Construction method and application of drosophila model for researching systemic infection of candida auricula

Abstract

The invention provides a construction method of a drosophila model for researching systemic infection of Candida auricula, and the method adopts wild type drosophila oreR as a basic model to construct an ideal animal model which is very suitable for researching the pathogenic mechanism of Candida auricula and the immune escape mechanism of Candida auricula in a host.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a fruit fly model for studying systemic infection of Candida auris and an application thereof. Background Art

[0002] Candida auris is an opportunistic fungal pathogen that has been globally recognized as a serious health threat due to its widespread innate and acquired resistance to antifungal drugs and its widespread spread throughout the world. In hospital settings, C. auris can colonize both biological and non-biological surfaces, which increases the risk of infection in immunocompromised people and often leads to severe systemic infections. As one of the most widely used model organisms in scientific research, Drosophila has been commonly used to study host-pathogen interactions, which is attributed to the conservation of Drosophila and mammals in evolution. Therefore, Drosophila can be used to study the pathogenic mechanisms of C. auris.

[0003] The existing fruit fly model of Candida auris infection uses the Toll immune-deficient fruit fly [1], which does not have a complete immune system, and therefore cannot clearly understand how the host immune system responds to pathogenic fungal invasion. Studies have shown that the adaptive immune response has a limited effect on the host's defense against Candida auris infection [2,3]. At the same time, in the systemic infection animal model of mice, Candida auris can persist in the mouse host and cannot be effectively recognized and eliminated by the host's innate immune system [4]. Therefore, wild-type fruit flies that have no acquired immunity and rely only on innate immune responses to defend against pathogens are suitable models for studying Candida auris infection. Summary of the invention

[0004] The present invention develops a Drosophila animal model for studying systemic infection with Candida auris and evaluates its feasibility and reproducibility, in order to provide a simple and cost-effective alternative model for studying the potential pathogenic mechanisms of Candida auris.

[0005] In a first aspect of the present invention, a method for constructing a Drosophila model for studying systemic infection with Candida auris is provided, the method comprising the following steps:

[0006] (1) providing a fruit fly to be infected, wherein the fruit fly is a wild-type fruit fly oreR;

[0007] (2) Infecting the fruit flies to be infected with Candida auris, with the infection amount being 100-10,000 Candida auris per fruit fly.

[0008] In another preferred embodiment, the fruit fly model is used for:

[0009] (a) studying the pathogenicity of Candida auris; and / or

[0010] (b) To study the immune escape mechanism of Candida auris in the host.

[0011] In another preferred embodiment, the fruit flies to be infected are adult fruit flies.

[0012] In another preferred embodiment, the fruit flies to be infected are raised in an environment of 25°C.

[0013] In another preferred embodiment, in step (2), Candida auris is injected into the fruit fly to be infected by microinjection, and the injection dose is 100-10000 Candida auris per fruit fly.

[0014] In another preferred embodiment, in step (2), the injection dose is 10,000 Candida auris per fruit fly.

[0015] In another preferred embodiment, in step (2), the injection dose is 5000 Candida auris per fruit fly.

[0016] In another preferred embodiment, in step (2), the injection dose is 1000 Candida auris per fruit fly.

[0017] In another preferred embodiment, in step (2), the injection dose is 100 Candida auris per fruit fly.

[0018] In another preferred embodiment, in step (2), each fruit fly is only injected and infected with Candida auris once.

[0019] In another preferred embodiment, the construction method further comprises step (3): raising the fruit flies injected with and infected with Candida auris in an environment of 29°C.

[0020] In another preferred embodiment, in the construction method, the fruit flies used are raised under a 12h light / 12h dark cycle to ensure the normal biological rhythm of the fruit flies.

[0021] The second aspect of the present invention provides a use of wild-type Drosophila oreR for constructing an animal model for studying systemic infection of Candida auris.

[0022] In another preferred embodiment, the research on Candida auris infection includes:

[0023] (a) studying the pathogenicity of Candida auris; and / or

[0024] (b) To study the immune escape mechanism of Candida auris in the host.

[0025] The third aspect of the present invention provides a method for studying systemic infection of Candida auris, the method comprising: conducting research using the Drosophila model obtained by the construction method described in the first aspect of the present invention.

[0026] In another preferred embodiment, the research on Candida auris infection includes:

[0027] (a) studying the pathogenicity of Candida auris; and / or

[0028] (b) To study the immune escape mechanism of Candida auris in the host.

