Application of quorum sensing quenching enzyme in prevention and control of vibrio corallinum pathogens

Abstract

The invention discloses application of quorum sensing quenching enzyme in prevention and control of vibrio corallinum pathogens. The quorum sensing quenching enzyme can remarkably inhibit coral bacterial diseases with different symptoms caused by different vibrio infections, can be used for preparing drugs or biological products for preventing and treating coral vibrio infections, and is used for preventing and controlling coral early pathogens.

Description

Technical Field

[0001] The present invention relates to the field of marine biotechnology, and in particular to the application of quorum sensing quenching enzyme in the prevention and control of Vibrio corallii pathogens. Background Art

[0002] The coral reef ecosystem has important value both in ecology and economy. In ecology, it provides habitats for many fish, algae and even microorganisms on the seabed, and is an important breeding ground for them. In economics, in addition to creating tourism value, it also makes important contributions to coastal commercial and fishery development.

[0003] However, with global warming, rising temperatures and the continuous expansion of human living space, the coral reef ecosystem is facing threats. There have been many large-scale coral bleaching events around the world, making it urgent to curb coral bleaching with economic and environmentally friendly means.

[0004] The cause of coral bleaching is believed to be the release of zooxanthellae caused by rising temperatures. Recent studies have shown that in addition to high temperature stress, the symbiotic flora of corals also undergo significant changes with environmental changes such as temperature, leading to coral bacterial diseases. Coral bleaching or pigment abnormalities have become one of the main symptoms. The symbiotic flora of corals provides the necessary nutritional source for corals. Regulating the flora through quorum sensing can effectively delay the occurrence of coral bacterial diseases. Coral diseases are related to the presence of opportunistic pathogens in coral symbionts. Opportunistic pathogens in the waters around corals colonize in coral symbionts through chemotaxis and other methods to form biofilms, which is also inseparable from the regulatory role of quorum sensing.

[0005] Vibrio species are the main pathogens that cause coral bleaching and other diseases. It has been reported that V. corallilyticus causes bleaching of Acropora horribilis; V. natriegens and V. parahaemolyticus cause white ulcer spot disease of Porites; V. corallilyticus and V. shiloi cause bleaching of Piper coral, etc. Our team found that the abundance of Vibrio such as Vibrio fortis increased significantly in the symbiotic bacteria of bleached and pigmented corals. The subsequent coral infection experiments also proved that the inoculation of V. fortis not only affected the pigment composition of Porites, but also caused the disease of Acropora horribilis and Porites. According to Koch's postulates, this result proves that V. fortis is the pathogen that causes coral bleaching. Similar coral bacterial diseases also occur in coral species such as Acropora, Pocillopora, and Capybara that are infected with Vibrio pathogens such as Vibrio corallilyticus and Vibrio parahaemolyticus. During the research, our team also found that Vibrio infection caused two different lesions in corals: bleaching and tissue ulceration. The colonization of pathogens in hosts is generally believed to have a dose effect, that is, the more doses, the greater the harm. However, our team's research results found that low concentrations of Vibrio produce stronger toxicity and lead to more obvious coral bleaching symptoms, while high concentrations of Vibrio produce nutritional competition and lead to nutritional imbalance of coral symbionts. This trend does not conform to the traditional understanding of the dose effect of bacterial colonization, and it is necessary to analyze and treat the different bacterial symptoms caused by different doses of Vibrio infection.

[0006] In summary, in view of the colonization of Vibrio pathogens in coral symbionts and the infection of coral hosts, it is urgent to develop targeted biological control technology for Vibrio pathogens, and develop a product that can simultaneously alleviate different coral lesions, reduce the symptoms of coral diseases caused by high-concentration and low-concentration Vibrio infections, and reduce the impact of Vibrio infections on coral health. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art, provide the application of quorum sensing quenching enzyme in the prevention and control of Vibrio corallii pathogens, and solve the problems in the above-mentioned background technology.

[0008] One of the technical solutions adopted by the present invention to solve its technical problem is: providing application of quorum sensing quenching enzyme in the prevention and control of Vibrio corallii pathogens.

[0009] The quorum sensing quenching enzyme refers to a class of enzyme substances that can inhibit the synthesis and accumulation of signal molecules, or degrade and modify signal molecules, destroy the bacterial quorum sensing system, and thus block the adverse effects of spoilage bacteria or pathogenic bacteria. The quorum sensing quenching enzyme of the present invention can be the lipase YtnP (whose amino acid sequence is as shown in SEQ ID NO: 1) or the AiiA enzyme (whose amino acid sequence is as shown in SEQ ID NO: 2) in the quorum sensing quenching enzyme.

[0010] SEQ ID NO: 1:

[0011] METLNIGNISLTWLDGGVTHMDGGAMFGVVPKPLWSRKYPVNDKNQIELRTDPILIRKKGLNILVDAGIGRGKFSDKQKRNYGVTQESNLKQSLRDLGLSCEDIHIAAMTHLHFDHACGLTEYSGEELVSVFPNARIITS ATEWREMRNPNIRSKNTYWRENWEAVQDQVETFENEYQLTEGITMHHTGGHSDGHSVIVLEDGGEAAVHFADLMPTNAHKNPLWVLAYDDYPMTSIPEKQKWQTFAAEKDAWLIFYHDAIYRAVKWGEDGEVAASVKREKK

[0012] SEQ ID NO: 2:

[0013] MTVKKLYFIPAGRCMLDQSSVNSTLTAGNLLNLPVWCYLLETEEGPILVDTGMPESAVHNENLFEGTFAEGQILPKMTEEDRIVTILKRVGYKPEDLLYIISSHLHFDHAGGNGAFSNTPIIIQR AEYEAAQYREEYLKECILPNLNYKIIEGDYEVVPGVQLLYTPGHSPGHQSLLIETEKSGLVLLTIDASYTKENFEGEVPFAGFDSELALSSIKRLKEVVMKEKPIVFFGHDIEQEKGCKVFPEYI

[0014] Preferably, the quorum sensing quenching enzyme is used in the preparation of drugs or biological products for preventing and treating Vibrio corallii infection.

[0015] Preferably, the concentration of the quorum sensing quenching enzyme is not less than 1 ng / L.

[0016] The implementation of the present invention has the following beneficial effects:

[0017] The present invention has the following beneficial effects: the quorum sensing quenching enzyme of the present invention can regulate the growth, reproduction and physiological and biochemical activities of environmental flora by degrading AHL signal molecules in the microenvironment, including the pathogenicity of opportunistic pathogens such as Vibrio, and the nutritional metabolism of symbiotic flora in the environment. Although the principles of the symbiotic interactions of these environmental flora are not yet completely clear, quorum sensing regulation can undoubtedly inhibit low or high concentrations of Vibrio infection in coral symbionts, inhibit the formation of biofilms of Vibrio in symbionts, and thus inhibit the colonization and growth of Vibrio in coral symbionts; in addition, the intervention of quenching enzymes can also alleviate the expression of Vibrio virulence genes, and alleviate the influence of Vibrio on the community structure of other environmental symbiotic bacteria and symbiotic microorganisms such as zooxanthellae by inhibiting the pathogenicity of Vibrio. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0019] Figure 1 This is the SDS-PAGE electrophoresis of the quorum sensing quenching enzyme YtnP. M: protein marker; 1: cell disruption solution of E. coli empty plasmid; 2: YtnP crude enzyme; 3: YtnP pure enzyme.

[0020] Figure 2 This is the SDS-PAGE electrophoresis of the quorum sensing quenching enzyme AiiA. M: protein marker; 1: pET28a whole protein; 2: AiiA whole protein; 3: AiiADH82 purification.

[0021] Figure 3 The effect of AHL and YtnP on the growth curve and biofilm formation of Vibrio robustus, where (a) is the growth curve and (b) is the biofilm.

[0022] Figure 4 The effect of Vibrio robustus infection and QQ intervention on the health status and effective fluorescence production of corals. (a) is the bleaching degree of Acropora hornense; (b) is the bleaching degree of Pocillopora porifera; (c) is the effective fluorescence production of Pocillopora hornense and Pocillopora porifera.

[0023] Figure 5 This is a diagram showing the changes in the coral symbiotic bacterial community structure caused by Vibrio robustus infection. (A) is a heat map analysis of the bacterial community structure; (B) is the difference in Vibrio abundance; (C) is the difference in Bacillus abundance.

[0024] Figure 6 The effect of exogenous quenching enzyme treatment under heat stress on the bleaching of Acropora ceratospora caused by Vibrio robustus infection. (A) is 25℃; (B) is 28℃ heat stress.

[0025] Figure 7is the chlorophyll fluorescence quantum yield of the coral symbiont (Fv' / Fm).

[0026] Figure 8 This is a density map of zooxanthellae in the coral symbiosis.

