Autographiviridae phage HH109 uses capsular polysaccharide for infection of Vibrio alginolyticus.

To identify host receptors of the phage HH109, we assembled a library of approximately 30,000 random transposon insertion mutants derived from V. alginolyticus E110. Eleven mutants were selected as phage-resistant upon initial screening and confirmed for significant reduction of lysis by monitoring the turbidity (OD 600 ) of the bacteria cultures in the presence of the phage and spot testing assays ( Figures S1 A and S1B). Transposon insertion sites determination by sequencing revealed that seven genes ( ugd , wecA , wzc , wzb , argE , JYG29_07705 , and JYG29_22310 ) were involved in transposon insertions in those eleven mutants, with two genes ( ugd and wecA ) represented in multiple mutants ( Figure S1 C). To further validate the screening data, we generated in-frame deletion mutants of seven genes and tested their effects on the phage HH109 infection. Bacterial growth and phage titer assays showed that individual deletion of the four genes, ugd , wecA , wzc , and wzb were not lysed, and phage titer decreased ( Figures 1 A and 1B). In contrast, complementation of those genes restored the phenotype of the wild-type strain ( Figure 1 ). However, deletions of JYG29_22310 , argE , and JYG29_07705 did not affect bacterial lysis caused by the phage HH109 infection ( Figure S1 D), which was inconsistent with the result of transposon insertion, indicating that polar effect occurred due to transposon insertion. Notably, the four genes ( ugd , wecA, wzc , and wzb ) are all annotated to be related to CPS biosynthesis ( Figure S1 C); however, wzc and wzb are not located within the CPS synthesis gene cluster. These data indicated bacterial CPS may be necessary for the phage HH109 infection. Figure 1 Several CPS genes of V. alginolyticus strain E110 are involved in the phage HH109 infection (A) Growth assays of wild-type, deletion mutants, and complemented strains as indicated with or without the phage HH109 infection at 5 h. (B) Phage titer assays in wild type, deletion mutants, and complemented strains. Data are displayed as the means ± SD from three independent experiments. Ns > 0.05, ∗∗∗, p  < 0.0001. Several CPS genes of V. alginolyticus strain E110 are involved in the phage HH109 infection (A) Growth assays of wild-type, deletion mutants, and complemented strains as indicated with or without the phage HH109 infection at 5 h. (B) Phage titer assays in wild type, deletion mutants, and complemented strains. Data are displayed as the means ± SD from three independent experiments. Ns > 0.05, ∗∗∗, p  < 0.0001. To further elucidate whether CPS is involved in the phage HH109 infection, we identified the entire CPS gene cluster from V. alginolyticus E110 based on whole-genome sequencing (GenBank: PRJNA842900). The remaining thirty-four genes within the CPS loci, except ugd and wecA , were then individually or tandemly deleted. The susceptibility of those mutants to the phage was evaluated based on bacterial growth and phage spot testing assays. The results showed that deletions of gt1 , gt6 , and orf2 made the bacterial cells resistant to the phage HH109 infection, while in trans expression of gt1 , gt6 , and orf2 in their deletion mutants restored the susceptibility ( Figures 2 A and S2 C). By comparison, disruption of other genes did not impact phage infection ( Figures S2 A and S2B). The gt1 and gt6 encode glycosyltransferase, and the orf2 encodes an ATP-grasp fold amidoligase family protein. To address why only some of the CPS genes were important for phage HH109 infection, we further analyzed the capsules of both phage-resistant and phage-sensitive mutants using qualitative and quantitative methods. Take ugd as an example, capsule staining showed that the wild-type and complemented strains (Δ ugd : BBR- ugd ) exhibited a halo surrounding a cell, indicating the presence of capsules in the peripheral part; on the contrary, the Δ ugd mutant did not show any halo ( Figure 2 B, upper panel); Meanwhile, scanning electron microscopy (SEM) observation also revealed that the surface of the wild-type and complemented strains was rough whereas the Δ ugd mutant was smooth ( Figure 2 B, upper panel). Similar phenotypes were also observed in the wecA , gt1 , gt6 , and orf2 deletion mutants ( Figure S3 A). As a control, the Δ galU and Δ mbL mutants that remained sensitive to the phage infection were randomly selected for capsule staining and SEM observation, and the data showed the two strains have identical phenotypes to the wild-type strain ( Figure 2 B, lower panel). Furthermore, the quantitative analysis also confirmed that the five mutants (Δ ugd , Δ wecA , Δ gt1 , Δ orf2 , and Δ gt6 ) yielded much lower CPS contents than the wild-type, Δ mbL , and Δ galU strains ( Figure 2 C). Taken together, these results indicated that the deletions of ugd , wecA , gt1 , gt6 , and orf2 significantly reduced CPS yield of V. alginolyticus E110 and in consequence caused diminishing susceptibility to the phage HH109. In contrast, the deletions of the remaining within the CPS loci barely changed the CPS amount. Figure 2 CPS in V. alginolyticus strain E110 functions as the receptor for the phage HH109 adsorption (A) Growth assays of wild type, deletion mutants, and complemented strains as indicated with or without the phage HH109 infection at 5 h. (B) Cell morphology observation of different strains by light microscopy after capsule staining or scanning electron microscopy. The halo surrounding the cells indicates the presence of capsules. Strain names labeled on the top left corner of each picture. (C) Quantitative assay of CPS. Surface capsular polysaccharides produced by each strain as indicated were measured and compared with the wild-type strain. (D) Phage adsorption assays. The number of phages that remained in the supernatant was determined after 10 min adsorption of the phage HH109 on the wild type, deletion mutants, and complemented strains as indicated. Data are displayed as the means ± SD from three independent experiments. Ns > 0.05, ∗∗∗,  p  < 0.0001. CPS in V. alginolyticus strain E110 functions as the receptor for the phage HH109 adsorption (A) Growth assays of wild type, deletion mutants, and complemented strains as indicated with or without the phage HH109 infection at 5 h. (B) Cell morphology observation of different strains by light microscopy after capsule staining or scanning electron microscopy. The halo surrounding the cells indicates the presence of capsules. Strain names labeled on the top left corner of each picture. (C) Quantitative assay of CPS. Surface capsular polysaccharides produced by each strain as indicated were measured and compared with the wild-type strain. (D) Phage adsorption assays. The number of phages that remained in the supernatant was determined after 10 min adsorption of the phage HH109 on the wild type, deletion mutants, and complemented strains as indicated. Data are displayed as the means ± SD from three independent