[0029] In another preferred embodiment, the method specifically comprises the following steps:

[0030] (i) providing a fruit fly to be infected, wherein the fruit fly is a wild-type fruit fly oreR;

[0031] (ii) injecting the fruit flies to be infected with Candida auris, with the injection dose being 100-10000 Candida auris per fruit fly;

[0032] (iii) The fruit flies injected with and infected with Candida auris are raised in an environment of 29° C., and the effect of Candida auris on the host is studied by detecting the transcriptome of the fruit flies 6 hours to 24 hours after infection. For example, the effect of Candida auris on the host is studied by detecting the transcriptome of the fruit flies 6 hours and / or 24 hours after infection.

[0033] In a fourth aspect of the present invention, a fruit fly model for studying systemic infection of Candida auris is provided, wherein the fruit fly model is constructed using the construction method described in the first aspect of the present invention.

[0034] In another preferred embodiment, the Drosophila model is used to study the pathogenic mechanism of Candida auris.

[0035] In another preferred embodiment, the Drosophila model is used to study the immune escape mechanism of Candida auris in the host.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure shows the establishment of the fruit fly model infected with Candida auris. Among them, a shows the schematic diagram of the process of establishing the fruit fly model infected with Candida auris systemically; b shows the survival curve of fruit flies after systemic infection with Candida auris, and the infection dose is 10,000 Candida auris per fruit fly; c shows the survival curve of fruit flies infected with different infection doses; d shows the fungal load in fruit flies after systemic infection with Candida auris, and the infection dose is 10,000 Candida auris per fruit fly.

[0038] Figure 2The changes in RNA levels of some antimicrobial peptides downstream of the Toll pathway in the whole body of fruit flies after infection with Candida auris are shown to characterize the activity of the Toll pathway. Among them, a shows the expression of Drosomycin 6 hours after infection with Candida auris; b shows the expression of Drosomycin (drs) 24 hours after infection with Candida auris; c shows the expression of Metchnikowin (mtk) 6 hours after infection with Candida auris; d shows the expression of Metchnikowin (mtk) 24 hours after infection with Candida auris. DETAILED DESCRIPTION

[0039] Through extensive and in-depth research, the inventors have obtained a method for constructing a fruit fly model for studying systemic infection of Candida auris, which uses wild-type fruit flies oreR as a basic model to construct an ideal animal model that is very suitable for studying the pathogenic mechanism of Candida auris and the immune escape mechanism of Candida auris in the host. On this basis, the present invention was completed.

[0040] the term

[0041] Wild-type Drosophila oreR

[0042] OreR is a wild-type fruit fly recognized in the art and is commonly used in immunology-related research. It can be obtained from the Bloomington Drosophila Stock Center under the number 5.

[0043] Method of the present invention

[0044] One aspect of the present invention provides a method for constructing a fruit fly model for studying systemic infection with Candida auris, the method comprising infecting wild-type fruit flies oreR with Candida auris, wherein the infection amount of Candida auris is 100-10,000 Candida auris per fruit fly. The infection is carried out by microinjection, and each fruit fly is injected only once; specifically, 100-10,000 Candida auris are injected into the fruit fly using a microinjector. Before infection with Candida auris, the fruit flies are kept in an environment of 25°C. After infection with Candida auris, the fruit flies are kept in a constant temperature environment of 29°C.

[0045] Another aspect of the present invention provides a method for studying systemic infection of Candida auris, the method comprising: conducting research using a fruit fly model obtained by the construction method described in the first aspect of the present invention. The research comprises (a) studying the pathogenic mechanism of Candida auris; and / or (b) studying the immune escape mechanism of Candida auris in the host. In a specific embodiment of the present invention, the effect of Candida auris on the host is studied by detecting the transcriptome of fruit flies.

[0046] As used herein, the terms "Candida auris" and "C. auris" are used interchangeably. Candida auris, also known as Candida auris, is a species of Candida. It is a new pathogenic fungal species first discovered in Japan in 2009 and was named because it was first isolated from external auditory canal secretions.

[0047] Drosophila model of the present invention

[0048] The present invention also provides a fruit fly model for studying systemic infection of Candida auris, and the fruit fly model is constructed by the construction method described in the first aspect of the present invention. Based on the wild-type fruit fly oreR, the present invention constructs a fruit fly model for studying systemic infection of Candida auris by studying the survival rate of the wild-type fruit fly oreR after infection with Candida auris, and studying the activation of signal pathways in the fruit fly after infection, and verifies the feasibility of the fruit fly model in studying the pathogenic mechanism of Candida auris and / or the immune escape mechanism of Candida auris in the host.