[0027] Sg, Seriatoporaguttatus; Sg_A group: blank group; Sg_B group: quenching enzyme group; Sg_C group: low concentration Vibrio group; Sg_D group: low concentration Vibrio quenching enzyme AiiA treatment group; Sg_E group: high concentration Vibrio group; Sg_F group: high concentration Vibrio quenching enzyme AiiA treatment group.

[0028] Fig. 9 This is a diagram of the zooxanthellae community structure analysis in the coral symbiosis. (a) is the analysis of zooxanthellae α; (b) is the zooxanthellae group analysis and structural composition.

[0029] Sg, Seriatoporaguttatus; Sg_A group: blank group, Sg_B group: quenching enzyme group, Sg_C group: low concentration Vibrio group, Sg_D group: low concentration Vibrio quenching enzyme AiiA treatment group, Sg_E group: high concentration Vibrio group, Sg_F group: high concentration Vibrio quenching enzyme AiiA treatment group.

[0030] Fig.10 This is a bar graph of the alpha diversity of symbiotic bacteria in Porphyroblastoma.

[0031] Sg, Seriatoporaguttatus; Sg_A group: blank group, Sg_B group: quenching enzyme AiiA group, Sg_C group: low concentration Vibrio group, Sg_D group: low concentration Vibrio quenching enzyme AiiA treatment group, Sg_E group: high concentration Vibrio group, Sg_F group: high concentration Vibrio quenching enzyme AiiA treatment group.

[0032] Fig.11 This is a community analysis diagram of symbiotic bacteria in the coral reef. (a) is the community composition of the bacterial community at the genus level in the coral symbiont; (b) is the Lefse analysis diagram of the difference in bacterial community structure between the blank group and the quenching enzyme group.

[0033] Sg, Seriatoporaguttatus; Sg_A group: blank group, Sg_B group: quenching enzyme AiiA group, Sg_C group: low concentration Vibrio group, Sg_D group: low concentration Vibrio quenching enzyme AiiA treatment group, Sg_E group: high concentration Vibrio group, Sg_F group: high concentration Vibrio quenching enzyme AiiA treatment group.

[0034] Fig.12The genus level analysis diagram of the bacterial community with significant differences among groups in the coral symbionts. Among them, (a) is Sulfitobacter; (b) is Acinetobacter; (c) is Stenotrophomonas; (d) is Corynebacterium; (e) is Sphingobacterium; (f) is Methylobacterium

[0035] blank: blank group, QQ: quenching enzyme AiiA group, LV: low concentration Vibrio group, LV_QQ: low concentration Vibrio quenching enzyme AiiA treatment group, HV: high concentration Vibrio group, HV_QQ: high concentration Vibrio quenching enzyme AiiA treatment group.

[0036] Fig.13 The differential bacterial communities are linearly correlated with the Vibrio infection concentration. Among them, (a) is Sphingomonas; (b) is Rhizobium; (c) is Brevundimonas; (d) is Burkholderia; (e) is Haliangium; (f) is Epibacterium; (g) is Enterococcus

[0037] blank: blank group, LV: low concentration Vibrio group, HV: high concentration Vibrio group.

[0038] Fig.14 The significantly different bacterial groups are only associated with high-concentration Vibrio infection. Among them, (a) is Acidibacter; (b) is Sphingobacterium; (c) is Renibacterium; (d) is Haliangium; (e) is Devosia; (f) is Rhodococcus; (g) is Vicinamibacteraceae

[0039] blank: blank group, QQ: quenching enzyme AiiA group, LV: low concentration Vibrio group, LV_QQ: low concentration Vibrio quenching enzyme AiiA treatment group, HV: high concentration Vibrio group, HV_QQ: high concentration Vibrio quenching enzyme AiiA treatment group.

[0040] Fig.15The horizontal box plot of bacterial genus associated with low-concentration Vibrio infection. Among them, (a) is Amaricoccus; (b) is Cognatishimia; (c) is f_Rhodobacteraceae; (d) is Lentilitoribacter; (e) is Chryseobacterium; (f) is Rubrobacter; (g) is o_S-olirubribacterales; (h) is c_Sphingobacteriales; (i) is o_Chloroplast; (j) is p_Chloroflexi; (k) is c_Ktedonobacteria

[0041] blank: blank group, QQ: quenching enzyme AiiA group, LV: low concentration Vibrio group, LV_QQ: low concentration Vibrio quenching enzyme AiiA treatment group, HV: high concentration Vibrio group, HV_QQ: high concentration Vibrio quenching enzyme AiiA treatment group.

[0042] Fig.16 The following are the bacterial community structure analysis diagrams of typical opportunistic pathogens. Among them, (a) is Serratia; (b) is Pseudomonas; (c) is Alteromonas; (d) is Pseudoalteromonas; (e) is Staphylococcus

[0043] blank: blank group, QQ: quenching enzyme AiiA group, LV: low concentration Vibrio group, LV_QQ: low concentration Vibrio quenching enzyme AiiA treatment group, HV: high concentration Vibrio group, HV_QQ: high concentration Vibrio quenching enzyme AiiA treatment group.

[0044] Fig.17 The diagram shows the changes in the free bacterial community structure in the water environment. (a) is the α analysis of the bacterial community in the water environment; (b) is the genus level analysis of the bacterial community in the water environment.

[0045] water, pore coral water sample; water_A group: blank group, water_B group: quenching enzyme AiiA group, water_C group: low concentration Vibrio group, water_D group: low concentration Vibrio quenching enzyme AiiA treatment group, water_E group: high concentration Vibrio group, water_F group: high concentration Vibrio quenching enzyme AiiA treatment group.

[0046] Fig.18 Analysis of the Vibrio community structure in coral symbionts. (a) shows the differences at the genus level; (b) shows the differences at the robust Vibrio; and (c) shows the Vibrio community structure.

[0047] Sg, S. guttatus; Sg_A group: blank group, Sg_B group: quenching enzyme AiiA group, Sg_C group: low concentration Vibrio group, Sg_D group: low concentration Vibrio quenching enzyme AiiA treatment group, Sg_E group: high concentration Vibrio group, Sg_F group: high concentration Vibrio quenching enzyme AiiA treatment group.

[0048] Fig.19 This is a picture of the symptoms of Vibrio coralligensis infecting clumps of helmet corals.

[0049] Fig. 20 Figure 2 shows bacterial diseases of clump-shaped corals caused by Vibrio corallii under different temperature stresses. (a) is the chlorophyll fluorescence quantum yield of corals at 26°C, (b) is the chlorophyll fluorescence quantum yield of corals at 32°C, (c) is the bleaching and ulceration rate of corals at 26°C, and (d) is the bleaching and ulceration rate of corals at 32°C. DETAILED DESCRIPTION

[0050] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Vibrio fortis S10-1 (MCCC 1H00104) was donated by the Marine Microbial Culture Collection Center of the Third Institute of Oceanography, Ministry of Natural Resources, and was isolated from sea cucumber samples in the coral reef area.

[0054] V. corallilyticus S4-1 was isolated from diseased Montipora sp.

[0055] The quorum sensing quenching enzymes AiiA and YtnP were cloned from the deep-sea strain Bacillus velezensis DH82 (GenBank: MK203035) by our research group in the previous work, and were constructed into the pET28a vector for recombinant expression.

[0056] Escherichia coli BL21(DE3):pET28a / aiiA and BL21(DE3):pET28a / ytnP carrying the recombinant expression plasmids of quorum sensing quenching enzyme AiiA and YtnP were constructed by our research group in previous work and are preserved in our research group.

[0057] The Pocillopora damicornis and Seriatoporaguttatus used in this experiment were provided by the Coral Conservation Laboratory of the Third Institute of Oceanography. They were coral fragments collected from field expeditions and cultivated through artificial indoor propagation. The coral culture environment was set at 25°C and cultivated in 20L of seawater with a salinity of 33.3‰.

[0058] Example 1: Recombinant expression and purification of quorum sensing quenching enzymes YtnP and AiiA

[0059] Experimental methods

[0060] 1. Strain recovery and cultivation

[0061] Use an inoculation loop to pick up the frozen bacterial liquid of Vibrio robusta S10-1, streak it on 2216E solid culture medium, and then invert it in a 37°C incubator for overnight culture. Pick a single colony on the solid culture medium, inoculate it into 2216E liquid culture medium, and culture it in a shaker at 37°C and 200 rpm until the end of the logarithmic growth phase, and collect the bacterial liquid for later use.

[0062] 2. Recombinant expression and purification of quorum sensing quenching enzymes YtnP and AiiA

[0063] (1) Induced expression: Take a sterile inoculation loop to pick up the preserved E. coli BL21 (DE3): pET28a / ytnP positive strain, add it to 50 mL of LB liquid medium containing 50 mg / L kanamycin (working concentration), and culture it overnight at 37°C and 200 rpm. The revived bacterial liquid is inoculated into fresh LB medium at an inoculum ratio of 1:100 and cultured until the OD 600 When nm was 0.5-0.6, IPTG (0.1 mmol / L) was added to induce expression. At the same time, the culture conditions were adjusted to 18°C ​​and 200 rpm. After inducing expression for 24 hours, the bacterial solution was collected for later use.