experiments. Ns > 0.05, ∗∗∗,  p  < 0.0001. Capsule constitutes the outmost layer of bacterial cells; therefore, we assume that the CPS of V. alginolyticus E110 is the receptor for phage infection. Consequently, we measured the adsorption rate of the phage HH109 on different deletion mutants. As expected, deletions of ugd , wecA , gt1 , orf2 , and gt6 caused a significant reduction in the phage adsorption compared with their corresponding complemented and wild-type strains ( Figure 2 D). Using a diverse set of mutants (Δ lpxM , Δ waaF , Δ waaA , Δ hldE , Δ rfbB , and Δ rfbA ) in the lipopolysaccharide (LPS) locus, we demonstrated that LPS is not essential for phage HH109 adsorption ( Figure S3 B). Similarly, phage HH109 does not use a protein receptor as proteinase K treatment of cells did not prevent phage binding ( Figure S3 C). Therefore, we suggest that phage HH109 uses the CPS of V. alginolyticus E110 as a receptor for adsorption. Although the phage HH109 causes rapid lysis of strain E110, we noticed regrowth of HH109 infected cultures after 6 h of incubation ( Figure 3 A). The phenomenon was supported by the spot plate assays after overnight incubation ( Figure 3 B), indicating the development of spontaneous phage-resistant mutants. Five colonies were picked, and their phage-resistant phenotypes were confirmed ( Figures S4 A and S4B). In addition, they displaced deficient CPS production ( Figure 3 C). As a result, the phage HH109 was unable to efficiently adsorb to their cell surface compared with the wild type ( Figure 3 D). Subsequently, we carried out a comparative genome analysis between the five phage-resistant isolates and the wild-type strain, showing that four resistant isolates all harbored mutations of single nucleotide substitution (EP1 and EP3), or a 10-bp deletion (EP2), or 19-bp insertion (EP4) in the coding region of wecA . At the same time, EP5 had a single nucleotide substitution in the coding region of orf2 ( Figure 3 E). Thus, our data indicate that phage resistance phenotype in the late stage of HH109 infection arose through the reduction or loss of CPS receptor that caused by gene frameshift in wecA and orf2 . These findings further confirmed that the CPS of the E110 strain is an adsorption receptor of phage HH109 and suggested that the capsule receptors are prone to change by a genetic mutation to develop resistance to phage infection. Figure 3 Mechanism for the emergence of phage-resistant V. alginolyticus isolates under the pressure of the phage HH109 infection (A) Growth curves of V. alginolyticus strain E110 with or without the phage HH109 infection over a 12 h time course. (B) Spot plate assay of strain E110 with the phage HH109 showed the emergence of some bacterial colonies within the large central lysis zone (red arrowheads) at overnight incubation. (C) Quantitative assay of CPS. Surface capsular polysaccharides produced by five phage-resistant isolates (EP1, EP2, EP3, EP4, and EP5) were measured and compared with those in the wild-type strains. (D) Phage adsorption assays. The number of phages in the supernatant was determined after 10 min adsorption of the phage HH109 on EP1, EP2, EP3, EP4, EP5, and WT. ∗∗∗, p  < 0.0001. (E) Mutations in the five phage-resistant isolates derived from V. alginolyticus strain E110. EP1, EP2, EP3, and EP4 all have mutations in the coding region of wecA by substitution, deletion or insertion, while EP5 has a single nucleotide substitution in the coding region of orf2 . Numbers represent gene nucleotide positions, and box colors represent different nucleotides. Blank, deletion; Caret, insertion; gray, stop codon. Mechanism for the emergence of phage-resistant V. alginolyticus isolates under the pressure of the phage HH109 infection (A) Growth curves of V. alginolyticus strain E110 with or without the phage HH109 infection over a 12 h time course. (B) Spot plate assay of strain E110 with the phage HH109 showed the emergence of some bacterial colonies within the large central lysis zone (red arrowheads) at overnight incubation. (C) Quantitative assay of CPS. Surface capsular polysaccharides produced by five phage-resistant isolates (EP1, EP2, EP3, EP4, and EP5) were measured and compared with those in the wild-type strains. (D) Phage adsorption assays. The number of phages in the supernatant was determined after 10 min adsorption of the phage HH109 on EP1, EP2, EP3, EP4, EP5, and WT. ∗∗∗, p  < 0.0001. (E) Mutations in the five phage-resistant isolates derived from V. alginolyticus strain E110. EP1, EP2, EP3, and EP4 all have mutations in the coding region of wecA by substitution, deletion or insertion, while EP5 has a single nucleotide substitution in the coding region of orf2 . Numbers represent gene nucleotide positions, and box colors represent different nucleotides. Blank, deletion; Caret, insertion; gray, stop codon. To identify RBPs of the phage HH109, Interpro ( https://www.ebi.ac.uk/interpro/ ) was utilized to predict the structural domains of phage tail-associated proteins. The tail fiber protein gp02 was identified in the genome since it harbored a pectin_lyase_fold/virulence homologous superfamily, and the domain is essential for the phage KP32 spike proteins gp37 and gp38 ( Figure 4 A), which acted as a depolymerase to degrade Klebsiella pneumoniae capsular polysaccharide via spot testing. 19 The protein structure of gp02 was predicted by AlphaFold2, which overlaps with the structure of phage KP32 spike protein gp38, further supporting the annotation of gp02 protein as a spike protein ( Figure S5 ). Therefore, we constructed and purified the recombinant protein gp02 (fused with His and TF tag) and generated anti-serum against the gp02. We found that when the phage HH109 was pretreated with the anti-gp02 serum, the titer of free phages went down by roughly 65% compared with the regular serum-pretreated and untreated group ( Figure 4 B), and protein competition assay revealed that the phage adsorption was reduced by about 60% after gp02 was blocked by the phage HH109 ( Figure 4 C), indicating that gp02 is the RBP. Notably, the binding of the gp02 to host CPS was observed by immunofluorescence staining ( Figure 4 D), showing that the wild-type strain, not the ugd deletion mutant, was labeled by the HIS-tag antibody (red fluorescence) after they were pre-incubated with the recombinant protein gp02. The wild-type strain was not labeled as a negative control after pre-incubating with the tag protein trigger factor (TF). These findings suggested that the gp02 directly interacts with the host receptor CPS and mediates HH109 infection. To make sure whether the gp02 has polysaccharide depolymerase activity, we quantified the CPS contents of different strains, showing that the content of E110 strain after gp02-treated was similar to that of Δ ugd but significantly lower than those of E110 after TF-treated or untreated ( Figure 4 E); meanwhile, an examination of those strains after capsular staining also revealed disappear of the halo around gp02-treated