Enzyme characterisation and gene expression profiling of Atlantic salmon chicken- and goose-type lysozymes.

Lysozymes represent important innate immune components against bacteria. In this study, Atlantic salmon (Salmo salar) goose (g-) and chicken (c-) types of lysozyme were subjected to protein characterisations and tissue expression analyses. Specific bacterial protein inhibitors of g- and c-type lysozymes were employed to discriminate between respective enzyme activities. Blood, gills and liver contained activities exclusive for the g-type lysozyme. Only haematopoietic organs (head kidney and spleen) contained enzyme activities of both g- and c-lysozyme enzymes and c-type activity was not found outside these organs. Gene transcript levels proportional to enzyme activity levels were detected for the g-type lysozyme but not for the c-type. In vitro studies revealed significant induction of c-type gene expression and enzyme activity in macrophages after incubation with lipopolysaccharide (LPS) while expression of the g-type lysozyme gene was unaffected. The activity of purified native c-type enzyme was profoundly reduced by divalent cations and displayed low tolerance to monovalent cations, while the native g-type lysozyme was stimulated by monovalent cations and tolerated low concentrations of divalent cations. Activities of both enzymes increased with temperature elevations up to 60°C. The native g-type lysozyme responses to temperature in particular are in apparent conflict to the ones for the recombinant salmon g-lysozyme. Our results imply separate expression regulations and different functions of c- and g-type lysozymes in salmon. LPS-induced expression of c-type lysozyme and broad constitutive tissue distribution of g-type lysozyme in salmon is different from findings in other studied fish species.

Role of lysozyme inhibitors in the virulence of avian pathogenic Escherichia coli.

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors.

Guards of the great wall: bacterial lysozyme inhibitors.

Peptidoglycan is the major structural component of the bacterial cell wall. It provides resistance against turgor and its cleavage by hydrolases such as lysozymes results in bacteriolysis. Most, if not all, animals produce lysozymes as key effectors of their innate immune system. Recently, highly specific bacterial proteinaceous lysozyme inhibitors against the three major animal lysozyme families have been discovered in bacteria, and these may represent a bacterial answer to animal lysozymes. Here, we will review their properties and phylogenetic distribution, present their structure and molecular interaction mechanism with lysozyme, and discuss their possible biological functions and potential applications.

Effects of arabinoxylan-oligosaccharides (AXOS) on juvenile Siberian sturgeon (Acipenser baerii) performance, immune responses and gastrointestinal microbial community.

Arabinoxylan-oligosaccharides (AXOS) are a newly discovered class of candidate prebiotics that exert different properties depending on their structure. In this study the effects of two different structures of AXOS, namely AXOS-32-0.30 (average degree of polymerization: 32, average degree of substitution: 0.30) and AXOS-3-0.25, were investigated on growth performance, immune responses, gut microbial fermentation and gut bacterial composition of juvenile Siberian sturgeon (Acipenser baerii). After a two weeks acclimation, fish (25.9 ± 0.9 g) were distributed over 24 aquariums (8 replicates per treatment) and fed a control diet or a diet containing 2% AXOS-32-0.30 or AXOS-3-0.25 for 12 weeks. Growth performance and feed utilization tend to improve in sturgeon fed on diets supplemented with AXOS-32-0.30, however not significant. Survival was high in all groups. Both AXOS preparations significantly enhanced the phagocytic activity of fish macrophages compared to the control group, while the alternative haemolytic complement activity and total serum peroxidase content improved only in the group fed AXOS-32-0.30 (P < 0.05). The lysozyme activity was not affected by AXOS addition. Simultaneously, the amount of short-chain fatty acids (SCFAs) was highest in the hind gut of sturgeon fed AXOS-32-0.30. The concentrations of acetate, butyrate and total SCFAs in fish fed AXOS-32-0.30 was significantly higher than in the groups fed the control diet or AXOS-3-0.25. Study of the bacterial community in the sturgeon hindgut using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that both preparations of AXOS induced changes in the bacterial composition. According to redundancy analysis (RDA), hindgut microbiota of each treatment group clustered apart from one another (P = 0.001). DNA sequencing of the dominant DGGE bands recovered from the different treatments showed that AXOS mainly stimulated the growth of lactic acid bacteria and Clostridium sp., with more pronounced effects of AXOS-32-0.30. It is concluded that AXOS improves sturgeon health through prebiotic action, but the induced effects depend on the specific structure of AXOS. A higher degree of polymerization of AXOS had a stronger beneficial impact in this sturgeon species.

Goose-type lysozyme inhibitor (PliG) enhances survival of Escherichia coli in goose egg albumen.

The goose-type lysozyme inhibitor PliG enhances the survival of Escherichia coli in goose but not in chicken egg white, which contains goose- and chicken-type lysozymes, respectively. These results indicate that both the type of host lysozyme and the type of bacterial lysozyme inhibitor may affect bacterium-host interactions.

Structure based discovery of small molecule suppressors targeting bacterial lysozyme inhibitors.

