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9 October 2022

Skin Mucus Proteome Analysis Reveals Disease-Resistant Biomarker Signatures in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) against Vibrio alginolyticus

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1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia (UPM), Serdang 43400, Malaysia
2
Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia (UPM), Serdang 43400, Malaysia
3
Department of Biology, Faculty of Science, Universiti Putra Malaysia (UPM), Serdang 43400, Malaysia
4
Malaysian Palm Oil Board, Persiaran Institusi, Bandar Baru Bangi, Kajang 43000, Malaysia
Fishes2022, 7(5), 278;https://doi.org/10.3390/fishes7050278
This article belongs to the Section Welfare, Health and Disease
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Abstract

Fish skin mucus is the first line of defense that provides physical and chemical barriers against pathogens and toxins. The mucus is produced continuously and sloughed off regularly from the skin to defend against infections through the skin. However, the molecular properties of the mucus content that prevent pathogen invasion are yet to be fully understood. In this study, a proteomic approach using liquid chromatography–mass spectrometry (LCMS) was applied to explore the changes in the mucus protein content of resistant and susceptible groupers in response to Vibrio alginolyticus. The Vibrio-resistant groupers showed no observable clinical sign of infection after the immersion challenge, while the Vibrio-susceptible groupers presented either hemorrhagic- or non-hemorrhagic ulceration of the skin. A comparative proteome analysis on the mucus samples yielded 1488 identified proteins. The immune-related proteins, namely Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1, that were significantly abundant in the resistant group responded to V. alginolyticus infection. Interestingly, there was an expression of immune-related proteins that possibly could be the non-invasive biomarkers, namely 3-hydroxybutyrate dehydrogenase type 2 and L-rhamnose-binding lectin SML.

1. Introduction

The world aquaculture production in 2018 surpassed capture fisheries with a record of 114.5 million tons of production. Fish protein primarily derived from aquaculture is expected to comprise 59% of human consumption by 2030 [1]. Grouper is one of the most abundant fishery species due to its significant commercial value that has been established in Singapore, Malaysia, Hong Kong, Thailand and Taiwan since the 1970s. Among the most common grouper species are the Epinephelus fuscoguttatus (Brown Marble grouper), Epinephelus coiodes (Orange Spotted grouper), Epinephelus lanceolatus (Giant grouper), Epimetheus tauvina (Greasy grouper), Mycteroperca tigris (Tiger grouper) and Cromileptes altivelis (Mouse grouper). However, throughout the years, the transition of the aquaculture technique from extensive to semi-intensive and intensive farming has led to the emergence and outbreak of infectious diseases. In order to mitigate the outbreak, several approaches have been taken to improve the quality of fish seeds produced in the hatchery. Hybridization, an approach to crossbreeding between two species, has been practiced in aquaculture to improve the quality of the fish seeds. The hybrid offspring inherited the desirable traits of the parental species, such as a superior growth rate, robustness and disease resistance [2,3,4]. The hybrid of E. fuscoguttatus × E. lanceolatus represents 70% of the total cultured grouper population in Indonesia, and this hybrid has become the second target species for aquaculture production in Hong Kong [5,6,7,8].
Although the novel hybrid of E. fuscoguttatus × E. lanceolatus yielded a significant and superior growth performance over its parental species [9], the inheritance of the desirable disease-resistant trait requires further evaluation [9,10,11]. Notwithstanding the reported improved immunological parameters in this hybrid grouper [9], the aquaculture of the hybrid species is still being affected by the prevailing infectious diseases, such as grouper iridovirus [12], vibriosis [13] and marine leech infestation [14]. Vibriosis is among the most common infectious diseases that affect grouper aquaculture. This disease is caused by the Gram-negative Vibrio sp., which includes V. alginolyticus, V. vulnificus, V. harveyi and V. parahaemolyticus [15]. Dark skin, pale gills, excessive mucus production, hemorrhage and ulceration of the affected areas, such as body, mouth and fins, are the common clinical signs of infection. The outbreak of vibriosis commonly resulted in a high mortality rate of the affected farms, causing an inevitable slump in marine aquaculture production [16,17].
Proteomics has been widely applied in studies related to infectious diseases, which include drug discovery, biomarker identification, disease pathogenesis, vaccine development and disease diagnosis, as well as in fundamental research to comprehend the host’s physiological and immunological responses against pathogens [18,19]. The characterization of the immunological responses against pathogen invasion using the proteomics approach facilitates the identification of novel immune-related proteins and potential biomarkers for disease diagnosis and therapy [20,21]. This approach has successfully identified differentially expressed proteins in different immune organs of the disease-resistant/tolerant and the susceptible fish [22]. The fish skin mucus is rich in biochemicals, especially proteins and metabolites [23,24]; hence, a comparative analysis of the mucus has become of great interest in understanding the immunological responses of skin- and mucosal-associated lymphoid tissues. The functional skin mucus proteome of disease-resistant grouper is hypothesized to provide new insights into the disease-resistant/tolerant phenotype, which could assist in the screening of broodstock/fish seed quality. This study aimed to identify the proteome changes in the grouper skin mucus in response to Vibrio alginolyticus infection and the potential protein biomarkers that correspond to the disease-resistant phenotype.