cells ( Figure 4 F), indicating that the gp02 can depolymerize CPS of E110. Furthermore, the conclusion was supported by SDS-PAGE analysis that CPS degradation required the gp02 in a dose-dependent manner when the purified gp02 and the extracted CPS were incubated together in vitro ( Figure 4 G). In summary, we concluded that the phage HH109 uses the tail spike gp02 to recognize and degrade host CPS to facilitate infection specifically. Figure 4 GP02 acts as RBP to recognize the CPS in V. alginolyticus strain E110 (A) Schematic diagram of phage tail proteins gp02, gp37, and gp38. Gp02, a phage HH109 tail protein, includes a pectin lyase fold/virulence (360–733) homologous superfamily. gp37 and gp38 are both from Klebsiella phage KP32 and have the Pectin lyase fold/virulence homologous superfamily (located in 295–505 and 67–299, respectively). (B) Antibody blocking assay. Anti-gp02 serum was mixed with the phage HH109 for 10 min at RT and then added into the V. alginolyticus strains E110 for 1 h to assess phage titer. The normal serum was used as a control. The phage titer in the supernatant was determined. Data are displayed as the means ± SD from three independent experiments, and significance was determined by a Student’s t test. ∗∗∗, p ≤ 0.0001. (C) Protein competition assay in phage adsorption. V. alginolyticus strain E110 was pre-incubated with the recombinant protein gp02 (with tag TF) or the Tag TF for 10 min and then added into the phage HH109 to evaluate the phage adsorption rate. LB group (without protein pre-incubation) was used as a control, and the adsorption rate was set at 100%. Data are displayed as the means ± SD from three independent experiments, and significance was determined by a Student’s t test. ∗∗∗, p ≤ 0.0001. (D) Immunofluorescence assay showing the binding of GP02 to CPS of V. alginolyticus strain E110. Strains E110 and Δ ugd were pre-incubated with the recombinant protein GP02 (with tag TF) for 30 min and then subjected to an immunofluorescence assay using the HIS antibody as the primary antibody and the Alexa Fluor 594 goat anti-rabbit IgG as second antibody. As a control, the tag protein TF was also included to pre-incubate with strain E110. All samples were examined under a fluorescence microscope at 100×magnification, and red fluorescence indicates the presence of the recombinant protein GP02. (E) Quantitative assay of CPS. CPS was extracted from V. alginolyticus strain E110 after incubation with the recombinant protein GP02 (with tag TF) or the tagger protein TF and quantified by measuring the concentration of uronic acid. The deletion mutant Δ ugd was included as a negative control. Data are displayed as the means ± SD from three independent experiments. ∗∗∗, p  < 0.0001. (F) CPS staining and observation. V. alginolyticus strain E110 was incubated with the recombinant protein GP02 (with tag TF) or the tag protein TF and then visualized under light microscopy after capsule staining. (G) CPS degradation in the dose-dependent effect of GP02. CPS was extracted from V. alginolyticus strain E110 and then incubated with 2-fold serial dilutions of the recombinant protein GP02 (with tag TF) or the tag protein TF. All samples were subjected to SDS-PAGE analysis and alcian blue staining. GP02 acts as RBP to recognize the CPS in V. alginolyticus strain E110 (A) Schematic diagram of phage tail proteins gp02, gp37, and gp38. Gp02, a phage HH109 tail protein, includes a pectin lyase fold/virulence (360–733) homologous superfamily. gp37 and gp38 are both from Klebsiella phage KP32 and have the Pectin lyase fold/virulence homologous superfamily (located in 295–505 and 67–299, respectively). (B) Antibody blocking assay. Anti-gp02 serum was mixed with the phage HH109 for 10 min at RT and then added into the V. alginolyticus strains E110 for 1 h to assess phage titer. The normal serum was used as a control. The phage titer in the supernatant was determined. Data are displayed as the means ± SD from three independent experiments, and significance was determined by a Student’s t test. ∗∗∗, p ≤ 0.0001. (C) Protein competition assay in phage adsorption. V. alginolyticus strain E110 was pre-incubated with the recombinant protein gp02 (with tag TF) or the Tag TF for 10 min and then added into the phage HH109 to evaluate the phage adsorption rate. LB group (without protein pre-incubation) was used as a control, and the adsorption rate was set at 100%. Data are displayed as the means ± SD from three independent experiments, and significance was determined by a Student’s t test. ∗∗∗, p ≤ 0.0001. (D) Immunofluorescence assay showing the binding of GP02 to CPS of V. alginolyticus strain E110. Strains E110 and Δ ugd were pre-incubated with the recombinant protein GP02 (with tag TF) for 30 min and then subjected to an immunofluorescence assay using the HIS antibody as the primary antibody and the Alexa Fluor 594 goat anti-rabbit IgG as second antibody. As a control, the tag protein TF was also included to pre-incubate with strain E110. All samples were examined under a fluorescence microscope at 100×magnification, and red fluorescence indicates the presence of the recombinant protein GP02. (E) Quantitative assay of CPS. CPS was extracted from V. alginolyticus strain E110 after incubation with the recombinant protein GP02 (with tag TF) or the tagger protein TF and quantified by measuring the concentration of uronic acid. The deletion mutant Δ ugd was included as a negative control. Data are displayed as the means ± SD from three independent experiments. ∗∗∗, p  < 0.0001. (F) CPS staining and observation. V. alginolyticus strain E110 was incubated with the recombinant protein GP02 (with tag TF) or the tag protein TF and then visualized under light microscopy after capsule staining. (G) CPS degradation in the dose-dependent effect of GP02. CPS was extracted from V. alginolyticus strain E110 and then incubated with 2-fold serial dilutions of the recombinant protein GP02 (with tag TF) or the tag protein TF. All samples were subjected to SDS-PAGE analysis and alcian blue staining. To explore the therapeutic potential of phage HH109 in disturbing V . alginolyticus biofilms, we investigated the dynamic process of biofilm formation by V. alginolyticus . Crystal violet staining assays showed that the biofilm of V . alginolyticus E110 reached its highest growth value at 12–20 h with mature biofilm ( Figure 5 A). Subsequently, the mature biofilm of E110 strain was exposed to the phage HH109 diluted by a 10-fold gradient. As shown in Figure 5 B, biofilm was significantly dispersed by phage HH109 at different concentrations (1.2×10 9 , 1.2×10 8 , and 1.2 × 10 7 pfu/mL); after 8 h, phage HH109 degraded 88%, 64%, and 45% of the biofilm biomass at concentrations 1.2×10 9 , 1.2×10 8 , and 1.2 × 10 7 pfu/mL respectively, when compared to control (no phage). The aforementioned results indicate that the Vibrio phage HH109 could act as a potential biocontrol agent. Figure 5 Degradation of V. alginolyticus biofilm by phage HH109 (A) Biofilm-forming capacity of V. alginolyticus E110. Bacterial overnight cultures were diluted in fresh LB with 2% NaCl, transferred into a 24-well plate, and incubated at 30°C to allow biofilm formation. After 4, 8, 12, 20, 24, and 36 h, the biofilms were measured at OD 595 nm. (B) Degradation of mature biofilm by phage HH109. 