G-type Lysozyme Research Articles

Evolution of g-type lysozymes in metazoa: insights into immunity and digestive adaptations

Exploring the evolutionary dynamics of lysozymes is critical for advancing our knowledge of adaptations in immune and digestive systems. Here, we characterize the distribution of a unique class of lysozymes known as g-type, which hydrolyze key components of bacterial cell walls. Notably, ctenophores, and choanoflagellates (the sister group of Metazoa), lack g-type lysozymes. We reveal a mosaic distribution of these genes, particularly within lophotrochozoans/spiralians, suggesting the horizontal gene transfer events from predatory myxobacteria played a role in their acquisition, enabling specialized dietary and defensive adaptations. We further identify two major groups of g-type lysozymes based on their widespread distribution in gastropods. Despite their sequence diversity, these lysozymes maintain conserved structural integrity that is crucial for enzymatic activity, underscoring independent evolutionary pathways where g-type lysozymes have developed functionalities typically associated with different lysozyme types in other species. Specifically, using Aplysia californica as a reference species, we identified three distinct g-type lysozyme genes: two are expressed in organs linked to both feeding and defense, and the third exhibits broader distribution, likely associated with immune functions. These findings advance our understanding of the evolutionary dynamics shaping the recruitment and mosaic functional diversification of these enzymes across metazoans, offering new insights into ecological physiology and physiological evolution as emerging fields.

Uncovering the antimicrobial activity of G-type lysozyme 2 derived from Cyprinus carpio mucus against bacterial and fungal pathogens

This study aimed to explore the antimicrobic potential of mucus samples collected from Cyprinus carpio and identify the specific antimicrobial peptides responsible for its activity. The crude extract was tested against various bacterial and fungal pathogens, and its protein content and profile were analyzed. Purification steps, including gel filtration chromatography, were employed to isolate the most active fraction (peak IV), which was further identified via liquid chromatography and mass spectroscopy. The results revealed varying degrees of antimicrobial activity of the crude extract against different bacterial and fungal strains, with Leclercia adecarboxylata, Candida glabrata, and Candida parapsilosis showing the highest susceptibility. SDS-PAGE analysis demonstrated the existence of multiple low molecular weight protein bands in the crude extract, while fraction IV obtained from gel filtration chromatography exhibited the strongest antimicrobial activity. Peak IV displayed a range of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values against the tested pathogens, spanning from 0.038 to 4.960 mg/mL. Further investigation identified the purified peptide derived from peak IV as G-type lysozyme 2, characterized by a molecular weight of 21 kDa. These findings shed light on the existence of a highly effective antimicrobial peptide, G-type lysozyme 2, within the mucus of Cyprinus carpio. This peptide demonstrates notable activity against diverse bacterial and fungal pathogens. The insights from this study enhance our understanding of the fish's antimicrobial defense mechanisms and hold promise for developing novel antimicrobial agents.

Lysozyme Inhibitors as Tools for Lysozyme Profiling: Identification and Antibacterial Function of Lysozymes in the Hemolymph of the Blue Mussel.

Lysozymes are universal components of the innate immune system of animals that kill bacteria by hydrolyzing their main cell wall polymer, peptidoglycan. Three main families of lysozyme have been identified, designated as chicken (c)-, goose (g)- and invertebrate (i)-type. In response, bacteria have evolved specific protein inhibitors against each of the three lysozyme families. In this study, we developed a serial array of three affinity matrices functionalized with a c-, g-, and i-type inhibitors for lysozyme typing, i.e., to detect and differentiate lysozymes in fluids or extracts from animals. The tool was validated on the blue mussel (Mytilus edulis), whose genome carries multiple putative i-, g-, and c-type lysozyme genes. Hemolymph plasma of the animals was found to contain both i- and g-type, but not c-type lysozyme. Furthermore, hemolymph survival of Aeromonas hydrophila and E. coli strains lacking or overproducing the i- type or g-type lysozyme inhibitor, respectively, was analyzed to study the role of the two lysozymes in innate immunity. The results demonstrated an active role for the g-type lysozyme in the innate immunity of the blue mussel, but failed to show a contribution by the i-type lysozyme. Lysozyme profiling using inhibitor-based affinity chromatography will be a useful novel tool for studying animal innate immunity.