2. Materials and Methods

2.1. Fish and Bacterial Infection, Mucus Sample Collection and Preparation

All animal handling and experimental protocol were performed in accordance with the ethics guidelines approved by Universiti Putra Malaysia Animal Ethics Committee (Approval number: UPM/IACUC/AUP-R054/2022). Hybrid grouper fingerlings (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) in 9.6 ± 0.4 cm size were obtained in April 2021 from a local hatchery in Kuala Selangor, Malaysia, and transported to the laboratory using plastic bags containing oxygen. The fingerlings were screened for health issues, from which only healthy fish were subjected to acclimatization under laboratory conditions for 2 weeks in 25 ppt saltwater with continuous aeration and fed with commercial feed twice daily on an ad libitum basis. The water temperature was maintained at 27 to 34 °C throughout the day.
Vibrio alginolyticus isolate was obtained from the Laboratory of Fish Health and Disease, Department of Aquaculture, Universiti Putra Malaysia. The isolate was cultured on marine agar for 24 h at 16 °C. A single colony was inoculated in 10 mL marine broth, and then the culture was up-scaled to 1 L. Bacterial cells were then collected through centrifugation at 11,000× g for 15 min. Bacterial cell suspension of 9.4 × 107 CFU/mL was prepared in 25 ppt saltwater for subsequent experimental infection in grouper fingerlings.
Eighty healthy grouper fingerlings with a mean total body length of 9.6 ± 0.4 cm were randomly selected. For experimental infection, 60 fingerlings were exposed to V. alginolyticus at a dose of 9.4 × 107 CFU/mL by immersion for 4 h, whereas the control group consisted of 20 fingerlings were not exposed to V. alginolyticus. The fingerlings were then transferred to clean saltwater with continuous aeration for observation, and the control group was kept in a separated aquarium. On day 7 post-infection, fingerlings were categorized into Vibrio-susceptible group according to the presence of clinical signs of infection that include red spots, hemorrhagic and/or non-hemorrhagic ulceration on the body, while the Vibrio-resistant fingerlings presented no observable clinical sign of infection. The fish were fasted 12 h before harvesting.
In the pre-harvesting process, all (46 samples) of the fish mucus from resistant and susceptible groups, including those from the dead fish, were spread individually on marine agar and thiosulfate citrate bile salts sucrose agar (TCBS: selective media for Vibrio sp.) to confirm the presence of Vibrio. The bacteria from the dead fish spleen were also detected by plating on the designated agar. All the subjected samples showed a positive Vibrio growth on the TCBS agar and thus confirmed the presence of Vibrio through the immersion method.
Fish were anesthetized using tricaine methanesulfonate (MS222) before skin mucus sampling. Skin mucus (n = 5) was collected by gently scraping the body using a sterile glass slide avoiding the collection of blood and the anal area. The 5 mucus samples of fish from the same group were pooled to represent one biological replicate due to the minute amount of mucus from each fish. All samples were stored at −80 °C until further use. The mucus samples were reconstituted in 1× phosphate-buffered saline and sample buffer (7 M Urea, 2 M Thiourea, 2 mM PMSF). The samples were mixed well and sonicated on ice at 20% amplitude for 3 min (10 s pulses on, 15 s pulses off). The samples were then centrifuged at 15,000× g for 15 min to collect supernatant containing skin mucus total protein which concentration was quantified using Bradford assay (Solarbio, Beijing, China) before subsequent analysis. The total protein in 9 µg from each sample was electrophoretically separated on 12% SDS polyacrylamide gel and double stained with Coomassie-silver.

2.2. Liquid Chromatography–Mass Spectrometry (Hybrid Quadrupole-Orbitrap Technology)

Nine samples of 3 biological replicates from each group were submitted to proteomic analysis using LCMS. Prior to LCMS, protein samples of 75 µg were loaded into 12% SDS polyacrylamide gel. Protein bands were then excised and in-gel digested using trypsin gold (Promega, Madison, WI, USA). Subsequently, clean-up of the digested samples was performed using Ziptip (Millipore, Burlington, MA, USA). LCMS analysis of the peptide samples was conducted according to the previous report [25]. In brief, peptide samples were reconstituted in 30 μL of 0.1% formic acid and 5% acetonitrile, before 2 μL of the samples were loaded onto an Acclaim PepMap 100 C18 column (2 μm, 0.075 × 150 mm) (Thermo Scientific, Waltham, MA, USA). The reverse phase column was equilibrated with 0.1% aqueous formic acid (mobile phase A) and 80% acetonitrile containing 0.1% formic acid (mobile phase B). Elution of gradient 5–35% mobile phase B was performed at a flow rate of 0.3 mL/min for 75 min using EASY-nano liquid chromatography (EASY-nLC) 1200 System (Thermo Scientific, MA, USA). An online Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer system (Thermo Scientific, MA, USA) was used to generate the peptide ions with a spray voltage of 1900 V in positive mode. A precursor ion scan was conducted with a resolution of 70,000 and a mass range of m/z 310–1800. Precursors containing charge states from 2+ to 8+ were further fragmented via collision induced and high-energy collision induced (CID and HCD) at normalized energy of 28%. Precursor mass was scanned at the range of 110–1800 m/z.