1 mL phage (1.2×10 9 , 1.2×10 8 , and 1.2 × 10 7 pfu/mL) was added to a 24-well plate with mature biofilm; biofilm was measured after 2, 4, 6, and 8 h. Data represent the mean of at least five biological replicates ±SD, analyzed in three technical repetitions. Degradation of V. alginolyticus biofilm by phage HH109 (A) Biofilm-forming capacity of V. alginolyticus E110. Bacterial overnight cultures were diluted in fresh LB with 2% NaCl, transferred into a 24-well plate, and incubated at 30°C to allow biofilm formation. After 4, 8, 12, 20, 24, and 36 h, the biofilms were measured at OD 595 nm. (B) Degradation of mature biofilm by phage HH109. 1 mL phage (1.2×10 9 , 1.2×10 8 , and 1.2 × 10 7 pfu/mL) was added to a 24-well plate with mature biofilm; biofilm was measured after 2, 4, 6, and 8 h. Data represent the mean of at least five biological replicates ±SD, analyzed in three technical repetitions.

Adsorption is the first step in phage infection and is one of the critical factors responsible for determining host range. The successful attachment of phages to the host surface depends on their host receptors and RBPs. The host receptor utilized by phages can be any component of the bacterial surface, including CPS, LPS residues in gram-negative bacteria, flagella, pili, or outer membrane proteins. 2 Meanwhile, the RBPs of phages are usually named tail fibers, tail spikes, and tail tips. Here, we illustrate the mechanism by which HH109 recognizes host V. alginolyticus E110, which has been rarely reported in Vibrio . Mutation analysis demonstrates that five genes involved in the CPS synthesis of V. alginolyticus have mutated, resulting in phage resistance. Deletion of glycosyltransferases (gt1 and gt2) may destroy glycosidic bonds in V . alginolyticus CPS leading to impaired synthesis of the CPS, which in turn affects the ability of phage HH109 to infect the host E110. These results were partially consistent with a previous study that found loss-of-function of gt2 in the host, reducing the adsorption capacity of phage EFap02. 20 Deleting the gene wecA in V. alginolyticus abolishes the synthesis of GlcNAc, indispensable for phage HH109 adsorption and subsequent infection. Similarly, previous studies have shown that the inactivation of WecA results in the loss of polymer and “primer” consisting of undecaprenyl pyrophosphoryl (und-PP)-linked GlcNAc, inhibits the Drexlerviridae phage NJS1 adsorption to the O-antigen of host. 21 The hypothetical protein-encoding gene orf2 is predicted to belong to the ATPgrasp_TupA protein family, which is involved in many different biological processes. 22 Thus, the lack of synthesis of transferase encoded by orf2 may affect the amidation of a downstream enzyme during the synthesis of V. alginolyticus CPS, resulting in impaired phage HH109. Deletion of the ugd gene prohibits the synthesis of UDP GlcA, the prominent component of CPS, preventing HH109 adsorption to host bacteria, and further biochemical experiments are needed to verify this assumption. In addition to genes on the CPS loci of V. alginolyticus E110, the genes wzb and wzc located outside the CPS loci also regulate susceptibility to phage HH109 by disrupting capsule synthesis. Previous reports indicated that three consecutive genes, wza , wzb , and wzc constitute an essential exportation system in group 1 and 4 capsules in E. coli . 23 In the present study, it is worth explaining that genes wza , wzb , and wzc are sequentially located on chromosome II of E110. Therefore, we speculate that wzb and wzc deletion hindered the synthesis and export of CPS and further affected phage HH109 adsorption to the host. However, this gene wza has not yet been identified. The aforementioned studies indicate that the CPS of host V. alginolyticus is involved in the infection of phage HH109. Bacteria can quickly adapt to external pressures and evolve strategies to circumvent phage infection. 24 For example, spontaneous phage-resistant strains of V. cholerae were found to delete 14 glutamine residues in the protein polyQ, resulting in phage VP1 resistance. 16 Meanwhile, Acinetobacter baumannii loses function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of Ackermannviridae phage ΦCO01 adsorption. 6 Similarly, our results found that the CPS biosynthesis genes wecA and orf2 underwent frameshift, reducing the adsorption of phage HH109. Thus, we attribute the emergence of phage HH109 resistance to the loss of V. alginolyticus ’s capsule. The tailed phages typically use tail spikes or tail fiber proteins to recognize the bacterial surface at the early stage of infection by specifically digesting the polysaccharides, the primary receptors for phages. 25 , 26 For example, the Autographiviridae phage T7 uses tail fiber gp17 interactions with the surface polysaccharide LPS of E. coli at the initial adsorption. Unlike T7, Autographiviridae phage K1-5 adsorbs the CPS receptor using the tail spike. 2 Here, we found that tail protein gp02, predicted to encode a depolymerase in the phage HH109 tail, was the RBP interacting with the CPS of E110. Biofilms cause most bacterial infections and conventional antibiotics cannot penetrate biofilms; therefore, they are ineffective in treating biofilm-related infections. 27 Previous studies reported the potential of phages and their derivatives have anti-biofilm potential against Vibrio . 28 , 29 In this study, phage HH109 exhibited a strong ability to disrupt biofilms, which is consistent with the report by Gao et al. 30 However, Yin et al.’s report indicated that the phage is expected to prevent biofilm formation but will not destroy the biofilm that has already formed. 31 The aforementioned evidence suggests that Vibrio phage HH109 could act as a potential biocontrol agent. In summary, we elucidated that Autographiviridae phage HH109 utilizes the tail protein gp02 to recognize and degrade the host E110’s CPS to initiate infection. Concurrently, we also confirmed that disrupting the CPS biosynthesis gene in E110 leads to the rapid development of phage-resistant strains. Moreover, the phage HH109 can disperse the biofilm of V. alginolyticus E110. Our research contributes to the understanding of the early interaction mechanism between Autographiviridae phages and pathogens but also serves as a crucial reference for the application of phages in biofilm eradication. Although our study indicates that phage HH109 uses the tail protein gp02 to recognize the CPS of the host V. alginolyticus for initiating infection. We also observed that phage HH109 exhibits inefficient infection against CPS mutants. Further experiments are required to verify whether this phage utilizes other types of adsorption receptors.