[0064] (2) Cell disruption: The collected bacterial suspension was centrifuged at 6000 × g for 10 min, the filtrate was discarded, and the suspension was resuspended in PBS, and repeated twice; in an ice bath, the resuspended bacterial suspension was ultrasonically disrupted at 400 W for 20 min (2 s of ultrasonication followed by a 4 s pause), and then centrifuged at 4°C and 6000 × g for 20 min. The supernatant was carefully aspirated with a pipette to obtain the crude enzyme of YtnP, which was then sterilized by filtering with a 0.22 μm filter membrane.

[0065] (3) Protein purification: The crude enzyme was purified using the NI-NTA column produced by Changzhou Tiandirenhe. The crude enzyme solution was mixed with a dissolution buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.4) and then loaded with imidazole elution buffer (300 mM NaCl, 200 mM imidazole, 50 mM NaH 2 PO 4 , pH 7.4) for washing, and collecting samples according to the protein absorption peak at 280nm of the protein purifier. The collected protein purification solution was dialyzed and desalted with a dialysis bag and then stored in a -20℃ refrigerator for later use.

[0066] Experimental Results

[0067] After the induction expression of recombinant YtnP and AiiA Escherichia coli and the separation and purification of the enzyme, Figure 1 , Figure 2 As shown in the figure, the comparison of electrophoresis bands in the figure shows that the quorum sensing quenching enzymes YtnP and AiiA have been successfully purified. After sequencing, the amino acid sequence of the quorum sensing quenching enzymes YtnP and AiiA is shown in SEQ ID NO:1.

[0068] SEQ ID NO: 1:

[0069] METLNIGNISLTWLDGGVTHMDGGAMFGVVPKPLWSRKYPVNDKNQIELRTDPILIRKKGLNILVDAGIGRGKFSDKQKRNYGVTQESNLKQSLRDLGLSCEDIHIAAMTHLHFDHACGLTEYSGEELVSVFPNARIITS ATEWREMRNPNIRSKNTYWRENWEAVQDQVETFENEYQLTEGITMHHTGGHSDGHSVIVLEDGGEAAVHFADLMPTNAHKNPLWVLAYDDYPMTSIPEKQKWQTFAAEKDAWLIFYHDAIYRAVKWGEDGEVAASVKREKK

[0070] SEQ ID NO: 2:

[0071] MTVKKLYFIPAGRCMLDQSSVNSTLTAGNLLNLPVWCYLLETEEGPILVDTGMPESAVHNENLFEGTFAEGQILPKMTEEDRIVTILKRVGYKPEDLLYIISSHLHFDHAGGNGAFSNTPIIIQR AEYEAAQYREEYLKECILPNLNYKIIEGDYEVVPGVQLLYTPGHSPGHQSLLIETEKSGLVLLTIDASYTKENFEGEVPFAGFDSELALSSIKRLKEVVMKEKPIVFFGHDIEQEKGCKVFPEYI

[0072] Example 2: Vibrio robustus causes disease (bleaching) in Pocillopora porifera and Acropora hornbeam, and the symptoms are alleviated by the intervention of the quenching enzyme YtnP

[0073] Experimental methods

[0074] 1. Growth curve determination of Vibrio robustus S10-1

[0075] (1) Resuscitation strain: S10-1 was inoculated into 2216E and cultured at 37°C and 200 rpm until the end of the exponential phase (OD 600 nm value is about 1.0), and the bacterial solution is diluted with fresh 2216E medium to OD 600 The nm value is approximately 0.1.

[0076] (2) Expanded culture: 5 mL of diluted bacterial solution was taken and mixed with 5 mL of YtnP enzyme solution (mother solution concentration 0.5 mg / mL) and exogenous AHL signal molecule (3-O-C6-HSL, Merck-Sigma product number K3255) respectively, and fresh 2216E liquid culture medium was added. At the same time, the enzyme solution or AHL signal molecule was replaced with an equal amount of distilled water as a control group. Each group was set up in parallel 3 times. The bacterial solution of each group was cultured at 37°C and 200 rpm. 1.5 mL of bacterial solution was taken at 1, 2, 4, 6 and 12 h, and its OD was measured. 600 The remaining samples were stored at -80 °C for later use.

[0077] 2. Biofilm experiment of Vibrio robustus S10-1

[0078] (1) Resuscitation strains

[0079] (2) Biofilm culture: dilute to OD 600The S10-1 resuscitation bacterial solution with a nm value of about 0.1 was inoculated into 3 mL of biofilm culture medium at a ratio of 1:100, and 500 μL of purified YtnP enzyme solution (working concentration of about 0.5 μg / mL) was added and mixed evenly, and dispensed into 12-well plates. At the same time, an equal volume of sterile water was set to replace the enzyme solution as a control group. Three parallels were set for each of the above groups of samples. The plates were placed in a 30°C incubator and cultured for 3 days.

[0080] (3) Biofilm staining (crystal violet staining): Aspirate the upper bacterial solution from each well, slowly add 4 mL PBS and let stand for 3 min, completely aspirate the PBS, and repeat three times; add 200 μL crystal violet solution, mix well and let stand for 10 min, slowly add 4 mL PBS and let stand for 3 min, completely aspirate the PBS, and repeat three times; add 200 μL ethanol (75%) and mix thoroughly to completely dissolve the biofilm on the plate, and let stand at room temperature for 15 min.

[0081] (4) Biofilm assay: 200 μL of the mixed solution was added to a 96-well plate and placed in a microplate reader to measure its absorbance at 595 nm. The relative amount of biofilm was calculated as the ratio of the absorbance of the bacterial solution stained with crystal violet to the absorbance of the blank bacterial solution at a wavelength of 595 nm.

[0082] 3. Vibrio infection coral experiment

[0083] (1) Water cultivation: Before the Vibrio infection experiment, the live rocks from the coral nursery of the Third Marine Institute were separated into small pieces and randomly placed in aquariums filled with seawater. They were pre-cultured separately at 25°C for 7 days. The microbial communities (the main functional microorganisms are nitrifying bacteria and algae) loaded on the live rocks were used to cultivate the water in order to obtain clean and stable seawater quality suitable for coral growth and to establish a stable free microbial flora.

[0084] (2) Coral cutting: Use coral scissors to cut corals from the same mother coral into coral fragments about 80 mm in length, randomly place them in an aquarium, and continue to culture them in seawater with stabilized water quality (remove live rocks) for 5 days. After the coral tissue at the cut of the coral fragment has recovered, conduct subsequent experiments.

[0085] (3) Experimental grouping: A total of 6 aquariums were set up, and 3 groups of treatments were set up for Acropora hornbeams and Pocillopora porifera (the two corals were mixed in the same tank). In each group, 5-6 coral fragments were placed as biological replicates, and each group was set up with 2 parallel replicates as technical replicates, namely: Group A was a blank group, and Group B was a Vibrio infection experimental group (the final concentration of Vibrio was 10 5 CFU / mL, and group C was the quorum sensing intervention group under Vibrio infection (final concentration of Vibrio was 10 5 CFU / mL, the final concentration of quenching enzyme was 0.05 μg / mL).

[0086] (4) Vibrio infection and quenching enzyme intervention: The coral fragments were immersed in diluted S10-1 bacterial solution or YtnP enzyme solution (the concentration of the bacterial solution and enzyme solution was 100 times the final concentration) in a 200 mL beaker for 10 minutes for bacterial infection or quenching intervention (make sure the coral fragments are completely immersed in the liquid surface, and pour the immersion solution into the aquarium to make the inoculation volume reach the final concentration). Then, the treated coral fragments were moved back to the aquarium and continued to be cultured at 25°C, and the changes in coral health were observed.

[0087] 4. Data Collection and Sample Preparation

[0088] The health status of corals was recorded by a digital camera, and the changes in the health status of corals were judged according to the color reference of the color bar marked by PANTONE colorManager software V2.2.0. The health status of corals was recorded every 4 hours until the occurrence of coral bleaching and other diseases, and the experiment was stopped and samples were collected. The photos of each group of corals were taken with a digital camera (Canon EOS 600D, 50mm lens, Japan), with an aperture parameter of F / 3.5, an exposure time of 1 / 50s, and a sensitivity of ISO 200.