2.3. Data Processing and Protein Annotation

Mass spectra of the peptides were acquired using Tune (Ver. 2.11 QF1 Build 3006; Thermo Scientific, Waltham, MA, USA) and deconvoluted with Proteome Discoverer Ver. 2.4 (Thermo Scientific, Waltham, MA, USA) to create the peptide mass list. SEQUEST HT search engine, incorporated in the Proteome Discoverer, was used to match the generated mass list against Epinephelus FASTA sequences downloaded from NCBI. Mass tolerance for the peptides and their fragments was fixed at 10 ppm and 0.02 Da, respectively. Trypsin was indicated as the digestion enzyme with up to two miscleavages allowed during the search. Carbamidomethylation (CAM) modification on cysteine residues was set as a static modification, while variable amino acid modifications were set as deamidation for asparagine and glutamine residues, and oxidation for methionine residues. The mass list was also searched against a decoy database generated from the randomized protein sequences. The identified proteins were accepted with at least a Rank 1 peptide and a false discovery rate of 1%. Spectra that matched the sequences were further validated with the Percolator algorithm (Ver. 2.04) where q-value at 1% false discovery rate (FDR).

2.4. Statistics and Protein Enrichment

The statistical analysis was performed using Perseus [26] to identify differentially expressed proteins. One-way ANOVA was applied and the comparison between groups was analyzed by applying a two-tailed Student’s t-test (p ≤ 0.05). Proteins with fold change ≥2 were identified to be differentially expressed [27], and the differentially expressed proteins identified were mapped to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analyses. Fisher’s exact test was applied to perform enrichment analysis (p-value ≤ 0.05).

3. Results

3.1. Experimental Infection of Hybrid Grouper with Vibrio alginolyticus

The hybrid grouper fingerlings were exposed to V. alginolyticus by immersion in the saltwater of 25 ppt containing approximately 9.4 × 107 CFU/mL of V. alginolyticus for 4 h. On day 7 post-infection, 10 mortalities out of 60 infected fingerlings were recorded (16.7% of cumulative mortality), 28 fingerlings (46.7%) were resistant to V. alginolyticus and 18 fingerlings (30%) were susceptible to the infection, exhibiting clinical signs of infection where ulceration and red spots were observed on the mouth, body and fins (Figure 1). The resistant fingerlings recorded no observable clinical signs of infection.
Figure 1. Development of ulcer and lesions on the body (a), mouth (b), anal fin (c), dorsal fin (d) and caudal fin (e) of hybrid grouper that were susceptible to Vibrio alginolyticus experimental infection, comparable to the resistant hybrid grouper where no observable ulcer and lesion were recorded.
The infection of V. alginolyticus on the selected healthy hybrid grouper was confirmed by applying the bacteria plating method. The fish mucus from resistant and susceptible groups was spread individually on marine agar and TCBS agar to confirm the presence of Vibrio in the fish. All the subjected samples, including the spleen of the dead fish, showed positive for Vibrio infection on the TCBS agar (Appendix A Figure A1). However, the bacterial load was not quantitatively analyzed. The immersion in the same concentration of bacterial suspension in controlled conditions and exposure time might have given similar bacterial exposure to the fish.
The evaluation on the skin mucus protein of the susceptible and resistant groups showed viscosity variation. The susceptible group secreted much more and a viscous mucus than the resistant fish. The protein composition of the skin mucus between the infected group and control group was significantly different with the former showing more variable intensities of the protein bands (Appendix A Figure A2).

3.2. Skin Mucus Proteome Profiling and Differential Expression Analysis

The label-free shotgun proteomic analysis using LCMS identified a total of 1488 proteins in the skin mucus samples. Out of the 1488 proteins, 3, 2 and 8 proteins were found to be exclusively expressed in the control, resistant and susceptible groups, respectively (Figure 2). The identified putative proteins are listed in Table 1.
Figure 2. Venn diagram that summarized the total number of putative proteins identified in control, resistant and susceptible groups, respectively, in response to Vibrio alginolyticus experimental infection.
Table 1. Putative proteins were found to be exclusively expressed in the control, resistant and susceptible groups, respectively.

3.3. Differentially Expressed Proteins (DEPs) in Response to V. alginolyticus Experimental Infection

A pairwise comparison between the control, resistant and susceptible groups revealed different numbers of DEPs in response to the V. alginolyticus experimental infection (Figure 3). A comparison of the protein abundance between the control and resistant groups reveals 99 DEPs (Table 2), where 47 upregulated and 52 downregulated proteins were from the resistant group, while 60 upregulated and 42 downregulated proteins were identified in the susceptible group compared to the control group (Table 3). The comparison between the resistant and susceptible groups revealed 94 DEPs (Table 4), wherein 28 upregulated and 66 downregulated proteins were recognized in the resistant group. Based on the list of DEPs, the immune changes in V. alginolyticus infection could be deciphered. These can be seen through the alteration of the expression of an immune-related protein, namely Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1, Annexin A1, 3-hydroxybutyrate dehydrogenase type 2 and L rhamnose-binding lectin SML.
Figure 3. The volcano plot showed a pairwise comparison of mucus proteome for the identification of significant differentially expressed proteins (p ≤ 0.05). (a) Control group versus resistant group; (b) control group versus susceptible group; (c) susceptible group versus resistant group.
Table 2. Differentially expressed protein was found to be resistant when compared to the control.
Table 3. Differentially expressed protein was found to be susceptible when compared to the control.
Table 4. Differentially expressed proteins were found to be in resistant when compared to susceptible.