DNA vaccine incorporated poly (lactic-co-glycolic) acid (PLGA) microspheres offer enhanced protection against Aeromonas hydrophila infection

Encapsulation of DNA vaccines onto carriers enhances the immunogenicity of an antigen. Specifically, biodegradable polymers offer sustained release of vaccines which is crucial for any targeted delivery approach. Poly (lactic-co-glycolic) acid (PLGA) microspheres were used to load a DNA vaccine having a targeted gene of outer membrane protein (OMP) of Aeromonas hydrophila to clone and construct a DNA vaccine using a eukaryotic expression vector system (pVAX1-OMP DNA) and delivery in Carassius auratus against A. hydrophila infection. PLGA microspheres were prepared by emulsion technique oil-in-water and characterized by a High-Resolution Scanning Electron Microscope (HR-SEM). The results of PLGA-pVAX1-OMP DNA microspheres shows that average of 100–150 μm particle size and a loading efficiency (LE) of 68.8 %. Results indicate that C. auratus fed with PLGA-pVAX1-OMP DNA microspheres revealed a significant improvement in innate immune response, which includes, myeloperoxidase activity, respiratory burst and total immunoglobulin level compared with control group fish. The immune-related gene, IL1β, IL10, TGF, c-type, and g-type lysozyme also showed significantly higher expression after immunization. Furthermore, dietary supplementation of the PLGA-pVAX1-OMP DNA (G III) group exhibited a significantly higher survival rate (78 %) than the control group of fish. These results help us to understand the of mechanism of DNA vaccine administrated feed through PLGA nanoparticles resistance to infection by regulating systemic and innate immunity in Carassius auratus.

Gene expression of Lysozyme C and Lysozyme G in diploid and triploid Astyanax lacustris experimentally infected with Aeromonas jandaei

Triploid fish are becoming increasingly popular in commercial aquaculture due to their sterility, which results in improved meat quality and better growth rates as a result of the lack of sexual maturation. However, triploid fish are generally perceived as being more susceptible to diseases and less able to cope with suboptimal environmental conditions. The present study aimed to evaluate the effects of triploidy on the expression of C-type and G-type lysozymes (LysC and LysG) in lambaris (Astyanax lacustris) that were challenged with a pathogenic strain of Aeromonas jandaei. The animals were induced to triploidy as described in the literature. We infected 10 diploid and 10 triploid fish with 104 CFU of A. jandaei in 0.1 mL of PBS by intraperitoneal injection, a dose capable of triggering an immune response in the animals. Fish from the control groups were inoculated with 0.1 mL of sterile PBS. Molecular cloning of Lysozyme C and Lysozyme G was performed to characterize these genes, and the analysis of gene expression for LysC and LysG of A. lacustris diploids and triploids was conducted using qPCR. The collected data revealed that there were no significant differences in basal expression between diploid and triploid uninfected fish. However, the challenged fish of both ploidies exhibited an increase in expression compared to the control group. The results of study indicate that infected diploid fish exhibit significantly higher lysozyme expression in the spleen compared to infected triploid fish. While basal lysozyme expression levels between diploid and triploid fish remain unaffected, our findings suggest that triploidy may lower lysozyme expression in response to infection, with diploid fish exhibiting higher levels of expression.

Immune characterization and expression analysis of a goose-type lysozyme gene from Pinctada fucata martensii

In the present study, a g-type lysozyme was successfully screened and cloned from Pinctada fucata martensii (designated as PmlysG). The cDNA has a length of 973 bp with an open reading frame (ORF) of 769 bp, encoding a protein of 255 amino acids. The PmlysG transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with the highest expression being in the hepatopancreas. Additionally, the temporal expression of PmlysG mRNA in the hepatopancreas after in vivo stimulation with pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic acid (PolyI:C) was detected by qRT-PCR. Although PmlysG responded to all three stimulation modes, it rapidly responded to PGN stimulation. Meanwhile, the recombinant protein of g-type lysozyme of P.f. martensii (rPmlysG) was used for antibacterial function analysis, and the results showed that rPmlysG has antibacterial function against Vibrio parahaemolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa. Overall, these study results suggest that the identified PmlysG participates in the innate immune responses of P.f. martensii against pathogen infection.

