Methods for suppressing or promoting bacterial infection in fish, and compositions therefor.

Abstract

To facilitate the suppression or promotion of bacterial infections in fish. [Solution] A method for suppressing bacterial infection in fish includes administering the omcin5 protein to the fish, and a method for promoting bacterial infection in fish includes suppressing the expression of the omcin5 protein in the fish.

Description

Technical Field

[0001] The present invention relates to a method for suppressing or promoting bacterial infection in fish, and a composition therefor.

Background Art

[0002] Fish utilize innate immunity and acquired immunity for defense against pathogens, and the molecules utilized for defense are common between fish and mammals. Such molecules are known, for example, immunoglobulins, major histocompatibility complex (MHC), recombination activating genes, Toll-like receptors (TLRs), etc. There are possibilities that fish are infected with multiple bacteria such as Escherichia coli, Shigella, Aeromonas, Salmonella, Mycobacterium, etc.

[0003] Among fish, zebrafish are often used by researchers as an infection model. Not only to elucidate the mechanism of immunity against bacterial infection conserved among vertebrates, but the knowledge obtained from these studies can also be applied to other fields such as infection control in laboratories and fish farms (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] While antibiotics and other molecules for treating or preventing bacterial infections in fish are already known, there is still a need for new substances that can improve the fish's defense against bacterial infections. Furthermore, researchers are seeking ways to easily suppress or promote bacterial infections in fish.

[0006] The present invention has been made in view of the above-mentioned problems, and its object is to provide a method and composition that can easily suppress or promote bacterial infection in fish. [Means for solving the problem]

[0007] To achieve the aforementioned objective, the inventors, through diligent research, have discovered new factors involved in bacterial infections in fish, and as a result, have completed the present invention.

[0008] Specifically, a method for suppressing bacterial infection in fish according to one embodiment of the present invention is characterized by comprising administering the omcin5 protein to fish.

[0009] According to one embodiment of the present invention, a method for suppressing bacterial infection in fish involves administering omcin5 protein, which has been discovered by the inventors as a novel substance that suppresses bacterial infection, and thus an inhibitory effect on bacterial infection in fish can be obtained.

[0010] Furthermore, a method for suppressing bacterial infection in fish according to another embodiment of the present invention is characterized by including the promotion of omcin5 protein expression in fish.

[0011] Another embodiment of the present invention provides a method for suppressing bacterial infections in fish, which includes promoting the expression of the omcin5 protein, a novel substance discovered by the inventors to suppress bacterial infections, and thus an inhibitory effect on bacterial infections in fish can be obtained.

[0012] Another embodiment of the present invention provides a method for promoting bacterial infection in fish, characterized by comprising suppressing the expression of the omcin5 protein in fish.

[0013] Another embodiment of the present invention provides a method for promoting bacterial infection in fish, which involves suppressing the expression of the omcin5 protein, a novel substance discovered by the inventors to suppress bacterial infection, and thus can achieve a promotion effect on bacterial infection in fish.

[0014] In the method according to the above embodiment, the fish may be a zebrafish. Also, the bacteria may be Escherichia coli.

[0015] Another embodiment of the present invention, a composition for suppressing bacterial infection in fish, is characterized by containing the omcin5 protein.

[0016] Another embodiment of the present invention provides a composition for suppressing bacterial infections in fish, which contains the omcin5 protein, a novel substance discovered by the inventors to suppress bacterial infections, and therefore can be used to achieve an inhibitory effect on bacterial infections in fish.

[0017] In a composition for suppressing bacterial infection in fish according to another embodiment of the present invention, the fish may be a zebrafish. The bacteria may also be Escherichia coli. [Effects of the Invention]

[0018] A method for suppressing or promoting bacterial infection in fish, and a composition therefor, can easily suppress or promote bacterial infection in fish. [Brief explanation of the drawing]

[0019] [Figure 1] This graph shows the results of measuring the survival rate of zebrafish embryos when reared in the presence (dotted line) or absence (solid line) of E. coli in the examples. [Figure 2] This graph shows the results of measuring the expression level of Omcin5 when zebrafish embryos were reared in the presence or absence of E. coli in the examples. [Figure 3]This is a graph showing the results of measuring the survival rate of zebrafish embryos with Omcin5 knocked out when reared under normal culture conditions (solid line) or under sterile conditions (dotted line) in an example. [Figure 4] This is a graph showing the results of measuring the survival rate of wild-type zebrafish embryos when reared under normal culture conditions (dotted line) or under sterile conditions (solid line) in an example. [Figure 5] This is a graph showing the results of measuring the survival rate of wild-type (solid line) or Omcin5-knocked-out (dotted line) zebrafish embryos when reared in the presence of Escherichia coli. [Figure 6] This is a photograph showing the state of wild-type or Omcin5-knocked-out zebrafish when reared in an aquarium without a water circulation system.