3.4. Gene Ontology Analysis of DEPs

In order to gain further insight into the DEPs, they were categorized according to their biological process, molecular and cellular functions (Figure 4). The number of proteins annotated in most of the GO terms for the resistant group was relatively higher than the susceptible group, except for the developmental process (GO: 0032502), ATP-dependent activity (GO: 0140657), molecular function regulator (GO: 0098772) and transporter activity (GO: 0005215). The five GO terms annotated with the highest number of proteins were the cellular process, metabolic process, binding, cellular anatomical entity and protein-containing complex.
Figure 4. Gene Ontology annotations of the differentially expressed protein in the mucus of Vibrio-resistant and -susceptible hybrid groupers based on the PANTHER classification system (p ≤ 0.05), which were categorized into biological process, molecular function and cellular function. y-axis: number of proteins; x-axis. GO terms. a: response to stimulus (GO: 0050896); b: signaling (GO: 0023052); c: developmental process (GO: 0032502); d: cellular process (GO: 0009987); e: metabolic process (GO: 0008152); f: locomotion (GO: 0040011); g: biological regulation (GO: 0065007); h: localization (GO: 0051179); i: molecular adaptor activity (GO: 0060090); j: binding (GO: 0005488); k: structural molecule activity (GO: 0005198); l: ATP-dependent activity (GO: 0140657); m: molecular function regulator (GO: 0098772); n: catalytic activity (GO: 0003824); o: transporter activity (GO: 0005215); p: cellular anatomical entity (GO: 0110165); q: protein-containing complex (GO: 0032991).

3.5. KEGG Enrichment

To ascertain the pathway-enriched proteins, the DEPs entailed in different biological pathways were annotated using the KEGG reference database. The DEPs were significantly (p-value ≤ 0.05) enriched in 17 pathways, with most of the DEPs highly associated with metabolic pathways followed by oxidative phosphorylation, glycolysis and carbon metabolism (Table 5).
Table 5. KEGG pathways enrichment analysis of differentially expressed proteins that were associated with V. alginolyticus experimental infection.
More details of the differentially expressed proteins are presented in the appendix tables.

3.6. Identification of Potential Biomarker Candidates (PBCs)

The potential biomarkers that correspond to the Vibrio-resistant phenotype were identified and listed in Table 6. Thirteen proteins were found to be significantly altered in the resistant group when compared to the control, whereas no significant difference in these proteins was detected in the susceptible fish. This suggests disease-resistant properties against V. alginolyticus infection could be due to these identified non-invasive biomarkers.
Table 6. Potential biomarker candidates.

