In this study, the bacteria, plasmids, and primers were listed in Table 1. V. alginolyticus strain HY9901 was separated from a sick maricultured grouper (Epinephelus coioides) at Zhanjiang, China, and stored in our laboratory. V. alginolyticus was grown at 28°C on Tryptic soya broth (TSB) medium supplemented with 2% NaCl (Haling, China). Escherichia coli (E. coli) strains were cultured at 37°C in Luria broth (LB) (Haling, China). Ampicillin (Amp, 100 μg/ml) or chloramphenicol (Cm, 25 μg/ml) were supplemented when the optimal antibiotics were needed.

Pearl gentian groupers (♀Epinephelus fuscoguttatus × ♂ Epinephelus lanceolate, 30.0 ± 3.0 g) were purchased from a local fish farm from Donghai island, Zhanjiang and maintained in seawater at a temperature of 28 ± 1°C in a recirculation system for 2 weeks. Fishes were considered healthy by bacteriological recovery tests as described previously (Pang et al., 2022).

Before the extraction of tissues after the cessation of the experiment, fishes were anesthetized with MS222 (tricaine methanesulfonate, 100 ng/ml). All the fish experiments were approved by the respective Animal Research and Ethics Committees of the Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals.

A pair of primers pBAD-VAGM001033-F/pBAD-VAGM001033-R were designed as shown in Table 1 according to V. alginolyticus genomic sequences (GenBank number: CP072781-CP072782). PCR has conducted following the conditions: denaturation at 95°C for 5 min, followed by 38 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 30 s in a Thermocycler (Bio-Rad, Hercules, CA, United States). The PCR product was examined on 1% agarose gel, then was purified and ligated into the pBAD-HisA vector. The recombinant plasmid was transformed into E. coli DH5α and subsequently E. coli BL21. The inserted fragment was sequenced by Sangon Biological Engineering Technology & Services Corporation, Ltd. (Guangzhou, China) using the primers pBAD-TF/pBAD-TR. Finally, the recombinant pBAD-VAGM001033 was inoculated in Congolese red LB agar plates containing different concentrations of inducing agent L-arabinose. The assay was conducted in triplicates.

The ΔVAGM001033 mutant was constructed by homologous recombination techniques described by Chen et al. (2019b). Briefly, the upstream homologous fragment of 524 bp was amplified using a primer (primers: VAGM001033-UF and VAGM001033-UR) and the downstream homologous fragment of 493 bp was amplified using a primer (primers: VAGM001033-DF and VAGM001033-DR) from V. alginolyticus HY9901 genomic DNA. Both fragments contained a 20 bp overlapping sequence were used to generate an in-frame deletion of the VAGM001033 gene by overlap extension PCR. Then, the overlap PCR product was ligated into a suicide T-vector plP12 (Luo et al., 2015), a universal genetic tool for rapid and efficient deleting mutation in Vibrio species, which is based on carrying a novel counterselectable vmi480 marker. The recombinant plP12-ΔVAGM001033 was transformed into E. coli DH5α and subsequently transformed into E. coli β2163. After homologous recombination two times, positive clones were selected and used for the conjugation with V. alginolyticus, and ΔVAGM001033 mutant was successfully constructed using a PCR identification (primers: VAGM001033-TF and VA001033-TR).

The antimicrobial susceptibility tests were conducted using the Kirby–Bauer disc diffusion method (Bauer, 1966) on TSA nutrient agar plates according to Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute [CLSI], 2013) and also following manufacturer’s guidelines. The antibiotic impregnated discs were purchased from Binhe Microbial Reagent Corporation, Ltd., Hangzhou, China, namely, polymyxin B, cefoxitin, cefuroxime, kanamycin, oxacillin, gentamicin, vancomycin, cefuroxime, amikacin, cefoperazone, spectinomycin, ceftriaxone, chloramphenicol, SMZ/TMP, clarithromycin, tetracycline, norfloxacin, ofloxacin, cefixime, cefotaxime, and nitrofurantoin. Vernier calipers were used to scale the diameters of the inhibition zones. The experiments were conducted in triplicate.

For transcriptome sequencing, V. alginolyticus HY9901 and ΔVAGM003125 mutant were cultured in TSB media at 28°C overnight. According to the method of Pang et al. (2022), the bacterial cells were harvested and dissolved in TRIzol (Takara). The followed experiments, namely, RNA extraction, RNA fragmentation, cDNA synthesis, RNA-Seq libraries construction, and bioinformatics analysis were conducted by Gene Denovo Biotechnology Corporation, Ltd. (Guangzhou, China). The results of the transcriptome sequencing have been deposited at the SRA database under accession numbers PRJNA794348.

Vaccination experiments were performed to evaluate the immunoprotectivity of ΔVAGM001033 mutant as a live attenuated vaccine candidate against V. alginolyticus as the previous study of Chen et al. (2019c). Fishes were randomly divided into two groups with 90 fishes per group. Fishes in the ΔVAGM001033 mutant group were injected intraperitoneally with 100 μl 1.0 × 10⁵ CFU/ml ΔVAGM001033 mutant. Control fishes were injected intraperitoneally with 100 μl sterile PBS. The experiment was conducted in triplicates.

