A Novel Antimicrobial Peptide Spampcin56–86 from Scylla paramamosain Exerting Rapid Bactericidal and Anti-Biofilm Activity In Vitro and Anti-Infection In Vivo

The abuse of antibiotics leads to the increase of bacterial resistance, which seriously threatens human health. Therefore, there is an urgent need to find effective alternatives to antibiotics, and antimicrobial peptides (AMPs) are the most promising antibacterial agents and have received extensive attention. In this study, a novel potential AMP was identified from the marine invertebrate Scylla paramamosain and named Spampcin. After bioinformatics analysis and AMP database prediction, four truncated peptides (Spa31, Spa22, Spa20 and Spa14) derived from Spampcin were screened, all of which showed potent antimicrobial activity with different antibacterial spectrum. Among them, Spampcin56–86 (Spa31 for short) exhibited strong bactericidal activity against a variety of clinical pathogens and could rapidly kill the tested bacteria within minutes. Further analysis of the antibacterial mechanism revealed that Spa31 disrupted the integrity of the bacterial membrane (as confirmed by scanning electron microscopy observation, NPN, and PI staining assays), leading to bacterial rupture, leakage of cellular contents (such as elevated extracellular ATP), increased ROS production, and ultimately cell death. Furthermore, Spa31 was found to interact with LPS and effectively inhibit bacterial biofilms. The antibacterial activity of Spa31 had good thermal stability, certain ion tolerance, and no obvious cytotoxicity. It is worth noting that Spa31 could significantly improve the survival rate of zebrafish Danio rerio infected with Pseudomonas aeruginosa, indicating that Spa31 played an important role in anti-infection in vivo. This study will enrich the database of marine animal AMPs and provide theoretical reference and scientific basis for the application of marine AMPs in medical fields.


Introduction
Antimicrobial peptides (AMPs), also known as host defense peptides, are widely distributed in animals, plants, and microorganisms. It is a class of small-molecule peptides with broad antimicrobial activity and involved in physiological processes such as immune regulation, inflammation, wound repair, etc. It is a key component of host defense against bacteria, fungi, viruses, and parasites [1]. Most AMPs are cationic peptides, containing 2 to 13 positive charges, with hydrophobic residues in the majority [2]. In addition to natural sources, AMPs can also be obtained through various computational algorithm design methods and synthesis strategies. As reported, a total of 23, 253 AMPs were collected in LAMP2 database, including 7824 natural AMPs and 15, 429 synthetic AMPs [3]. Some of these 153 amino acids, without a signal peptide (GenBank accession NO. MW388709) ( Figure S1A). The mature peptide of Spampcin has a molecular weight of 16.1 kDa and an estimated isoelectric point (pI) of 9.44, which showed high possibility to be classified as an AMP (Table 1). Quantitative real-time PCR (qPCR) results showed that the Spampcin gene was widely distributed in different tissues of female adult crabs, with relatively high expression in the ovaries ( Figure S1B) (primers used for qPCR were listed in Table S1). In male adult crabs, the Spampcin gene was predominantly expressed in the gills ( Figure S1C). We further investigated the expression profiles of the Spampcin gene in the hepatopancreas of male adult crabs following a bacterial challenge (Vibrio alginolyticus or S. aureus). The results showed that the Spampcin gene expression was significantly induced at 3 h after V. alginolyticus infection. In response to an S. aureus challenge, the Spampcin gene was downregulated at 3 h and upregulated at 24 h ( Figure S1D,E).

Truncated Peptides Derived from Spampcin Show Potent and Broad-Spectrum Antimicrobial Activity
Based on bioinformatics analysis, physicochemical properties, and AMP database predictions, a truncated peptide containing 31 amino acids derived from the mature peptide of Spampcin (a region from 56th to 86th) was found to have a high AMP potential (Table 1), named Spampcin 56-86 (Spa31 for short). The net charge and hydrophobicity of Spa31 is +7.5 and 42% ( Figure 1A). Spa31 was then chemically synthesized and subjected to antimicrobial activity evaluation. It was found that Spa31 exhibited potent antibacterial and antifungal activity against Gram-negative bacteria (such as P. aeruginosa, Escherichia coli, Acinetobacter baumannii), Gram-positive bacteria (such as S. aureus, Listeria monocytogenes, Enterococcus faecalis) and fungi (such as Fusarium graminearum, Fusarium solani) with MIC values ranging from 1.5-3 µM and MBC or MFC values lower than 6 µM (Table 2).

Effective Bactericidal Activity of Spa31 against S. aureus and P. aeruginosa
The bactericidal efficiency of Spa31 was evaluated using a time-killing kinetic assay. The results showed that when incubated with S. aureus and P. aeruginosa, 6 µM Spa31 rapidly killed more than 99.9% of S. aureus within 2 min and eliminated 99.9% of P. aeruginosa within 6 min ( Figure 3A,B).
SEM observations revealed that after Spa31 treatment, S. aureus exhibited cell rupture, disruption of membrane integrity, and even leakage of cellular contents ( Figure 3C); the surface of P. aeruginosa was obviously rough, filamentous substances appeared, and finally the cell contents flowed out ( Figure 3C). SYTO 9 and PI staining analysis further confirmed the disruption of bacterial membrane integrity by Spa31. As shown in Figure 4A,C, the fluorescence signal of PI was obviously enhanced after Sap31 treatment compared with the control group, and together with the evidence provided by flow cytometry (Figure 4B,D) indicated that Spa31 significantly increased the bacterial inner membrane permeability of S. aureus and P. aeruginosa.