4. Discussion

Skin mucus is known to be the first line of defense against diseases through the non-specific immune system [28]. Through proteome profiling, one can gain an understanding of molecular changes under certain events. Hence, it has been proven to be beneficial in gaining intuition about an organism’s biological response [29,30,31]. However, the immune response of hybrid groupers toward Vibrio sp. infection is poorly understood, owing to the little understanding of the underlying molecular mechanisms. This study assessed the acute inflammatory response of hybrid groupers by proteome analysis on skin mucus which allowed a better understanding of the fish immunity as well as to determine the possible disease biomarkers.
Currently, the execution of Vibrio sp. on hybrid groupers marked a positive effect on the innate immune response. Protein profiling on skin mucus showed several identified proteins in the susceptible group compared to the resistant group, which indicates a positive response to the infection [32]. The majority of DEPs were also found to be perturbed in the resistant and susceptible groups involved in the cellular process based on the GO classification and the metabolic and oxidoreductase phosphorylation activity as in the KEGG enrichment. In fish, skin mucus is crucial in providing respiratory and metabolic function [33]. In this regard, the present study exhibits most of the DEPs being enriched in metabolic pathways which could be classified as enzyme energy metabolisms, such as NADH dehydrogenase and ATP synthase. Moreover, two crucial proteins that may be involved in the pathological immune response, 3-hydroxybutyrate dehydrogenase type 2 (BDH2) and L rhamnose-binding lectin SML (L-RBL), were also observed.
It is notable that the expression of BDH2, a regulatory molecule that was found to be enriched in metabolic pathways and the synthesis and degradation of ketone bodies pathways, is increased only in the resistant group. BDH2 is a key component of ketone bodies, an endogenous molecule that is responsible for maintaining cellular energy and modulating the signaling cascade through the cellular process. The cell can derive energy from BDH2 during disease infection [34,35]. A slight increase in BDH2 in concentrations was modulated by signaling pathways as they became involved in the cell growth, proliferation and oxidative stress resistance [36]. BDH2 can be activated in immune cells that lead to an anti-inflammatory effect [37]. The present findings support that BDH2 is involved in providing the disease-resistant properties of hybrid groupers, which is advantageous as a basis for developing the non-invasive biomarkers.
In addition, the L-RBL protein was also discovered in this study. It was anticipated that this immune-related protein would have a distinct role in providing disease resistance. Focusing on its high expression in resistant fish, it can offer a non-invasive biomarker during the segregation of disease-resistant and -susceptible groupers. L-RBL is a member of the rhamnose-binding lectin proteins (RBLs) that belong to the animal lectin group, a sugar-specific binding protein that recognizes the carbohydrate structure domain. RBLs are widely distributed in viruses, prokaryotes and eucaryotes [38], exclusively found in teleost and aquatic invertebrates which have been previously identified in eggs, serums and skin mucus [39,40,41]. It has been reported that RBLs can provide innate immunity as they inhibit proliferation, cytotoxicity and opsonize nonself-cells and particles [42]. The present study is consistent with the reported data on the clearance of Gram-positive and -negative bacteria by phagocytosis, which is enhanced by the expression of RBLs in sea bass [40].
The proteomic analysis also revealed some immune-related DEPs which are Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1. These DEPs have provided us with an understanding of the molecular changes during Vibrio infection.
Cystatin B was found to be upregulated in the infected fish only. This suggests the idea of fish with naive immunity may produce this protein in response to pathogen invasion to maintain their physiological state. The role of Cystatin B is consistent with the previous study [43], as the overexpression of Cystatin B in mice interacted with various types of proteins to activate the JAK/STAT signaling pathway for the removal of pathogens and the killing of infected cells. Cystatin B may also be present and translocated into mitochondria, which helps to protect the mitochondria activity and increase the ROS generation, enhancing the activity of pathogen removal [44,45]. Another protein that shares the same pattern of expression with Cystatin B is Complement component C6 and Complement factor 1. As components of the complement system, they are believed to play an important role in forming the membrane attack complex (MAC) and could assist in the cytolytic killing of the pathogen [46,47].
Other immune-related proteins were also found to be upregulated after infection in the resistant compared to the susceptible group. Allograft inflammatory factor 1 (AIF-1), Deleted in malignant brain tumors protein (DMBT1) and MHC class 1 alpha were detected. In this study, the resistance to infection was associated with high levels of Allograft inflammatory factor 1 (AIF-1) expression. AIF-1 is a 17 kDa type II interferon (IFN-y), for which expression is most likely limited to macrophages and monocytes. The high expression of AIF-1 was previously found in the spleen of E. awaora treated with lipopolysaccharides [48]. The present findings agree with the previous studies wherein AIF-1 was noted to provide innate immunity in various ways. According to a previous report [49], the presence of AIF-1 on the body wall of the infected area showed the migration and recruitment of numerous counts of macrophages after the lipopolysaccharides’ stimulation. AIF-1 is also claimed to be involved in wound healing and shell repair after the tissue injury of abalone [50]. Theoretically, the protein recognition receptor on the surface of Gram-negative bacteria is able to recognize the molecular pattern associated to the pathogen and hence initiate the defense mechanism of the cellular process. AIF-1 also has been proven to be involved in the innate immunity in the head kidney, spleen and various body tissues of vertebrates, but it was first established in the skin mucus of an aquatic organism [50,51,52].
Another detected protein is DMBT1 which belongs to a scavenger receptor protein that can bind to both Gram-negative and -positive bacteria. DMBT1 expression is obvious upon inflammation and epithelial cell differentiation and is involved in innate immunity. This protein was previously found to be upregulated in intestinal epithelial cells after lipopolysaccharide or tumor necrosis factor induction. However, it can also inhibit LPS-induced NF-B activation and cytokine secretion, leading to anti-inflammatory functions that can trigger the acquired immunity later [53,54].
MHC class 1 is a major histocompatibility complex class 1. The overexpression of MHC class I in a resistant group compared to the control that involved cell adhesion molecule and phagosome pathways was observed in the present study. In teleost, MHC class 1 normally displays and presents itself as a surface antigen-presenting cell on an infected cell. Upon entering the environment, an unknown pathogen will infect the cell which will then be degraded by innate immunity. As a result, MHC class 1 is produced within the reticulum and the pathogen is marked to trigger the cytotoxic T-cell wherein this process will help in the killing of the pathogen [55,56]. This finding is in line with the previous report in which the V. alginolyticus was found to be cytotoxic and lethal to fish cell lines and able to degrade the mucus of sea bream [57].
The present finding also reveals AnxA1 as the significantly expressed protein detected in the resistant group after infection. The AnxA1 protein was found to have dual functions in inducing inflammation. Over 100 types of annexins have been discovered in various tissues of vertebrates and invertebrates. The binding properties of this protein are influenced by cytoskeleton interactions, membrane fusion, anticoagulation, intracellular signaling and phospholipase inhibition. Molecular studies of annexin in humans are sparse, but some research could be found on teleost [58,59]. In humans, the expression of AnxA1 is a pro-inflammatory and anti-inflammatory response increased by exogenous and endogenous glucocorticoids, respectively. It is capable of controlling cell apoptosis in various pathways [59,60]. In the infected human, the cell will undergo apoptosis that may cause inflammation in the host [61]. Therefore, AnxA1 serves in apoptotic cell removal by acting as a highly specific signaling ligand of an endogenous danger molecule to enhance phagocytic clearance without causing any inflammation [61,62,63,64,65]. AnxA1 has been detected upregulated in the gill mucus of Atlantic salmon infected with amoebic gill disease with no observed lesion [66]. However, this protein was also expressed in serum and provided immunity to Chinook salmon with skin lesions [60]. The anti-inflammatory role of AnxA1 is still largely unknown, even though its pro-inflammatory-induced protein role is well studied in fish

5. Conclusions

This study has successfully generated new knowledge which strongly suggests that 3-hydroxybutyrate dehydrogenase type 2 and L-rhamnose-binding lectin SML protein, as new non-invasive biomarkers, could be used in the discrimination between Vibrio-resistant and -susceptible groupers. In addition, the biological context exhibited by the mucus is also provided, likely to aid in enhancing the understanding of fish immunity under natural infection. Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1 protein are signified as having distinct functions and roles toward V. alginolyticus infection.

