Gp8 mediates adsorption of bacteriophage vB_VpP_21JZSM01 to the host Vibrio parahaemolyticus

doi:10.21203/rs.3.rs-8267643/v1

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Gp8 mediates adsorption of bacteriophage vB_VpP_21JZSM01 to the host Vibrio parahaemolyticus

https://doi.org/10.21203/rs.3.rs-8267643/v1

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In this study, we aimed to identify the principal tail protein responsible for mediating adsorption of bacteriophage vB_VpP_21JZSM01 to its host bacterium Vibrio parahaemolyticus, a significant pathogen in aquaculture. The structure and function of three candidate proteins—the tail tubular protein Gp8, tail completion protein Gp20, and tail fibre protein Gp41 — were predicted through bioinformatic analysis. Recombinant plasmids were constructed and the three tail proteins were successfully expressed and purified. Competitive adsorption assays demonstrated an 81.3% reduction in bacteriophage adsorption efficiency following pre-treatment with rGp8, whereas rGp20 and rGp41 showed no statistically significant effects (p > 0.05). Labelling experiments using enhanced green fluorescent protein (EGFP) fusions revealed that only the group treated with rGp8-EGFP exhibited specific fluorescence signals. Furthermore, tight binding of rGp8 to the surface of host bacteria was directly visualised by transmission electron microscopy. Analysis of environmental factors indicated that the adsorption efficiency of rGp8 was optimal at 4 °C (approximately 80%), decreasing to 30% at 75 °C. Neutral pH supported the highest adsorption efficiency (70 – 80%), while strongly acidic (pH ≤ 5) or alkaline (pH ≥ 9) conditions markedly inhibited adsorption, reducing it to below 20%. Our findings identify the tail tubular protein rGp8 as the core functional determinant for host cell adsorption of bacteriophage vB_VpP_21JZSM01, with its adsorption efficiency modulated by temperature and pH.

adsorption

bacteriophage vB_VpP_21JZSM01

phage-host interaction

tail tubular protein

Vibrio parahaemolyticus

Vibrio parahaemolyticus is one of the principal pathogens responsible for seafood-associated diarrhoeal diseases worldwide (Li et al. 2019). Its pathogenicity arises from multifactorial virulence mechanisms, including adhesion, invasion, and immune-evasion strategies (Ghenem et al. 2017). This marine bacterium predominantly resides in coastal waters, seabed sediments, and consumable marine organisms, with seasonal prevalence peaking during summer months (Lopatek et al. 2018). Infections typically manifest as acute gastroenteritis characterised by abdominal cramps, nausea, vomiting, and fever, with immunocompromised individuals (e.g., those with diabetes or liver disease) facing elevated risks of septicaemia (Pazhani et al. 2021). In recent years, the overuse and/or misuse of antibiotics have resulted in the global emergence of an increasing number of multidrug-resistant strains of V. parahaemolyticus. These strains carry genes that mediate multidrug resistance mechanisms, presenting significant challenges to antibiotic treatment (Y. Li et al. 2025). Globally, drug resistance profiles of V. parahaemolyticus show significant geographical variation, with relatively high resistance rates in Asian countries, such as China, Malaysia, and South Korea. Coastal areas of China, with their industrial-scale aquaculture operations, face a serious V. parahaemolyticus multidrug resistance problem attributable to the extensive use of antibiotics (Zhao et al. 2018). Therefore, finding an environmentally friendly, highly efficient alternative to antibiotics has become a significant research focus. Bacteriophages (phages), which are viruses that specifically infect bacteria, may offer a solution to the growing issue of multidrug-resistant bacteria resulting from excessive antibiotic use (Jia et al. 2023).