Effective Bactericidal Activity of Spa31 against S. aureus and P. aeruginosa
The bactericidal efficiency of Spa31 was evaluated using a time-killing kinetic assay. The results showed that when incubated with S. aureus and P. aeruginosa, 6 μM Spa31 rapidly killed more than 99.9% of S. aureus within 2 min and eliminated 99.9% of P. aeruginosa within 6 min ( Figure 3A,B). SEM observations revealed that after Spa31 treatment, S. aureus exhibited cell rupture, disruption of membrane integrity, and even leakage of cellular contents ( Figure 3C); the surface of P. aeruginosa was obviously rough, filamentous substances appeared, and finally the cell contents flowed out ( Figure 3C). SYTO 9 and PI staining analysis further confirmed the disruption of bacterial membrane integrity by Spa31. As shown in Figure 4A,C, the fluorescence signal of PI was obviously enhanced after Sap31 treatment compared with the control group, and together with the evidence provided by flow cytometry ( Figure 4B,D) indicated that Spa31 significantly increased the bacterial inner membrane permeability of S. aureus and P. aeruginosa.

Antibacterial Mechanism of Spa31 against P. aeruginosa
To further investigate the antibacterial mechanism of Spa31 against P. aeruginosa, NPN staining assay was conducted to reveal the effect of Spa31 on the outer membrane of P. aeruginosa. As shown in Figure 5A, as the concentration of Spa31 treatment increased, the NPN fluorescence intensity was obviously enhanced. LL-37 treatment (12 μM) also had similar results to the 24 μM Spa31 treatment group, while the antibiotic PMB treat-

Antibacterial Mechanism of Spa31 against P. aeruginosa
To further investigate the antibacterial mechanism of Spa31 against P. aeruginosa, NPN staining assay was conducted to reveal the effect of Spa31 on the outer membrane of P. aeruginosa. As shown in Figure 5A, as the concentration of Spa31 treatment increased, the NPN fluorescence intensity was obviously enhanced. LL-37 treatment (12 µM) also had similar results to the 24 µM Spa31 treatment group, while the antibiotic PMB treatment showed low fluorescence intensity ( Figure 5A). The results indicated that Spa31 could cause disruption of the outer membrane of P. aeruginosa in a dose-dependent manner. Whether Spa31 interacts with bacterial surface components such as LPS prior to outer membrane penetration deserves exploration. Without the addition of LPS, Spa31 at a concentration of 1 × MIC completely inhibited the growth of P. aeruginosa within 24 h ( Figure  5B). However, when 3 μg/mL P. aeruginosa-derived LPS was added to the medium, P. Whether Spa31 interacts with bacterial surface components such as LPS prior to outer membrane penetration deserves exploration. Without the addition of LPS, Spa31 at a concentration of 1 × MIC completely inhibited the growth of P. aeruginosa within 24 h ( Figure 5B). However, when 3 µg/mL P. aeruginosa-derived LPS was added to the medium, P. aeruginosa began to grow exponentially at approximately 21 h of incubation ( Figure 5B). As the increase of the concentration of LPS (6, 12, 24 µg/mL), the exponential growth time decreased in a dose-dependent manner ( Figure 5B).
The results have demonstrated that Spa31 disrupted the inner and outer membranes of P. aeruginosa, and we also found that Spa31 treatment resulted in the release of ATP ( Figure 6A) and the production of ROS ( Figure 6B) in a dose-dependent manner. Flow cytometry analysis showed that a 12 µM Spa31 treatment caused 98.34% ROS production, compared with 0.64% and 87.19% in the control and 12 µM LL-37-treated groups, respectively ( Figure 6C).

Anti-Biofilm Activity of Spa31 against P. aeruginosa
Biofilm formation is a strategy that is well developed for P. aeruginosa to adapt to th stresses of environment and antibiotics, which are more difficult to eliminate. Since Spa exerted rapid and efficient bactericidal activity against planktonic P. aeruginosa, the effe of Spa31 on P. aeruginosa biofilm formation or mature biofilm was also evaluated. Spa3 at a concentration of 1.5 μM, significantly inhibited biofilm formation by approximate 10% (p < 0.001) ( Figure 7A,B). Especially, when the concentration of Spa31 reached 12 μM the biofilm mass reduced by 90% (p < 0.0001) ( Figure 7A,B). Furthermore, Sap31 cou eradicate mature biofilms of P. aeruginosa in a dose-dependent manner, with an eradic tion rate of 84.4% at 48 μM (p < 0.0001) ( Figure 7C,D).