Six weeks post-immunization, fishes (n = 30) were anesthetized and challenged separately by 100 μl 1 × 10⁸ CFU/ml of V. alginolyticus HY9901 in each group. The cumulative mortality was recorded for 14 days after the challenge. The relative percent survival (RPS) was calculated according to the formula: RPS = 100% [1 – (mortality of the ΔVAGM001033 mutant group/mortality of the PBS control group)]. The bacteria were reisolated from the liver and spleen of all the dead fishes and identified by 16s rDNA. The experiment was conducted in triplicates.

During the experimental period from 1 to 6 weeks post-vaccination, approximately 200 μl of blood from individual fish (3 fish per group) in the PBS group and the ΔVAGM001033 mutant group are collected. Serum was collected following centrifugation at 15,000 g for 5 min to measure antibody levels using ELISA as the method described by Chen et al. (2019b). Briefly, microtiter plate wells were coated with 100 μl of formalin-killed V. alginolyticus by overnight incubation at 4°C. Excess cells were discarded and wells were blocked with 100 μl of PBS containing 2% bovine serum albumin (BSA) for 3 h at 22°C. After removing the blocking solution and washing three times with PBS added with 0.05% Tween-20 (PBST), the wells were incubated for 3 h at 22°C with 100 μl of serially diluted fish serum. Antibody binding to the antigen was detected using mouse antipearle gentian grouper IgM polyclonal antibody (1:10,000), followed by rabbit antimouse IgG-HRP (Wuhan Boster, Wuhan, China) at 1:20,000 dilutions, and color was developed with a chromogenic reagent tetramethylbenzidine (TMB) (Ameresco, Ltd., Framingham, MA, United States) for 20 min with the reaction being stopped by the addition of 2.0 M H₂SO₄. The plates were then read at 450 nm with a microplate reader (Bio-Rad). The experiment was conducted in triplicates.

After 42 days post-immunization, liver and intestine tissues (3 fish per group) were taken from the PBS group, the ΔVAGM001033 mutant group, and the V. alginolyticus HY9901 group. According to the previous study (Xie et al., 2020), the tissues were fixed in Davidson’s fixative (Shanghai Tarui Bioscience, China) at room temperature for 24 h. Tissues were dehydrated with a series of different concentrations of ethanol, cleared in xylene, and embedded into paraffin. Paraffin blocks of tissues were cut at 5 μm thickness on decontaminated glass slides. After deparaffinization, samples were stained with H&E and observed under an optical microscope (Leica, Germany).

According to the method described by Wei et al. (2020), liver, spleen, head, and kidney (3 fishes per group) were taken from individual fish in the PBS group and the ΔVAGM001033 mutant group at 42 days post-immunization. The real-time quantitative PCR (qPCR) was performed according to standard protocols with the use of the SYBR Green qPCR Supermix Kit (TransGen, China). The final reaction volume of 20 μl contained 1 μl cDNA, 10 μl of 2 × SYBR Green qPCR SuperMix, 1 μl of each primer (10 μM), and 7 μl of ddH₂O. The reaction was performed under the following conditions: 95°C 30 s; 40 cycles for 95°C 15 s, 55°C 20 s, and 72°C 30 s in LightCycler^® 96 (Roche, United States). A melting curve analysis was performed to access the amplification of specific products. The primers for interleukin-16 (IL-16), IL-1, IgM, MHC-Iα, and tumor necrosis factor-α (TNF-α) are shown in Table 1. β-actin was employed as an internal reference. The IL-16, IL-1, IgM, MHC-Iα, and TNF-α gene expressions were normalized to β-actin as relative expression values using the 2^–ΔΔCt method (Pang et al., 2022).

SPSS software version 21.0 was used for statistical analysis (SPSS Incorporation, United States). The data were analyzed using one-way ANOVA. Duncan’s test was used to test mean comparisons. For identifying significant differences between groups, data are reported as mean ± SD and analyzed by the Student’s t-test. When p < 0.05, the differences were judged significant.

The recombinant vector pBAD-VAGM001033 was successfully constructed and confirmed by PCR identification and DNA sequencing as shown in Figure 1. The VAGM001033 gene consists of an ORF of 1,035 bp, encoding 344 amino acids with a GGDEF domain from amino acid 184–344. With the increase of the concentration of the L-arabinose, the red-stained, dry, and rough colonies were observed (Figures 2A–C), indicating that VAGM1033 synthesizes c-di-GMP as a diguanylate cyclase.

The VAGM001033 gene (locus in Chr1: 1104559-1105593) consists of an ORF of 1,035 bp, encoding 344 amino acids with a predicted GGDEF domain from amino acid 184-344. The ΔVAGM001033 mutant was constructed by deleting the total ORF of the ΔVAGM001033 gene based on genetic tools. The result of the successfully constructed ΔVAGM001033 mutant was achieved without altering the remaining sequences by a PCR identification (Figure 3) and direct DNA sequencing.