[0049] Uses of the present invention

[0050] The present invention also provides the use of wild-type fruit fly oreR. The present invention first discovered that wild-type fruit fly oreR is particularly suitable for constructing an animal model for studying systemic infection of Candida auris. Wild-type fruit fly oreR has a complete innate immune system and can be used not only to study the pathogenic mechanism of Candida auris in the host, but also to study the immune escape mechanism of Candida auris in the host.

[0051] The main advantages of the present invention include:

[0052] (1) Compared with the existing animal models of systemic infection of Toll-immunodeficient fruit flies and mice, the wild-type fruit fly oreR model constructed using the method of the present invention is a simple and low-cost animal model for studying Candida auris infection.

[0053] (2) The Drosophila model constructed using the method of the present invention can be used not only to study the pathogenic mechanism of Candida auris, but also to study the immune escape mechanism of Candida auris in the host.

[0054] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where no detailed conditions are specified are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0055] Experimental methods

[0056] Fruit fly rearing

[0057] The fruit flies used in the experiment of the present invention were all raised in an environment of 25°C, fed with standard corn culture medium, and raised in small tubes with an inner wall diameter of 2 cm or large bottles with a diameter of 5.5 cm. After infection, the fruit flies were raised in a constant temperature incubator at 29°C. All fruit flies used in the experiment were raised under a 12h light / 12h dark cycle to ensure the normal biological rhythm of the fruit flies.

[0058] Standard corn culture medium formula: 1L system contains 1L distilled water, 10.6g agar, 32.19g Angel yeast, 0.726g calcium chloride, 31.62g sucrose, 63.2g glucose, 77.7g corn flour, 2g potassium sorbate, and 15ml Nicotine (100g ethyl parahydroxybenzoate dissolved in 500ml, 95% pure ethanol).

[0059] Preparation of infection solution

[0060] (1) Take out the frozen Candida auris strain from the -80℃ freezer, streak it on a YPD solid plate, and incubate it in a 30℃ incubator for two days for activation. Pick a single colony from the plate and inoculate it into 5mL YPD liquid medium, incubate it at 200rpm / min overnight, then transfer it to a conical flask and culture it to the logarithmic phase.

[0061] (2) Transfer the bacterial solution to a 50 mL centrifuge tube and centrifuge at 4000 rpm / min for 5 min at room temperature.

[0062] (3) Remove the supernatant, resuspend the Candida auris pellet in an appropriate amount of sterile PBS, vortex and mix, and then centrifuge at 4000 rpm / min for 5 min at room temperature. Repeat the operation two or three times.

[0063] (4) Remove the supernatant, resuspend the Candida auris pellet in an appropriate amount of sterile PBS, and transfer to a sterile 1.5 mL centrifuge tube.

[0064] (5) Dilute the above bacterial solution with sterile PBS, take 10 μL of the diluted bacterial solution to a blood cell counting plate, count under a microscope, take the average of the two counting results, and calculate the average value × dilution factor × 10 4 The bacterial concentration is calculated and then the bacterial solution is diluted to the concentration required for infection.

[0065] (6) The infected bacterial solution was appropriately diluted with sterile PBS, vortexed to mix, and 100 μl was pipetted onto a Sabouraud solid plate. The plate was placed in an incubator at 30°C and incubated for two days before counting to verify the exact concentration of the infected bacterial solution.

[0066] Fruit fly infection

[0067] The experiment used NANOJECT II (Drummond Scientific Company) to perform microinjection infection on fruit flies. The selected experimental fruit flies were anesthetized with carbon dioxide and placed on a fruit fly plate. Adjust the knob to slowly insert the needle tip downward into the chest of the fruit fly. Press the inject key once, and the prompt sound will be heard. The machine will begin to vibrate slightly until it stops, and an injection is completed. The injection volume is 50.6nL (containing approximately 10,000 Candida auris). Fruit flies injected with PBS served as a blank control group. Each fruit fly was infected only once in each independent experiment, and at least 2 tubes (20 male fruit flies / tube) were injected in the experiment. After the injection, carefully place the fruit flies into the fruit fly tube with a brush, and place the fruit fly tube horizontally before the fruit flies wake up. When all the fruit flies have woken up, place the fruit flies in a constant temperature and humidity incubator at 29°C for culture.