Anti-Biofilm Activity of Spa31 against P. aeruginosa
Biofilm formation is a strategy that is well developed for P. aeruginosa to adapt to the stresses of environment and antibiotics, which are more difficult to eliminate. Since Spa31 exerted rapid and efficient bactericidal activity against planktonic P. aeruginosa, the effect of Spa31 on P. aeruginosa biofilm formation or mature biofilm was also evaluated. Spa31, at a concentration of 1.5 µM, significantly inhibited biofilm formation by approximately 10% (p < 0.001) ( Figure 7A,B). Especially, when the concentration of Spa31 reached 12 µM, the biofilm mass reduced by 90% (p < 0.0001) ( Figure 7A,B). Furthermore, Sap31 could eradicate mature biofilms of P. aeruginosa in a dose-dependent manner, with an eradication rate of 84.4% at 48 µM (p < 0.0001) ( Figure 7C,D).

In Vitro Determination of Spa31 Properties and In Vivo Anti-P. aeruginosa Infection Analysis
In vitro determination of various properties of AMPs, including cytotoxicity, hemolytic activity, thermostability, and ionic tolerance, will provide a scientific basis for their future applications. In this study, we found that Spa31 was not toxic towards several cell lines (including RAW264.7, HEK-293T, and HepG2 cells), even at a concentration of 48 µM ( Figure 8A-C). Furthermore, Spa31 also exhibited no hemolytic activity on mouse erythrocytes, even at a high concentration of 250 µg/mL with the hemolysis rates lower than 5% ( Figure 8D). In addition, it had good thermal stability, and its antibacterial activity against S. aureus, P. aeruginosa, and E. coli was still maintained after a 30 min treatment in a 100 • C water bath ( Figure S2A-C). It exhibited a certain ionic tolerance, and the addition of 10 mM to 80 mM NaCl did not affect its antibacterial activity ( Figure S2D-F).

In Vitro Determination of Spa31 Properties and In Vivo Anti-P. aeruginosa Infection Analysis
In vitro determination of various properties of AMPs, including cytotoxicity, hemolytic activity, thermostability, and ionic tolerance, will provide a scientific basis for their future applications. In this study, we found that Spa31 was not toxic towards several cell lines (including RAW264.7, HEK-293T, and HepG2 cells), even at a concentration of 48 μM ( Figure 8A-C). Furthermore, Spa31 also exhibited no hemolytic activity on mouse erythrocytes, even at a high concentration of 250 μg/mL with the hemolysis rates lower than 5% ( Figure 8D). In addition, it had good thermal stability, and its antibacterial activity against S. aureus, P. aeruginosa, and E. coli was still maintained after a 30 min treatment To investigate whether Spa31 has an anti-infective effect in vivo, the zebrafish Danio rerio is commonly used as an animal model. It was found that 10 µg Spa31 treatment significantly improved the survival of zebrafish infected with P. aeruginosa (p = 0.017) ( Figure 8E). After 168 h of treatment, the PBS group had a survival rate of 50%, compared with 79% in 5 µg Spa31 treatment group or 85% in 10 µg Spa31 treatment group ( Figure 7E). To investigate whether Spa31 has an anti-infective effect in vivo, the zebrafish Da rerio is commonly used as an animal model. It was found that 10 μg Spa31 treatment s nificantly improved the survival of zebrafish infected with P. aeruginosa (p = 0.017) (Figu 8E). After 168 h of treatment, the PBS group had a survival rate of 50%, compared w 79% in 5 μg Spa31 treatment group or 85% in 10 μg Spa31 treatment group ( Figure 7E)

Discussion
In studying the marine invertebrate S. paramamosain, many efforts and exciting p

