Vibrio alginolyticus HY9901 was isolated from a diseased red snapper (Lutjanus sanguineus) in Zhanjiang Port, Guangdong Province (37). The strains and plasmids used in this study are in Table 1. All primers were designed as shown in Table 2 according to the V. alginolyticus gene sequence (GenBank Number: GU074526.1) and the pykF gene (GenBank Number: OK376642). Healthy zebrafish (Danio rerio) were purchased from Zhanjiang Aquatic Market, with an average body length of 4 cm and weight of 0.2 g. The zebrafish were performed by bacteriological recovery tests and temporary rearing in seawater in a circulation system at 28°C for 2 weeks before the experiment.

The protocol of LS-MS/MS analysis was performed by following the protocol described in previous studies (38, 39). Purified PykF proteins were fractionated on a 4–20% SDS-PAGE gel. For in-gel tryptic digestion, gel pieces were destained in 50 mM/L NH₄HCO₃ in 50% acetonitrile (v/v) until clear. Gel pieces were dehydrated with 100 μl of 100% acetonitrile for 5 min, the liquid removed, and the gel pieces rehydrated in 10 mM/L dithiothreitol and incubated at 56°C for 60 min. Gel pieces were again dehydrated in 100% acetonitrile, liquid was removed and gel pieces were rehydrated with 55 mM/L iodoacetamide. Samples were incubated at room temperature, in the dark for 45 min. Gel pieces were washed with 50 mM/L NH₄HCO₃ and dehydrated with 100% acetonitrile. Gel pieces were rehydrated with 10 ng/μl trypsin resuspended in 50 mM/L NH₄HCO₃ on ice for 1 h. Excess liquid was removed and gel pieces were digested with trypsin at 37°C overnight. Peptides were extracted with 50% acetonitrile /5% formic acid, followed by 100% acetonitrile. Peptides were dried to completion and resuspended in 2% acetonitrile/0.1% formic acid.

The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.). The gradient was comprised of an increase from 7 to 25% solvent B (0.1% formic acid in 98% acetonitrile) over 18 min, 25 to 38% in 6 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 450 nl/min on an EASY-nLC 1000 UPLC system.

The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in OrbitrapFusion (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1,550 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 60,000. Peptides were then selected for MS/MS using NCE setting as 35 and the fragments were detected in the Orbitrap at a resolution of 15,000. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The peptides eluted from the HPLC column/electrospray source were subjected to MS survey scans. Raw MS/MS data were used to search a user-defined amino acid sequence database with the Proteome Discoverer 1.3 program. Cysteine Alkylation was used as a fixed modification, while lysine acetylation was set as the variable modification.

The gene of pykF and their variants were cloned into the pET-28a plasmid with 6 xHis-tag and transformed into BL21 (DE3) cells (CD601, TransGen Biotech, Beijing, China) for expression and purified using BeyoGold™ His-tag Purification Resin (P2218, Beyotime, Shanghai, China) according to the manufacturer's recommended procedures. Site-directed mutagenesis of the pykF was performed using a Fast MultiSite Mutagenesis System kit (FM201-01, TransGen Biotech, Beijing, China) following manufacturer's protocol.

According to previous studies (40, 41), all deletion mutants and complemented strains were made. Primer pairs used for plasmid construction were included in Table 2. Overlap extension PCR was applied to generate an in-frame deletion of the pykF gene on the V. alginolyticus wild-type HY9901 chromosome. Two about 500 bp PCR fragments corresponding to genomic sequences flanking pykF for chromosomal in-frame deletions. Chromosomal in-frame deletions were cloned into a suicide plasmid (pLP12) by using standard cloning procedures followed by DNA sequencing. The resulting constructs were individually transformed into E. coli S17-1 λpir and introduced by conjugation into V. alginolyticus. Deletion mutants were selected on 10% sucrose TSA plates. Its presence was subsequently confirmed by PCR using primers located inside of the deleted sequence and subsequent sequencing of the PCR product.