[0089] The chlorophyll fluorescence quantum yield (Qy, Fv' / Fm) of the coral symbiont was determined by the average light measured at the tip and middle of the coral fragment using a pulse amplitude modulation fluorimeter (PAM, FluorPen FP100, Czech Republic). Measurements were taken every 4 hours, and at each measurement, 3 points in the middle of the coral fragment and 3 points at the tip of the coral were randomly selected to measure the average value and standard deviation of each coral branch. The results were analyzed by ANOVA for statistical differences.

[0090] Experimental Results

[0091] Growth curve and biofilm methods were used to verify the effect of quorum sensing quenching enzyme on the growth of S10-1. Figure 3 (a) It can be seen that the addition of exogenous YtnP and AHL had no significant effect on the growth curve of S10-1. Figure 3 As shown in (b), exogenous YtnP enzyme significantly inhibited the biofilm formation of S10-1 (the P value of the T test was 0.009259, extremely significant difference).

[0092] The results of this experiment showed that under nutrient-rich conditions (2216E medium), Vibrio robustus mainly bioaccumulated in the form of free bacteria, and its biomass proliferation had exceeded the range of quorum sensing regulation, so the induction of exogenous AHL and the inhibition of the quenching enzyme YtnP had no significant effect on its growth curve. However, under oligotrophic biofilm culture conditions, the formation of S10-1 biofilm was regulated by its quorum sensing, and the exogenous YtnP enzyme preparation significantly inhibited the formation of bacterial biofilm by degrading AHLs signal molecules. Although the genes related to the LuxM / LuxN pathway mediated by AHLs have not been retrieved from the genome sequencing results, the results of this biofilm experiment also proved the existence of the AHLs-mediated quorum sensing pathway of the S10-1 strain from the perspective of the physical and chemical properties of the strain, which can be used as the main way to intervene in the quorum sensing of the strain in the future.

[0093] In order to further verify the pathogenicity of Vibrio robustus S10-1 to different types of corals, and the feasibility of targeted regulation of their quorum sensing to inhibit bacteria, we set up Acropora hornii and Pocillopora as infection models in the indoor infection experiment. Figure 3 As shown in the figure, under Vibrio infection, both coral model organisms showed significant bleaching after 30 hours of Vibrio invasion ( Figure 4 (a) with Figure 4 (b)), the effective fluorescence yield Qy value also decreased significantly; under the intervention of YtnP quorum sensing regulation, although the Acropora hornii and Pocillopora radiata with exogenous quenching enzyme showed a similar Qy value decline trend as the Vibrio infection group, the bleaching degree was significantly alleviated, and the effective fluorescence yield of the coral symbiont was significantly enhanced ( Figure 4 (c)), indicating that the pathogenicity of Vibrio robustus to coral bleaching is regulated by quorum sensing, proving the feasibility of Vibrio quorum sensing inhibition in alleviating coral bleaching.

[0094] The quorum sensing response of strain S10-1 was preliminarily verified by growth curve and biofilm experiments. The growth curve did not seem to be significantly different, but the biofilm control group and the YtnP treatment group showed significant differences, indicating that the addition of YtnP had an effect on the biofilm formation of strain S10-1, which could inhibit the growth of S10-1 and regulate it.

[0095] Vibrio robustus infection also affected the coral symbiotic microecology: 16S rRNA high-throughput sequencing results showed that Vibrio infection led to a significant increase in the abundance of Proteobacteria and Actinobacteria, and a significant decrease in Firmicutes in the symbiotic microorganisms of Porifera spp. At the genus level, the abundance of Bacillus was significantly reduced, while Vibrio, Rhodococcus, Ralstonia and Burkholderia carinii were significantly increased; the water quality in the coral culture system changed significantly, the microbial community in the live rock was less affected, and there was a quantitative difference between the Vibrio in the coral symbiotic state (biofilm) and the free state in the water ( Figure 5 ). These results indicate that Bacillus may play a key role in coral health, Vibrio may cause synergistic pathogenicity with other symbiotic bacteria, and that pathogenicity is mainly related to the bacterial community (biofilm state) in the symbiont. This is the main basis for selecting Vibrio biofilm morphology as an important indicator of pathogenicity in this project, and for selecting Bacillus and symbiotic Vibrio as the main targets for exploring bacterial community interactions.

[0096] Example 2: Vibrio robustus caused more severe bleaching of Porifera spp. under temperature stress, but this was significantly alleviated by the intervention of the quenching enzyme YtnP

[0097] In the temperature environmental factor response experiment, we found that the bleaching phenomenon of Acropora hornii infected with Vibrio robusta under heat stress was more significant, and the effective fluorescence output was significantly reduced, while the regulation of the quenching enzyme YtnP inhibited the bleaching of the coral ( Figure 6 ), indicating that the pathogenicity of Vibrio robustus to corals may be related to temperature, and that quorum sensing regulation is not affected by heat stress.

[0098] Example 3: Different concentrations of Vibrio robustus infection caused different lesions in Porifera porifera, and all of them were alleviated under the intervention of quenching enzyme AiiA

[0099] Experimental methods

[0100] 1. Vibrio infection coral experiment

[0101] (1) Water conservation

[0102] (2) Coral cutting

[0103] (3) Experimental grouping: A total of 12 aquariums were set up and divided into 6 groups. In each group, 5 to 6 coral fragment samples were placed as biological replicates. Each group was set up with 2 parallels as technical replicates, namely: Group A was a blank group, Group B was a quenching enzyme control group, and Group C was a low-concentration Vibrio infection group (the final concentration of Vibrio was 10 3 CFU / mL), group D was the low-concentration Vibrio quenching enzyme intervention group (the final concentration of Vibrio was 10 3 CFU / mL, the final concentration of quenching enzyme AiiA was 0.05 μg / mL, and group E was a high-concentration Vibrio infection group (the final concentration of Vibrio was 10 5CFU / mL), and group F was the intervention group with high concentration of Vibrio quenching enzyme AiiA (final concentration of Vibrio was 10 5 CFU / mL, the final concentration of quenching enzyme AiiA was 0.05 μg / mL).

[0104] (4) Vibrio infection and intervention with quenching enzyme AiiA

[0105] 2. Data Collection and Sample Preparation

[0106] A record of coral health.

[0107] Determination and statistical analysis of chlorophyll fluorescence quantum yield in coral symbionts.

[0108] After the experiment was stopped, the coral fragments were sampled and quickly frozen in liquid nitrogen for later use; the seawater samples in each group were filtered with a 0.22 μm filter membrane (Millipore, USA), and the filter membrane samples with filtered microorganisms were collected. The pre-treated coral fragments and filter membrane samples were then stored at -80°C for further research.

[0109] 3. Preparation of coral tissue homogenate

[0110] (1) Flushing coral tissue: Place a coral fragment sample in a sterile sealed bag, use an Eluan air pump to provide high-pressure air (set the turbine speed to 66.8 rpm), and a Kamoer peristaltic pump connected to sterile water to provide high-pressure water flow (set the water output rate to 26.7 mL / min). Combined with high-pressure water vapor, flush the coral tissue on each side until the tissue on the coral skeleton is cleaned, forming a coral tissue suspension in a sterile sealed bag.

[0111] (2) Tissue homogenization: Grind the washed coral tissue with a tissue grinder for 1 min to fully disperse the tissue suspension in a sealed bag to form a tissue homogenate. Dilute the homogenate with sterile water to 50 mL to obtain coral tissue homogenate, which is then stored in an ice bath until testing.

[0112] 4. Zooxanthellae density detection

[0113] (1) Calculate the surface area of ​​coral: Insert the coral skeleton into the plasticine and fix it, scan it with the Jeatch3D scanner to obtain images of each surface of the coral skeleton, and use the supporting software to calculate the surface area of ​​the coral skeleton.

[0114] (2) Microscopic counting: Take 100 μL of the coral tissue homogenate obtained above and drop it into a plankton cell counting plate. Let it stand for 3 to 5 min. The diluent will fill the entire chamber of the counting plate through liquid penetration. Count the zooxanthellae under a microscope at a magnification of 400 times. Calculate the average value of the zooxanthellae counts in the five chambers in the middle and the edge. After conversion according to the dilution factor, divide it by the surface area of ​​the coral skeleton to obtain the zooxanthellae density of the coral sample.

[0115] 5. DNA Extraction and Amplification

[0116] DNA was extracted using a soil DNA extraction kit (MP Biomedicals, USA), and 1% agarose gel was prepared as a DNA extraction solution. The DNA concentration and purity were determined using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Scientific, USA). The PCR Master Mix kit (Takara, China) was used to amplify the highly variable region V3-V4 of bacterial 16S rRNA and the zooxanthellae ITS2 sequence (upstream and downstream primers are shown in Table 1) using total genomic DNA as a template. The 16S rRNA PCR amplification method was as follows: initial denaturation at 95°C for 3 minutes; denaturation at 98°C for 30 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 15 seconds; single extension at 72°C for 5 minutes; and amplification was terminated at 4°C. The PCR products of 400–450 bp were detected by electrophoresis on 2% agarose gel, and the samples were extracted from 2% agarose gel, purified using a DNA gel extraction kit (Omega, China) according to the manufacturer’s instructions, and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).

