Method for suppressing or promoting bacterial infection in fish, and composition therefor

Abstract

This method for suppressing bacterial infection in fish involves administering omcin5 protein to fish, and this method for promoting bacterial infection in fish involves suppressing expression of omcin5 protein in fish.

Description

Method for suppressing or promoting bacterial infection in fish and composition therefor

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

[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, immunoglobulin, major histocompatibility complex (MHC), recombination activating gene, Toll-like receptor (TLR), etc. There are possibilities that fish are infected by a plurality of 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 elucidating the mechanism of immunity against bacterial infection conserved among vertebrates, but the knowledge obtained from these studies is also applicable to other fields such as infection control in laboratories and fish farms (Non-Patent Document 1).

[0004] Matthews JL. Methods Cell Biol. 2004;77:617 - 643. doi:10.1016 / s0091 - 679x(04)77033 - 8

[0005] Molecules such as antibiotics for treating or preventing bacterial infection in fish have been known so far, but there is still a demand for new substances that improve the defensive ability against bacterial infection. Also, in the research field, it is required to be able to easily suppress or promote bacterial infection in fish.

[0006] The present invention has been made in view of the above problems, and an object thereof is to provide a method and a composition capable of easily suppressing or promoting bacterial infection in fish.

[0007] To achieve the above object, as a result of intensive studies, the present inventor newly found factors related to bacterial infection in fish, and as a result, 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 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 includes 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 provides a composition for suppressing bacterial infections in fish, characterized by comprising 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.

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

[0019] 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. This graph shows the results of measuring the expression level of Omcin5 when reared in the presence or absence of E. coli in the examples. This graph shows the results of measuring the survival rate of zebrafish embryos with Omcin5 knocked out when reared under normal culture conditions (solid line) or sterile conditions (dotted line) in the examples. This graph shows the results of measuring the survival rate of wild-type zebrafish embryos when reared under normal culture conditions (dotted line) or sterile conditions (solid line) in the examples. This graph shows 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 E. coli. This is a photograph showing the condition of wild-type or Omcin5 knocked out zebrafish when reared in a tank without a water circulation system.

[0020] The following describes embodiments for carrying out the present invention. The following description of preferred embodiments is essentially illustrative and is not intended to limit the present invention, its application methods, or its uses.

[0021] The present invention utilizes 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 it 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. Yet another embodiment is a method for promoting bacterial infection in fish by suppressing the expression of the omcin5 protein in fish. Yet another embodiment is a composition containing the omcin5 protein for suppressing bacterial infection in fish.

[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 several 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. Of these, 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 omcin5 protein is not particularly limited as long as it is a composition that can be administered to fish, but for example, it is in the form of a solution in which omcin5 protein is dissolved. In addition to 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 omcin5 protein to the fish, but for example, it can 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 methods 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.

[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 then 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 added E. coli (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 5 dpf zebrafish embryos cultured in or without E. coli. Specifically, total RNA was isolated from 5 dpf 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 primer / probe mixture for omcin5 or actb1 (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]

[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 test, p = 0.029). These findings suggest that omcin5 is associated with E. coli infection.

[0037] To further investigate the function of omcin5, an omcin5 KO (omc5- / -) zebrafish strain was obtained 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 aseptic 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 omcin5 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 in age from six months to one year, were reared in a tank equipped with a water circulation system. They were then transferred to a tank without a water circulation system. Figure 6 shows the fish after two days of incubation in such a tank.

[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 omcin5 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 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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