Phages primarily consist of two major blocks: a head and a tail. The head is composed of a protein capsid that safeguards the internal nucleic acid from damage, while the tail comprises essential structures including a tail tube, tail sheath, tail fibres, and tail spikes, which facilitate attachment and penetration of the host bacteria (Bhella et al. 2023). Following the recognition and adsorption of the bacteriophage tail proteins to the receptors on the host surface, the viral genetic material is injected into the periplasm of the host bacterium. Subsequently, the phage hijacks the host's biosynthetic machinery to drive viral replication and assemble progeny virus particles. These virus particles are then released into the extracellular environment, resulting in host cell lysis (Stone et al. 2019). The tail protein structures and functions of different bacteriophages vary significantly. Therefore, the specificity of a bacteriophage depends on the binding specificity between its tail proteins and host receptors (Gaborieau et al. 2024). The tail fibre assembly (Tfa) proteins derived from the Escherichia coli phages Mu and P2 mediate fibre folding and remain at the distal end of the fibre, becoming a component of the mature phage particle and binding to lipopolysaccharides (LPS) on the bacterial surface (North et al. 2021). In phage T4, the tail fibres exhibit dual receptor specificity, initiating host adsorption via binding to the outer membrane protein OmpC in E. coli K12 strains and to LPS receptors in E. coli B strains (Taslem Mourosi et al. 2022). Hu et al. (2020) demonstrated that tail tubular proteins A (TTPA) and tail tubular proteins B (TTPB) of the Vibrio phage OWB mediate bacteriophage adsorption, enabling the adsorption to the transmembrane protein Vp0980 on the V. parahaemolyticus strain ATCC17802. Andres et al. (2010) showed that the initial adhesion of P22 to Salmonella is mediated by the interaction between LPS and the spike protein of the bacteriophage. In summary, among the molecular determinants of phage – host specificity, the tail proteins play pivotal roles in receptor recognition and adsorption.

We isolated vB_VpP_21JZSM01, a virulent long-tailed bacteriophage infecting V. parahaemolyticus, from seafood samples. Morphological analysis revealed an icosahedral head approximately 55 nm in diameter and a non-contractile tail about 90 nm in length. The phage exhibited a broad lytic spectrum, with an optimal multiplicity of infection (MOI) of 0.01, a latent period of 25 minutes, and a burst size of 180 PFU cell⁻¹. It demonstrated remarkable stability under various temperatures, pH conditions, and chloroform exposure. Additionally, vB_VpP_21JZSM01 significantly inhibited biofilm formation by its host bacterium. Further studies confirmed that vB_VpP_21JZSM01 primarily adsorbs to host cells by recognising surface polysaccharides.

Here, we systematically investigated the structural and functional roles of the three predicted tail proteins (Gp8, Gp20, and Gp41) of the Vibrio phage vB_VpP_21JZSM01. Bioinformatics tools were used to elucidate the molecular signatures of these proteins through evolutionary conservation analysis and functional site prediction. A primary goal of functional genomics is to assign precise biological roles to genes predicted from sequence data. In this study, we addressed this challenge in the context of the phage vB_VpP_21JZSM01 genome, focusing on the functional characterisation of its structural components to elucidate the initial step of infection: host adsorption. Recombinant versions of these tail proteins were then produced using prokaryotic expression vectors and affinity purification. Finally, the dominant roles of these key tail proteins in host adsorption were explored using the double-layer agar plate method, fluorescence protein labelling, and transmission electron microscopy (TEM), revealing their sensitivity to environmental conditions such as temperature and pH. The results of this study offer a theoretical framework for understanding the mechanism underlying phage – host interactions.

Bacterial strains, phages, and plasmids

V. parahaemolyticus Vp1 was cultured at 37 °C in Luria-Bertani (LB) medium supplemented with 3.5% (w/v) NaCl. Bacteriophage vB_VpP_21JZSM01 is preserved in our laboratory (GenBank accession number: OR734989.1). The pCold-GST plasmid (Takara Bio, Dalian, China), which tags proteins with glutathione S-transferase, was used for recombinant tail protein expression, while pET-28a-EGFP (Abiowell Biotechnology, Changsha, China), a plasmid encoding enhanced green fluorescent protein (EGFP), served as the fluorescent protein expression system.

Bioinformatics analysis of phage tail protein

The amino acid sequences of tail proteins were analysed using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for homology searches (Kuznetsov and Bollin 2021). Multiple sequence alignment was performed using the ClustalW algorithm implemented in MEGA 11, followed by phylogenetic reconstruction using the neighbor-joining (NJ) method with 1000 bootstrap replicates. Evolutionary conservation analysis was conducted using SnapGene, while sequence similarity assessment and heatmap visualization were achieved using TBtools-II. Structural characterisation was performed using CavityPlus (http://www.pkumdl.cn/cavityplus) to predict potential ligand-binding pockets and to identify key amino acid residues constituting the putative binding sites (Wang et al. 2023).

