A lysozyme isolated from rainbow trout acts on mastitis pathogens

Abstract The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.     

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin

Summary The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymesc and α-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymesc and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.     

Molecular characterization of lysozyme type II gene in rainbow trout (Oncorhynchus mykiss): evidence of gene duplication

Rainbow trout (Oncorhynchus mykiss) have two types of lysozyme. Type II lysozyme differs from type I by only one amino acid, but only type II lysozyme has significant bactericidal activity. Due to this novel antibacterial property, lysozyme type II appears to be a candidate gene for enhancing disease resistance in fish as well as livestock species. Using polymerase chain reaction the lysozyme type II gene was amplified from genomic DNA isolated from rainbow trout. Two amplified fragments of 2041 and 2589 bp were observed. Sequencing revealed both amplicons were lysozyme genes having nearly identical nucleotide sequences, except the longer fragment has 548 base pairs inserted in intron 2 at nucleotide position 513 and a few point mutations within intron 2. Both versions of trout lysozyme type II gene were comprised of four exons and three introns. We also demonstrated that trout lysozyme is most likely encoded by these two different genes.     

Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata

The gut of the adult soft ticks Ornithodoros moubata displays high lytic activity against the bacteria Micrococcus luteus. The activity differed in the range of two orders of magnitude among individual animals and increased on average 4 fold during the first week following ingestion. In homogenates of first instar nymphs the activity was much lower increasing exponentially as nymphs neared the first molt. The protein responsible for this activity was purified out of gut contents of adult ticks by means of affinity adsorption on magnetic-chitin followed by two chromatography steps on cation exchange FPLC column MonoS. The homogeneous active protein has a mass of 14006 +/- 20 Daltons as determined by MALDI-TOF mass spectrometry. The N-terminal amino-acid sequence of this protein is K-V-Y-D-R-C-S-L-A-S-E-L-R with the highest similarity to the lysozyme from liver of rainbow trout and to lysozymes from digestive tracts of several mammals. The motif DRCSLA is specific for the digestive lysozymes of several dipteran insects. Based on this evidence, we have identified the protein as the tick gut lysozyme. The tick gut lysozyme has a pI near 9.7 and retains its full activity after treatment at 60 degrees C for 30 minutes. The pH optimum of the tick lysozyme was in the range from pH 5-7. Only marginal activity could be detected at pH > 8 which raises the question about the function of lysozyme in anti-bacterial defense in the environment of the tick gut.     

Purification, amino acid sequence, and some properties of rabbit kidney lysozyme

The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.     

The probiotic potential against vibriosis of the indigenous microflora of rainbow trout

The antibacterial properties of the indigenous microflora of rainbow trout ( Oncorhynchus mykiss Walbaum) and the potential use of inhibitory bacteria as fish probiotics were investigated. A total of 1018 bacteria and yeasts were isolated on tryptone soy agar (TSA) from skin, gills and intestine. Forty-five of these inhibited growth of the fish pathogenic bacterium Vibrio anguillarum in a well diffusion assay. The antagonism was most prominent among Pseudomonas spp., as 28 (66%) of the antagonistic bacteria belonged to this genus, despite constituting only 15% of the total tested flora. As pseudomonads are typically siderophore producers, chrome azurol S (CAS) agar was used as a semi-selective medium for isolation of antagonistic bacteria. On this medium, 75% of the iron-chelating strains were inhibitory to V. anguillarum . Eight strains out of a subset of 11 antagonists caused a 3–6 log unit reduction in the density of V. anguillarum [measured by polymerase chain reaction (PCR) detection in a most probable number (MPN) regimen] in a broth co-culture assay. Survival of rainbow trout infected with vibriosis was improved 13–43% by six out of nine antagonistic strains tested in vivo. All disease-protecting strains were pseudomonads, isolated from CAS plates, whereas two Carnobacterium spp. that were antagonistic in in vitro well diffusion assays did not alter the accumulated mortality of rainbow trout. The addition of live bacterial cultures to fish-rearing water may thus improve survival of the fish; however, in vitro antagonism could not completely predict an in vivo effect. Further studies on the underlying mechanism of activity are required to design appropriate selection criteria for fish probiotic bacteria.     

Production of lysozyme by staphylococci and its correlation with three other extracellular substances