Author Contributions

Conceptualization, A.C.; Data curation, B.Y.C.L. and L.C.F.; Formal analysis, N., B.Y.C.L. and L.C.F.; Funding acquisition, A.C. and I.S.I.; Investigation, N., W.M.S.W.S. and L.C.F.; Methodology, N. and L.C.F.; Project administration, I.S.I.; Supervision, A.C., W.M.S.W.S., I.S.I. and L.C.F.; Visualization, A.C.; Writing—original draft, N.; Writing—review & editing, I.S.I. and L.C.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Education (grant number TRGS/1/2020/UPM/02/1/3).

Institutional Review Board Statement

The study was conducted in accordance with the ethics guidelines approved by Universiti Putra Malaysia Animal Ethics Committee (Approval number: UPM/IACUC/AUP-R054/2022).

Data Availability Statement

Supporting data will be provided upon acceptable request.

Acknowledgments

This research was supported by the grant TRGS/1/2020/UPM/02/1/3. We thank the Institute of Biology System (INBIOSIS) for providing the experiment facilities and PROMET Laboratory, and MPOB for the research collaboration on the Orbitrap-MS System.

Conflicts of Interest

Authors declare that they have no conflict of interest.

Appendix A

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Figure A1. Bacteria isolated on the TCBS agar from the mucus and spleen of dead fish (a), mucus of the resistant (b), and susceptible (c) hybrid groupers.
Figure A1. Bacteria isolated on the TCBS agar from the mucus and spleen of dead fish (a), mucus of the resistant (b), and susceptible (c) hybrid groupers.
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Figure A2. Evaluation on mucus protein from different hybrid grouper groups by electrophoretically separated on 12% SDS polyacrylamide gel and double stained with Coomassie-silver. C1,C2,C3—control, S1,S2,S3—susceptible, and R1,R2,R3—resistant.
Figure A2. Evaluation on mucus protein from different hybrid grouper groups by electrophoretically separated on 12% SDS polyacrylamide gel and double stained with Coomassie-silver. C1,C2,C3—control, S1,S2,S3—susceptible, and R1,R2,R3—resistant.
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Table A1. Differentially expressed protein-enriched in KEGG pathways.
Table A1. Differentially expressed protein-enriched in KEGG pathways.
Pathway IDDescription Protein Count p-ValueProtein
Accession Name
1100Metabolic pathways300.000834906005glyceraldehyde-3-phosphate dehydrogenase
295792242nucleoside diphosphate kinase B
328677247glyceraldehyde 3-phosphate dehydrogenase isoform, partial
221048061fructose-bisphosphate aldolase
834904058malate dehydrogenase
1832677686NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial
834904842phosphoglycerate kinase
1832683648nucleolin
18326872253-hydroxybutyrate dehydrogenase type 2
1832613275cytochrome c oxidase subunit 5A, mitochondrial
197725770mitochondrial cytochrome C oxidase subunit Vb precursor
1832617105NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial
1832614309very-long-chain 3-oxoacyl-CoA reductase-A
1832627296isovaleryl-CoA dehydrogenase, mitochondrial
1832669039aldo-keto reductase family 1 member B1 isoform X1
1832625753glyceraldehyde-3-phosphate dehydrogenase
343459175ATP synthase, H+ transporting, mitochondrial F0 complex, partial
295792326mitochondrial cytochrome c oxidase subunit 7C
1393214856type I glyceraldehyde-3-phosphate dehydrogenase
1832641526uroporphyrinogen decarboxylase
1832664977cytochrome c oxidase subunit 6B1
1832644368peroxiredoxin-6
1042045193glycine dehydrogenase (aminomethyl-transferring)
1832671044deoxyribose-phosphate aldolase
1832640714L-threonine dehydrogenase
1832608120cytochrome c oxidase subunit 4 isoform 1, mitochondrial
2067150115Thioredoxin domain-containing protein 12
343459105NADH dehydrogenase 1 alpha, partial
1832680021betaine--homocysteine S-methyltransferase 1
295792244fructose-bisphosphate aldolase A
190Oxidative phosphorylation90.0001832613275cytochrome c oxidase subunit 5A, mitochondrial
197725770mitochondrial cytochrome C oxidase subunit Vb precursor
1832608120cytochrome c oxidase subunit 4 isoform 1, mitochondrial
343459105NADH dehydrogenase 1 alpha, partial
343459175ATP synthase, H+ transporting, mitochondrial F0 complex, partial
295792326mitochondrial cytochrome c oxidase subunit 7C
1832617105NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial
1832664977cytochrome c oxidase subunit 6B1
1832677686NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial
10Glycolysis/Gluconeogenesis70.000834906005glyceraldehyde-3-phosphate dehydrogenase
1393214856type I glyceraldehyde-3-phosphate dehydrogenase
1832625753glyceraldehyde-3-phosphate dehydrogenase
295792244fructose-bisphosphate aldolase A
328677247glyceraldehyde 3-phosphate dehydrogenase isoform, partial
221048061fructose-bisphosphate aldolase
834904842phosphoglycerate kinase
1200Carbon metabolism80.000834906005glyceraldehyde-3-phosphate dehydrogenase