Construction of prokaryotic expression plasmids

Using vB_VpP_21JZSM01 genomic DNA as template, the tail genes g8, g20, and g41 were amplified using PCR. The amplified products were purified, double-digested with restriction enzymes, and ligated into the pCold-GST expression vector using T4 DNA ligase overnight at 16 °C. The resulting recombinant plasmids, designated pCold-GST-g8, pCold-GST-g20, and pCold-GST-g41, were verified by sequencing. The PCR primers used in this experiment are listed in Table 1.

Table 1 PCR primer sequences used to amplify tail genes.

Primer name	Base sequence (5’→3’)	Tm (°C)	Amplicon Size (bp)
g8-F	GGAATTCCATATGATGAGAAAGTACAACGAAGATTATGC (Nde I)	59.2	1965
g8-R	CCGGAATTCTTAATTCTGGTCAAATGTCTTGTAAAC (EcoR I)	58.6	1965
g20-F	GGAATTCCATATGATGCTTGATATTATTGAGCTAAACAAA (Nde I)	57.4	567
g20-R	ACGCGTCGACTTATATCACGATAGGGTCGGTAG (Sal I)	65.1	567
g41-F	GGAATTCCATATGATGTCTGATGTAATGCGCAAGATAG (Nde I)	60.8	948
g41-R	CCGGAATTCTTAAACTCGCGCAACAACAATAACG (EcoR I)	62.8	948

Bold underlining indicates restriction sites for the enzymes in parentheses.

Expression and purification of recombinant tail proteins

Plasmids pCold-GST-g8, pCold-GST-g20, and pCold-GST-g41 were transformed into E. coli BL21 (DE3) cells, expressed, and verified by restriction enzyme analysis. For recombinant protein purification, the cells were cultured in liquid LB medium supplemented with 50 μg ml^-1 ampicillin at 37 °C with shaking until the OD_{600 nm} reached 0.6, then induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol l^-1 and incubating at 16 °C for 18 h. The bacterial cells were harvested by centrifugation (4 °C, 12,000 × g, 20 min) and the pellets were resuspended in 5–10 ml Binding Buffer (1% Triton X-100, 150 mmol l^-1 NaCl, 50 mmol l^-1 Tris-HCl pH 7.5, 1 mmol l^-1 EDTA, 1 mmol l^-1 DTT, and protease inhibitors) (Chatzileontiadou et al. 2021). The cell suspension was subjected to ultrasonication on ice at 300 W under pulse mode (2 s working and 3 s stopping) for 15 min (K. Zhang et al. 2021). The supernatant was collected and subjected to affinity purification using Ni-Agarose Resin (Beyotime, Shanghai, China). The bound proteins were eluted with Elution Buffer (10 mmol l^-1 reduced glutathione, 50 mmol l^-1 Tris-HCl pH 8.0). The eluted fractions containing the purified recombinant proteins (rGp8, rGp20, and rGp41) were collected, and their concentrations were determined using a BCA Protein Concentration Assay Kit (Solarbio, Beijing, China).

Western Blot (WB) analysis of recombinant proteins

WB analysis was performed following Tsuji’s method with minor modifications (2020). Briefly, purified recombinant proteins rGp8, rGp20, and rGp41 were separated by SDS-PAGE and subsequently transferred onto methanol-activated PVDF membranes at 180 mA for 1.5 h. After the transfer, PVDF membranes were blocked with 4% (w/v) skimmed milk at room temperature for 2 h. They were then incubated overnight at 4 °C with the primary antibody, ProteinFind® Anti-His Mouse Monoclonal Antibody (TransGen Biotech, Beijing, China). Following washing with TBST (Tris-buffered saline with 0.1% Tween 20), the membranes was incubated with an HRP-conjugated goat anti-mouse IgG (H+L) (Sangon Biotech, Shanghai, China) secondary antibody for 1–2 h at room temperature. After furthe washing with TBST, protein bands were visualised using an enhanced chemiluminescence substrate. To confirm the integrity and position of the recombinant proteins, a parallel SDS-PAGE gel was run with identical samples and stained with BeyoBlue™ Coomassie Blue Super Fast Staining Solution (Beyotime, Shanghai, China).