Discussion
In studying the marine invertebrate S. paramamosain, many efforts and exciting progress have been made in discovering new AMPs. For example, the scygonadin gene and its homolog SCY2-5, which were all predominantly expressed in the ejaculatory ducts of adult male crabs, have been shown to participate in fertilization and reproduction immunity of mud crabs [12][13][14]. Interestingly, a further in-depth study found that the SCY2-interacting protein Scyreprocin, screened using yeast two-hybrid technology, has also been proven to be an AMP with broad-spectrum antibacterial, antifungal, and anti-biofilm activities [15]. Recently, the newly identified AMP Sparanegtin, which was highly expressed in the testis, was responsive to LPS and V. alginolyticus stimulation, and exerted immunomodulatory effects to improve the survival of V. alginolyticus-infected crabs [16]. Similarly, in the study, Spampcin was also a novel functional gene, which was widely distributed in the tissues of adult crabs and was significantly regulated by V. alginolyticus and S. aureus challenge, indicating that it also played a role in the immune response of S. paramamosin. Further studies are needed to obtain its recombinant protein and confirm its immune-related functions in vitro and in vivo. Other novel AMPs, such as Sparamosin   [17], Sphistin [7,18], SpHyastatin [19], Sp-NPFin [20], etc., including the above AMPs as mentioned, all play an important role in the innate immunity of S. paramamosin and have good prospects for future applications in aquaculture and medicine. Here, in this study, another novel functional gene with high AMP potential, named Spampcin, was characterized from S. paramamosain and its derived truncated peptide Spa31 had been confirmed to exhibit broad-spectrum and potent antibacterial and antifungal activity, and several shortened Spa31-derived peptides (Spa20, Spa22, and Spa14) also had certain antimicrobial activity. Further analysis of the in vitro properties, antibacterial mechanism, and in vivo anti-infection effect of Spa31 will lay the foundation for the application of marine animal-derived AMPs in the future.
At present, there are various databases that provide computational tools for the prediction of AMPs, such as CAMP R3 , AntiBP2 [21], ClassAMP [22], APD3 [23], AMPer [24], etc., and their predictions are based on different algorithms. For example, in the CAMP R3 database, according to the conserved sequence signatures of AMPs (captured as patterns and Hidden Markov Models), 4 different prediction methods are involved, including support vector machine (SVM), random forest (RF), artificial neural network (ANN), and discriminant analysis (DA), which have become useful tools to discover and design novel AMP candidates [25]. In the study, we designed and synthesized four truncated peptides with high AMP potential derived from Spampcin (Spa31, Spa20, Spa22, and Spa14) through CAMP R3 database prediction, and compared and analyzed their antibacterial activities.
Previous studies have demonstrated that peptide hydrophobicity, amphipathicity, helicity, and net charge number are important factors affecting the biological activities (such as antimicrobial activity, hemolytic activity, etc.) of AMPs [26][27][28][29]. Within a certain range, the higher the positive charge and hydrophobicity, the stronger the antibacterial activity of α-helical AMPs [26,27]. However, beyond the optimum window, a decrease in antimicrobial activity and an increase in hemolytic activity were observed [26]. In particular, the net charge and hydrophobicity are inseparable from the secondary structures of AMPs (such as α-helical, β-sheet, linear/cyclic, irregularly coiled or mixed extended structures) [30]. Many efforts have been made to improve the antibacterial activity, enhance the stability, and reduce the hemolytic activity of AMPs, such as increasing the number of positively charged residues on the polar face [27], replacing L-type amino acids with D-enantiomers [29], incorporating unnatural amino acids (such as L-Dab, L-Dap) [31] or "specificity determinants" (such as Lys residues) [28], and multimerization of AMP [32], etc. [30]. In the study, it was found that the three peptides (Spa31, Spa20, and Spa22) all had good antibacterial activities, among which Spa31 had relatively stronger antibacterial and antifungal activity with the MBC range of 3-6 µM or 6-12 µM, which had the greatest hydrophobicity (42%) and the highest positive net charge (+7.5). We speculated that their net charge and hydrophobicity might fall within the optimal range as mentioned above. In addition, the 3D structure predictions indicated that all four synthetic peptides had a mixed extended structure, including α-helix, which needs to be confirmed by further evidence (e.g., circular dichroism analysis). The differences in the second structure might be attributed to their different antibacterial activity spectrum, and the underlying mechanism deserves in-depth investigation. Among them, Spa14 only showed antibacterial activity against two bacteria, with the MBC value of 12-24 µM against L. monocytogenes and 48-96 µM against A. baumannii. However, Spa14 still had certain antifungal activity (such as C. neoformans, A. niger, F. graminearum, and F. solani). Compared with other synthetic pep-tides, Spa14 had the shortest peptide length and the least positive charge but is similar in hydrophobicity to Spa22 and Spa31. Although Spa14 has weak antibacterial activity, it may still be used as an antifungal agent targeting fungi in the future. For most bacteria tested, Spa31 is always the best choice, but for some bacteria (such as A. baumannii, C. neoformans), the shortened peptides Spa20, Spa22, or Spa14 can be used to reduce application costs. Therefore, they all have potential application prospects.
Spa31 was further selected to reveal its antibacterial mechanism. We first observed the disruptive effect of Spa31 on bacterial morphological structure by SEM and confirmed the penetration of Spa31 into the bacterial outer and inner membrane by NPN, SYTO9, and PI staining using flow cytometry and confocal microscopy. Most AMPs can directly damage bacterial cell membranes [33][34][35]. For example, AMPR-11 causes changes in cell membrane permeability and cell rupture in carbapenem-resistant P. aeruginosa and methicillin-resistant S. aureus [36]. Similarly, after Spa31 treatment, the results showed that the inner membranes of S. aureus and P. aeruginosa were damaged, and a large amount of PI dye entered the cells. Both bacteria aggregated, had rough surface, shrank, and the contents flowed out, and filamentous substances appeared on the surface of P. aeruginosa. The human cathelicidin LL-37 is commonly used as a positive control, which has been demonstrated to exert antibacterial activity through disrupting bacterial membranes [37]. It is well known that the outer membrane of Gram-negative bacteria is their first barrier against environmental stress and is also the target of many antimicrobial agents. LPS is an important component of the outer membrane of Gram-negative bacteria and is negatively charged. Studies have shown that cationic AMPs, such as SNAPP, Brevicidine, and Laterocidine [38,39], can bind to LPS through electrostatic interactions, which is the first critical driver of their ability to target cell membranes. When AMPs accumulate to a threshold concentration on the bacterial surface, they can cause cell membrane lysis, thereby killing bacteria [40,41]. In this study, the addition of LPS reduced the anti-P. aeruginosa activity of Spa31 in a concentrationdependent manner, which indirectly indicated that Spa31 (also as a cationic AMP) could interact with LPS, and eventually disrupt the bacterial outer cell membrane (enhanced NPN fluorescence intensity was observed). After the permeability of the bacterial cell membrane changes, intracellular substances are released or leaked out of the cell. ATP is an energy substance produced by bacterial intracellular metabolism, and the release or leakage of this substance will affect the life activities of bacteria. Following AS-hepc3 (48)(49)(50)(51)(52)(53)(54)(55)(56) or α-helical AMP II treatment, the content of ATP in the bacterial extracellular environment was significantly increased in a concentration-dependent manner [42,43]. Likewise, in the study, Spa31 also caused an increase in extracellular ATP levels.
In addition, it is generally believed that excess ROS (reactive oxygen species) can rapidly oxidize various biological macromolecules such as DNA, proteins, and lipids in cells, thereby causing damage to organisms [44]. Many studies have shown that AMPs can induce excessive ROS production, leading to bacterial oxidative damage. For example, Cathelicidin can significantly increase ROS levels and induce the expression of E. coli ArcA, OxyR, gshA, and other genes, which play key roles in resistance to ROS damage [45]. In the study, 12 µM Spa31 treatment resulted in a 98.34% increase in ROS compared to the control group (0.64%) and LL-37 treatment group (87.19%), indicating that Spa31 caused severe oxidative stress in P. aeruginosa. This might be another strategy for Spa31 to effectively kill bacteria, in addition to disrupting cell membranes. Notably, some proline-and argininerich AMPs, such as A3-APO and Arasin 1, have multiple antibacterial mechanisms, and they interact with intracellular targets. For example, they bind to the heat shock protein DnaK, which causes protein misfolding and aggregation in cells, inhibition of biosynthesis, etc., and eventually leads to bacterial death [46]. Whether Spa31 has a similar bactericidal mechanism deserves further study. Furthermore, it is worth mentioning that the rapid bactericidal process of AMPs and the mechanism of action on cell membranes make it difficult to induce bacterial resistance [43,47]. In the study, Spa31 was able to kill S. aureus and P. aeruginosa within minutes and had anti-biofilm activity against P. aeruginosa. As reported, the antibiofilm mechanism of AMPs varies and involves different biofilm for-mation processes. For example, LL-37 can inhibit the formation of P. aeruginosa biofilm by reducing bacterial adhesion and disrupting the quorum-sensing systems [48]. Human β-defensin 3 regulated the expression of biofilm formation-related genes, such as binding protein transport genes [49,50]. Nisin A and esculentin  have been demonstrated to eliminate mature biofilms through disrupting bacterial membranes [51,52]. Peptide G3 may be involved in the entire biofilm formation processes, such as inhibiting bacterial attachment, destroying mature biofilm structure through interacting with extracellular DNA, and dispersing it [53]. Similarly, Spa31 could inhibit both P. aeruginosa biofilm formation and mature biofilm, and whether it exerts its antibiofilm effect through a mechanism similar to the aforementioned AMPs needs to be confirmed by further studies. In addition, whether Spa31 and the other three peptides exhibit antibacterial activity against clinical drug-resistant bacteria and does not induce bacterial resistance requires further evidence.
Due to the potential application value of Spa31, it is necessary to reveal whether its antibacterial activity is affected by various factors (such as temperature, cation, pH, etc.). BAMP derived from Bacillus paralicheniformis has a wide temperature (4-125 • C) and pH (2.0-9.0) tolerance, and is not easily degraded by trypsin, pepsin, and protease K. [54]. The AMP HSPE3 retained its antibacterial activity against Bacillus cereus regardless of high temperature steam heating or dry heating [55]. In this study, the results showed that Spa31 maintained antibacterial activity against S. aureus, P. aeruginosa, and E. coli after 30 min of treatment in boiling water, indicating it had good thermal stability ( Figure S2A-C). Furthermore, the electrostatic attraction between cationic AMPs and negatively charged bacterial surfaces was found to be easily shielded or disrupted in the presence of high concentrations of cations [56]. Under high salinity conditions, many AMPs have been reported to be inactivated, such as human β-defense-1 (inactivated by 125 mM of NaCl) and LL-37 (completely inactivated by 100 mM NaCl) [57][58][59]. However, some AMPs have good ionic tolerance. For example, human β-defensin 3 (hBD3) has antibacterial activity against various pathogens in the presence of 150 mM Na + . Among them, the C-terminal "RRKK" motif of hBD3 is thought to play a role in its cation tolerance mechanism [60]. In the study, Spa31 also showed a certain ion tolerance against S. aureus, P. aeruginosa, and E. coli under the condition of 80 mM Na + (Figure S2D-F). We speculate that Spa31 contains a high positive charge and binding activity to LPS, which might contribute to its resistance to cations. Taken together, the novel AMP Spa31 has good in vitro thermal stability and ion tolerance, laying a foundation for future application in food preservation, antibacterial agents, or feed additives. In addition, the stability of Spa31 under other environmental conditions, such as pH, serum, and various enzymes still needs to be further clarified.
It is believed that AMPs may have strong hemolytic activity due to their high positive charge number, which limits their application in animals. It has been reported that peptides with more than 9 positive charges can cause damage to blood cells. For example, AMP MAP-36 with 14 positive charges produces hemolysis at a concentration of 4 µM, while AMP RI12 with 6 positive charges shows hemolytic activity at a concentration of 128 µM [61]. In this study, Spa31 possessed biocompatibility and did not cause hemolysis of mouse erythrocytes at concentrations up to 250 µg/mL (72 µM), whose therapeutic index would exceed 24 against P. aeruginosa (the MIC value was 3 µM) (calculated by the ratio of hemolytic activity and MIC as described previously [29]). Meanwhile, the cytotoxicity of Spa31 was evaluated, and it did not affect cell viability under the concentration of 48 µM, which laid a foundation for subsequent development and application. Based on this, an in vivo model of bacterial infection in zebrafish D. rerio was established in this study to explore whether Spa31 had an anti-infective effect in vivo. The results showed that injecting a certain amount of Spa31 (10 µg per fish) significantly improved the survival rate of D. rerio infected with P. aeruginosa, indicating that Spa31 had a significant immunoprotective effect in vivo. The doses of Spa31 used in zebrafish were selected with reference to those used in actual clinical applications [62] and in mouse models [43,63,64] (5-20 mg/kg). Other AMPs have also used zebrafish infection models to evaluate their in vivo effects, such as Scyreprocin, epinecidin-1, and fish-derived cathelicidins, with effect doses of 1-8 µg, 1-10 µg, and 10 µg per fish, respectively [15,65,66]. We believe that the 10 µg Spa31 dose determined in this study provides a convincing reference dose for future clinical applications. Taken together, in this study, the preliminary analysis of the immune effect of the Spa31 in vitro and in vivo will lay a foundation for further in-depth discussion of the molecular mechanism of this AMP and provide reference for the clinical application of this peptide in the future.

