G-type Lysozyme Research Articles

Characterization of an antiserum against Atlantic cod ( Gadus morhua L.) g-type lysozyme

In this study we describe the production and characterization of an antiserum against recombinant g-type lysozyme derived from Atlantic cod. This is also the first initial analyses of g-type lysozyme protein expression in tissues of Atlantic cod. Recombinant expression and purification of cod g-type lysozyme was used for immunization to rabbit and the rabbit sera were analysed for anti g-type lysozyme antibodies using enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. ELISA results showed that antibody titres were mounted between 12,800 and 25,600 as measured at an optical density corresponding to 50% of the maximal level. By Western blot analysis the rabbit immune serum detected a single ∼23 kDa band representing the size of the injected antigen, in both spleen and head kidney homogenates from the Atlantic cod. Immunohistochemisrty detected the native folded g-type lysozyme in tissues and revealed that g-type lysozyme positive cells were observed in haematopoietic tissue of the head kidney and in red pulp of spleen. In conclusion, the rabbit anti g-type lysozyme immune sera was developed and is effectively utilized for ELISA, Western analysis as well as for immunohistochemistry. This has allowed us to obtain new knowledge about this protein regarding localization and distribution in cod tissue.

Identification of a bacterial inhibitor against g-type lysozyme.

Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyze peptidoglycan. Bacteria have evolved protective mechanisms that contribute to lysozyme tolerance such as the production of lysozyme inhibitors, but only inhibitors of chicken (c-) and invertebrate (i-) type lysozyme have been identified. We here report the discovery of a novel Escherichia coli inhibitor specific for goose (g-) type lysozymes, which we designate PliG (periplasmic lysozyme inhibitor of g-type lysozyme). Although it does not inhibit c- or i-type lysozymes, PliG shares a structural sequence motif with the previously described PliI and MliC/PliC lysozyme inhibitor families, suggesting a common ancestry and mode of action. Deletion of pliG increased the sensitivity of E. coli to g-type lysozyme. The existence of inhibitors against all major types of animal lysozyme and their contribution to lysozyme tolerance suggest that lysozyme inhibitors may play a role in bacterial interactions with animal hosts.

Infection-induced changes in expression of antibacterial and cytokine genes in the gill epithelial cells of Atlantic cod, Gadus morhua during incubation with bacterial pathogens

The molecular processes of immune responses in mucosal tissues, such as the gills, during infection with bacterial pathogens are poorly understood. In the present study, we analyzed the transcriptional profiles of selected antibacterial genes and cytokines in the gills of a cold-water fish, Atlantic cod, Gadus morhua following in vitro infection with bacterial pathogens, Vibrio anguillarum and atypical Aeromonas salmonicida using semi-quantitative RT-PCR. There was significant upregulation in the transcripts of the antibacterial genes: bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme, transferrin, metallothionein, galectin and hepcidin at 3h post-incubation with the two pathogens. The expression of cathelicidin in the gills was significantly enhanced by A. salmonicida, but not by V. anguillarum. At 24h post-incubation, most of these genes were still significantly upregulated, although some genes returned to their basal expression levels. The transcription levels of cytokines such as interleukin (IL)-1beta, IL-8 and interferon (IFN)-gamma significantly increased at 3h post-incubation with the pathogens. IL-22 and CC-chemokine type 1 transcripts were enhanced by A. salmonicida, but not by V. anguillarum. There was down-regulation of expression in CC-chemokine type-2 and -3 by V. anguillarum, while the expression levels of IL-10 remained unchanged upon infection with either of the two bacterial pathogens. The early upregulation of antibacterial genes in the gills could signal the onset of the acute phase response following bacterial infection and the differential modulation of some cytokine genes could be related to host-pathogen interactions that trigger immune response cascades in mucosal tissues of the host.

Thermodynamics and structure of a salmon cold active goose-type lysozyme

Atlantic salmon goose-type lysozyme (SalG) was previously shown to display features of cold-adaptation as well as renaturation following heat treatment. In this study differential scanning calorimetry (DSC) was carried out to investigate unfolding and potential refolding, while X-ray crystallography was used to study structural factors contributing to the temperature-related characteristics. The recombinant SalG has a melting temperature (T(m)) of 36.8 degrees C under thermal denaturation conditions and regains activity after returning to permissive (low) temperature. Furthermore, refolding is dramatically reduced in solutions with high SalG concentrations, coupled with significant protein precipitation. The structural features of SalG closely resemble those of other g-type lysozymes. However, the N-terminal region of SalG is less anchored to the rest of the molecule due to the absence of disulphide bonds, thus, contributing significantly to the low T(m) of SalG. The absence of disulphide bonds and the distribution of salt bridges may at the same time ease refolding leading to renaturation.

Lysozymes in the animal kingdom

Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the beta-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme types have been identified--the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.

Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection

Edwardsiella tarda is an important Gram-negative bacterium that causes systemic infections in a wide range of hosts including fish. The pathogenic mechanisms in this disease are still poorly understood in fish. Indian major carp, Labeo rohita were intraperitoneally challenged with a pathogenic isolate of E. tarda to measure sequential changes in immunity level. A significant decrease in the superoxide production, myeloperoxidase, alternative complement activity, total protein levels and antiprotease activity of serum was marked in the infected fish. However, the serum lysozyme activity and haemagglutination titre were raised in the infected fish. Similarly, a significant rise in specific antibody titre was noticed on and after 10 days post-challenge. This study also elucidates the changes in the relative expression of some immune-related genes viz., interleukin 1-beta (IL-1β), inducible nitric oxide synthase (iNOS), complement component C3, β 2-microglobulin, CXCa, tumor necrosis factor-alpha (TNFα), and C-type and G-type lysozymes during the infection. Significant up-regulation of IL-1β, iNOS, C3, CXCa and expression of both types of lysozyme genes was noticed at 6–12 h post-challenge (h.p.c.) whereas down-regulation of β 2-microglobulin and TNFα genes was observed after 48 h p.c. The results obtained here strengthen the understanding on molecular pathogenesis of edwardsiellosis in L. rohita.

Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyngodon idellus

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. We identified two genes encoding g-type and c-type lysozymes from grass carp Ctenopharyngodon idellus by the RACE method. The deduced amino acids of both lysozymes possessed typical structural residues and conserved catalytic sites similar to their counterparts across the species. In contrast, there was only 8.6% similarity of amino acid sequence between these two lysozymes. Phylogenetic analyses revealed that these two genes evolved at different rate. C-type lysozyme of grass carp was diverged early in the evolutionary history. Moreover, the expression patterns of these two genes differed. The mRNA levels of both genes were increased after bacterial infection, but the up-regulation of g-type lysozyme was much stronger than that of c-type lysozyme. We also showed that the c-type and g-type recombinant lysozymes possessed different lytic activities against fish bacterial pathogens. These results confirmed that both lysozymes play important roles in the defense of grass carp against bacterial infections. The g-type lysozyme may be induced for the defense against bacterial infections, while c-type lysozyme might be the main molecule for the house-keeping defense under normal conditions. These two types of lysozymes likely use different mechanisms to regulate their expressions.

Expression profiles of genes associated with immune response and oxidative stress in Atlantic cod, Gadus morhua head kidney leukocytes modulated by live and heat-inactivated intestinal bacteria

The molecular mechanisms of immune response and antioxidant defense in Atlantic cod Gadus morhua head kidney (HK) leukocytes to live and heat-inactivated intestinal bacteria were investigated by transcriptome analyses. The HK leukocytes were incubated with Pseudomonas sp. (GP21) and Psychrobacter sp. (GP12), which are intestinal bacteria of Atlantic cod. The responses of the defense-associated genes at 3 and 24h post-incubation (hpi) were assayed by semi-quantitative RT-PCR. Live and heat-inactivated GP21 caused a significant increase in the transcript levels of bacterial defense genes in the HK leukocytes: BPI/LBP and g-type lysozyme were highest at 24hpi. The levels of BPI/LBP were significantly upregulated at 24hpi by live GP12 but not by the heat-inactivated type. The expression of g-type lysozyme was significantly elevated regardless of the type of GP12. IL-1beta was significantly upregulated by live GP21 and GP12, with maximum expression observed at 3hpi. In contrast, the expression levels of IL-8 in the HK leukocytes were not augmented by both types of GP21 and GP12. A significant upregulation of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) was observed with live GP12 at 3hpi, whereas in the case of GP21 such a change was noted only with the heat-inactivated type at 24hpi. A definite pattern of granzyme expression was not observed with both the live and heat-inactivated GP21 and GP12. The levels of antioxidant genes (catalase and GSH-Px) remained unchanged except in cells incubated with heat-inactivated GP21, where a significant elevation of GSH-Px was seen at 24hpi. Thus, this in vitro study has revealed that the defense mechanisms in the HK leukocytes can be modulated by the commensal intestinal bacteria of Atlantic cod. The extent of this activation is dependent on the bacterial species and its viability.

Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole ( Solea senegalensis): Potential effects on the immune response

Stocking density and ration size are two major factors influencing aquaculture production. To evaluate their effects on growth and immune system in Senegalese sole ( Solea senegalensis) juveniles, a 2 × 2 experimental design using two rations (1.0% and 0.25% of the total fish biomass) and two different initial stocking densities (7 and 30 kg m −2) was performed throughout a 60 days culture period. Soles fed 1.0% showed a higher specific growth rate (SGR) than those fed 0.25% (3.3-fold). No differences in SGR at 60 days were found between densities in spite of reduced values were detected at high density after 20 days (soles fed 0.25%) and 40 days (soles fed 1%) suggesting a compensatory growth. Physiologically, plasma cortisol levels were elevated in soles at high density (45-fold higher than at 7 kg m −2) whereas no differences associated to the feeding ration were observed. To assess the effects at a molecular level, the mRNA levels of genes involved in cellular stress (heat shock proteins HSP70 and HSP90), growth (insulin-like growth factors IGF-I, the spliced variants IGF-Ia and IGFI-b, and IGF-II) and innate immune system (g-type lysozyme and hepcidin (HAMP1)) were quantified. No differences in HSP90 expression were detected between densities or rations. In contrast, IGF-I, IGF-Ia and IGF-II showed reduced transcript levels in liver and HSP70 in liver and kidney at high density. Finally, g-type lysozyme and HAMP1 expression was greatly affected by both factors exhibiting an important reduction in the transcript levels at high density and low ration. Overall, our results show that S. senegalensis juveniles might exhibit satisfactory SGR at high density although the high plasma cortisol levels indicate a crowding stress that could negatively affect the expression levels of some of the genes studied.

Antimicrobial peptides derived from goose egg white lysozyme

Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.

Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism.

Crystal structures of Atlantic cod lysozyme have been solved with and without ligand bound in the active site to 1.7 and 1.9 A resolution, respectively. The structures reveal the presence of NAG in the substrate binding sites at both sides of the catalytic Glu73, hence allowing the first crystallographic description of the goose-type (g-type) lysozyme E-G binding sites. In addition, two aspartic acid residues suggested to participate in catalysis (Asp101 and Asp90) were mutated to alanine. Muramidase activity data for two single mutants and one double mutant demonstrates that both residues are involved in catalysis, but Asp101 is the more critical of the two. The structures and activity data suggest that a water molecule is the nucleophile completing the catalytic reaction, and the roles of the aspartic acids are to ensure proper positioning of the catalytic water.

Profiling gene expression in the spleen of Atlantic cod, Gadus morhua upon vaccination with Vibrio anguillarum antigen

The mechanisms of physiological responses in Atlantic cod, Gadus morhua following vaccination with a heat-killed Vibrio anguillarum were investigated by transcriptome analysis of spleen tissues. Semi-quantitative RT-PCR of several genes involved in immune defense, inflammation, antioxidant defense and glucose transport were determined in vaccinated fish at 1, 3, 7 and 10 days after vaccination (dpv)and compared with sham-injected fish. Transcript levels of the selected genes involved in bacterial defense such as the bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin, were significantly upregulated (P < 0.05) throughout the duration of sampling (1-10 dpv). There was differential expression of the genes involved in antiviral activity, cellular immunity, antioxidant defense and glucose transport, while the pro-inflammatory cytokines remained relatively unchanged in both the vaccinated and sham-vaccinated fish. The expressions of interferon stimulated gene-15 (ISG-15) and interferon regulatory factor-1 (IRF-1), which are involved in viral defense, were significantly enhanced (P < 0.05) after vaccination. Likewise, the transcript levels of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) and granzyme A/K, which are components of the cell-mediated immunity were upregulated. Among the antioxidants, the transcript levels of catalase and phospholipid hydroperoxide glutathione peroxidase (GSH-Px) significantly increased (P < 0.05) following vaccination, while glucose transporter-4 (GLUT-4) was enhanced among the genes involved in glucose transport. Our results indicate that the spleen of Atlantic cod is able to mount a potent physiological response through enhanced transcription of at least the mentioned genes, upon exposure to a bacterial antigen. These genes work synergistically to protect the fish during subsequent infection.

A novel peptidoglycan recognition protein containing a goose-type lysozyme domain from the Pacific oyster, Crassostrea gigas

Peptidoglycan recognition protein (PGRP) is considered an essential molecule for effective immunity in invertebrates by its detection and clarification of invading bacteria. Bivalve mollusks also possess PGRP systems for self-defense, however, their functions in bivalves remain to be understood. In the present study, cDNA of a novel PGRP was identified from the Pacific oyster, Crassostrea gigas, using EST-based RACE PCR. This novel PGRP is homologous to short PGRPs and the presence of a signal peptide was predicted. The PGRP is classified into the short PGRP group, although its molecular weight was estimated as 54 kDa, close to that of long PGRP groups. A conserved domain search detected amidase_2/PGRP and goose-type ( g-type) lysozyme domains in this PGRP structure, and thus this novel PGRP was designated as CgPGRP-L. Catalytic residues for PGRP and g-type lysozyme are well conserved, suggesting that CgPGRP-L may have both binding and lytic functions against bacteria. Reverser transcription PCR (RT-PCR) detected CgPGRP-L mRNA expression in circulatory hemocytes, and quantitative real-time RT-PCR revealed that its expression increased after Marinococcus halophilus and Vibrio tubiashii exposure. These results indicate that CgPGRP-L is expressed in hemocytes by bacterial invasion, and then may play roles of a short PGRP and bacterio-lytic lysozyme.