For the construction experiments of the complemented strain, the pykF and its variants were cloned into expression vector pMMB207, which incorporates a C-terminal His-tag by PCR. The Fast MultiSite Mutagenesis System kit was used to perform site-directed mutagenesis of pykF with the corresponding primers (Table 2). These constructs were fully sequenced to check their inserts and then introduced by conjugation into the appropriate mutant strains. Then, all the resulting target mutations were confirmed by DNA sequencing. In addition to this, His Tag Mouse Monoclonal Antibody was used to confirm the normal expression by Western blot (described below).

Purified PykF and its variants were quantified using the detergent compatible Bradford protein assay kit (P0006, Beyotime, Shanghai, China). Pyruvate kinase enzymatic activity was measured using the pyruvate kinase Activity Detection Kit (BC0540, Solarbio, Beijing, China) according to the manufacturer's instruction. Pyruvate kinase catalyzes phosphoenolpyruvate and ADP to generate ATP and pyruvate, and lactate dehydrogenase further catalyzes NADH and pyruvate to generate lactate and NAD⁺. The decrease rate of NADH can be measured at OD340nm to reflect PykF activity.

For in vitro tests, the protocol of the deacetylation assay was performed by following described in previous studies (22–24), the reaction of the deacetylation was performed in the buffer containing 50 mM HEPES (pH 7.0), 5 mM MgCl₂, 1 mM NAD⁺, 1 mM DTT, and 10% glycerol. The deacetylation reaction was initiated by mixing 5 μg PykF, 5 μg CobB proteins in a total volume of 100 μL and incubated at 37°C for 1 h. The acetylation level of the treated proteins was analyzed by Western blot (described below).

For in vivo tests, the acetylation level of the native PykF purified from the WT:pykF or ΔcobB:pykF of V. alginolyticus grown in TSB medium was determined by Western blot (described below).

For in vitro tests, the protocol of the acetylation assay was performed by following described in previous studies (22–24), the reaction of the acetylation was performed in the buffer containing 50 mM HEPES (pH 7.0), 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 10 mM sodium butyrate. The acetylation reaction was initiated by mixing 5 μg PykF varied concentrations of AcP in a total volume of 100 μL and then incubated at 37°C for 1 h. The acetylation level of the treated proteins was analyzed by Western blot (described below).

The method of detection of growth was performed by following described in previous studies (41). All strains were incubated separately in TSB overnight and all diluted to (OD₅₉₅ = 0.2), then inoculated at 28°C at a ratio of 1:100 and determined OD₅₉₅ every hour. All measurements were repeated three times per group.

The method of detection of extracellular protease activity was performed by following described in previous studies (41). All strains were coated on TSA plates coated with sterile cellophane, and cultured at 28°C at 24 h, washed with sterile cooled PBS, centrifuged at 4°C for 30 min, and the supernatant filtered to obtain extracellular products. All measurements were repeated three times per group.

The LD₅₀ evaluation was performed by following described in previous studies (41). The injection concentrations used for the dose response of wild-type strain HY9901, ΔpykF, and all complemented strains were 10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ CFU/mL. A total of 930 fish were randomly divided into seven groups (Table 3). The water temperature was adjusted to 28°C. The experiment was repeated three times. Five microliter of bacterial solution was injected into fish by intramuscular injection. The control group was injected with equivalent volumes of PBS. Fish were monitored for 14 days or until no morbidities occurred.

Protein concentrations were determined by Bradford Protein Assay (P0006C, Beyotime, Shanghai, China). For Western blot, purified PykF and its variants were fractionated on a 4–20% SDS-PAGE gel and transferred to the PVDF membrane by Trans-Blot Turbo (Bio-rad, USA). The samples were blocked with QuickBlock™ Blocking Buffer for Western blot (P0239, Beyotime, Shanghai, China) overnight at 4°C and then incubated with the horseradish peroxidase (HRP)-conjugated acetyl lysine antibody (9441S, Cell Signaling Technology, USA) as primary antibodies. The solutions were diluted at a ratio of 1: 2000 in QuickBlock™ Primary Antibody Dilution Buffer for Western blot (P0239, Beyotime, Shanghai, China) for 2 h at room temperature. Afterward, the membranes were washed three times in TBST and incubated with HRP-labeled goat anti-rabbit IgG (H+L) (A0208, Beyotime, Shanghai, China) at room temperature for 1 h. Antigen–antibody complexes were detected via the enhanced chemiluminescence method (P0018, Beyotime, Shanghai, China).