AMP Prediction and Truncated Peptide Synthesis
After obtaining the full-length cDNA sequences of Spampcin gene (details are presented in Supplementary Materials), the AMP possibilities of its deduced amino acid sequences were predicted by the antimicrobial peptide database CAMP R3 , as well as the antimicrobial region within peptides. In CAMP R3 , 4 different prediction methods are involved, including support vector machine (SVM), random forest (RF), artificial neural network (ANN), and discriminant analysis (DA), and the closer the value is to 1, the higher the probability of AMP. Interestingly, we found that both the Spampcin protein and its derived region from position 56th to 86th showed high AMP potential (Table 1), named Spampcin 56-86 (Spa31 for short) (RRAAHGLLPRLRAPPPFHKRCVCLCRTAPPP). Afterwards, several truncated peptides derived from Spa31 were also selected for antimicrobial activity detection, including Spampcin 56-75 (Spa20 for short) (RRAAHGLL-PRLRAPPPFHKR), Spampcin 65-86 (Spa22 for short) (RLRAPPPFHKRCVCLCRTAPPP), and Spampcin 65-78 (Spa14 for short) (RLRAPPPFHKRCVC). Their AMP prediction results were summarized in Table 1. Four peptides were further chemically synthesized by GL Biochem (Shanghai, China) with a purity of >95%, and verified by high-performance liquid chromatography and mass spectrometry (Figures S3-S6). The total net charges and hydrophobic ratios of the four peptides were calculated by APD3. The 3D structures of four peptides were created using the Pep-Fold structure prediction tool for de novo peptides (https://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#forms::PEP-FOLD3 (accessed on 26 September 2022)).