Modes for Carrying Out the Invention

[0020] Hereinafter, modes for carrying out the present invention will be described. The following description of the preferred embodiments is merely illustrative in nature and is not intended to limit the present invention, its application method, or its use.

[0021] The present invention uses the omcin5 protein to suppress or promote bacterial infection in fish. Specifically, one embodiment of the present invention is a method for suppressing bacterial infection in fish by administering the omcin5 protein or a composition containing the same to fish. Another embodiment of the present invention is a method for suppressing bacterial infection in fish by promoting the expression of the omcin5 protein in fish. Still another embodiment is a method for promoting bacterial infection in fish by suppressing the expression of the omcin5 protein in fish. Furthermore, another embodiment is a composition for suppressing bacterial infection in fish containing the omcin5 protein.

[0022] As will be explained later in the examples, omcin5 was found to be a factor involved in bacterial infection through the diligent research of the inventors. Omcin5 is a gene identified in zebrafish, registered as zgc153932 in a well-known gene database. The omcin5 protein, as a gene product, consists of 1121 amino acids. The nucleotide sequence of omcin5 is as shown in Sequence ID No. 1 below, and the amino acid sequence of its gene product, the omcin5 protein, is as shown in Sequence ID No. 2 below.

[0023]

[0024]

[0025] In the zebrafish genome, zgc153932 has multiple similar genes in a region of approximately 400KB on chromosome 12, which the inventors have named omcin1 to omcin15. These genes are presumed to constitute a gene family with high sequence similarity to one another. Among them, the omcin5 protein has amino acid sequence similarity to the human uromodulin and GP2 proteins, which are thought to control bacterial infections in the urinary tract and intestinal tract, and has five D10C domains, an EGF IV domain, and a Zona Pellucida (ZP) module.

[0026] In each of the above embodiments, the term "fish" is not particularly limited as long as it is generally classified as a fish, but for example, it is a zebrafish.

[0027] In each of the above embodiments, the term "bacteria" is not particularly limited as long as it is generally classified as a bacterium, but for example, it refers to Escherichia coli.

[0028] In each of the above embodiments, the composition containing the omcin5 protein is not particularly limited as long as it is in a form that can be administered to fish, but for example, it is in the form of a solution in which the omcin5 protein is dissolved. In addition to the omcin5 protein, the composition may also contain various commonly used additives such as excipients, suspending agents, emulsifiers, preservatives, pH adjusters, and flavorings. Furthermore, the method of administering the composition to fish is not particularly limited as long as it can administer the omcin5 protein to the fish, but for example, it may be done by adding the composition to the water in which the target fish are raised.

[0029] In each of the above embodiments, methods for promoting or suppressing the expression of the omcin5 protein in fish can be those commonly used in the field of genetic engineering. For example, the expression of the omcin5 protein can be promoted or suppressed by performing a well-known knock-in or knock-out of the omcin5 gene, which is represented by the nucleotide sequence of SEQ ID NO: 1. [Examples]

[0030] The following are examples illustrating in detail the method for suppressing or promoting bacterial infection in fish according to the present invention.

[0031] First, we investigated the effects of E. coli infection on zebrafish. To this end, we cultured several zebrafish embryos from 1 dpf (days post-fertilization) to 7 dpf in culture dishes with or without E. coli (NIHJ JC-2 strain) added. E. coli was cultured in LB medium at 37°C in a shaker and added to 20 mL of egg water so that the OD600 of the LB medium containing E. coli was 0.03. For the control without E. coli, we used the same amount of LB medium without E. coli added to egg water. The embryos were cultured in the respective egg waters at 28°C, and the number of viable embryos was counted daily to calculate the percentage of viable embryos. Embryos were considered dead if no heartbeat was observed.

[0032] As a result, as shown in Figure 1, 100% of zebrafish embryos survived up to 6 dpf in a solution without E. coli (solid line in Figure 1). On the other hand, in a solution with E. coli added (dotted line in Figure 1), some embryos died before 6 dpf, and the survival period was shorter compared to the case without E. coli (Log-rank test, p<0.001).

[0033] Next, we investigated the changes in omcin5 expression levels upon exposure of zebrafish embryos to E. coli. To this end, we performed ddPCR (Droplet digital PCR) using RNA extracted from 5pdf zebrafish embryos cultured in or without E. coli. Specifically, total RNA was isolated from 5dpf zebrafish embryos using Nucleospin RNA XS (Macherey-Nagel) and reverse transcribed using the PrimeScript RT reagent kit (Takara) according to the manufacturer's instructions. The assay was performed using the Bio-Rad QX200 Droplet Digital System (Bio-Rad Laboratories). 1 μL of cDNA sample, 1 μL of omcin5 or actb1 primer / probe mixture (designed and synthesized by Bio-Rad), 10 μL of 2×ddPCR supermix for the probe, and 8 μL of RNase-free water were mixed with 20 μL of reaction mixture. The sequences of the omcin5 and actb1 primers and probes used are as follows.