A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium Ralstonia sp. A-471: cloning, sequencing, and expression

In this study, we cloned the gene encoding goose-type (G-type) lysozyme with chitinase (Ra-ChiC) activity from Ralstonia sp. A-471 genomic DNA library. This is the first report of another type of chitinase after the previously reported chitinases ChiA (Ra-ChiA) and ChiB (Ra-ChiB) in the chitinase system of the moderately thermophilic bacterium, Ralstonia sp. A-471 and also the first such data in Ralstonia sp. G-type lysozyme gene. It consisted of 753 bp nucleotides, which encodes 251 amino acids including a putative signal peptide. This ORF was modular enzyme composed of a signal sequence, chitin-binding domain, linker, and catalytic domain. The catalytic domain of Ra-ChiC showed homologies to those of G-type lysozyme (glycoside hydrolases (GH) family 23, 16.8%) and lysozyme-like enzyme from Clostridium beijerincki (76.1%). Ra-ChiC had activities against ethylene glycol chitin, carboxyl methyl chitin, and soluble chitin but not against the cell wall of Micrococcus lysodeikticus. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the enzyme, the second and third glycosidic linkage from the non-reducing end were split producing (GlcNAc)2 + (GlcNAc)4 and (GlcNAc)3 + (GlcNAc)3 of almost the same concentration in the early stage of the reaction. The G-type lysozyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3 + (GlcNAc)3 predominating over that to (GlcNAc)2 + (GlcNAc)4. Thus, Ra-ChiC was found to be a novel enzyme in its structural and functional properties.

Changes in selected stress and immune-related genes in Atlantic cod,Gadus morhua, following overcrowding

Using Atlantic cod, Gadus morhua, as a model for the stress response in gadoid fish, the changes in the expression of some stress and immune genes as well as the profiles of plasma cortisol were examined. Adult fish were kept at a density of ca. 100 kg m−3 by lowering the water level in the rearing tank for 1 h and this short-term crowding stress was repeated thrice over a 12-h interval period. Blood samples were collected before exposure and at 2, 24 and 72 h post crowding. Plasma cortisol level significantly increased at 2 h post crowding but returned to pre-crowding levels 24 h after exposure. The relative expression of the stress response genes, glucose transporter-3 and a putative heat shock protein 70 significantly increased at 2 and 24 h post crowding respectively. Significant up-regulation of the pro-inflammatory cytokines, interleukin (IL)-1β and IL-8, as well as anti-bacterial genes, g-type lysozyme and bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP) was also observed at 2 h and the levels were maintained until 72 h post exposure, except for BPI/LBP which had maximum up-regulation at 24 h. The present observations have implications with respect to fish welfare and assessment of the health status of the farmed fish.

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein-protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the beta domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein-protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.

Role of disulfide bonds in goose‐type lysozyme

The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme.

Molecular characterisation of a goose-type lysozyme gene in Atlantic cod ( Gadus morhua L.)

Lysozymes are antibacterial enzymes important in the innate immune defense of several animal phyla. An Atlantic cod goose-type (g-type) lysozyme EST was identified in a suppression subtractive hybridisation (SSH) cDNA library and the full-length cDNA ( codg1) was obtained by RACE-PCR. The lysozyme gene is organised in five exons and four introns similar to g-type lysozyme genes in other fish species. Two different cod lysozyme transcripts, named codg1 and codg2, seem to be produced by the use of alternative transcription start sites (TSS) in the lysozyme gene. The alternative TSS cause a different exon I usage where exon Ia transcripts possess a putative signal peptide ( codg1) while exon Ib transcripts ( codg2) lack this feature. Lysozyme without the signal peptide was produced recombinantly in Escherichia coli and displayed muramidase activity against Micrococcus luteus cells at an unusually low pH. Gene expression analysis of codg1 and codg2 showed that both were expressed in several tissues with highest expression in the head kidney, peritoneum and spleen. Codg1 and codg2 were differentially expressed in some tissues. In the non-immunised control group, codg2 was expressed significantly higher in the head kidney compared to codg1, while an opposite expression profile was observed in the gills. Compared to non-immunised fish, a significant up-regulation of codg2 transcripts was observed in the peritoneum and gills after injection of formalin inactivated Listonella anguillarum indicating a role for g-type lysozyme in the innate defense system of cod.

Enhanced C‐Type Lysozyme Content of Wood Duck (Aix sponsa) Egg White: An Adaptation to Cavity Nesting?

Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.

Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill ( Scophthalmus rhombus)

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill ( Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24 h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24 h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.