Antimicrobial Assay
The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of four synthesized peptides were determined in triplicate on separate occasions following a previously described protocol [67]. Briefly, four peptides were serially diluted with Milli-Q water and placed on ice until use. All strains were harvested in logarithmic growth phase, washed with 10 mM Dulbecco's phosphate buffered saline (DPBS, pH 7.4), and adjusted to a concentration of 1 × 10 6 CFU mL −1 (bacteria) or 1 × 10 4 cells mL −1 (mold spores) in Mueller-Hinton (MH) broth. After incubation with an equal volume of peptides for 24 h (bacteria) or 48 h (mold spores), MIC and MBC values were determined. The effect of Spa31 on the germination of mold spores was further observed by light microscope (Leica Dmi1, Wetzlar, Germany). Experiments were performed in triplicate and repeated at least three times independently.

Zone of Inhibition Assay
S. aureus, P. aeruginosa, and E. coli were selected to conduct the zone of inhibition assay as previously described [68] with some modifications. Briefly, exponentially growing bacteria were harvested and diluted to a concentration of 1 × 10 6 CFU mL −1 in MH broth and 100 µL of bacterial solution were spread evenly on a sterile agar plate. Then 4 blank antimicrobial susceptibility discs (Bkmam, Changde, China) were pasted on the surface of the plate, and 20 µL of Spa31 or LL-37 at a concentration of 1 µg/µL were added to different discs. Meanwhile, 20 µL of Milli-Q water and 1 µg/µL of BSA at were used as controls. They were incubated overnight at 37 • C. The plate was observed the next day and the size of the inhibition zone was measured by a vernier caliper (Meinaite, Shanghai, China). Experiments were performed in triplicate and repeated at least twice independently.

Time-Killing Kinetics
S. aureus and P. aeruginosa were further selected to determine the bactericidal kinetics of Spa31 following a previous report [20]. Briefly, the final concentration of Spa31 was adjusted to 3 µM or 6 µM (1 × MIC or 1 × MBC for both bacteria). After incubating Spa31 with bacteria (prepared as described above for antimicrobial assay) for a period of time (within 30 min), bacterial suspensions were diluted and spread on nutrient broth plates at different time points and incubated at 37 • C for 24 h. Bacterial colony counts were recorded, and the bactericidal kinetic curves were drawn using GraphPad Prism 8.3.0 version (San Diego, CA, USA). Experiments were performed in triplicate and repeated at least three times independently.

