhibit the motility and spread of Vibrio splentospira.

[0069] Depend on Figure 7 It can be seen that 10, 50, and 100 μM 5-iodoindole can significantly inhibit the relative protease activity of Vibrio splenium.

[0070] Depend on Figure 8 It can be seen that 10, 50, 80 and 100 μM of 5-iodoindole can reduce the hemolytic activity of Vibrio splenium, and the inhibitory effect is more significant as the concentration of 5-iodoindole increases.

[0071] Example 2: Detection of the effect of 5-iodoindole on reducing the virulence of Vibrio splenita infection in sea cucumber.

[0072] The sea cucumbers used in this embodiment were purchased from a sea cucumber breeding base in Dalian, Liaoning Province. The sea cucumbers were uniformly 5-10cm in length. They were temporarily raised for 2 days before the experiment. During this period, poorly performing and dead sea cucumbers were screened out. After the overall condition stabilized, they were divided into groups: Vibrio splendidus group, 5-iodoindole group, 5-iodoindole + Vibrio splendidus group, and control group. Each group had 3 replicates, and each replicate contained 30 sea cucumbers. The concentration of 5-iodoindole was 20μM.

[0073] The revived Vibrio brevicornuate culture was mixed with 2216E liquid medium at a ratio of 1:1000 and incubated in a shaker at 28°C until OD600 = 1.0 (CFU = 1 × 10⁻⁶). 9 Add 1% (by volume) of Vibrio brilliance bacterial suspension (after centrifugation) to the aquaculture water to achieve a challenge concentration of 1×10⁻⁶. 7 CFU / mL, while 20 μM of 5-iodoindole was added to the experimental group.

[0074] During the experiment, it is important to keep all conditions consistent except for the experimental variables. Disinfect and clean the breeding environment with chlorine dioxide in advance, introduce sufficient oxygen, control the breeding temperature at around 16℃, maintain semi-dark lighting conditions, observe twice a day, and promptly remove feces and dead individuals, and record data.

[0075] Depend on Figure 9 , 10 It is evident that 20 μM 5-iodoindole has a significant protective effect against sea cucumbers infected with Vibrio splenium, and can significantly reduce the virulence of Vibrio splenium infection and the incidence of disease in sea cucumbers.

[0076] In summary, this invention provides a method for inhibiting the virulence factors of Vibrio splenium with 5-iodoindole. By co-culturing 5-iodoindole with Vibrio splenium, the expression of virulence factors of Vibrio splenium can be effectively inhibited. This method weakens the infectivity of Vibrio splenium, thereby reducing the infection and disease incidence of Vibrio splenium in sea cucumber farming and alleviating infection symptoms.

[0077] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1,5-Iodoindole One application, characterized in that, The stated use is in the preparation of products that inhibit the virulence of Vibrio brilliance.

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