1393214856type I glyceraldehyde-3-phosphate dehydrogenase
1832625753glyceraldehyde-3-phosphate dehydrogenase
295792244fructose-bisphosphate aldolase A
221048061fructose-bisphosphate aldolase
834904058malate dehydrogenase
834904842phosphoglycerate kinase
1042045193glycine dehydrogenase (aminomethyl-transferring)
1230Biosynthesis of amino acids60.000834906005glyceraldehyde-3-phosphate dehydrogenase
1393214856type I glyceraldehyde-3-phosphate dehydrogenase
1832625753glyceraldehyde-3-phosphate dehydrogenase
295792244fructose-bisphosphate aldolase A
221048061fructose-bisphosphate aldolase
834904842phosphoglycerate kinase
4260Cardiac muscle contraction60.0001832606995tropomyosin alpha-4 chain-like isoform X4
1832613275cytochrome c oxidase subunit 5A, mitochondrial
197725770mitochondrial cytochrome C oxidase subunit Vb precursor
1832608120cytochrome c oxidase subunit 4 isoform 1, mitochondrial
295792326mitochondrial cytochrome c oxidase subunit 7C
1832664977cytochrome c oxidase subunit 6B1
3010Ribosome50.001183261257960S ribosomal protein L27a
1832640175FAU ubiquitin-like and ribosomal protein S30 fusion a
183267325360S ribosomal protein L8
183261061060S ribosomal protein L39
157929900ribosomal protein L23
30Pentose phosphate pathway30.001295792244fructose-bisphosphate aldolase A
1832671044deoxyribose-phosphate aldolase
221048061fructose-bisphosphate aldolase
4672Intestinal immune network for IgA production30.001380006108MHC class II antigen
326632479MHC class II antigen
62255674immunoglobulin mu heavy chain
51Fructose and mannose metabolism30.001295792244fructose-bisphosphate aldolase A
221048061fructose-bisphosphate aldolase
1832669039aldo-keto reductase family 1 member B1 isoform X1
260Glycine, serine and threonine metabolism30.0021832680021betaine--homocysteine S-methyltransferase 1
1042045193glycine dehydrogenase (aminomethyl-transferring)
1832640714L-threonine dehydrogenase
4514Cell adhesion molecules (CAMs)40.005380006108MHC class II antigen
161935902MHC class I alpha antigen
326632479MHC class II antigen
1832656308neural cell adhesion molecule 1a isoform X4
4145Phagosome40.007380006108MHC class II antigen
161935902MHC class I alpha antigen
326632479MHC class II antigen
62255674immunoglobulin mu heavy chain
5168Herpes simplex virus 1 infection40.014380006108MHC class II antigen
161935902MHC class I alpha antigen
1832633330protein phosphatase 1, catalytic subunit, alpha isozyme a
326632479MHC class II antigen
630Glyoxylate and dicarboxylate metabolism20.015834904058malate dehydrogenase
1042045193glycine dehydrogenase (aminomethyl-transferring)
270Cysteine and methionine metabolism20.029834904058malate dehydrogenase
1832680021betaine--homocysteine S-methyltransferase 1
72Synthesis and degradation of ketone bodies10.04718326872253-hydroxybutyrate dehydrogenase type 2
Table A2. Differentially expressed proteins pathways annotated in KEGG Automatic Annotation Server.
Table A2. Differentially expressed proteins pathways annotated in KEGG Automatic Annotation Server.
Accession Name KEGG Orthology
1832702676von Willebrand factor A domain-containing protein 5A-like isoform X1 K24510
1832606995tropomyosin alpha-4 chain-like isoform X4 K10373
1832633330protein phosphatase 1, catalytic subunit, alpha isozyme a K06269
1832613275cytochrome c oxidase subunit 5A, mitochondrial K02264
1832681058antithrombin-III K03911
1832628613EH domain-containing protein 4K12477
1832617553small nuclear ribonucleoprotein Sm D1K11087
1832634797transmembrane emp24 domain-containing protein 2 isoform X1 K20347
1832661391complement factor IK01333
1832671044deoxyribose-phosphate aldolase K01619
1832645324arginine--tRNA ligase, cytoplasmic K01887
1832640714L-threonine dehydrogenase K15789
183261061060S ribosomal protein L39 K02924
183268549239S ribosomal protein L45, mitochondrialK17426
1832700793TATA-binding protein-associated factor 2N isoform X1 K13098
18326872253-hydroxybutyrate dehydrogenase type 2 K25939
1832690325uncharacterized protein LOC117256382 isoform X3K08870
1832685958arachidonate 15-lipoxygenase B-like isoform X1 K18684
1832683648nucleolin K11294
145105480interleukin enhancer-binding factor 2 K13089
161935902MHC class I alpha antigen K06751
1832684689trypsin-3K23011
1832608120cytochrome c oxidase subunit 4 isoform 1, mitochondrial K02263
1832640175FAU ubiquitin-like and ribosomal protein S30 fusion a K02983
343459105NADH dehydrogenase 1 alpha, partial K03948
1832688344ADP/ATP translocase 1 K05863
1832620903cystatin-BK13907
1832667755elongation factor 1-alpha 1a K03231
327239696complement component C6, partial K03995
1832674019deleted in malignant brain tumors 1 protein-like isoform X1K13912
1832691617ubiquinol-cytochrome-c reductase complex assembly factor 2 K17682
1832614309very-long-chain 3-oxoacyl-CoA reductase-A K10251