Scanning Electron Microscope (SEM) Observation
The effects of Spa31 on S. aureus and P. aeruginosa were observed using SEM. The steps to prepare SEM samples refer to a previous study [17]. Briefly, 6 µM of Spa31 was incubated with the bacteria (1 × 10 7 CFU mL −1 ) first. After incubation for a period of time (10 min or 30 min), they were fixed with pre-cooled 2.5% glutaraldehyde at 4 • C for 2 h, dehydrated in a series of concentrations of ethanol in a critical point dryer (EM CPD300, Leica, Wetzlar, Germany), coated with gold, and finally observed under a scanning electron microscope (Zeiss SUPRA 55, Oberkochen, Germany).

SYTO 9 and PI Staining Assay
The permeability of Spa31 on the bacterial inner membrane of S. aureus and P. aeruginosa was analyzed. The collection of exponential phase bacteria was adjusted to a final concentration of 1 × 10 7 CFU mL −1 . The SYTO 9/PI (propidium iodide) mixture was prepared following the instructions of the LIVE/DEAD ® BacLight TM Bacterial Viability Kit (Invitrogen, Waltham, MA, USA). Equal volumes of bacteria and Spa31 (diluted to a final concentration of 6 µM) were incubated at 37 • C for 30 min, followed by the addition of the dye mix and incubated at room temperature for 15 min in the dark. The fluorescence intensities of SYTO 9 and PI in bacteria were detected by flow cytometry (CytoFLEX, Beckman, Brea, CA, USA) and observed by confocal laser scanning microscope (CLSM, Zeiss Lsm 780 NLO, Oberkochen, Germany). FlowJo_v10.7.1 software (Brea, CA, USA) was used for data analysis. Experiments were performed in triplicate and repeated at least three times independently.

NPN, LPS Inhibition, ATP Release, ROS Production Assays
To further reveal the antibacterial mechanism of Spa31 against P. aeruginosa, various assays were designed, including NPN, LPS inhibition, extracellular ATP release, and ROS production assays. P. aeruginosa and Spa31 were prepared as described above. Experiments were performed in triplicate and repeated at least three times independently.

NPN Staining Assay
The outer membrane permeability of Spa31 against P. aeruginosa was determined using the probe N-phenyl-1-naphthylamine (NPN) (Sigma-Aldrich, St. Louis, MO, USA) as previously described [43]. Briefly, the exponential growing P. aeruginosa was harvested, resuspended in HEPES buffer (5 mM HEPES, 5 mM glucose, pH 7.4), and diluted to 1 × 10 8 CFU mL −1 . NPN was then added to the diluted bacterial solution to a final concentration of 10 µM and placed in a 96-well black microplate at room temperature in the dark for 5 min. Afterwards, Spa31 was added to the mixture at final concentrations of 0, 3, 6, 12, and 24 µM. LL-37 (12 µM) and polymyxin B (PMB, 1 µg/mL) were used as controls. Fluorescence intensities (excitation 350 nm, emission 420 nm) were measured every 2 min (30 min total) in a microplate reader (Tecan, Männedorf, Switzerland).

LPS Inhibition Assay
The exponentially growing P. aeruginosa was harvested and adjusted to 1 × 10 6 CFU mL −1 in MH broth and added to a 96-well plate. Spa31 and P. aeruginosa-derived LPS (Sigma-Aldrich, Männedorf, Switzerland) were then added at final concentrations of 3 µM of Spa31 (1 × MIC) and 0, 3, 6, 12, 24 µg/mL of LPS. The plate was placed in a microplate reader and detected every 0.5 h for 24 h. The absorbance at 595 nm was recorded and the curve was plotted using GraphPad Prism 8.3.0 version (San Diego, CA, USA).

ATP Release Assay
P. aeruginosa suspensions at 1 × 10 8 CFU mL −1 in MH broth were incubated with a series of concentrations of Spa31 (0, 3, 6, 12, and 24 µM) at 37 • C for 10 min. Then, the mixture was centrifuged, and the supernatant was collected and subjected to analysis of the extracellular ATP using a GloMax ® 20/20 Luminometer E5311 (Promega, Madison, WI, USA) according to the instructions of the Enhanced ATP Assay Kit (Beyotime, Shanghai, China).

Biofilm Inhibition Assays
The biofilm inhibition assays were carried out as previously described [69,70] with some modifications. Experiments were performed in triplicate and repeated at least three times independently.

Biofilm Formation Inhibition Assay
Exponentially growing P. aeruginosa were harvested, adjusted to 1 × 10 8 CFU mL −1 in MH broth and placed in a 96-well plate. A series of concentrations of Spa31 (0, 1.5, 3, 6, 12, 24, and 48 µM) were added and incubated at 37 • C for 24 h. After incubation, planktonic cells were removed, and the wells were gently washed 3 times. The plate was then placed in a 60 • C oven for 30 min to fix the biofilm and stained with crystal violet (0.1%, w/v). The absorbance at 595 nm were recorded using a microplate reader (Tecan, Männedorf, Switzerland).