Morphology was observed by transmission electron microscope (TEM) and no discernible morphological difference was observed between the wild type and ΔVAGM001033 mutant. Both the wild type and ΔVAGM001033 mutant had a rod shape and a single polar flagellum (Figure 4). ΔVAGM001033 mutant showed a similar growth curve with the wild-type strain when cultured in the TSB medium (Figure 5). Wild type and ΔVAGM001033 mutant grew in the exponential growth phase for 0–6 h and reached the stationary phase at 15 h (OD₆₀₀ ≈ 2.0).

The swarming circle diameter of ΔVAGM001033 mutant (39.33 ± 0.57 mm) was significantly bigger than that in the wild type (Figure 6 and Table 2), indicating that the swarming ability of ΔVAGM001033 mutant was significantly enhanced (p < 0.01). However, biofilm formation of VAGM001033 mutant (0.29 ± 0.02) had significantly decreased when compared with that of the wild type (p < 0.01) (Table 2). The extracellular protease activity of ΔVAGM001033 mutant (0.188 ± 0.02) was significantly lower than that of the wild type (0.308 ± 0.02) (p < 0.05) (Table 2). The LD₅₀ value of ΔVAGM001033 mutant was 100-fold higher than that of the wild type (Table 2), indicating that the virulence of ΔVAGM001033 mutant was significantly decreased (p < 0.01). All the dead groupers revealed symptoms of vibriosis specifically characterized by hemorrhaging, skin lesions, and swelling in the liver and kidney. Re-isolated bacteria taken from the liver and spleen tissues were identified as V. alginolyticus by 16S rDNA. None of the fish died or were diseased in the control group.

The results of the antimicrobial susceptibility are given in Table 3. Both the wild type and ΔVAGM001033 mutant were sensitive to spectinomycin, ceftriaxone, chloramphenicol, SMZ/TMP, clarithromycin, tetracycline, norfloxacin, ofloxacin, and cefixime but resistant to polymyxin B, cefoxitin, cefuroxime, kanamycin, oxacillin, gentamicin, vancomycin, amikacin, and cefoperazone. In contrast, the mutant was sensitive to cefotaxime and nitrofurantoin, while the wild type was intermediate.

To compare the transcriptomic profiles of the wild-type strain and ΔVAGM001033 mutant, RNA-seq was conducted. A total of 756 differentially expressed genes (DEGs) were identified between these two strains. Of these DEGs (sequence accession number PRJNA794348), 109 were upregulated and 647 were downregulated in ΔVAGM001033 mutant (p < 0.05, more than 2-fold enrichment, Figure 7A). Genes associated with flagellar assembly were identified as the most enriched pathway according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (Figure 7B). Two-component system, ATP-binding cassette (ABC) transporters, and peptidoglycan biosynthesis were also significantly enriched (Figure 7B). Transcriptomic profiling analysis indicated that VAGM001033 regulated multiple biological pathways, namely, flagellar motility, two-component system, ABC transporters, and peptidoglycan biosynthesis.

Enzyme-linked immunosorbent assay experiment was performed to measure the specific antibody titer of V. alginolyticus in fish of ΔVAGM001033 mutant and the PBS group. The result showed that specific antibodies in the ΔVAGM001033 mutant group could be detected at the first week after the vaccination. The specific antibody titers of fish immunized with ΔVAGM001033 mutant were significantly higher than those in the control group. During weeks 1 to 4 post-vaccination, log₂ (antibody titers) in the sera of ΔVAGM001033 mutant group all reached above 4.0 and the maximum reached 9.0, while that of the PBS group was always only 2.0 to 3.0. Comparison of the means of log₂ (antibody titers) between ΔVAGM001033 mutant and PBS control groups revealed a statistically significant difference (p < 0.05) (Figure 8).

Groupers injected with V. alginolyticus exhibit sluggish behavior, inactive movements, curve or twirl their body. No clinical signs were observed in the ΔVAGM001033 group and the PBS group. Histopathological analysis was conducted for the further observation that no histological abnormality was detected between the ΔVAGM001033 mutant group and the PBS group. Nevertheless, distinct histological changes were observed in the V. alginolyticus HY9901 group (Figure 9). The intestine section of the V. alginolyticus HY9901 group revealed degeneration and necrosis of the mucosal epithelium, destruction of the intestinal microvilli, and cell debris in the lumen (Figure 9A3). The liver section of the V. alginolyticus HY9901 group showed vacuolation, hepatocyte disruption, and hepatic necrosis (Figure 9B3).

Grouper were challenged with V. alginolyticus HY9901 at 42 days post-vaccination. The results revealed that the survival rate in the ΔVAGM001033 mutant group was 84% with an RPS of 82% (Figure 10), which was significantly higher than as observed in the PBS group (p < 0.01).

In this study, qPCR was conducted to evaluate the transcription levels of the immune-related genes. The immune-related genes of pearl gentian grouper, namely, IgM, MHC-Iα, IL-1β, IL-16, and TNF-α was significantly upregulated in the ΔVAGM001033 group compared with those in the control group after vaccination (Figure 11).