Molecular characterization and antibacterial activities of a goose-type lysozyme gene from roughskin sculpin (Trachidermus fasciatus)

Lysozymes, acting as antimicrobial molecules, play a vital role in the host's innate immune response to pathogen infections. In the present study, a g-type lysozyme gene termed Tf-LyzG from roughskin sculpin, Trachidermus fasciatus was firstly reported. The deduced amino acid sequence of Tf-LyzG contained 188 residues and possessed conserved catalytic residues (Glu71, Asp84, and Asp95). Gene expression analysis revealed that Tf-LyzG was widely distributed in the tested eleven tissues with the highest expression in the gill and could be significantly induced post lipopolysaccharide (LPS) challenge. The lysozyme activity of the purified recombinant protein (rTf-LyzG) was found to be most active at pH 5.5 and 37 °C. rTf-LyzG exhibited a wide spectrum of potent bacteriolytic activity against four Gram-positive bacteria and six Gram-negative bacteria. It also displayed a high affinity to polysaccharides on bacteria surfaces including LPS, lipoteichoic acid (LTA), and peptidoglycan (PGN). rTf-LyzG was capable of binding and agglutinating all nine bacteria. Flow cytometry assay further revealed that rTf-LyzG could disrupt the membrane of Micrococcus lysodeikticus which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. In summary, these data indicate that Tf-LyzG is of great importance in the fish immune response against pathogens invasion.

Molecular cloning and functional characterization of HMGB1 and HMGB2 in large yellow croaker Larimichthys crocea

High mobility group box 1 (HMGB1) and HMGB2 have been demonstrated to be key regulators not only in DNA recombination, replication, gene transcription, but also in host inflammation and immune responses. In the present study, orthologs of HMGB1 and HMGB2 named Lc-HMGB1 and Lc-HMGB2 were characterized in large yellow croaker (Larimichthys crocea). The ORFs of Lc-HMGB1 and Lc-HMGB2 are 621 bp and 648 bp, encoding proteins of 206 aa and 215 aa, with the putative Lc-HMGB1 and Lc-HMGB2 proteins both contain two HMG domains, respectively. The genome organizations of Lc-HMGB1 and Lc-HMGB2 are both composed of four exons and three introns, which are conserved in vertebrates. Lc-HMGB1 and Lc-HMGB2 were identified as cell nucleus localized proteins, and were ubiquitously distributed in the examined organs/tissues. Additionally, Lc-HMGB1 was significantly up-regulated under LPS and PGN stimulation, whereas the stimulation of poly I:C, LPS, PGN, and Pseudomonas plecoglossicida infection could significantly induce Lc-HMGB2 expression in vivo. Notably, both Lc-HMGB1 and Lc-HMGB2 overexpression could significantly up-regulated the expression of diverse immune-related genes, including IFN1, IRF3, ISG15, ISG56, RSAD2, g-type lysozyme, and TNF-α. Moreover, overexpression of Lc-HMGB1 could also induce the expression of IRF7 and Mx. These results collectively indicate that Lc-HMGB1 and Lc-HMGB2 play important roles in host immune responses against pathogen infection.

Efficacy of recombinant subunit OMP and hly vaccines against Aeromonas hydrophila in Rohu (Labeo rohita).

The main aim of the current study was to clone and express a new outer membrane protein (OMP) and haemolysin (hly) from a pathogenic Aeromonas hydrophila and to investigate their potential as a vaccine candidate against A. hydrophila infection in Rohu (Labeo rohita). The OMP and hly genes were cloned in pET-30b vector and recombinant plasmids pET-30b-OMP and pET-30b-hly were constructed, which were then transferred into Escherichia coli BL21 (DE3). The recombinant E.coli BL21 (DE3) was induced by IPTG, and the OMP and hly proteins were expressed highly. The proteins OMP and hly were estimated in 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weights were found to be 40 kD and 68 kD. The expressed proteins OMP and hly were purified by Ni-NTA His-Bind Resin column, and the immunogenicity was confirmed by Western blotting. The fishes (L.rohita) were divided into IV groups, and the group I fishes were treated with phosphate saline, the II and III group were immunized with the purified OMP and hly recombinant proteins, and the fishes were treated IV group with combined OMP and hly for 10 days. After 10 days of treatment, the fishes of all the four groups were challenged with virulent A.hydrophila. The results revealed that vaccinated fish showed significantly improved haematological profile, phagocytic activity, myeloperoxidase activity and total immunoglobulin levels on the 5th and 10th days. The non-vaccinated group (Group I) showed 100% mortality, whereas the mixture of recombinant OMP (r-OMP) and hly (r-hly) protein-treated groups (Group IV) exhibited higher survival rate (80%). Relatively, expression of pro- and anti-inflammatory cytokines (IL-1β, IL-10 and TGF-β), c-type and g-type lysozymes were significantly up-regulated in heart and kidney of vaccinated groups compared with the non-vaccinated group. Our results revealed that OMP and hly genes were effective vaccine candidates in the aquaculture system and could be used as recombinant subunit vaccine for diseases caused by pathogenic A.hydrophila.