Western blot images were quantified using Image J software. The experimental data were analyzed by single factor analysis of variance (ANOVA) with SPSS17.0 software and GraphPad Prism 8. The results in the figures are displayed as the mean ± standard deviation. Differences were considered statistically significant if the P-value was smaller than 0.05. Significance was indicated as ^*P ≤ 0.05; no ^*, P > 0.05, no significance.

The acetylation signal of PykF was detected by Western blot, and then 11 acetylated lysine residues were identified via the previous acetylome profiling of V. alginolyticus and the mass spectrometry data (Supplementary Figures 1–11). Subsequently, 11 lysine residues, namely, K13, K19, K52, K59, K68, K145, K317, K319, K340, K368, and K382 were identified and analyzed to uncover the effects of deacetylated lysine residues on PykF.

Acetylation levels of PykF and its acetylation and deacetylated variants were evaluated. Compared to the acetylation levels of PykF, the acetylation mimicking status of those sites were not significantly different from PykF (Figure 1A top line). But the effect of deacetylation at different sites on acetylation level was different. Compared to the acetylation levels of PykF, the deacetylation mimicking status of K52, K68, K317, and K382 significantly decreased acetylation level, but other deacetylation mimicking status were no significant difference (Figure 1B top line).

Pyruvate kinase activities of PykF and its acetylation and deacetylated variants were evaluated. Compared to pyruvate kinase activities of PykF, the acetylation mimicking status of those sites were not significantly different from PykF (Figure 1A bottom line). But the effect of deacetylation at different sites on pyruvate kinase activities was different. Compared to the pyruvate kinase activities of PykF, the deacetylation mimicking status of K13, K368, or K382 significantly increased the activity, while acetylation at K145 or K319 only had no significantly change. Furthermore, the deacetylation mimicking status of K52 or K317 lost about 80% of pyruvate kinase activity, while the deacetylation mimicking status of K19, K59, K68, or K340 almost eliminated its activity (Figure 1B bottom line).

The principle of lysine site selection for the next experiments was that deacetylated variants had significantly lower acetylation levels and activity than PykF. According to the results 3.1, K52, K68, and K317 were selected to study the changes in pyruvate kinase activity. In this study, WT: pykF expression strain, ΔpykF mutant strain (Figure 6 of Supplementary Files), ΔpykF: K52R, ΔpykF: K68R, ΔpykF: K317R site-directed mutagenesis strains were successful constructed (Figure 2A). Pyruvate kinase activity of ΔpykF was decreased by about 60% compared to wild-type (Figure 2B). Site-directed mutagenesis complemented strains such as ΔpykF:K52R, ΔpykF:K68R and ΔpykF:K317R showed significantly decreased pyruvate kinase activity compared to ΔpykF:pykF complemented strain (Figure 2C). Therefore, the deacetylation status of K52, K68, and K317 was required for pyruvate kinase activity of V. alginolyticus.