[0117] Table 1 Primer sequences used

[0118] Primers sequence 338F 5'-ACTCCTACGGGAGGCAGCAG-3' (such as SEQ ID NO: 2) 806R 5'-GGACTACHVGGGTWTCTAAT-3' (such as SEQ ID NO: 3) ITS-DINO 5'-GTGAATTGCAGAACTCCGTG-3' (such as SEQ ID NO: 4) ITS2Rev2 5'-CCTCCGCTTACTTATATGCTT-3' (such as SEQ ID NO: 5)

[0119] 6. High-throughput sequencing

[0120] The purified amplicons were subjected to equal molecular weight and paired-end sequencing on the Illumina MiSeq PE300 platform / NovaSeq PE250 platform (Illumina, San Diego, USA) according to the standard protocol of Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China). The 16S rRNA and ITS2 sequencing reads of the original bacterial community were demultiplexed, and the sequencing data results were quality filtered using fastp version 0.20.0 software. The sequences were spliced ​​using FLASH version 1.2.7 software according to the following criteria: (1) Within the 50 bp sliding window, 300 bp fragments at any site with an average quality score less than 20 were truncated, and fragments shorter than 50 bp and fragments containing ambiguous characters were discarded; (2) Only overlapping sequences with a length greater than 10 bp were assembled according to the overlapping sequences, and the maximum mismatch ratio of the overlapping region was 0.2. Reads that could not be assembled were discarded; (3) Samples were distinguished based on barcodes and primers, and the sequence direction was adjusted based on exact barcode matching or a maximum of two nucleotide mismatches in primer matching.

[0121] UPARSE version 7.1 software was used to cluster operational taxonomic units (OTUs) with a 97% similarity cutoff, and chimeric sequences were identified and removed. RDP Classifier version 2.2 software was used to classify and analyze representative sequences of each OTU according to the SILVA ribosomal RNA database and the algal symbiont genome resource database, with a confidence threshold of 0.7. After removing rare sequences, the Alpha diversity index (including observed OTUs) and richness estimation indices (such as Ace, Chao, Shannon, and Simpson indices) were calculated based on the frequencies of OTUs and genera in the specified sequence collection. T-tests were used to analyze OTUs and compare the abundance differences at the genus level.

[0122] Experimental Results

[0123] The chlorophyll fluorescence quantum yield of coral symbionts is an important indicator of the photosynthetic efficiency of coral symbionts. Therefore, this indicator is also commonly used to show the health of corals. When testing healthy corals under light conditions, this indicator is usually between 0.4 and 0.6. The higher the value, the healthier the coral. When the value decreases and exceeds the normal range, it often indicates that the symbiotic algae in the coral symbionts are under environmental stress, resulting in a decrease in photosynthetic efficiency. Figure 7As shown in the figure, at the beginning of the experiment, the Qy value of the coral fragments in each experimental group fluctuated with the regular intervals of light. Except for the samples in group C, they all recovered to the normal level of about 0.45 in the afternoon of the second day, while the Qy value of the corals in group C infected with low concentrations of Vibrio only recovered to 0.323±0.052. As the experiment progressed, 42 hours after Vibrio infection, the Qy value of the corals in the blank group remained at 0.340±0.009, and both groups CE infected with Vibrio showed varying degrees of bleaching symptoms.

[0124] Interestingly, the severity of coral bleaching did not show a direct concentration effect with Vibrio infection. High concentrations of Vibrio led to subhealthy coral symbionts with a Qy of 0.215±0.032 (P value of 0.120), while low concentrations of Vibrio infection led to more severe bleaching with a Qy value of 0.174±0.024 (P value of 0.017, significantly different from the blank group). As predicted, although the QQ intervention slightly led to a decrease in the Qy value of group B corals, it did not affect the health of the corals (P value of 0.461 compared with the blank group). However, in terms of inhibiting the pathogenicity of Vibrio, the quenching intervention treatment of AiiA alleviated the bleaching symptoms of the symbionts at two different Vibrio infection concentrations.

[0125] These results indicate that the health of the coral symbiont is highly correlated with the invading Vibrio, possibly because the symbiotic zooxanthellae are subjected to biological stress under Vibrio infection, resulting in a decrease in photosynthesis efficiency; while low concentrations of Vibrio show stronger pathogenicity. Considering the known regulatory mechanism of Vibrio QS on its pathogenicity, this phenomenon may be due to the fact that Vibrio QS plays a major regulatory role in pathogenicity at low concentrations.

[0126] We then used fluorescence microscopy to calculate the number of zooxanthellae in the coral symbiosis and obtained the density of the coral symbiotic zooxanthellae per unit area. Figure 8 As shown in the figure, although Vibrio infection has caused stress to the coral symbiotic zooxanthellae, there is no difference in the density of zooxanthellae in the symbionts of each group of corals, indicating that the zooxanthellae have not yet been freed from the symbionts. This indicates that in the initial stage of bacterial infection, the biological stress caused by Vibrio infection is still in a reversible state. It may be possible to relieve the stress on zooxanthellae by inhibiting the Vibrio pathogens to restore the health of corals. This also indirectly proves the feasibility of the quorum sensing quenching intervention strategy proposed in this project for biological control of coral pathogens.

[0127] Based on the ITS2 sequence of zooxanthellae characteristics, we analyzed the zooxanthellae structure in the coral symbiont, such as Fig. 9As shown in the results of α analysis in (a), the diversity and richness of zooxanthellae in the symbionts did not change significantly under Vibrio infection and QQ treatment. However, further analysis of the species and structure of the symbiotic zooxanthellae community based on community abundance, such as Figure 7 As shown in the figure, although the zooxanthellae in the blank group of porphyroblasts were mainly C-type, and did not show significant changes under the influence of low-concentration Vibrio infection, under high-concentration Vibrio infection (Group E), the zooxanthellae of D-type in individual coral fragments showed a certain increase in proportion. At the same time, the intervention of quenching enzyme AiiA alleviated this trend.

[0128] The analysis results of zooxanthellae structure also confirm the inference based on the previous analysis of zooxanthellae density, that is, the Vibrio infection in this experiment has no significant effect on the structure of zooxanthellae in the coral symbiont.

[0129] We performed 16S rRNA high-throughput sequencing on the total DNA of the coral symbiotic microorganisms in each group, used RDP 2.2 classifier software to classify and analyze the representative sequences of each OTU, and compared the community structure of coral symbiotic microorganisms at the genus level. According to the annotation results of OTU, ANOVA was used to compare multiple groups of samples, and T test was used to compare two groups of samples to analyze the statistical differences between the treatments of each group. The results are indicated by * 0.01≤P≤0.05, ** 0.001≤P≤0.01, ** P≤0.001, and *** P≤0.001.

[0130] According to the bacterial 16S rRNA sequencing results, Fig.10 As shown in the data, the diversity of the bacterial community of coral symbionts decreased slightly under the inhibition of the quorum sensing quenching enzyme AiiA, but the difference was not significant. The Shannon index decreased from 4.38±0.74 to 4.08±1.35, and the Simpson index increased from 0.06±0.04 to 0.09±0.10, indicating that the diversity of coral symbionts decreased slightly under the quenching enzyme treatment; the Ace index increased from 423.25±87.25 to 431.43±434.47, and the Chao index increased from 428.22±91.88 to 437.32±437.90, both indicating that the richness increased slightly after the quenching enzyme treatment.

[0131] The effect of Vibrio infection was more serious than that of quenching enzyme treatment, and its diversity showed a concentration-dependent decrease, while richness did not. The results showed that biodiversity decreased significantly and was concentration-dependent, with the Shannon index being 2.68±0.85 (P value was 0.01542) at low concentrations and 2.14±0.31 (P value was 0.005528) at high concentrations. Interestingly, the effect on bacterial richness was independent of concentration. At low Vibrio concentrations, the Ace and Chao indices were significantly reduced to 226.10±91.15 (P value was 0.01395) and 226.65±91.58 (P value was 0.0149), respectively, while at high Vibrio concentrations, the Ace and Chao indices were 269.50±198.03 and 269.50±198.03, respectively.

[0132] Predictably, treatment with the quenching enzyme AiiA alleviated the damage of Vibrio infection at both concentrations, with Shannon, Simpson, Ace and Chao indices all showing an improvement trend, with no significant difference compared with the blank group.

[0133] The results of alpha analysis of the symbiotic bacterial community showed that the impact of Vibrio infection on bacterial diversity showed a concentration effect, that is, the higher the Vibrio concentration, the greater the impact; Vibrio infection had little effect on the richness of symbiotic bacteria; intervention with the quenching enzyme AiiA did lead to a decrease in the abundance of some bacterial communities, but it was more effective in reducing the damage caused by Vibrio infection.