[0068] Statistics and analysis of fruit fly mortality

[0069] Observe and record the death of fruit flies in each tube regularly every day, and transfer the fruit flies to new fruit fly tubes for cultivation. Draw a curve of the survival rate of fruit flies after infection.

[0070] Detection of fungal load after Drosophila injection infection

[0071] Inject and infect fruit flies as described above. According to the sampling time point of the experiment, all fruit flies in the fruit fly tubes were anesthetized and placed on the fruit fly plate. After mixing, the samples were placed in a 1.5 mL centrifuge tube, 200 μL PBS and three times the volume of fruit flies were added with porcelain beads (diameter 0.5 mm), and the mixture was fully ground in a hybrid grinder. Then 800 μL PBS was added, and the mixture was diluted appropriately. 100 μL was drawn and spread on the Sabouraud antibiotic solid plate, and counted after incubation at 30°C for two days.

[0072] Example 1: Construction of Drosophila model infected with Candida auris

[0073] To investigate whether wild-type Drosophila can be used to study Candida auris, a Candida auris systemic infection model was established using wild-type Drosophila oreR ( Figure 1 a). First, determine whether Candida auris can work in Drosophila by injection infection. Use a dose of 10,000 Candida auris per Drosophila to infect Drosophila and observe the survival curve of Drosophila, such as Figure 1As shown in b, on the first day after infection, the survival rate of fruit flies reached about 50% 24 hours after infection, and as the infection time prolonged, the survival rate of fruit flies showed a gradual downward trend. The results show that Candida auris can exert certain virulence in the wild-type fruit fly host through systemic infection and eventually lead to its death. In order to determine the effect of the infection dose on the survival rate of fruit flies, fruit flies were infected with different infection doses, from low to high, 100, 1000, 5000, and 10,000 Candida auris per fruit fly, respectively. The results are shown in Figure 1 As shown in Figure c, as the infection concentration increases, the survival rate of fruit flies will decrease within a certain period of time, showing a certain concentration dependence.

[0074] Example 2: Feasibility analysis of the Candida auris infection fruit fly model

[0075] According to the results of the survival curve ( Figure 1 b), Candida auris has a killing effect on wild-type fruit fly OreR, and the effect on fruit flies is most significant 1-2 days after infection. Therefore, the inventors verified the fungal load in fruit flies at different times after infection to determine whether there is a causal relationship between the significant mortality 1-2 days after infection and the bacterial load.

[0076] The fruit flies were infected with 10,000 C. auris per fruit fly and samples were taken at different time points to detect the fungal load in the fruit flies. Figure 1 As shown in Figure d, 24 hours after infection, the number of fungi in the fruit fly reached a peak, and then the bacterial load gradually decreased and finally maintained at a certain level. This result shows that the fungal load in the body is directly related to the survival rate of the fruit fly. This shows that the number of Candida auris in each fruit fly increased from the initial 10 4 Increased to 10 24 hours after infection 5 , within 24 hours after infection, the fruit fly's immune system cannot completely inhibit the growth of Candida auris; and as the infection time goes by, the bacterial load in some fruit flies gradually decreases. The results show that the fruit fly's immune system can effectively eliminate the invading pathogenic fungi, but the fruit fly's immune system cannot completely eliminate Candida auris, but can only reduce the abundance of bacteria, thus forming a long-term confrontation between the host and Candida auris. This also shows that Candida auris may have an immune escape mechanism in the fruit fly.

[0077] Studies have shown [4] that regardless of whether mice were systemically infected with high or low doses of Candida auris (BJCA001) and Candida albicans (SC5314), all mice infected with Candida auris survived from beginning to end and showed normal clinical manifestations, indicating that Candida auris can escape immunity in mammals, which may increase the risk of disease in immunocompromised individuals. However, systemic infection of wild-type fruit flies with Candida auris can cause the death of fruit flies, indicating that Candida auris has a certain degree of virulence in wild-type fruit flies. Therefore, fruit flies can be used to study the pathogenic mechanism of Candida auris. In the early stage of infection, Candida auris can proliferate in the fruit fly, and in the later stage of infection, it can persist in the fruit fly ( Figure 1 d), indicating that the Drosophila immune system cannot completely eliminate C. auris, which is helpful for studying the immune escape mechanism of C. auris in the host.

[0078] Based on the above characteristics, the wild-type fruit fly established in the present invention is expected to become an ideal model for studying Candida auris.