Bacteriophages (phages), the most abundant living entities on Earth, are recognized as critical components in food web ecology and major evolutionary drivers of bacterial communities. 1 Various components, including proteins to polysaccharides, have been identified in gram-negative bacteria as phage host receptors, such as capsular polysaccharides (CPS). 2 Capsules are surface polysaccharide structures that provide protective roles from physical and chemical stresses for many bacteria and also are essential for the virulence of pathogenic bacteria. 3 CPS made up of repeating linear or branched oligosaccharide units by joining linkages is highly diverse and has been used as a bacterial strain classification. Phages use CPS as receptors as well. 4 For example, Stephanstirmvirinae phage PNJ1809-36 reversibly binds to CPS for the initial adsorption, then irreversibly the host’s second receptor LPS. 5 It was also reported that Ackermannviridae phage ΦCO01 adsorption to the host Acinetobacter baumannii requires the presence of an intact CPS. 6 It is worth noting that only a limited number of Autographiviridae phages, such as Klebsiella pneumoniae phage KP32 and Escherichia coli phage K1-5 have been identified as possessing receptor-binding proteins that specifically recognize CPS. 7 , 8 Phage encoded receptor-binding proteins (RBPs) are key factors determining host range, mediating initial contact with host cell surface receptors. For example, in Autographiviridae phage KP32, tail tubular protein A is responsible for host-cell recognition, attachment, and initiation of infection. 7 The phage K1-5 encodes two distinct hydrolyzed tail fiber proteins for initial contact with their host receptors. One is the endosialidase protein, allowing the phage K1-5 to bind to and degrade K1 polysaccharide capsules. The other protein, nearly identical to the lyase, enables the phage to attach to and degrade K5 polysaccharide capsules. 8 Besides, Autographiviridae phage T7 possesses a lower nozzle encased by six or twelve tail fibers or tail spikes. The initial interaction of T7 with host membrane proteins facilitates further interaction between gp17 tail fibers and LPS. This interaction leads to a conformational change in the tail fibers, aligning them perpendicular to the cell surface and initiating protein and DNA ejection. 9 , 10 In summary, diverse phage groups use different mechanisms to interact with host cells due to their enormous structural diversity. Vibrio species are ubiquitous and abundant in brackish water ecosystems worldwide, and some of them are zoonotic pathogens that can cause fatal diseases in aquatic animals and humans. 11 , 12 From a phage therapy point of view, many studies on Vibrio -specific phages have been widely reported, and phage receptors are also of concern. For example, Vibrio phage OWB utilizes the outer membrane protein (OMP) vp0980 as a receptor for adsorbing the host V. parahaemolyticus . In contrast, Vibrio phage VP3 employs TolC or LPS to adsorb the host V. cholerae 13 , 14 Additionally, V . cholerae phages vp882 and VP1 utilize the transcription factors VqmA and OMP PolyQ as a receptor, respectively. 15 , 16 Like OWB and VP1, phage KVP40 infects V. anguillarum using OMP as the host receptor. 17 Despite the presence of CPS on the surface of Vibrio species, there are few reports that CPS serves as a receptor for Vibrio phages. 18 In our previous work, Autographiviridae phage HH109 could exclusively infect and efficiently lyse host strain V. alginolyticus E110. However, the mechanism of host recognition by phage HH109 to host E110 remains unknown. In this study, we identified CPS as the receptor of phage HH109 and the phage tail protein that binds to CPS was also confirmed. Additionally, we confirmed the destructive impact of phage HH109 on the mature biofilm of host E110. In summary, our work will be necessary for understanding the mechanisms of early interaction between Vibrio phages and pathogens and determining the application of phages in antimicrobial therapy.

REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and v irus strains E. coli SM10 λpir Jiao Lab CAS: ZC1038 E. coli GEB883 Deng Lab Nguyen et al. 32 E. coli DH5a Vazyme CAS: 9057 E. coli BL21(DE3) Vazyme CAS: 9126 E. coli S17-1λpir This Lab Milton et al. 33 V. alginolyticus E110 This Lab GenBank no. PRJNA842900 V. alginolyticus EP1-5 This Lab GenBank no. PRJNA1009694 pSW7848 Deng Lab Val et al. 34 pBBR1MCS-1 Deng Lab Kovach et al. 35 pCold-TF Takara CAS: 3365 Biological samples Seawater Guangzhou N/A Chemicals , p eptides, and r ecombinant p roteins 2, 6-Diaminopimelic acid Merck Millipore CAS: 583-93-7 Chloramphenicol Merck Millipore CAS: 56-75-7 Kanamycin Sangon Biotech CAS: 25389-94-0 Ampicillin Sangon Biotech CAS: 69-52-3 D-glucose Merck Millipore CAS: 50-99-7 L-arabinose Merck Millipore CAS: 5328-37-0 Ethylenediaminetetraacetic acid Merck Millipore CAS: 60-00-4 3-sulfopropyltetradecyldimethylbetaine Sangon Biotech CAS: 14933-08-5 hydrochloric acid Merck Millipore CAS: 7647-01-0 Ammonium sulfamate Merck Millipore CAS: 7773-06-0 Sodium tetraborate Merck Millipore CAS: 1330-43-4 3-hydroxyldiphenol Merck Millipore CAS: 580-51-8 Glucuronic acid Merck Millipore CAS: 6556-12-3 Proteinase K Merck Millipore CAS: 39450-01-6 Isopropyl- β -d-thiogalactopyranoside Merck Millipore CAS No.: CAS: 367-93-1 BugBuster® Master Mix Merck Millipore CAS: 70584-M Ni-NTA Agarose Qiagen CAS: 30210 Paraformaldehyde Merck Millipore CAS: 30525-89-4 HIS antibody Cell Signaling Technology RRID: AB_10786151 Alexa Fluor® 594 goat anti-rabbit IgG Cell Signaling Technology RRID: AB_2650602 Critical c ommercial a ssays FastPure Bacteria DNA Isolation Mini Kit Vazyme DC112-01 Capsular stain kit Solarbio G1130-50 BCA protein assay kit Biosharp PC0020 Deposited d ata NCBI database The genome sequence https://www.ncbi.nlm.nih.gov/genome/ Oligonucleotides Primers for identification of phage bacterial receptors, see Tables S4 and S5 This paper N/A Primers for identification of phage receptor-binding proteins, see Table S6 This paper N/A Software and a lgorithms Structural domains Web https://www.ebi.ac.uk/interpro/ Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zhe Zhao ( [email protected] ). All unique/stable reagents in this study are available from the lead contact without restriction. • The whole genome sequence for V. alginolyticus E110 obtained through the Illumina Miseq platform was deposited in GenBank under BioProject number PRJNA842900. The genome sequence-associated data for five spontaneous HH109-resistant mutants were deposited in GenBank under BioProject number PRJNA1009694. These data are publicly available as of the date of publication. • This paper does not report the original code. • Additional information required to reanalyze the data in this paper is available from the lead contact upon request. The whole genome sequence for V. alginolyticus E110 obtained through the Illumina Miseq platform was deposited in GenBank under BioProject number PRJNA842900. The genome sequence-associated data for five spontaneous HH109-resistant mutants were deposited in GenBank under BioProject number PRJNA1009694. These data are publicly available as of the date of publication. This paper does not report the original code. Additional information required to reanalyze the data in this paper is available from the lead contact upon request. No experimental models were used in this study. The bacterial strains and plasmids used in this study are summarized in Tables S1 , S2 , and S3 . V. alginolyticus strains were cultured at 37°C in Luria-Bertani (LB, Huankai, China) broth supplemented with 2% NaCl or Thiosulfate-Citrate-Bile Salts-sucrose-agar (TCBS, Huankai, China). E. coli strains were cultured in LB broth or agar plate at 37°C. E. coli SM10 strain bearing the plasmid pSC189 was used to construct the transposon mutation library of the V. alginolyticus E110 strain. E. coli GEB883 growth was cultured in an LB medium with 50 μg/mL DAP (2, 6-Diaminopimelic acid) and used for conjugation in gene deletion experiments. Suicide plasmid pSW7848 was used to generate gene deletion mutants in V. alginolyticus strains, and the low-copy vector pBBR1MCS-1 was used for complementation experiments. pCold-TF vector was used for the protein expression of gp02. Unless otherwise specified, Antibiotics were used at the following concentrations: Chloramphenicol (Cm), 25 mg/mL; Kanamycin (Kan), 100 mg/mL; Ampicillin (Amp), 100 mg/mL. To create a random mutant library, donor bacterial strain SM10 containing transposon pSC189 was conjugated with recipient strain E110 on LB at a 1:1 donor-to-recipient ratio. After culture overnight, 100 μL of the transformed bacterial suspension was streaked on TCBS plates containing 100 μg/mL kanamycin and incubated at 30°C overnight. Subsequently, individual colonies were picked into 96-deep-well plates and cultured for 6-8 h in 600 μL LB broth with 2% NaCl per well supplemented with 60 μg/mL Kan. The resulting mutant pool was then stored at -80°C. The pool consists of 30,760 mutants in 321 microplates and represents sixfold coverage of the genome (i.e., averages 6 insertions per gene) with greater than 99% assurance of covering the entire genome. To screen phage-resistant mutants, two rounds of screening were performed as described previously with a slight modification. 5 For the first round, each mutant with approximately 10 8 CFU/mL was infected with the phage HH109 at an MOI of 1 in a 96-well plate and incubated at 37°C, shaking at 120 rpm for 4 h. As a control, the wild-type E110 was also included as performed under the same conditions. Then, the turbidity (OD 600 ) for each well was quantified using a Microplate reader (SPARK; Tecan). For the second round, suspected phage-resistant colonies obtained from the first screening were further confirmed using spot testing. In brief, after the 20 μL bacterial suspensions (∼10 8  CFU/mL) were air-dried on an LB agar plate containing 2% NaCl, 3 μL phage (10 8 PFU/mL) was added to the center of the bacterial lawn and then cultured at 37°C for 4 h. In principle, the phage HH109 can cause a clearing of the bacteria encompassing the original spot in the wild-type strain, whereas not in phage non-susceptible mutants. Tail-PCR products determined the transposon-insertion site of these 11 phage HH109-resistant mutants. Genomic DNA from the stored phage HH109-resistant mutants were isolated using a FastPure Bacteria DNA Isolation Mini Kit (Vazyme, China) per manufacturer instructions. All primers used in this section are listed in Tables S4 and S5 . After a round of PCR amplification using the mutant strain DNA, the PCR product used for the second round of PCR is used as the template. The PCR products were sequenced (Sangon Biotechnology, China) and performed to BLAST in the NCBI database to determine the transposon insertion site. All gene deletion strains were generated using homologous recombination, as described previously. 