[0134] The analysis results of the bacterial community structure showed that the dominant bacterial community changed under the action of quenching enzyme treatment and Vibrio infection. Fig.11 As shown in (a), the original bacterial community in the blank group of polyp symbionts was mainly composed of the following active bacterial genera (average abundance > 1.0%), including Paracoccus (8.18%), Acinetobacter (7.47%), Shimia (6.96%), Tropicobacter (6.92%), Delftia (4.21%), Stenotrophomonas (2.08%), Bacillus (1.80%), Pseudomonas (1.49%), Alteromonas (1.13%), and Epibacterium (1.10%).

[0135] Fig.11(b) Lefse analysis was performed on the samples of the blank group and the quenching intervention group. The results are as follows: under the intervention of the quenching enzyme AiiA, the proportion of Shimia (P value was 0.0342) and Sphingomonas (P value was 0.0373) in the symbiotic bacteria of the pore coral decreased significantly, indicating that these two bacterial groups are easily affected by external quorum sensing regulation and can be used as biomarkers for fluctuations in the bacterial community structure under the intervention of the quenching enzyme.

[0136] It is worth noting that although the intervention of quenching enzyme AiiA brought some effects on the bacterial community, compared with the regulation of quenching enzyme, Vibrio infection had a greater impact on the structure of symbiotic bacterial community. Moreover, under different concentrations of Vibrio infection, the changes in the structure of symbiotic bacterial community showed two different trends.

[0137] like Fig.12 As shown in the figure, the statistical results of one-way ANOVA analysis of variance showed that compared with the proportion of bacterial abundance in the blank group: in the low-concentration Vibrio group (group C), the genus Sulfitobacter increased from 0.9189±1.174 to 2.833±4.5581, while in the high-concentration Vibrio group (group E), the genus Sulfitobacter further increased to 42.16 (P value was 0.005337); in the low-concentration Vibrio group, the genus Acinetobacter decreased from 7.468±0.9822 to 5.57±5.461, and in the high-concentration Vibrio group, it was 1.129±1.036 (P value was 0.0001617), and the genus Stenotrophomonas decreased from 2.079±0.495 to 0.56 in group C, respectively. 91±0.7222 and 0.3321±0.3375 in group E (P value was 0.006209), Corynebacterium decreased from 1.702±0.3852 to 0.4015±0.4284 in group C and 0.2596±0.222 in group E (P value was 0.003435), Sphingobacterium m) decreased from 0.434±0.1239 to 0.1857±0.2208 in group C and 0.08001±0.1228 in group E (P value was 0.0353), and Methylobacterium decreased from 0.3±0.07197 to 0.1223±0.2004 in group C and 0.01962±0.0398 in group E (P value was 0.002809). The above-mentioned genera with changes in the proportion of bacterial community structure all showed a gradient change trend with the increase of S10-1 infection concentration, and the change value was the largest under high-dose Vibrio infection.

[0138] In addition, the change ratio of some microorganisms showed a concentration-dependent decreasing trend with the concentration of Vibrio infection, e.g. Fig.13 As shown, there were significant differences in the genera Sphingomonas, Rhizobium, Brevundimonas, Burkholderia, Haliangium, Epibacterium, and Enterococcus.

[0139] and strains that differed significantly only when infected with high concentrations of Vibrio spp., such as Fig.14 As shown, in addition to the above-mentioned Sphingobacterium, it also includes Acidibacter, Renibacterium, Haliangium, Devosia, Rhodococcus and an unannotated genus in the Vicinamibacteraceae family.

[0140] Interestingly, not all transferred bacterial abundances varied with the concentration of infecting Vibrio , e.g. Fig.15 As shown in the figure, Amaricoccus, Cognatishimia and another unclassified genus in the Rhodobacteraceae family, Lentilitoribacter, Chryseobacterium, Rubrobacter and an unclassified genus in the Solirubrobacterales and an unclassified genus in the Sphingobacteriales, only the changes in the bacterial community structure of these bacterial groups were closely related to low-concentration Vibrio infection after T-test analysis. Fig.15 As shown in the figure, the abundance of several photoautotrophic bacteria also decreased significantly, including an unclassified genus belonging to the class Chloroplast in the phylum Cyanobacteria, and two unclassified genera belonging to the classes Chloroflexia and Ktedonobacteria in the phylum Chloroflexi. The decrease in their abundance was more correlated with low-concentration Vibrio infection, and was consistent with the changing trend of chlorophyll fluorescence quantum yield of coral symbionts mentioned above, indicating that the photosynthetic efficiency of these photosynthetic bacteria was also affected by Vibrio infection. Considering their ecological functions, the changes in these bacterial groups affected the health of corals.

[0141] In addition, if Fig.16 As shown, the proportion of Serratia, Pseudomonas, Alteromonas, Pseudoalteromonas and Staphylococcus also increased with the infection of Vibrio, and the difference was greater when the concentration of Vibrio was low. Although they did not show significant differences, the data showed that these groups showed large standard deviations in different parallel samples, which may be related to individual differences in coral fragments, but these trends indicate that the invasion of alien Vibrio has led to secondary infection of typical opportunistic pathogens.

[0142] In each experimental group, except for Sulfitobacter, the abundance changes and abnormal proportions of most bacterial groups caused by Vibrio infection were alleviated by the intervention of quenching enzymes, and there was a recovery trend compared with the blank group. These results indicate that the invasion of Vibrio caused a series of coral-related bacterial diseases, among which the significantly increased bacteria may be involved in nutritional metabolism proportional to the invasion concentration of Vibrio, and the known conditional pathogens associated with the pathogenicity of the invasive strains may have the risk of causing secondary bacterial diseases.

[0143] These results suggest that the invasion of S10-1 induces a range of coral-associated bacterial diseases, among which the significantly increased bacteria may be involved in nutrient metabolism proportional to the invasion concentration of Vibrio, and known opportunistic pathogens associated with the pathogenicity of the invading strain may have the risk of initiating secondary bacterial diseases.

[0144] Considering that the bacterial community in aquaria mainly comes from coral symbionts, in addition to exogenous inoculated Vibrio, changes in planktonic bacteria in the water could also reflect the interactions of bacterial communities in the coral symbiosis, either through excretion by coral polyps or through recognition by the immune system.

[0145] like Fig.17As shown in the figure, the effect of quenching enzyme AiiA did not significantly affect the diversity and richness of the excreted bacterial community in the blank group. In terms of diversity, the Shannon and Simpson indexes of the CE group did not change significantly compared with the blank group, but the Shannon index of the DF group was significantly reduced and the Simpson index was significantly increased, indicating that Vibrio infection had no significant effect on the diversity of the excreted bacterial community, while the quenching enzyme may inhibit the pathogenicity or competitiveness of Vibrio, resulting in a decrease in the diversity of bacteria that were freed into the water body due to Vibrio infection. In terms of the richness mainly reflected by the ACE and Chao indexes, the CE group showed a downward trend that was positively correlated with the amount of Vibrio inoculation, and the excreted bacteria decreased with the increase of the amount of Vibrio inoculation, which was consistent with the trend of bacterial community changes in the symbiont, indicating that Vibrio infection did reduce the richness of the symbiotic bacterial community; while the QQ treatment alleviated this phenomenon.

[0146] In terms of the composition of the bacterial community structure, the communities with significant differences in planktonic bacteria between the groups included: Pseudoalteromonas (P value was 4.14E-07), Cognatishimia (P value was 7.34E-06), Acinetobacter (P value was 2.57E-05), Pseudomonas (P value was 0.001234), Brevundimonas (P value was 0.001341), Epibacterium (P value was 0.008066), Vibrio (P value was 0.008139), and Pseudomonas (P value was 0.001697). The trend of community structure changes among the three bacterial groups was consistent with that of coral symbionts, which also confirmed the impact of exogenous Vibrio infection on the bacterial community structure in the coral microecological environment from another perspective.

[0147] As the key effector strain of the experiment, we also evaluated and analyzed the community structure of symbiotic Vibrio under the infection and quenching enzyme regulation intervention of S10-1. Fig.18(a) The genus-level one-way ANOVA showed that although there was no significant difference in the abundance of Vibrio among the groups, an interesting trend was presented, that is, after Vibrio infection, the increase in the relative abundance of Vibrio did not show a linear relationship with the inoculum amount of S10-1: except for the original indigenous strong Vibrio in the blank group, the relative abundance of Vibrio increased in both groups CE after infection with two different concentrations (i.e., doses) of exogenous Vibrio, but the proportion was higher in the low-concentration Vibrio group (group C). In the blank group and the low-concentration Vibrio group, the treatment of quenching enzyme inhibited their proportion, while in the high-concentration group, the treatment of quenching enzyme promoted the increase in the relative abundance of the Vibrio group.