Jay, James M. (Wayne State University, Detroit, Mich.). Production of lysozyme by staphylococci and its correlation with three other extracellular substances. J. Bacteriol. 91:1804-1810. 1966.-Lysozyme production was determined on plates containing 1 mg/ml of Lysozyme Substrate in Heart Infusion Agar with incubation at 37 C for 48 hr. Its production was compared with that of alpha-hemolysin and sheep hemolysin and egg-yolk precipitation, by use of both coagulase-positive and coagulase-negative strains of staphylococci. Of 126 coagulase-positive strains tested, 120 or 95.2% produced lysozyme, 117 or 92.9% produced alpha-hemolysin, 108 or 85.7% precipitated egg yolk, and 102 or 81% produced sheep hemolysin. Of the 49 coagulase-negative strains (which included 22 pathogens), only 4 or 8.1% produced lysozyme, 14 or 28.6% produced alpha-hemolysin, 13 or 26.5% produced sheep hemolysins, and 5 or 10.2% precipitated egg yolk. Only two of the six coagulase-positive strains which failed to produce lysozyme showed any consistent patterns in relation to the four characteristics determined. The four coagulase-negative strains which produced lysozyme were inconsistent for the other characteristics measured. It is suggested that lysozyme production is more a property of coagulase-positive staphylococci, and therefore a better ancillary test of pathogenicity, than either production of alpha-hemolysin or egg-yolk precipitation, because the incidence of lysozyme producers is higher among this group than among those producing the other substances and because fewer coagulase-negative staphylococci produced lysozyme than hemolysins or egg-yolk precipitation. Of 16 other species of bacteria and yeasts tested, all were found negative except Bacillus subtilis. Lysozyme production by staphylococci in heavily contaminated foods was not inhibited on plates containing sodium azide, whereas media containing 7.5% salt and sorbic acid were unsuitable. The possible relationship of lysozyme production to staphylococcal pathogenicity is discussed.     

Identification and expression analysis of three c-type lysozymes in Oreochromis aureus

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.     

Crystal structures of K33 mutant hen lysozymes with enhanced activities

Using random mutagenesis, we previously obtained K33N mutant lysozyme that showed a large lytic halo on the plate coating Micrococcus luteus. In order to examine the effects of mutation of K33N on enzyme activity, we prepared K33N and K33A mutant lysozymes from yeast. It was found that the activities of both the mutant lysozymes were higher than those of the wild-type lysozyme based on the results of the activity measurements against M. luteus (lytic activity) and glycol chitin. Moreover, 3D structures of K33N and K33A mutant lysozyme were solved by X-ray crystallographic analyses. The side chain of K33 in the wild-type lysozyme hydrogen bonded with N37 involved in the substrate-binding region, and the orientation of the side chain of N37 in K33 mutant lysozymes were different in the wild-type lysozyme. These results suggest that the enhancement of activity in K33N mutant lysozyme was due to an alteration in the orientation of the side chain of N37. On the other hand, K33N lysozyme was less stable than the wild-type lysozyme. Lysozyme may sacrifice its enzyme activity to acquire the conformational stability at position 33.     

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering.

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Streptococcus iniae type II infections in rainbow trout Oncorhynchus mykiss

Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout.     

Studies on the “Salvage” Synthesis of Ribonucleosides and their 5′-Phosphates by Microorganisms

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (ΔG) at 20°C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout,coho and Atlantic salmon

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.     

A genetic analysis of the London strain of rainbow trout

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.     

Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae Larvae

ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran.     

New variant of quail egg white lysozyme identified by peptide mapping

Two lysozymes were purified from quail egg white by cation exchange column chromatography and analyzed for amino acid sequence. The enzymes showed the same pH optimum profile for lytic activity with broad pH optima (pH 5.0-8.0) but had difference in mobility on native-PAGE. The native-PAGE immunoblot showed one or two lysozymes present in individual egg whites. The established amino acid sequence of quail egg white lysozyme A (QEWL A) was the same as quail lysozyme reported by Kaneda et al. [Kaneda, M., Kato, I., Tominaga, N., Titani, K., Narita, K., 1969. The amino acid sequence of quail lysozyme. J. Biochem. (Tokyo). 66, 747-749] and had six amino acid substitutions at position 3 (Phe to Tyr), 19 (Asn to Lys), 21 (Arg to Gln), 102 (Gly to Val) 103 (Asn to His) and 121 (Gln to Asn) compared to hen egg white lysozyme. QEWL A and QEWL B showed one substitution, at the position 21, Gln replaced by Lys, plus an insertion of Leu between position 20 and 21, being the first report that QEWL B had 130 amino acids. The amino acid differences between two lysozymes did not seem to affect antigenic determinants detected by polyclonal anti-hen egg white lysozyme, but caused them to separate well from each other by ion exchange chromatography.     

Antibacterial proteins in rainbow trout, Oncorhynchus mykiss

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.     

Purification, characterization and comparison of reptile lysozymes

Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle (Trionyx sinensis), ASTL from Asiatic soft shelled turtle (Amyda cartilagenea) and GSTL from green sea turtle (Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 degrees C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila.     

Lysozyme Production as an Aid for Identification of Potentially Pathogenic Strains of Staphylococci

Lysozyme production is a frequent property of potentially pathogenic staphylococci. In the present study, 1,186 strains of human origin, 85 strains of animal origin, and 156 strains of Staphylococcus albus (epidermidis) were tested. Of 1,114 coagulase-positive strains of human and animal origin, 1,098 were lysozyme-positive (98.5%). On the other hand, of 157 coagulase-negative strains which, based on further investigations, belong to the potentially pathogenic staphylococci, all were lysozyme-positive. All of the 156 strains (100%) belonging to the species S. albus (epidermidis) were lysozyme-negative. We conclude that lysozyme production is a better index of potentially pathogenic staphylococci than the measurement of free coagulase, especially in cases of strains of animal origin. It is possible that lysozyme production allows a differentiation between pathogenic and nonpathogenic coagulase-negative staphylococci.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.