The acetyl on the ε-amino group of lysine residues is stable, and its removal needs a category of enzymes called lysine deacetylases (42). So far, only one lysine deacetylase has been identified in bacteria: the deacetylase CobB, a NAD⁺-dependent sirtuin class deacetylase (43, 44). we have verified the function of the CobB protein in V. alginolyticus (45). To confirm the effect of CobB on PykF, Western blot was used to detect the deacetylation of CobB to PykF, and then its enzyme activity was determined. The results showed that the CobB expressed and purified from E.coli BL21 (DE3) cells can deacetylate PykF with the participation of NAD⁺ (Figure 3A top line), and deacetylation of PykF significantly enhanced pyruvate kinase activity (Figure 3A bottom line). For in vivo tests, the acetylation level of the native PykF purified from the WT:pykF or ΔcobB:pykF was determined by Western blot. Deletion of the cobB gene increased the acetylation level of PykF (Figure 3B top line), but it was no significant difference in pyruvate kinase activity (Figure 3B bottom line). In conclusion, CobB can regulate the acetylation level of PykF and pyruvate kinase activity.

So far, it is believed that there are two mechanisms of lysine acetylation in bacteria, the acetyl-CoA-dependent enzymatic process, and the AcP-dependent chemical reaction (11, 12, 14, 31, 46, 47). In order to study the acetylation mechanism of PykF, PykF expressed and purified from BL21 (DE3) cells was treated with AcP at concentrations of 200 μM, 3 mM, and 12 mM, corresponding to estimated intracellular AcP concentrations at the exponential phase, the stationary phase, and the ΔackA background, which accumulates AcP, respectively (30, 48, 49). The immunoblot with anti-AcK antibody and the detection of pyruvate kinase activity indicated that AcP can chemically acetylate PykF in a dose-dependent and time-dependent manner in vitro (Figure 4 top line). Furthermore, with the increase of incubation time, the acetylation level of PykF increased and its enzyme activity decreased correspondingly (Figure 4 bottom line). This suggested AcP can acetylate PykF, resulting in a decrease in enzyme activity of PykF.

To determine the effect of delete or overexpress pykF gene and site-directed mutagenesis at the K7, K52, and K317 on the biological function of V. alginolyticus. WT, ΔpykF mutant strain, and WT:pykF, ΔpykF:pykF complemented strain, and ΔpykF:K52R, ΔpykF:K68R and ΔpykF:K317R site-directed mutagenesis complemented strains were subjected to determine growth and extracellular protease activity. The results showed that the growth rate of the WT:pykF overexpression strain was similar to that of the wild-type strain, while the growth rate of the ΔpykF mutant strain was significantly reduced (Figure 5A). In addition, the growth rate of site-directed mutagenesis complemented strains had no significant change compared to the ΔpykF:pykF complemented strain (Figure 5B). These results indicate that deletion of pykF gene significantly weakened the growth of V. alginolyticus, whereas overexpression of pykF gene and site-directed mutagenesis complemented strains had no significant effect on the growth of V. alginolyticus.

Extracellular protease, as metabolites of bacteria, play an important role as virulence factors in the process of infecting the host. Extracellular proteins have a variety of protease activities, including lecithin, amylase, lipase, and casein. The extracellular protease activity was reduced in the ΔpykF mutant strain compared to the wild-type strain (Figure 5C). Compared to the ΔpykF:pykF complemented strain, site-directed mutagenesis complemented strains ΔpykF:K52R and ΔpykF:K68R showed decreased extracellular protease activity, but extracellular protease activity of ΔpykF:K317R had no significant change (Figure 5D). Among them, site-directed mutagenesis complemented strains ΔpykF:K52R and ΔpykF:K68R showed about a 50% reduction in extracellular protease activity, indicating that deacetylation of these two sites has an important role in protease activity inhibition.

Zebrafish from quarantined stocks recognized as disease-free were used as models to assess the virulence of WT, ΔpykF, and all complemented strains. The results showed that LD₅₀ of ΔpykF was 6 times higher than that of WT strain. Compared with ΔpykF: pykF complementary strains, ΔpykF: K52R and ΔpykF: K68R had about 3 and 4 times of LD₅₀, while ΔpykF: K317R had no significant change (Tables 3, 4). The results showed that the deletion of pykF gene weakened the virulence of V. alginolyticus, and the deacetylation of K52 and K68 sites also weakened the virulence of V. alginolyticus, while the deacetylation of K317R did not significantly affect the virulence of V. alginolyticus.