[0148] Subsequently, we found the OTUs corresponding to Vibrio robustus from the total OTU list corresponding to the sequencing results according to the sequence BLAST results, and statistically analyzed their proportions and presented them in Fig.18 (b) The results of its abundance ratio were consistent with the trend of total Vibrio. In the blank group, Vibrio robustus accounted for 0.03019% of the bacterial community, while all Vibrio groups accounted for 0.2245%; with the infection of S10-1, the proportion of Vibrio robustus increased to 1.766% in the low-concentration Vibrio group (this percentage is the total percentage of Vibrio, and other Vibrio groups were not detected); while in the high-concentration Vibrio group, Vibrio robustus only increased to 0.1585%, and all Vibrio groups accounted for 0.1661% of the bacterial community. This result shows that the increase in the abundance of Vibrio may be mainly caused by the introduction of Vibrio robustus; in addition, the invasion of Vibrio robustus seems to have caused interspecies competition with local Vibrio, and it is more serious in the low-concentration Vibrio group; on the other hand, the quenching enzyme treatment seems to have alleviated the competitive inhibition effect. These experimental phenomena can prove that there is potential competition between exogenous strong Vibrio and native coral symbiotic Vibrio groups. The infection of S10-1 forms a competitive inhibition on the native Vibrio groups and shows stronger competitiveness at low concentrations, which is consistent with the inference in the previous article that low concentrations of Vibrio bring higher toxicity.

[0149] To confirm the inference of interspecies competition among Vibrio, we then listed all OTUs annotated to the genus Vibrio and compared their community abundance percentages. Fig.18As shown in (c), the native Vibrio community in the blank group was composed of V. fortis, V. parahemolyticus, V. aphrogenes, V. tubiashii, V. coralliilyticus, V. mediterranei and V. alginolyticus; treatment with quenching enzyme AiiA reduced the proportion of V. parahemolyticus, V. aphrogenes, V. tubiashii and V. coralliilyticus, and increased the proportion of V. mediterranei. However, under the infection of exogenous Vibrio robustus, this species occupied the entire Vibrio community and completely inhibited other Vibrio under low-concentration infection (none of them were detected); under high-concentration infection, exogenous Vibrio robustus also had a significant inhibitory effect on most native Vibrio (none of them were detected). Although the only remaining Vibrio alginolyticus showed an increase in relative abundance during the competition, from 1.64E-05 in the blank group to 1.64E-06, it was still at a disadvantage in terms of the proportion of the Vibrio community structure.

[0150] Similarly, treatment with the quenching enzyme AiiA appeared to alleviate the competitive inhibition of the alien Vibrio infection group. These results suggest that the Vibrio species associated with corals are conditional pathogens or even non-pathogenic bacteria, which may participate in symbiotic activities such as nutrient metabolism related to coral health; competitive inhibition of alien Vibrio may weaken these activities, leading to their opportunistic pathogenesis, thereby causing stress to symbiotic algae and affecting the health of corals.

[0151] Through the indoor controlled infection experiment of exogenous Vibrio robustus on the coral reef, we obtained the following results:

[0152] (1) Infection with different concentrations of Vibrio led to different structural changes in the symbiotic bacterial community.

[0153] From the above experimental results, it can be seen that different concentrations of Vibrio have different effects. Some bacterial communities show a linear change with the increase of Vibrio concentration, while the changes in some bacterial communities are related to the pathogenicity of Vibrio caused by the quorum sensing regulation mediated by AHLs at low concentrations of Vibrio.

[0154] The bacterial species positively correlated with the concentration of Vibrio infection included: Sulfitobacter, Sphingomonas, Rhizobium, Brevundimonas, Burkholderia, Haliangium, Epibacterium, and Enterococcus, and the strains that were significantly different only when infected with high concentrations of Vibrio included Acidibacter, Sphingobacterium, Renibacterium, Haliangium, Devosia, Rhodococcus, and an unclassified genus in the Vicinamibacteraceae family. It is reported that these strains mainly have nutrient metabolism functions such as N, P, and S, which may be related to the competition for nutrients brought by high concentrations of Vibrio. This indicates that with the increase of Vibrio infection dose, the rapid proliferation of Vibrio biomass may form competition with other symbiotic bacteria in the environment in nutritional metabolism, which inhibits the growth of nitrogen metabolism functional bacteria such as the above-mentioned Sphingomonas genus; at the same time, the imbalance of nutritional metabolism caused by competition may cause the symbiotic zooxanthellae to be subjected to biological stress, resulting in the production of osmotic pressure regulators such as DMSP, which indirectly causes the increase in the abundance of Bacillus sulfitum.

[0155] Some genera were independent of Vibrio concentrations and showed greater variation at low concentrations, including: Amaricoccus, Cognatishimia, and another unclassified genus of the Rhodobacteraceae family, Enhydrobacter, Lentilitoribacter, Chryseobacterium, Rubrobacter, and an unclassified genus of the Solirubrobacterales. An unclassified genus of Sphingobacteriales was only associated with low concentrations of Vibrio by T-test analysis; common opportunistic pathogens such as Serratia, Pseudomonas, Alteromonas, Pseudoalteromonas and Staphylococcus also showed an increasing abundance trend with low concentrations of Vibrio; in addition, photoautotrophic microorganisms such as cyanobacteria and Chloroflexus were also inhibited by low concentrations of Vibrio. This suggests that low concentrations of Vibrio infection may lead to different pathogenic mechanisms: the AHLs produced by Vibrio at low concentrations are still within the threshold range of quorum sensing regulation, resulting in higher pathogenicity (toxicity) for its colonization in coral symbionts, while also leading to the synergistic proliferation trend of other Gram-negative opportunistic pathogens.

[0156] (2) Vibrio infection causes biological stress to coral symbiotic zooxanthellae.

[0157] The escape of symbiotic zooxanthellae from the coral symbiont has been widely considered by scholars to be the main cause of coral bleaching. Previous research has focused on abiotic stresses caused by environmental changes, such as global warming, ocean acidification or environmental eutrophication. However, the biotic stress caused by other symbiotic microorganisms in the coral symbiont on zooxanthellae and their interaction mechanisms are not yet fully understood.

[0158] Studies on algal-microbial interactions have shown that phytoplankton-associated bacterial communities are composed of very few groups, including Rhodobacteraceae, Flavobacteraceae and Alteromonadaceae, which is consistent with the results in this study that the bacterial communities such as Sulfatile Bacillus, Pseudoalteromonas and Alteromonadaceae increased with Vibrio infection. This also shows that the algal-bacterial interaction formed by Vibrio infection may also affect the community structure of other symbiotic bacteria.

[0159] Symbiotic microalgae such as zooxanthellae are the main primary producers and the basis of the food web in coral-associated microecological systems. They participate in nutrient cycles and inevitable bacterial-microalgae interactions, including mutualism, competition, and antagonism. In this study, exogenous Vibrio robustus caused biological stress to zooxanthellae, which resulted in a decrease in the quantum yield of chlorophyll fluorescence, which was particularly significant under low concentrations of Vibrio infection. Therefore, we speculate that under the regulation of quorum sensing mediated by its own AHLs, Vibrio exhibited stronger pathogenicity and competitive inhibition on photoautotrophic microorganisms in the coral symbionts. These biological stresses not only acted on zooxanthellae with decreased photosynthetic efficiency, but also on photosynthetic bacteria such as cyanobacteria and Chloroflexus with reduced bacterial abundance. These factors may be the reasons that ultimately lead to the bleaching symptoms of corals.

[0160] As a member of the Rhodobacteraceae family, Sulfitobacter has been reported as a beneficial bacterium for diatoms, but it is also a harmful bacterium for dinoflagellates, such as the vigorous algae species Alexandrium, which produces algicidal compounds after absorbing algal DMSP. As representatives of the Gammaproteobacteria, Pseudoalteromonas and Alteromonas have been reported as harmful bacteria that harm phytoplankton by secreting proteases or other algicides. Considering that Vibrio also belongs to the Gammaproteobacteria and is commonly distributed on the surface of several algae, it is reasonable to speculate that Vibrio infection may cause biological stress to the symbiotic algae and affect their photosynthesis efficiency.

[0161] (3) Exogenous Vibrio infection leads to competition with native Vibrio flora.

[0162] Symbiotic Vibrio in the coral symbionts and changes in the free Vibrio community structure in the water environment all show competition between exogenous Vibrio and native Vibrio groups. Low concentrations of Vibrio infection, on the contrary, lead to a higher proportion of Vibrio community structure and a higher proportion of strong Vibrio groups in the flora, indicating that low concentrations of exogenous Vibrio bring greater pathogenicity, resulting in exogenous invading Vibrio having a greater advantage in the competition of symbiotic flora, and forming a dominant species. Referring to previous research reports, Vibrio will produce stronger toxicity in interspecies competition, which may also be one of the reasons why low concentrations of Vibrio in this experiment lead to more obvious coral bleaching symptoms.