[0034] [Table 1]

[0035] After the above mixing, droplets were generated using a QX200 droplet generator (Bio-Rad). The PCR cycle conditions were as follows: denaturation at 95°C for 10 minutes, denaturation at 94°C for 15 seconds, annealing and extension for 1 minute for 40 cycles, and the final step at 98°C for 10 minutes. The annealing temperature was 52°C for omcin5 and 54°C for actb1. The droplets were read with a droplet reader and analyzed using QuantaSoft (Bio-Rad Laboratories). The results are shown in Figure 2. Note that in the results, the omcin5 transcript was normalized with the actin (actb1) transcript.

[0036] As shown in Figure 2, the expression level of omcin5 (omcin5 / actin) was significantly elevated in the group exposed to E. coli compared to the control group not exposed to E. coli (Mann-Whitney U test, p=0.029). These findings suggest that omcin5 is associated with E. coli infection.

[0037] To further investigate the function of omcin5, we obtained an omcin5KO (omc5- / -) zebrafish strain from the Zebrafish International Resource Center (ZIRC). omc5- / - embryos and wild-type embryos were cultured under normal and sterile conditions, and their survival periods were examined. Under normal conditions, embryos were cultured in egg water. For the sterile condition, egg water was treated with 100 μg / ml ampicillin, 5 μg / ml kanamycin, and 250 ng / ml amphotericin for 4 hours, then further treated with 0.003% NaHCl for 20 minutes, and finally filtered through a 0.2 μm pore filter. The results are shown in Figures 3 and 4.

[0038] As shown in Figure 3, in the case of omc5- / - embryos, under normal culture conditions (solid line in Figure 3), death began after 7 dpf. When the embryos were sterilized at 1 dpf (dotted line in Figure 3), the survival period was extended (Log-rank, p<0.01). On the other hand, as shown in Figure 4, no effect of sterilization was observed in wild-type embryos (the solid and dotted lines overlap in Figure 4). These results suggest that the decrease in the survival period of omc5- / - embryos is due to bacteria present in the culture egg water. The bacterial species present in the culture egg water are likely to fluctuate, and the bacterial composition is expected to be unstable.

[0039] Next, to investigate the susceptibility of omc5- / - embryos to a single bacterial species, E. coli strain MG1655 expressing type I fimbria was added to the egg water after the above sterile procedure, and omc5- / - embryos or wild-type embryos were cultured in this egg water. The E. coli was cultured in LB medium at 37°C in a shaker, and 20 mL of the LB medium containing the E. coli was added to the egg water so that the OD600 was 0.0015. The embryos were cultured in the respective egg waters at 28°C, and the number of viable embryos was counted daily, and the percentage of viable embryos was calculated. If no heartbeat was observed, the embryo was considered dead.

[0040] As a result, as shown in Figure 5, the survival period of omc5- / - embryos (dotted line in Figure 5) was shorter compared to wild-type embryos (solid line in Figure 5) (Log-rank test, p<0.01), suggesting that omc5 confers resistance to bacteria, particularly Escherichia coli with type I fimbria, to the embryos.

[0041] Next, to analyze the bacterial susceptibility of adult fish, five male and five female zebrafish, ranging from 6 months to 1 year in the wild type and OMC5- / - group, were reared in a tank equipped with a water circulation system, and then transferred to a tank without a water circulation system. Figure 6 shows the fish after being cultured in such a tank for two days.

[0042] As shown in Figure 6, the wild-type fish showed no signs of disease, whereas, in contrast, all of the omc5- / - fish died within two days.

[0043] As described above, embryos and adult fish with omc5- / - showed reduced resistance to bacteria. Therefore, it can be said that omc5 is necessary for resistance to bacterial infections in fish.

Claims

1. A method for suppressing bacterial infections in fish, comprising administering omcin5 protein to fish.

2. A method for suppressing bacterial infection in fish, comprising promoting the expression of the omcin5 protein in fish.

3. A method for promoting bacterial infection in fish, comprising suppressing the expression of the omcin5 protein in fish.

4. The method according to any one of claims 1 to 3, wherein the fish is a zebrafish.

5. The method according to any one of claims 1 to 3, wherein the bacterium is Escherichia coli.

6. A composition containing the omcin5 protein for suppressing bacterial infections in fish.

7. The composition according to claim 6, wherein the fish is a zebrafish.

8. The composition according to claim 6 or 7, wherein the bacterium is Escherichia coli.

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