Preparation of phage lysate

Following the method of Asghar et al. (2022), with slight modifications, a 500 μl aliquot of bacteriophage vB_VpP_l stock (-80 °C) was inoculated into 2216E liquid medium containing 2 ml of V. parahaemolyticus at logarithmic growth phase (OD₆₀₀ _nm = 0.6-0.8). The mixture was incubated at 37 °C with shaking (180 rpm) for 5 h. Subsequent centrifugation (8,000 × g, 10 min, 4 °C) was performed to obtain the supernatant, followed by filtration through a 0.22 μm pore-size membrane filter. The resulting filtrate was collected as the amplified phage lysate.

Competitive adsorption testing of tail proteins

For competitive adsorption inhibition assays, 200 μl of bacterial suspension (10⁹ cfu ml^-1) was mixed with an equal volume of rGp8, rGp20, or rGp41 (diluted in PBS to a concentration of 1 mg ml^-1), or 200 μl sterile LB broth for the control group, and incubated at 37 °C for 30 min. The mixtures were centrifuged (8,000 × g, 4 °C, 5 min), and the resulting pellets were washed three times with PBS to remove unbound proteins before being resuspended in 200 μl PBS. An equal volume of phage proliferation solution was added, incubated at 37 ℃ for 10 min, and centrifuged. The phage titre in the supernatant was quantified using the double-layer agar plaque assay, and the phage adsorption rate was calculated (Gou et al. 2025).

Fluorescent labeling of tail proteins

To obtain recombinant tail proteins fused with EGFP, we ligated the PCR products g8, g20, and g41 in 2.3 with the vector pET-28a-EGFP to produce rGp8-EGFP, rGp20-EGFP, and rGp41-EGFP, respectively. A 100 μl aliquot of bacterial suspension was incubated with an equal volume of rGp8-EGFP, rGp20-EGFP and Gp41-EGFP at 37 ℃ for 30 min, using an untreated bacterial suspension as the control. The mixture was centrifuged (4 °C, 8,000 × g, 10 min), and the pellet was collected, washed three times with PBS, and fluorescence was measured using a spectrofluorometer (JEM1200EX, JEOL, Tokyo, Japan).

TEM of tail proteins

Protein adsorption assays were performed following a modified protocol reported by Li et al (2025). Briefly, the host bacteria were mixed with rGp8, rGp20, or rGp41 and incubated for 30 min. After centrifugation, the pellets were resuspended in 100 μl of PBS buffer. A pure bacterial suspension was used as the control. Copper grids were immersed in resuspension solution for 10 min, and filter paper was used to absorb excess liquid. The grids were then fixed overnight with 2.5% glutaraldehyde. Following removal of the excess liquid, the grids were negatively stained with 2% phosphotungstic acid for 2 min. The staining solution was aspirated, and the grids were air-dried at room temperature before observation using TEM (JEM-F200, JEOL, Tokyo, Japan) with an accelerating voltage of 80 kV.

Effect of temperature on adsorption of tail tubular protein to host bacteria

The temperature-dependent adsorption assay was conducted following a modified protocol adapted from Zhang et al (2018). Briefly, 200 μl aliquots of rGp8 were treated at 4 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃, or 75 ℃ for 10 min. Each sample was then combined with an equal volume of host bacterial suspension in the logarithmic growth phase and incubated for 30 min under static conditions to facilitate protein-mediated bacterial adsorption. An untreated bacterial suspension was used as the control. The suspensions were centrifuged (8,000 × g, 4 °C, 10 min), and the pellets were resuspended in 200 μl medium and mixed with an equivalent volume of phage suspension (7.96×10⁸ PFU ml^-1). After a further 10 min incubation at 37 °C, the mixtures were centrifuged, and the phage titre in the supernatant was quantified using the double-layer agar plaque assay.