The production of lysozyme inhibitors, competitively binding to the lysozyme active site, is a bacterial strategy to prevent the lytic activity of host lysozymes. Therefore, suppression of the lysozyme-inhibitor interaction is an interesting new approach for drug development since restoration of the bacterial lysozyme sensitivity will support bacterial clearance from the infected sites. Using molecular modelling techniques the interaction of the Salmonella PliC inhibitor with c-type lysozyme was studied and a protein-protein interaction based pharmacophore model was created. This model was used as a query to identify molecules, with potential affinity for the target, and subsequently, these molecules were filtered using molecular docking. The retained molecules were validated as suppressors of lysozyme inhibitory proteins using in vitro experiments revealing four active molecules.

Food applications of bacterial cell wall hydrolases.

Bacterial cell wall hydrolases (BCWHs) display a remarkable structural and functional diversity that offers perspectives for novel food applications, reaching beyond those of the archetype BCWH and established biopreservative hen egg white lysozyme. Insights in BCWHs from bacteriophages to animals have provided concepts for tailoring BCWHs to target specific pathogens or spoilage bacteria, or, conversely, to expand their working range to Gram-negative bacteria. Genetically modified foods expressing BCWHs in situ showed successful, but face regulatory and ethical concerns. An interesting spin-off development is the use of cell wall binding domains of bacteriophage BCWHs for detection and removal of foodborne pathogens. Besides for improving food safety or stability, BCWHs may also find use as functional food ingredients with specific health effects.

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.

The Rcs two-component system regulates expression of lysozyme inhibitors and is induced by exposure to lysozyme.

The Escherichia coli Rcs regulon is triggered by antibiotic-mediated peptidoglycan stress and encodes two lysozyme inhibitors, Ivy and MliC. We report activation of this pathway by lysozyme and increased lysozyme sensitivity when Rcs induction is genetically blocked. This lysozyme sensitivity could be alleviated by complementation with Ivy and MliC.

Detection of a lysozyme inhibitor in Proteus mirabilis by a new reverse zymogram method.

A reverse zymogram method for the detection of bacterial lysozyme inhibitors was developed. This method was validated by using a periplasmic protein extract of Escherichia coli containing a known inhibitor and subsequently led to the detection of a new proteinaceous hen egg white lysozyme inhibitor in Proteus mirabilis.

Role of the lysozyme inhibitor Ivy in growth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk.

Ivy is a lysozyme inhibitor that protects Escherichia coli against lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth of E. coli MG1655 and Pseudomonas aeruginosa PAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-type E. coli was able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 10(4) CFU ml(-1) of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of the ivy mutant to grow like the parental strain. P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.

A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria.

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.

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.

Inactivation of Escherichia coli by high-pressure homogenisation is influenced by fluid viscosity but not by water activity and product composition.

The inactivation of Escherichia coli MG1655 by high-pressure homogenisation (HPH) at pressures ranging from 100 to 300 MPa was studied in buffered suspensions adjusted to different relative viscosities (1.0, 1.3, 1.7, 2.7 and 4.9) with polyethylene glycol 6000 (PEG 6000). The water activity of these suspensions was not significantly affected by this high molecular weight solute. Bacterial inactivation was found to decrease with increasing viscosity of the suspensions, an effect that was more pronounced at higher pressures. To study the effect of water activity, series of E. coli suspensions having a different water activity (0.953-1.000) but the same relative viscosity (1.3, 1.7, 2.7 and 4.9) were made using PEG of different molecular weights (400, 600, 1000 and 6000), and subjected to HPH treatment. The results indicated that water activity does not influence inactivation. Finally, inactivation of E. coli MG1655 by HPH in skim milk, soy milk and strawberry-raspberry milk drink was found to be the same as in PEG containing buffer of the corresponding viscosity. These results identify fluid viscosity as a major environmental parameter affecting bacterial inactivation by HPH, as opposed to water activity and product composition, and should contribute to the development of HPH applications for the purpose of bacterial inactivation.

Moderate temperatures affect Escherichia coli inactivation by high-pressure homogenization only through fluid viscosity.

The inactivation of suspensions of Escherichia coli MG1655 by high-pressure homogenization was studied over a wide range of pressures (100-300 MPa) and initial temperatures of the samples (5-50 degrees C). Bacterial inactivation was positively correlated with the applied pressure and with the initial temperature. When samples were adjusted to different concentrations of poly(ethylene glycol) to have the same viscosity at different temperatures below 45 degrees C and then homogenized at these temperatures, no difference in inactivation was observed. These observations strongly suggest, for the first time, that the influence of temperature on bacterial inactivation by high-pressure homogenization is only through its effect on fluid viscosity. At initial temperatures > or =45 degrees C, corresponding to an outlet sample temperature >65 degrees C, the level of inactivation was higher than what would be predicted on the basis of the reduced viscosity at these temperatures, suggesting that under these conditions heat starts to contribute to cellular inactivation in addition to the mechanical effects that are predominant at lower temperatures. Second-order polynomial models were proposed to describe the impact of a high-pressure homogenization treatment of E. coli MG1655 as a function of pressure and temperature or as a function of pressure and viscosity. The pressure-viscosity inactivation model provided a better quality of fit of the experimental data and furthermore is more comprehensive and versatile than the pressure-temperature model because in addition to viscosity it implicitly incorporates temperature as a variable.