Chicken-type lysozyme is a major bacteriolytic enzyme in the blood of the banded houndshark Triakis scyllium

We examined lysozyme activities in the serum and the leukocyte extracts of the banded houndshark Triakis scyllium. The serum exhibited lytic activity, but not the leukocyte extracts. The lytic substance in the serum was of approximately 14 kDa and the N-terminal amino acid sequence was YVYSK. cDNA cloning identified a C-type lysozyme (TsLysC) gene and two G-type lysozyme (TsLysG) cDNA clones of different lengths. The TsLysC gene encodes 149 amino acids residues, and the sequence derived from the N-terminal amino acid sequencing was displayed at position 17–21. TsLysG, on the other hand, contains two ORFs that are homologous to the N- and C-terminal regions of G-type lysozyme of other fish species. TsLysC mRNA levels were high in the liver. TsLysG mRNA level was significantly lower than TsLysC mRNA in the liver.

A g-type lysozyme from the scallop Argopecten purpuratus participates in the immune response and in the stability of the hemolymph microbiota

Lysozymes are antimicrobial acid hydrolases widely distributed in nature. They are located inside the cells in lysosomes, or they are secreted to the extracellular space, where they can lyse the cell wall of certain species of bacteria via hydrolysis of the peptidoglycan. Thus, lysozymes are bacteriolytic enzymes and play a major biological role in biodefense, as these enzymes can act as antibacterial and immune-modulating agents. In this study, we characterized a g-type lysozyme from the scallop Argopecten purpuratus named ApGlys. The cDNA sequence comprises an open reading frame (ORF) of 600 nucleotides, codifying for a putative protein of 200 amino acids with a signal peptide of 18 amino acids. The deduced mature protein sequence displays a molecular weight of 20.07 kDa and an isoelectric point (pI) of 6.49. ApGlys deduced protein sequence exhibits conserved residues associated with catalytic activity and substrate fixation in other g-type lysozymes. The phylogenetic analysis revealed a high degree of identity of ApGlys with other mollusk g-type lysozymes, which form a restricted and separated clade from the vertebrate lysozymes. ApGlys transcripts were constitutively and highly expressed in the digestive gland, and it was induced in hemocytes and gills of scallops after an immune challenge. Furthermore, the ApGlys protein was located inside hemocytes of immunostimulated scallops, determined by immunofluorescence analysis. Finally, the transcript silencing of ApGlys by RNA interference led to an increase of total culturable bacteria from the scallop hemolymph. Furthermore, we detected a higher diversity of the bacterial community in ApGlys-silenced scallops and an imbalance of certain bacterial groups present in the hemolymph by 16S rDNA deep amplicon sequencing. Overall, our results showed that ApGlys is a new member of scallop lysozymes that is implicated in the immune response and in the microbial homeostasis of A. purpuratus hemolymph.

Investigation of putative antimicrobial peptides in Carassius gibel, revealing a practical approach to screening antimicrobials

Antimicrobial peptides (AMPs) and their mimics are rapidly gaining attention as a new class of antimicrobials due to their clinical potential. AMPs are widely distributed throughout nature and participate in the innate host defense. In this study, 18 AMPs, including 3 β-defensins, 3 hepcidins, 4 liver-expressed antimicrobial peptide 2 (LEAP-2) compounds, 4 g-type lysozymes, 2 c-type lysozymes, and 2 NK-lysins, were identified from the genome of Carassius auratus by a homologous search and were further classified based on their fundamental structural features and molecular phylogeny. C. auratus AMPs were found to be ubiquitously distributed in all tested tissues and showed similar expression profiles, with the exception of β-defensins, when RT-qPCR was used to investigate the tissue distribution of AMPs in healthy Carassius gibel. In addition, the expression levels of NK-lysin genes in the tested tissues tended to be upregulated upon bacterial and viral infection when representative NK-lysins were chosen to examine their relative expression levels in various tissues. Importantly, the synthetic peptide caNKL2102-119, which targets the functional domain of saposin B in caNK-lysins, could effectively counter Aeromonas hydrophila, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration (MIC) values of 3–6 μg/mL, as well as inhibit the proliferation of spring viraemia of carp virus (SVCV). These results provide potential targets for antibiotic-free breeding in the aquaculture industry.