Effect of pH on adsorption of tail tubular protein to host bacteria

To assess pH-dependent adsorption characteristics, aliquots of purified rGp8 were mixed with 100 μl of PBS buffer adjusted to pH values of 3, 4, 5, 6, 7, 8, 9, 10, and 11 using 1 mmol l^-1 NaOH or 1 mmol l^-1 HCl. The mixture were incubated for 10 min at room temperature. Each sample was then combined with an equal volume of bacterial suspension （10⁹ cfu ml^-1） and incubated for 30 min at 37 °C to facilitate adsorption. Unbound proteins were removed by centrifugation (8,000 × g, 4 °C, 10 min). The bacterial pellets were subsequently resuspended in 200 μl of medium and mixed with an equivalent volume of phage suspension. After a further10 min incubation at 37 °C, the samples were centrifuged, and the phage titre in the supernatant was quantified using a double-layer agar plaque assay.

Statistical analysis

All tests were carried out in triplicate (n = 3), with data presented as means ± standard deviation (SD). Statistical analysis was performed using SPSS version 29.0 with one-way analysis of variance. Mean values of individual groups were compared using Duncan's test to determine statistical significance, which was indicated by a p ‑ value < 0.05.

Bioinformatics analysis

Phylogenetic analysis of tail proteins was performed using homologous sequences identified via BLAST. In the phylogenetic tree constructed from these sequences, the tail tubular protein Gp8 (WPH64010.1) exhibited the closest evolutionary relationship with the corresponding protein from Vibrio phage VPMS1 (YP008239681.1) (Fig. 1a).

Comparative sequence analysis of proteins within the same phylogenetic clade demonstrated 77.22% amino acid identity between Gp8 and VPMS1 tail tubular protein (Fig. 1b). Multiple sequence alignment revealed high conservation in the C-terminal and central domains (Fig. S1), suggesting that these regions are critical for maintaining structural integrity and biological function. In contrast, the N-terminal region displayed significant sequence variability across species. We hypothesised that the conserved C-terminal domain represents the key recognition motif for host surface receptor binding, while the variable N-terminus contributes to host specificity.

The tail completion protein Gp20 was found to share the closest phylogenetic relationship with the tail completion protein of Vibrio phage 1.029.O.10N.261.55.A7 (AUR82863.1) (Fig. 2a). Sequence similarity between the two proteins reached 91.49% (Fig. 2b), but alignment of homologous sequences (Fig. S2) revealed low amino acid sequence conservation. This suggests that most amino acid residues have undergone evolutionary changes, with only potentially functionally important residues being retained.

The Gp41 tail fibre protein was found to exhibit the closest phylogenetic relationship with the putative tail fibre protein of Vibrio phage 1.056.O._10N.261.48.C11 (AUR84456.1) and that of phage 393E50-1 (CAH9015475.1) (Fig. 3a), with sequence similarities of 64.13% and 62.22%, respectively (Fig. 3b). Alignment of homologous sequences revealed low amino acid conservation (Fig. S3), suggesting that Gp41 may not play a key role in the adsorption of the phage to its host.

The active site of a protein refers to a specific region on its interior or exterior surface that typically adopts a defined tertiary structure, binds to a ligand, and constitutes a key component of its biological functions (Coleman and Sharp 2010). Identifying ligand-binding pockets on the protein surface is of considerable importance for the functional prediction, drug target selection, and drug design. Gp8 exhibited a strong druggability score of 1070, with pocket dimensions of 25 (X), 18 (Y), and 15.5 (Z) centred at coordinates 11 (X), 14.5 (Y), and 8.75 (Z) (Fig. 4a). In contrast, Gp20 showed a higher druggability score of 2747, with pocket dimensions of 13 (X), 16 (Y), and 13.5 (Z) centred at 184.5 (X), 157.5 (Y), and 140.75 (Z) (Fig. 4b). The corresponding amino acid sites are detailed in Table (Table. S1). No active site was predicted for Gp41.