Mature Biofilm Inhibition Assay
Similarly, exponentially growing P. aeruginosa was harvested, adjusted to 1 × 10 6 CFU mL −1 in MH broth, and placed in a 96-well plate for 24 h at 37 • C to allow biofilm maturation. Planktonic cells were removed, and the wells were gently washed 3 times. Then, Spa31 (at the final concentrations of 0, 1.5, 3, 6, 12, 24, 48 µM) and resazurin (at the final concentration of 0.1 mM) were added. The plate was incubated at 37 • C for 6 h and the absorbance at 560 nm and 620 nm were measured with a microplate reader (Tecan, Männedorf, Switzerland). The respiratory activity of cells in biofilms were evaluated by a modified resazurin assay as previously described [66]. The cytotoxicity of Spa31 on cell lines (including RAW 264.7, HEK-293T, and HepG2 cells) was evaluated using the MTS method as previously described [15]. The cell lines used in the study were all mycoplasma-negative, which were purchased from the Stem Cell Bank at the Chinese Academy of Sciences and also confirmed by the mycoplasma test kit according to the manufacturer's instructions (Solabio Life Science, Beijing, China) ( Figure S7). Briefly, cells were seeded at 10 4 cells well −1 in a 96-well plate overnight at 37 • C with 5% CO 2 . Then, the medium containing Spa31 was added at final concentrations of 0, 3, 6, 12, 24, and 48 µM and incubated for 24 h. Cell viability was determined following the instructions for the CellTiter 96 ® AQueous Kit (Promega, Madison, WI, USA). Experiments were performed in triplicate.

Hemolytic Activity Assay
The hemolytic activity of Spa31 on mouse erythrocytes was assessed as previously described [43]. Briefly, fresh mouse erythrocytes were collected, washed several times until the upper phase was clear, and adjusted to a concentration of 5 × 10 9 cells mL −1 . Then, Spa31 was added at the final concentration of 15.625, 31.25, 62.5, 125, 250 µg/mL (4.5, 9, 18, 36, 72 µM, respectively) and incubated at 37 • C for 1 h. PBS and 0.5% TritonX-100 treatment were used as negative and positive controls, respectively. Absorbance at 540 nm were recorded using a microplate reader (Tecan, Männedorf, Switzerland) and the hemolysis rates were calculated as previously described [43].
All animal procedures were carried out in strict accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Welfare and Ethics Committee of Xiamen University.

Evaluation of the In Vivo Infective Effect of Spa31 on Zebrafish D. rerio Infected with P. aeruginosa
To investigate the in vivo activity of Spa31, a zebrafish D. rerio bacterial infection model was used. After one week of temporary feeding in the laboratory, the fish were pre-injected intraperitoneally with P. aeruginosa at the final concentration of 3.7 × 10 6 CFU fish −1 . After 15 min of infection, Spa31 (5 µg fish −1 or 10 µg fish −1 ) was injected. And the control group was injected with the same volume of PBS. There were 24 zebrafish in each of the experimental groups and the control group. The survival rates of zebrafish were recorded at different time points and the survival curve was drawn with GraphPad Prism 8.3.0 version (San Diego, CA, USA). The independent assay was repeated at least twice.
All animal procedures were carried out in strict accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Welfare and Ethics Committee of Xiamen University.

Statistical Analysis
All data were presented as mean ± standard deviation (SD). All the statistical analyses were performed using GraphPad Prism 8.3.0 version (San Diego, CA, USA). For the extracellular ATP, ROS production, and biofilm inhibition assays, one-way ANOVA was employed to compare each treatment group with the control group. For the in vivo assay, survival comparisons were analyzed using the Log-rank (Mantel-Cox) test. Difference was considered as significant at p value < 0.05.

Conclusions
In summary, a new AMP, Spampcin 56-86 (Spa31 for short), was identified from Scylla paramamosain and three truncated peptides derived from Spa31 were also found to exhibit potent antibacterial activity with different spectra. Among them, Spa31 showed the strongest antibacterial activity against a variety of clinical pathogens (such as P. aeruginosa, E. coli, A. baumannii, S. aureus, L. monocytogenes, and E. faecalis) with the MBC values ranging from 1.5 µM-6 µM and could kill S. aureus and P. aeruginosa within minutes. Spa31 interacted with LPS, and directly disrupted the integrity of the inner and outer cell membrane of P. aeruginosa, resulting in the release of ATP and the production of ROS, which might be the bactericidal mechanism of Spa31. In addition, Spa31 had a significant anti-biofilm effect against P. aeruginosa, not only on biofilm formation but also on mature biofilms. The antibacterial activity of Spa31 had good thermal stability, certain ion tolerance, and had no obvious cytotoxicity in vitro, and Spa31 exhibited an anti-infective effect in vivo which could significantly improve the survival rate of zebrafish Danio rerio infected with P. aeruginosa. bactericidal concentration; MFC, minimum fungicidal concentration; MH, Mueller-Hinton broth; MIC, minimum inhibitory concentration; NPN, N-phenyl-1-naphthylamine; ORF, open reading frame; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PI, propidium iodide; PMB, polymyxin B; pI, isoelectric point; qPCR, real-time quantitative polymerase chain reaction; RACE, rapid-amplification of cDNA ends; RF, random forest; ROS, reactive oxygen species; SD, standard deviation; SEM, scanning electron microscope; SVM, support vector machine.