A g-Type Lysozyme from Deep-Sea Hydrothermal Vent Shrimp Kills Selectively Gram-Negative Bacteria.

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.

Parallel Independent Losses of G-Type Lysozyme Genes in Hairless Aquatic Mammals.

Lysozyme enzymes provide classic examples of molecular adaptation and parallel evolution, however, nearly all insights to date come from chicken-type (c-type) lysozymes. Goose-type (g-type) lysozymes occur in diverse vertebrates, with multiple independent duplications reported. Most mammals possess two g-type lysozyme genes (Lyg1 and Lyg2), the result of an early duplication, although some lineages are known to have subsequently lost one copy. Here we examine g-type lysozyme evolution across >250 mammals and reveal widespread losses of either Lyg1 or Lyg2 in several divergent taxa across the mammal tree of life. At the same time, we report strong evidence of extensive losses of both gene copies in cetaceans and sirenians, with an additional putative case of parallel loss in the tarsier. To validate these findings, we inspected published short-read data and confirmed the presence of loss of function mutations. Despite these losses, comparisons of selection pressures between intact g- and c-type lysozyme genes showed stronger purifying selection in the former, indicative of conserved function. Although the reasons for the evolutionary loss of g-type lysozymes in fully aquatic mammals are not known, we suggest that this is likely to at least partially relate to their hairlessness. Indeed, although Lyg1 does not show tissue-specific expression, recent studies have linked Lyg2 expression to anagen hair follicle development and hair loss. Such a role for g-type lysozyme would explain why the Lyg2 gene became obsolete when these taxa lost their body hair.

Oral delivery of pVAX-OMP and pVAX-hly DNA vaccine using chitosan-tripolyphosphate (Cs-TPP) nanoparticles in Rohu, (Labeo rohita) for protection against Aeromonas hydrophila infection

The present study examines the effectiveness of DNA vaccine against Aeromonas hydrophila through oral route using chitosan–tripolyphosphate (Cs-TPP) nanoparticles encapsulation. The virulent gene of outer membrane protein (OMP) and hemolysin (hly) related to pathogenicity of A. hydrophila was used to construct a DNA vaccine using pVAX1, and the construct was named as pVAX-OMP and pVAX-hly DNA vaccines. The pVAX-OMP and pVAX-hly DNA vaccines were encapsulated by Cs-TPP nanoparticles and size measured by field emission scanning electron microscopy (FE-SEM). The encapsulation efficiency of Cs-TPP nanoparticles was found to be 79.6% for pVAX-OMP DNA and 82.3% for pVAX-hly DNA binding with Cs-TPP nanoparticles. The stability and invitro release profile of plasmid DNA was also determined after encapsulation using DNase and chitosanase. DNA vaccines distribution in tissues was investigated in fish fed with the pVAX-OMP, pVAX-hly and pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles and confirmed by PCR and multiplex PCR. The results suggest that Cs-TPP nanoparticles encapsulated DNA vaccine delivered into fish by feeding. After oral vaccination of Labeo rohita were challenged with A. hydrophila by intraperitoneal injection. Relatively, gene expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (Interlukin-10 and Tumor Growth Factor β) was up-regulated in heart and kidney for pVAX-OMP+pVAX-hly vaccinated group. Moreover, fish fed with pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles had a significantly higher survival rate (76.2%) against A. hydrophila. This study concludes that pVAX-OMP and pVAX-hly DNA vaccines can be delivered orally using Cs-TPP nanoparticles for protection against A. hydrophilainfection.

Dietary Administration of Probiotic Aeromonas veronii V03 on the Modulation of Innate Immunity, Expression of Immune-Related Genes and Disease Resistance Against Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio).