Expression and purification of recombinant proteins

The genes g8, g20, and g41 of vB_VpP_l were PCR-amplified using the phage genome as a template and cloned into the pCold-GST expression vector (Fig. S4). Large-scale recombinant protein expression in E. coli was carried out, and the target proteins were purified from the culture supernatant. SDS-PAGE analysis confirmed the expected molecular weights of the recombinant proteins: Gp8, rGp8 (100 kDa), rGp20 (51 kDa), and rGp41 (62 kDa) (Fig. 5a). WB analysis further verified the presence of a single specific band corresponding to each recombinant protein (Fig. 5b).

Identification of the key tail protein through competitive adsorption testing

To investigate the role of each recombinant tail protein in phage adsorption, host bacterial cells were pre‑incubated with purified rGp8, rGp20, or rGp41 (1 mg ml^-1) for 10 min at 37 °C prior to exposure to phage vB_VpP_21JZSM01 at a titre of 7.96×10⁸ PFU ml^-1. Phage adsorption efficiency was quantified using the double-layer agar plate method. As shown in Fig. 6, compared with the untreated control, pretreatment with rGp8 significantly reduced phage adsorption by 81.3%, whereas phage adsorption to bacteria pretreated with rGp20 or rGp41 was largely unaffected. This finding indicates that rGp8 blocked phage vB_VpP_l adsorption and infection of host bacteria by occupying the phage‑binding sites, highlighting the critical role of the tail tubular protein rGp8 in this process.

Verification of rGp8 as the key tail protein through fluorescent labeling

The adsorption activity of the recombinant tail proteins was verified indirectly by fusing each with EGFP, which has an emission wavelength of 510 nm (Stepanenko et al. 2004). Following incubation of a suspension of host bacteria with rGp8-EGFP, rGp20-EGFP, or rGp41-EGFP fusion proteins, a distinct peak near the emission wavelength of EGFP would be expected for proteins involved in phage adsorption. As shown in Fig. 7, this fluorescence peak was only observed with rGp8-EGFP. Bacteria incubated with rGp20-EGFP or rGp41-EGFP exhibited fluorescence levels similar to those of the untreated control group, further confirming the tail tubular protein rGp8 as the receptor‑binding protein for host infection by phage vB_VpP_l.

TEM of host adsorption by recombinant tail proteins

TEM was used to observe the adsorption of rGp, rGp20, and rGp41 following incubation of each with host bacteria (Fig. 8). While clear morphological structures of the bacteria were visible in all TEM images, only rGp8 exhibited obvious adsorption to the surface of the host bacterial cells (Fig. 8a). The appearance of bacteria incubated with rGp20 (Fig. 8b) or rGp41 (Fig. 8c) was similar to that of the control bacteria (Fig. 8d), with no evidence of protein adsorption. These results further confirm that the tail tubular protein rGp8 is the key protein mediating phage vB_VpP_l adsorption to its host.

Effect of temperature on adsorption of tail tubular protein to host bacteria

To examine the effect of temperature on the ability of recombinant tail tubular protein rGp8 to influence the phage adsorption to host bacteria, aliquots of rGp8 were pre‑incubated at 4 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃, and 75 ℃ prior to incubation with a host bacterial suspension and subsequent exposure to phage. Results from the double‑layer agar plaque assay (Fig. 9) indicated that pre‑incubation of rGp8 at 4 ℃ resulted in a phage adsorption rate of 20.13%, which was not significantly different from that observed in the competitive adsorption inhibition assay (17.1%; Fig. 6). This suggests that rGp8 retained approximately 80% of its adsorption activity at 4 ℃. However, as the pre‑incubation temperature increased, the binding of rGp8 decreased while the phage adsorption rate rose. This indicates that occupancy of the bacterial surface by rGp8 was most effective at 4 ℃, thereby blocking phage adsorption and lysis of host cells. When rGp8 was pre‑incubated at 75 ℃, the phage adsorption rate reached 78.39%, corresponding to a reduction in rGp8 adsorption to about 20%. In summary, the tail tubular protein of phage vB_VpP_l exhibited the highest adsorption efficiency for host bacteria at 4 ℃, consistent with the findings of Zhang et al (2018).