1832627296isovaleryl-CoA dehydrogenase, mitochondrial K00253
1832635569annexin A1K17091
1832617105NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial K03939
1832631564E3 ubiquitin-protein ligase RBX1 K03868
197725770mitochondrial cytochrome C oxidase subunit Vb precursorK02265
1832626150C-reactive proteinK16143
2067150115Thioredoxin domain-containing protein 12K05360
1832680021betaine--homocysteine S-methyltransferase 1-like K00544
1832680525ubiquitin-conjugating enzyme E2 L3 K04552
1832606043ras-related protein Rab-27AK07885
1710371347electron transfer flavoprotein subunit beta K03522
1832702451LOW-QUALITY PROTEIN: apolipoprotein(a)-K01315
1832645146pigment epithelium-derived factor K19614
1832639949collagen alpha-1(X) chain K19479
1832701092latexin K23594
1832662114parvalbumin alpha K23926
1832699882peptidyl-prolyl cis-trans isomerase FKBP10 K09575
1832670505coatomer subunit gamma-2 K17267
209981964alpha-1-antitrypsin K04525
1832702196vasodilator-stimulated phosphoprotein isoform X1 K06274
834904058malate dehydrogenase K00026
1832656308neural cell adhesion molecule 1a isoform X4 K06491
1393214856type I glyceraldehyde-3-phosphate dehydrogenase K00134
306008591heat shock protein K04077
295792244fructose-bisphosphate aldolase A K01623
328677149hypothetical protein, partial K01315
343459175ATP synthase, H+ transporting, mitochondrial F0 complex, partialK02138
1832625753glyceraldehyde-3-phosphate dehydrogenase K00134
1832630592glucosidase 2 subunit beta K08288
1832669039aldo-keto reductase family 1 member B1 isoform X1 K00011
1832632244parvalbumin betaK23926
1832620905stefin-CK13907
1832677686NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial K03937
1832638111hyaluronan and proteoglycan link protein 1-like K06848
1832619571tetranectin-like K17520
1832616138testin K24270
1832604382histone H1K11275
834904842phosphoglycerate kinase K00927
1832645835ruvB-like 2 K11338
1832635997proteasome subunit beta type-7 isoform X1 K02739
1832641526uroporphyrinogen decarboxylase K01599
1832690337chitinase, acidic.1 K01183
183267325360S ribosomal protein L8 K02938
1832638138transcription factor BTF3 K01527
1832643512rho GTPase-activating protein 12-like isoform X1 K20636
1832644997coatomer subunit beta’ isoform X1 K17302
1832642084neurogenic differentiation factor 6-BK09080
1832685063hydroperoxide isomerase ALOXE3-like K18684
1832644368peroxiredoxin-6K11188
1832670971LSM8 homolog, U6 small nuclear RNA associatedK12627
1832638785leucine-rich alpha-2-glycoprotein-like K25431
1832664977cytochrome c oxidase subunit 6B1K02267
1832612090heat shock 70 kDa protein 4bK09489
1042045193glycine dehydrogenase (aminomethyl-transferring)K00281
1832622572acyl-CoA thioesterase 9, tandem duplicate 1 isoform X1 K17361
1832656769vesicle-associated membrane protein 2K13504
328677247glyceraldehyde 3-phosphate dehydrogenase isoform, partialK10705
1832679851drebrin-like b isoform X1K20520
1832650400prefoldin subunit 1 isoform X1 K09548
221048061fructose-bisphosphate aldolaseK01623
1832658469fibrinogen beta chain K03904
183261257960S ribosomal protein L27a K02900
222087999fibrinogen beta chain precursorK03904
1832689402fibrinogen gamma chainK03905
1832662038leukocyte cell-derived chemotaxin-2K25755
1832685624microfibril-associated glycoprotein 4K25409
1832634516keratin, type I cytoskeletal 13K07604
1832685241beta-2-glycoprotein 1K17305
1832623025apolipoprotein Eb K04524
405790938MHC class II antigen, partial K06752
295792326mitochondrial cytochrome c oxidase subunit 7CK02272
1832626146allograft inflammatory factor 1-like K18617
1832610590DNA-directed RNA polymerase I subunit RPA1 K02999
295792242nucleoside diphosphate kinase B K00940
1832702726serpin H1b K09501
1832665474sulfotransferase 2B1K01015
1832641369phosphotriesterase-related protein K07048
1832686354puromycin-sensitive aminopeptidase K08776
1832695641N-alpha-acetyltransferase 10 isoform X1 K20791
1832682447dedicator of cytokinesis protein 7 isoform X1K21852
1832667938sideroflexin-3 K23500
1832608362AP-1 complex subunit gamma-1 isoform X1 K12391
1832621671rRNA 2’-O-methyltransferase fibrillarin K14563
1832611868transmembrane 9 superfamily member 2 isoform X1K17086
1832671468coagulation factor VIIiK01320
1832618423class I histocompatibility antigen, F10 alpha chain-like isoform X1 K06751
62255674immunoglobulin mu heavy chain K06856
157929900ribosomal protein L23K02894
2022331196immunoglobulin M heavy-chain constant mu variant 1K06856
834906005glyceraldehyde-3-phosphate dehydrogenase K00134
1832604432erythroblast NAD(P)(+)--arginine ADP-ribosyltransferaseK19977
1832697311nucleobindin-2a isoform X1 K20371
1832604652fibrinogen alpha chain-likeK03903
326632479MHC class II antigenK06752
1832660271macrosialinK06501
380006108MHC class II antigenK06752
2022331198immunoglobulin M heavy-chain constant mu variant 2K06856

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