This study investigates the effects of dietary Aeromonas veronii V03 supplementation on growth performances, innate immunity, and expression of immune-related genes in lymphoid organs of Cyprinus carpio and resistance to Aeromonas hydrophila infection. Fish were fed for 4weeks with basal diet (BD; without probiotic), and experiment diet containing different doses of A. veronii V03 at 3.2 × 107 (DI) and 3.5 × 109 (DII) CFU g-1 of diet. At the end of the probiotic feeding trial, fish were challenged with A. hydrophila, and the percentage of survival rates was recorded over 7days. Results revealed that fish fed with A. veronii V03 demonstrated a significant improvement in growth and enhancement of innate immunity, including respiratory burst, myeloperoxidase, and lysozyme activities, and total immunoglobulin level compared with BD fed to fish. Relatively, expression of cytokines (MyD88, IL-1β1, IL-8, and IL-10) and c- and g-type lysozymes were significantly up- and downregulated in lymphoid organs of fish. Moreover, dietary supplementation of A. veronii V03 exhibited significantly (p < 0.001) higher survival rates of DI (90%) and DII (96.66%) compared with BD (53.33%) fed fish against A. hydrophila infection. These findings help to understand the effects of probiotic A. veronii V03 administrated feed influences on growth and ailment resistance to A. hydrophila infection by regulating innate and systemic immunity in common carp fish.

Dietary sodium propionate enhanced the growth performance, immune-related genes expression, and resistance against Ichthyophthirius multifiliis in goldfish (Carassius auratus)

Sodium propionate (SP) is a short-chain fatty acid that has long been used to improve the growth, physiological and immunological properties of aquatic animals. The aim of the current study was to assess the effect of SP intake on growth, immune-related genes, and resistance against Ichthyophthirius multifiliis in goldfish (Carassius auratus). The juvenile goldfish (initial weight = 8.61 ± 0.01 g) fed on diets supplemented with different levels of 0, 1.2, 2.5, and 5 g/kg diet) of SP for 50 days. At the end of the experiment, the treated fish were exposed to theronts stage of I. multifiliis at 5000 theronts per fish. Results showed that higher final weight, red blood cells (RBC), and white blood cells were seen in fish fed 5 g SP/kg than other treatments (P < 0.05), while no significant differences were seen in Na+ and Cl−1 electrolytes levels. With an increase in the level of SP in the diet, a significant up-regulation was observed in the expression of g-type lysozyme (gLys), interleukin-1β (IL-1ß), inducible nitric oxide synthase (iNOS), and tumor necrosis factor ɑ2 (TNF-ɑ2) genes (P < 0.05). Total numbers of parasite trophonts were also significantly decreased in fish treated with SP (P < 0.05). In addition, values of RBC and Cl−1 increased in the treated fish compared to control one, and lower glucose level was obtained in the treatment groups post-challenge with the parasite infection. These data revealed a positive effect of dietary SP on growth, immune response, and pro-inflammatory related genes and increased disease resistance towards I. multifiliis challenge in goldfish.

Expression and purification of ShLysG in Escherichia coli and initial characterization of its antimicrobial, antioxidant and anti-inflammatory activities

ShLysG is a novel g-type lysozyme isolated from Hippocampus abdominalis in 2016. However, its antibacterial, antioxidant and anti-inflammatory properties have not been investigated. In this study, a recombinant ShLysG (rShLysG) fused with a 6×His tag was produced in Escherichia coli with barely any hemolytic activity or cytotoxicity. It exhibited a broad antimicrobial spectrum against gram-positive and gram-negative bacteria with minimum inhibitory concentration (MIC) values between 20 and 60 μg/mL. The bioactivity of rShLysG was stable in high temperature and extreme acid base conditions. In addition, rShLysG was resistant to papain and pepsin, and it showed a concentration-dependent antioxidant activity. By inhibiting the phosphorylation of NF-κB, the inflammatory cytokines expression level, including TNF-α, IL-6, and IL-8 in RAW264.7 macrophage cells induced by LPS were decreased. Taken together, rShLysG is a promising substitute to antibiotics as an antimicrobial and immunoregulatory pharmaceutical peptide for clinical use.

Molecular characterization and expression analysis of the chicken-type and goose-type lysozymes from totoaba (Totoaba macdonaldi)

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60–90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes).

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1β (IL-1β), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.