[0079] Example 3: Candida auris infection in Drosophila activates the antifungal Toll signaling pathway

[0080] The Drosophila immune system is composed of humoral and cellular components. Fungal infection triggers the activation of the Toll signaling pathway. Intracellular signal transduction promotes the activation of transcription factors Dif and Dorsal. These transcription factors are translocated from the cytoplasm to the nucleus, causing the activation of Toll immune response genes, including genes encoding antifungal peptides Drosomycin (drs) and Metchnikowin (mtk). In order to verify the activity of the Toll signaling pathway in the whole body of wild-type Drosophila OreR after Candida auris infection, the whole body RNA of Drosophila at different time points after infection was extracted, and the expression levels of antimicrobial peptide genes downstream of the Toll pathway were detected by real-time fluorescence quantitative PCR.

[0081] like Figure 2 As shown in ad, 6 hours after infection, the expression level of Drosomycin (drs) increased 3 times after infection with Candida auris compared with the PBS control group, indicating that the immune system of the fruit fly has already taken effect at the early stage of infection and the Toll signaling pathway has been activated; 24 hours after infection, the expression level of Drosomycin (drs) in the host was 25 times that of the PBS control group; the expression level of Metchnikowin (mtk) in the infected group was 6 times that of the PBS group. At this stage, the activity of the Toll signaling pathway in the fruit fly reached the highest level, and the expression of antimicrobial peptide genes was rapidly regulated to resist the rapid proliferation of Candida auris.

[0082] In summary, wild-type Drosophila oreR can be used as an animal model to study the pathogenic mechanism of Candida auris.

[0083] References

[0084] [1]Wurster S, Bandi A, Beyda ND, et al. Drosophila melanogaster as a model to study virulence and azole treatment of the emerging pathogen Candidaauris[J]. Journal of Antimicrobial Chemotherapy, 2019, 74(7): 1904-1910.

[0085] [2]Johnson CJ,Davis JM,Huttenlocher A,et al.Emerging FungalPathogen Candida auris Evades Neutrophil Attack[J].mBio,2018,9(4):e01403-18.

[0086] [3] Bruno M, Kersten S, Bain JM, et al. Transcriptional and functional insights into the host immune response against the emerging fungal pathogen Candida auris[J]. Nature Microbiology, 2020, 5(12): 1516-1531.

[0087] [4]Wang Y, Zou Y, Chen X, et al. Innate immune responses against the fungal pathogen Candida auris[J]. Nature Communications, 2022,13:3553.

[0088] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for constructing a Drosophila model for studying systemic infection of Candida auris, characterized in that: The method comprises the following steps: (1) providing a fruit fly to be infected, wherein the fruit fly is a wild-type fruit fly oreR; (2) Infecting the fruit flies to be infected with Candida auris, with the infection amount being 100-10,000 Candida auris per fruit fly.

2. The method according to claim 1, characterized in that The Drosophila model is used to: (a) studying the pathogenicity of Candida auris; and / or (b) To study the immune escape mechanism of Candida auris in the host.

3. The method according to claim 1, characterized in that In step (2), the fruit flies to be infected are infected with Candida auris by microinjection, with the injection dose being 100-10,000 Candida auris per fruit fly.

4. The method according to claim 3, characterized in that In step (2), the injection dose is 10,000 Candida auris per fruit fly.

5. The method according to claim 1, characterized in that The construction method further comprises step (3): raising the fruit flies injected with and infected with Candida auris in an environment of 29°C.

6. A use of wild-type Drosophila oreR, characterized in that: Used to construct an animal model for studying Candida auris infection.

7. The use according to claim 6, characterized in that The studies on C. auris infections include: (a) studying the pathogenicity of Candida auris; and / or (b) To study the immune escape mechanism of Candida auris in the host.

8. A method for studying systemic infection of Candida auris, the method comprising: The Drosophila model obtained by the construction method as claimed in claim 1 was used for research.

9. The method according to claim 8, characterized in that The studies on C. auris infections include: (a) studying the pathogenicity of Candida auris; and / or (b) To study the immune escape mechanism of Candida auris in the host.

10. The method according to claim 8, characterized in that The method specifically comprises the following steps: (i) providing a fruit fly to be infected, wherein the fruit fly is a wild-type fruit fly oreR; (ii) injecting the fruit flies to be infected with Candida auris, with the injection dose being 100-10000 Candida auris per fruit fly; (iii) The fruit flies injected with C. auris were housed at 29°C, and the effect of C. auris on the host was studied by detecting the transcriptome of the fruit flies 6 to 24 hours after infection.

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