[0163] In summary, different concentrations of exogenous Vibrio infection may form different pathogenic mechanisms for coral hosts: low concentrations of Vibrio lead to stronger pathogenicity under quorum sensing regulation and interspecies competition, while high concentrations of Vibrio cause imbalance in the nutrient cycle in the coral symbiont due to nutrient competition. The quorum sensing quenching enzyme AiiA can inhibit both low and high concentrations of Vibrio robustus infection.

[0164] Example 4: Different concentrations of Vibrio corallilyticus caused bleaching and tissue ulceration in the clump of helmet corals, and the symptoms were alleviated under the intervention of the quenching enzyme AiiA

[0165] Experimental methods

[0166] 1. Vibrio infection coral experiment

[0167] (1) Water conservation

[0168] (2) Coral cutting

[0169] (3) Experimental grouping: A total of 12 aquariums were set up and divided into 12 groups. In each group, 5 to 6 coral fragment samples were placed as biological replicates, namely: Group AF was cultured at 26°C, Group A was a blank group, Group B was a quenching enzyme AiiA control group, and Group C was a low-concentration Vibrio infection group (the final concentration of Vibrio was 10 3 CFU / mL), group D was the low-concentration Vibrio quenching enzyme intervention group (the final concentration of Vibrio was 10 3 CFU / mL, the final concentration of quenching enzyme was 0.05 μg / mL, and group E was a high-concentration Vibrio infection group (the final concentration of Vibrio was 10 5 CFU / mL), and group F was the intervention group with high concentration of Vibrio quenching enzyme AiiA (final concentration of Vibrio was 10 5 CFU / mL, the final concentration of quenching enzyme was 0.05 μg / mL); the af group was subjected to 32℃ temperature stress, and except for the temperature difference, other treatments were consistent with AF.

[0170] (4) Vibrio infection and intervention with quenching enzyme AiiA

[0171] 2. Data Collection and Sample Preparation

[0172] A record of coral health.

[0173] Determination and statistical analysis of chlorophyll fluorescence quantum yield in coral symbionts.

[0174] After the experiment was stopped, the coral fragments were sampled and quickly frozen in liquid nitrogen for later use; the seawater samples in each group were filtered with a 0.22 μm filter membrane (Millipore, USA), and the filter membrane samples with filtered microorganisms were collected. The pre-treated coral fragments and filter membrane samples were then stored at -80°C for further research.

[0175] 3. Preparation of coral tissue homogenate

[0176] (1) Flushing coral tissue: Place a coral fragment sample in a sterile sealed bag, use an Eluan air pump to provide high-pressure air (set the turbine speed to 66.8 rpm), and a Kamoer peristaltic pump connected to sterile water to provide high-pressure water flow (set the water output rate to 26.7 mL / min). Combined with high-pressure water vapor, flush the coral tissue on each side until the tissue on the coral skeleton is cleaned, forming a coral tissue suspension in a sterile sealed bag.

[0177] (2) Tissue homogenization: Grind the washed coral tissue with a tissue grinder for 1 min to fully disperse the tissue suspension in a sealed bag to form a tissue homogenate. Dilute the homogenate with sterile water to 50 mL to obtain coral tissue homogenate, which is then stored in an ice bath until testing.

[0178] 4. Zooxanthellae density detection

[0179] (1) Calculate the surface area of ​​coral: Insert the coral skeleton into the plasticine and fix it, scan it with the Jeatch3D scanner to obtain images of each surface of the coral skeleton, and use the supporting software to calculate the surface area of ​​the coral skeleton.

[0180] (2) Microscopic counting: Take 100 μL of the coral tissue homogenate obtained above and drop it into a plankton cell counting plate. Let it stand for 3 to 5 min. The diluent will fill the entire chamber of the counting plate through liquid penetration. Count the zooxanthellae under a microscope at a magnification of 400 times. Calculate the average value of the zooxanthellae counts in the five chambers in the middle and the edge. After conversion according to the dilution factor, divide it by the surface area of ​​the coral skeleton to obtain the zooxanthellae density of the coral sample.

[0181] Experimental Results

[0182] The superficial phenomena of coral tissue can directly reflect the health status of coral. Fig.19 As shown in the figure, at 0h, the coral tissue on the surface of the clump-shaped helmet coral was intact and in a healthy dark brown state, mainly formed by the zooxanthellae in the symbiont. After the infection of Vibrio, the coral tissue gradually faded over time, showing obvious bleaching symptoms. In addition, the coral tissue showed increasingly severe tissue ulceration, gradually exposing the coral skeleton. By 48h when the experiment was terminated, the tissue ulceration area of ​​the diseased coral individuals had exceeded half, and the remaining part also showed obvious lesions.

[0183] The chlorophyll fluorescence quantum yield of the coral symbiont is an important indicator of the photosynthetic efficiency of the coral symbiont. As in the previous examples, this indicator is also used to show the health of the coral. When healthy corals are tested under light conditions, this indicator is usually between 0.4 and 0.6. The higher the value, the healthier the coral. When the value decreases and exceeds the normal range, it often indicates that the symbiotic algae in the coral symbiont are under environmental stress, resulting in a decrease in photosynthetic efficiency. We first tested the chlorophyll fluorescence yield of the coral symbiont in each group of samples in the experiment. The results are as follows: Fig. 20 As shown, at a normal 26℃ ( Fig. 20 a) Under culture, the fluorescence yield of corals in groups C and E decreased significantly after 40 hours, while the control group (AB group) and the quenching enzyme intervention group (DF group) still maintained a good level; under high temperature stress of 32°C, corals in group C showed more severe bleaching (the fluorescence yield decreased most significantly) after low concentration of Vibrio infection, indicating that under high temperature stress, low concentration of Vibrio may show stronger pathogenicity. Similarly, under the intervention of quenching enzyme, the disease degree of corals was significantly improved.

[0184] We then counted the percentage of coral bleaching and tissue ulceration samples, and the results were as follows: Fig. 20 c and Fig. 20 As shown in Figure d, bleaching only occurred in the experimental group infected with Vibrio lysate, and was alleviated to varying degrees under the intervention of quenching enzyme. Among them, at the normal culture temperature of 25°C, the quenching enzyme treatment effect was better; under the high temperature stress of 32°C, the bleaching rate was still high, but it also showed a certain degree of alleviation, while the bleaching rate caused by high concentration of Vibrio was not high, which was consistent with the results of effective fluorescence yield. For the symptoms of tissue ulceration, although there were a certain number of lesions in the control groups AB, after comparison, the proportion of tissue ulceration after Vibrio infection was higher, and it was most significant in the low concentration of Vibrio infection group. Since the coral samples were impacted by the water flow during the experiment, resulting in individual collisions and damage to the coral skeleton, we speculate that this may be one of the reasons for the occurrence of coral tissue ulceration in all groups (especially the blank control group). From the trend point of view, Vibrio infection caused two different lesions of bleaching and tissue ulceration in the helmet coral, among which low concentration of Vibrio showed stronger pathogenicity. Similarly, these lesions were significantly alleviated under the intervention of quenching enzyme.

[0185] These results show that the health of the coral symbiont is highly correlated with the invading Vibrio, which is not only reflected in the previously reported Vibrio parahaemolyticus and Vibrio schrenkii, but also in the embodiments of Vibrio robustus and Vibrio corallilyticus described in the present invention, indicating that pathogenic Vibrio infection of corals to cause disease is a common phenomenon, which may be due to the fact that the symbiotic zooxanthellae are subjected to biological stress under Vibrio infection, resulting in a decrease in photosynthesis efficiency; while low concentrations of Vibrio show stronger pathogenicity. Considering the known regulatory mechanism of Vibrio QS on its pathogenicity, this phenomenon may be due to the fact that Vibrio QS plays a major regulatory role in pathogenicity at low concentrations; at the same time, applying quorum sensing quenching enzymes to coral symbionts for biological control can not only achieve quorum quenching intervention of Vibrio pathogens, but also effectively inhibit the abnormal flora in the coral symbionts caused by Vibrio infection and secondary infection with other pathogens. Therefore, intervening and regulating Vibrio QS is an effective measure to solve coral bacterial diseases, and the quorum sensing quenching enzymes described in the present invention are feasible for the prevention and control of coral bacterial infections.

[0186] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. Application of quorum sensing quenching enzyme in the prevention and control of coral Vibrio pathogens.

2. Use of the quorum sensing quenching enzyme according to claim 1 in the preparation of medicines or biological products for preventing and treating Vibrio corallii infection.

3. The use according to claim 1 or claim 2, characterized in that: The working concentration of the quorum sensing quenching enzyme is not less than 1 ng / L.

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