Effect of pH on adsorption of tail tubular protein to host bacteria

To examine the effect of pH on the ability of the recombinant tail tubular protein rGp8 to influence phage adsorption to host bacteria, aliquots of rGp8 were treated at different pH values (3–11) for 10 min prior to incubation with a host bacterial suspension and subsequent exposure to phage. After removing unbound protein by centrifugation, double-layer agar plaque assays were performed (Fig. 10). At pH 6-8, the adsorption rate of the tail tubular protein ranged from 51.3% to 79.6%, indicating relatively effective adsorption under neutral conditions. The results showed that phage adsorption rates at pH 3-5 and pH 9-11 were not significantly different from those of the control group, suggesting that rGp8 exhibited minimal adsorption to the host bacteria under these pH conditions. This may be attributable to alterations in the charge distribution of rGp8 under acidic and alkaline conditions, where disruption of hydrogen and ionic bonds leads to protein denaturation or conformational changes that impair host recognition (Sahin et al. 2010).

Our findings provide a clear functional annotation for the g8 gene within the vB_VpP_21JZSM01 genome, transforming it from a hypothetical open reading frame into a mechanistically characterised host-recognition protein. This integrated approach—from genomic sequence to in vitro functional analysis—exemplifies how discrete genetic elements determine pivotal phenotypic outcomes in phage biology. Beyond its role as a structural component, Gp8 exhibits evolutionary conservation in its C-terminal domain, which is likely to harbour critical ligand-binding pockets essential for receptor recognition. Collectively, results from competitive adsorption assays, fluorescence labeling, and TEM with rGp8 demonstrated that rGp8 dominates the initial adsorption phase by occupying host surface receptors, effectively blocking phage attachment.

Environmental sensitivity testing of rGp8 indicated that its adsorption is optimal at 4 °C and neutral pH, but is severely impaired under high temperatures or extreme pH conditions, providing important insights for practical applications. These findings suggest that phage-based biocontrol strategies targeting V. parahaemolyticus in seafood or aquaculture should prioritise storage conditions that preserve rGp8 functionality. From an evolutionary perspective, the high conservation of rGp8 across Vibrio-infecting phages (e.g., VPMS1) indicates a shared receptor-binding strategy within this phage group. This raises intriguing questions about the identity of the host receptor—likely a conserved surface protein or polysaccharide—which warrants further structural and genetic investigation. To identify the host receptor for rGp8, future studies could employ cryo-electron microscopy or gene knockout approaches, thereby advancing precision in phage engineering. Furthermore, the methodology established here—namely competitive phage adsorption assays combined with fluorescence protein tracking—provides a robust framework for the rapid identification of adsorption proteins in other phage systems, accelerating the development of phage-based therapeutics against multidrug-resistant pathogens. Our findings elucidate the phage adsorption machinery while establishing a crucial link between fundamental virology and applied microbiology.

Acknowledgments

We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript.

Funding

This work was supported by the Basic Scientific Research Project of Liaoning Provincial Department of Education, P.R.China (LJ212510167019) and the Open Fund of Institute of Ocean Research, Bohai University (BDHYYJY2025002).

Competing Interests

No conflict of interest declared. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Author Contributions

Conceptualization, SX.L and DF.Z.; methodology, SX.L., J.L. and DF.Z.; software, XN.W. and K.L.; validation, SX.L. and K.L.; formal analysis, SX.L.; investigation, J.L. and M.Z.; resources, DF.Z. and JR.L.; data curation, M.Z. and CY.L.; writing—original draft preparation, SX.L. and XN.W.; writing—review and editing, DF.Z. and M.Z; visualization, XN.W. and CY.L.; supervision, K.L.; project administration, DF.Z., XP.L. and JR.L.; funding acquisition, DF.Z. and XP.L. All authors have read and agreed to the published version of the manuscript.

Data Availability

Data will be made available on request. All data generated in this study are available within this article and its supplementary information files.

Ethical Approval

No studies involving human participants or animals were conducted by the authors for this article.

Consent for publication

All authors have read and agreed to the final version of the manuscript. Furthermore, they consent to its submission for publication.

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Gp8 mediates adsorption of bacteriophage vB_VpP_21JZSM01 to the host Vibrio parahaemolyticus

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