36 Briefly, the upstream and downstream arms of the target gene were amplified by PCR using indicated primers ( Tables S4 and S5 ), and the two arms were then used as the templates to generate a gene deletion fragment, which was then cloned into the suicide vector pSW7848. PCR amplified the vector fragment pSW7848 with the primer pair pSW7848-F/R ( Table S4 ). The recombinant suicide plasmids were obtained by isothermal assembly and transformed into DAP-auxotrophic E. coli GEB883 cells. The positive colonies were detected on LB plates containing Cm 25 and 50 μg/mL DAP using the primers annotated Del-check-pSW7848-F/R. Then, the recombinant suicide plasmid was transferred by conjugation from strain GEB883 to V . alginolyticus wild-type strain E110 before allelic exchange. Transconjugants were first selected on TCBS plates containing 0.2% D-glucose and 5 μg/mL chloramphenicol and then selected on TCBS plates containing 0.2% L-arabinose to induce the suicide ccdB gene expression. The deletion of genes was confirmed by PCR ( Tables S4 and S5 ). The complete open reading frame with the promoter region of each indicated target gene was PCR-amplified for complementation experiments using primers containing cut sites for EcoRI (forward) and SalI (reverse) restriction enzymes and cloned into the pBBR1MCS-1 vector. As described above, the recombinant plasmids were transformed into the corresponding knockout mutants by conjugation using E. coli S17-1λpir as the donor strain. The transformed colonies were screened on TCBS plates supplemented with 5 μg/mL chloramphenicol and confirmed via PCR. The primers used in this part are listed in Tables S4 and S5 . Growth kinetics of wild-type, deletion mutants, and complemented strains were determined using the Bioscreen C (FP-1100-C, Bioscreen), measuring the turbidity OD 600 every 1 h interval with an MOI of 1. Spot testing was performed as described above. A small volume of HH109-containing diluent (10-fold serial dilutions) was added onto the dried bacterial lawn of the indicated strains on an LB agar plate with 2% NaCl. After 4 h of incubation at 37°C, clear zones were recorded to reflect the inhibition of bacterial growth. The adsorption capacities of the phage HH109 on the wild-type, deletion mutants, and complemented strains were conducted as described previously with slight modifications. 37 Briefly, bacterial cells with a 10 6 CFU/mL density were infected with the phage at an MOI of 0.01. The mixtures were incubated at 37°C for 10 min and then immediately centrifuged at 10000 g at 4°C for 1 min to pellet bacterial cells. Subsequently, free phage particles in the supernatant were quantified by the double-layer agar method to calculate the phage adsorption rate. 38 To observe the cell surface structure of the wild-type, deletion mutants, and complemented strains, scanning electron microscopy (SEM) was first used, and all samples were prepared as described previously. 39 Briefly, these strains were cultured overnight at 37°C and then fixed in 2.5% glutaraldehyde for 2 h at room temperature (RT). After being washed three times with 0.1 M PBS (pH 7.4), they were treated with 1% osmium tetroxide in 0.1 M PBS (pH 7.4) for 2 h at RT, followed by dehydration with sequential washes in 30, 50, 70, 80, 95 and 100% ethanol and dry using a critical-point dryer (K850, Quorum). All samples were coated with gold using an ion sputter coater (MC1000, Hitachi) and observed under a scanning electron microscope (SU8100, Hitachi). For further qualitative observation of capsules in those strains, capsule staining was performed using a capsular stain kit according to the manufacturer’s instructions (Solarbio, China). Concisely, a drop of bacterial suspension was smeared on a clean glass slide. After drying, the slide was flooded with crystal violet solution for 5 min, rinsed with copper sulfate solution, and blotted dry with filter paper. Slides were examined using light microscopy under oil immersion (Axio Vert.A1, Carl Zeiss). For quantitative analysis of CPS in various V. alginolyticus strains, as indicated, extraction and quantification of surface CPS were performed. The 500 mL cultures of V. alginolyticus were grown in LB broth containing 2% NaCl at 37°C until the OD 600 reached 0.8. The cells were then harvested by centrifugation (8000 g, 5 min, at 4°C) and washed with 100 mL of sterile saline solution (2% NaCl). After that, the cells were pelleted again (8000 g, 5 min, at 4°C) and resuspended in 100 mL of 50 mM EDTA (pH 8.0). The sample was incubated at 30°C for 1 h, and the precipitate was washed with 100 mL of sterile saline solution (2% NaCl). The washed precipitate was collected by centrifugation (8000 g, 5 min, at 4°C) and resuspended in 100 mL of sterile saline solution (2% NaCl) to which 20 mL of 1% 3-sulfopropyltetradecyldimethylbetaine (pH 2.0) was added. After mixing, the sample was heated at 50°C for 30 min and then centrifuged at 8000 g for 5 min at 4°C. The resulting supernatant (120 mL) was mixed with a chloroform-n-butanol mixture (5:1, 24 mL) by inverting and allowed to stand for 30 min at RT. The mixture was then centrifuged at 8000 g for 5 min at 4°C. The uppermost aqueous phase containing CPS was precipitated with four times the volume of cold 100% ethanol by inverting and incubated at 4°C for 12 h. The samples were pelleted by centrifugation (12,000 g, 10 min, at 4°C), washed with 70% ethanol, and lyophilized using the vacuum freeze dryer (Alpha-2LDPLUS, Christ). The purified CPS dry powder was reconstituted in 250 μL of 100 mM hydrochloric acid, and 100 μL of CPS solution was added to 5 μL of pre-cooled 4 M ammonium sulfamate and 600 μL of 25 mM sodium tetraborate. The mixture was vortexed vigorously and boiled for 15 min. After cooling to RT, 40 μL of 0.15% 3-hydroxyldiphenol was added, and the samples were aliquoted to a 96-well plate. The absorbance was measured at 520 nm, and standard curves were determined using 100 μL of glucuronic acid (0 to 40 μg/mL). The receptor properties of HH109 were measured as described previously. 40 Briefly, exponentially growing V. alginolyticus were treated with proteinase K (0.2 mg/mL; Qiagen) to destroy surface proteins at 37°C for 2 h. After that, the treated bacteria were washed thrice with fresh LB broth with 2% NaCl. Adsorption assays were performed for the various treated cells using double-layer agar plate assays. As performed in the spot testing assay above, 30 μL of the phage HH109 (approximately 10 5 PFU/mL in phage titer) were spotted onto bacterial lawns and incubated at 37°C overnight. When bacterial colonies regrew in the middle of lysis zones, several of them were randomly picked from different spots. These colonies were then streaked on LB agar plates containing 2% NaCl for purification by three rounds of single colony isolation, and the phage-resistant phenotypes were further confirmed by spot testing and growth curve assays. The whole-genome sequencing of the wild-type V. alginolyticus was performed using the Pacific Biosciences platform (PacBio, Menlo Park, CA) and the Illumina Miseq platform. According to the manufacturer’s instructions, the total DNA of V. alginolyticus E110 was extracted using the HiPure Bacterial DNA kit (Magen, Guangzhou, China). The quality of the extracted DNA was determined using Qubit (Thermo Fisher Scientific, Waltham, MA) and Nanodrop (Thermo Fisher Scientific, Waltham, MA). The library construction and data analysis were completed by Gene Denovo (Guangzhou, China), and the detailed information refers to previous reports. 41 The genome of five spontaneous HH109-resistant mutants and sixteen naturally occurring phage-resistant strains of V. alginolyticus were sequenced using a Novaseq 6000 owned by Guangzhou Gene Denovo Co., Ltd. Coding sequences (CDSs) in each draft genome were predicted using Prodigal 2.60 with the default settings. High-quality reads were filtered and obtained using FASTP (version: 0.20.0). 42 Coverage of the V. alginolyticus genome was evaluated using SOAPaligner ( http://soap.genomics.org.cn/soapaligner.html ), and reads were assembled using SOAPdenovo ( http://soap.genomics.org.cn/soapnovo.html ). Afterward, SNP mutations were obtained after sequence alignment using the software package MUMmer (version 3.23). 43 The initial InDels (less than 10 bp) were obtained for alignment using LASTZ and axtBest. 44 Reliable InDel sites were obtained using BWA software 45 to align the 150 bp (3sd) upstream and downstream of the reference sequence InDel sites and verified by sample sequencing. BLASTp was also used to explore the variation in phage-resistant strains, which was conducted by aligning the Nucleic acid sequence of our strains to the reference genome E110 ( CP098035.1 ). A large fragment of gp02 (covering C-terminal sequence from 1072 bp to 2820 bp) was amplified from the genome of phage HH109 using the primers pCold-TF-F/R ( Table S6 ) and cloned into the pCold-TF vector (Takara, Japan) using One Step Cloning Kit (Vazyme, China) to generate the recombinant plasmid pCold-TF/ gp02 . The construct was transformed into E. coli BL21(λDE3) plysS cells and the bacteria were then induced with 0.6 mM isopropyl- β -d-thiogalactopyranoside (IPTG) at 20°C for 24 h to express the recombinant protein gp02, which contained the tag proteins Trigger factor (TF) and 6× His. The induced cultures were harvested by centrifugation and resuspended in BugBuster® Master Mix (Millipore, USA) following manufacturer protocol to achieve cell lysis. The supernatant of cell lysates mixed with Ni-NTA Agarose (Qiagen, Germany) was incubated overnight at 4°C with gentle agitation. The mixture containing the recombinant protein was then loaded onto a column and purified using Ni-NTA affinity chromatography. The recovery and purity of protein in each fraction were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pooled. The protein concentration was estimated by using a BCA protein assay kit (Biosharp, China). As a control, empty vector pCold-TF was simultaneously performed following the same procedure as the pCold-TF/ gp02 . Anti-gp02 serum was obtained by immunizing New Zealand white rabbits with the purified recombinant protein gp02-TF (ABclonal, China). For antibody blocking assay, 200 μL anti-gp02 serum and normal serum were individually mixed with the phage HH109 at a concentration of 10 5 PFU/mL for 10 min at RT, and V. alginolyticus strain E110 was then added into the mixture to allow phage infection for 1 h, and the phage titer of HH109 was determined by the double-layer agar method. For protein competition assay, 2 mg/mL of the purified gp02-TF was incubated with an equal volume of E110 at a cell density of 10 8 CFU/mL for 10 min at RT. As a control, the tag protein TF produced from the pCold-TF vector was included to do the same thing. Then, 10 6 PFU/mL of phage HH109 was added to the above mixtures to calculate the adsorption rate. To detect the binding of gp02 to host CPS, immunofluorescence staining was carried out using the anti-gp02 serum. Overnight, the wild-type strain or Δ ugd strain was pre-incubated with equal volume of the purified gp02-TF (2 mg/mL) for 30 min at RT and pelleted by centrifugation. These strains were washed three times with sterile saline solution (2%NaCl) and fixed in 4% paraformaldehyde (Merck, Germany) for 15 min at RT. After blocking in 10% normal goat serum for 1 h, bacterial cells were incubated with the HIS antibody (1:500; Cell Signaling Technology, USA) in 1% normal goat serum for 2 h at RT, rinsed three times for 10 min each with sterile saline solution (2% NaCl), and then incubated with the Alexa Fluor® 594 goat anti-rabbit IgG (1:500; Cell Signaling Technology, USA) for 1 h at RT in the dark. Bacterial cells were then smeared onto a glass slide and examined under a fluorescence microscope (Axio Vert.A1, ZEISS). The tagged protein TF was also included to incubate with the wild-type strain as a negative control similarly. The degradation activity of purified gp02 was demonstrated using several qualitative and quantitative experiments. Firstly, the wild-type E110 strain was pre-treated with 500 ng of the purified gp02 or TF at RT for 15 min or untreated and then subjected to capsule staining and CPS quantification. Capsule staining was performed as described above, and stained cells were observed under light microscopy. Extraction and quantification of CPS were also carried out following the above protocol, and the Δ ugd , a capsule-deficient strain, was included in this assay as a negative control. Next, to visualize CPS degradation, aliquots of purified CPS (50 μg) were incubated with an equivalent volume of two-fold serial diluted gp02 (500 ng-62.5 ng) or 500 ng TF at RT for 1 h. Then, all samples were separated by 6-12% gradient SDS-PAGE and stained with Alcian Blue. 46 Briefly, the gel was washed in fixing solution (25% ethanol and 10% acetic acid in Milli-Q water) at 50°C for 15 min and stained with 0.125% Alcian blue in fixing buffer for 20 min at 50°C in the dark. Consequently, the gel was detained in fixing buffer overnight at RT and visualized. To evaluate the biofilm degradation efficacy of phage HH109, 1% of an overnight bacterial culture was inoculated into HI fresh broth containing 0.8% NaCl and cultured on 24-well-polystyrene plates for 12 h (n = 6) at 30°C. The biofilms were washed to remove the residual planktonic bacterial cells, and the phage with concentrations 1.2×10 9 , 1.2×10 8 , and 1.2×10 7 pfu/mL were treated for 2, 4, 6, and 8 h. Then, the supernatant was removed from the well and washed twice. The total biomass was quantified using a crystal violet staining assay. All data are presented as means ± the standard deviations(s.d.) from a minimum of three independent experiments. Statistical analysis was carried out using Student t tests or two-way ANOVA using GraphPad Prism v7 software. P ≤0.05 was considered significant. Significance is indicated in the figures by asterisks (∗, P ≤ 0.05; ∗∗, P ≤ 0.01; ∗∗∗∗, P ≤ 0.0001). No additional resources are involved.