Lysozyme-like Enzyme Research Articles

Recent Insights Into the Prognostic and Therapeutic Applications of Lysozymes.

Lysozymes are naturally occurring enzymes present in a variety of biological organisms, such as bacteria, fungi, and animal bodily secretions and tissues. It is also the main ingredient of many ethnomedicines. It is well known that lysozymes and lysozyme-like enzymes can be used as anti-bacterial agents by degrading bacterial cell wall peptidoglycan that leads to cell death, and can also inhibit fungi, yeasts, and viruses. In addition to its direct antimicrobial activity, lysozyme is also an important component of the innate immune system in most mammals. Increasing evidence has shown the immune-modulatory effects of lysozymes against infection and inflammation. More recently, studies have revealed the anti-cancer activities of lysozyme in multiple types of tumors, potentially through its immune-modulatory activities. In this review, we summarized the major functions and underlying mechanisms of lysozymes derived from animal and plant sources. We highlighted the therapeutic applications and recent advances of lysozymes in cancers, hypertension, and viral diseases, aiming toseeking alternative therapies for standard medical treatment bypassing side effects. We also evaluated the role of lysozyme as a promising cancer marker for prognosis to indicate the outcomes recurrence for patients.

Purification, crystallization, and preliminary analysis of the lysozyme-like enzyme SagA from Brucella abortus

Purification, crystallization, and preliminary analysis of the lysozyme-like enzyme SagA from Brucella abortus

Effect of Azadirachta indica (Sapindales: Meliaceae) Oil on the Immune System of Spodoptera frugiperda (Lepidoptera: Noctuidae) Immatures.

The insect immune system includes several mechanisms responsible for defending against pathogens, parasites, and parasitoids. Some botanical insecticides, such as Azadirachta indica oil, cause changes in the immune system of various insect species. Spodoptera frugiperda is an important agricultural pest; thus, knowledge about the effect of neem oil on the immune system of this species can assist in its management. This study aimed to evaluate the effect of A. indica oil on the immune system of S. frugiperda. Caterpillars (2–3 mg) were placed individually in containers (50 ml) with approximately 10 g of diet, containing 125, 250, and 500 ppm of neem oil with propanone; the control group received only the propanone diet. In four experiments, the total number of hemocytes, the phagocytic activity, the activity of lysozyme-like enzymes, and phenoloxidase activity were measured in caterpillars at the end of the sixth instar. The total number of hemocytes in insects exposed to neem oil was 21% lower than in the control group. The percentage of cells that phagocyted the latex beads was similar among the caterpillars that ingested the different concentrations. The mean diameter of cell lysis halos was reduced only at concentrations of 125 and 250 ppm. Absorbance did not differ between treatments. Knowing that this oil reduces the number of circulation cells and the activity of lysozyme-like enzymes is of great importance to design control strategies, once the neem oil could be added to other biological agents for mortality reducing the chances of this insect surviving in the environment.

Continuous Agrochemical Treatments in Agroecosystems Can Modify the Effects of Pendimethalin-Based Herbicide Exposure on Immunocompetence of a Beneficial Ground Beetle

Herbicide application for pest control can negatively affect soil biodiversity, mainly acting on species that are involved in ecosystem service. In this study, field and laboratory trials were designed to assay herbicide exposure effects on the constitutive immunity of Harpalus (Pseudoophonus) rufipes (De Geer, 1774), a beneficial carabid species that inhabits croplands. The circulating hemocytes (THCs) and plasmatic levels of basal and total phenoloxidase (PO), as well as lysozyme-like enzyme activities, were measured as markers of exposure. In laboratory tests, the exposure to realistic field doses of pendimethalin-based herbicides for two, seven and 21 days caused a reduction in enzyme activities in beetles from organic crops. In beetles from conventional fields, the THCs and total PO activity decreased significantly at two and seven days after the initial exposure, though no effects were recorded on basal PO and lysozyme like-enzyme activities. These differences in enzyme activities and THCs indicate that the interference of pendimethalin with immune parameters clearly depends on both the different field conditions from which the population comes and the cumulative effects of repeated applications over the time.

Harpalus (Pseudoophonus) rufipes as a model to study cellular and humoral immune defence strategies in coleopteran species

Carabids are of special interest as environmental quality assessment indicators of exposure to xenobiotic and for pest control. In agroecosystems, they can be exposed to a wide range of pathogens and environmental pollution exerting a stronger selection on their innate immune systems. Therefore, information on species-specific immunocompetence is necessary to complete the ecological framework of ground beetles. In this study, cellular and humoral responses were characterized in adults of Harpalus (Pseudoophonus) rufipes (De Geer, 1774) to define a baseline knowledge for future ecotoxicological studies. The circulating hemocytes were characterized by light and transmission electron microscopy and in vivo assay performed by injecting latex beads to identify phagocytizing hemocytes. Ultrastructural analyses revealed four morphologically distinct types of circulating hemocytes: prohemocytes, plasmatocytes, granular cells and oenocytoids. Differential hemocyte counts showed that plasmatocytes and granular cells were the most abundant circulating cell types and granular cells exhibited phagocytic activity following immune challenge with latex beads. Mitotic figures and non-differentiated hemocytes observed under light microscopy indicate a continuous cell turnover in the hemolymph. Melanotic nodules found 2h after the immune challenge were formed to immobilize the latex beads. Phenoloxidase (PO) assays revealed an increase of basal PO activity in hemolymph after immune system activation with lipopolysaccharide (LPS). However, the LPS-stimulated adults showed no significant variation in the lysozyme-like enzyme activity in hemolymph. Based on these results, H. rufipes displays a rapid, non-specific immune response involving cellular and humoral effectors that both sequester and clear pathogens.

Impact of agrochemicals on non-target species: Calathus fuscipes Goeze 1777 (Coleoptera: Carabidae) as model

Carabid beetles are important in the biological control of arable crop pests. Agricultural practices can produce over time a delayed toxic effect at the organismal and population levels and can compromise the survival on these species. In this research, we quantified the cumulative sublethal effect on body size, Malpighian tubules and immune responses in Calathus fuscipes adults living in the potato field and exposed to lambda-cyhalothrin and cymoxanil-based commercial formulates. Reductions of morphological parameters such as body, pronotum and elytron in both males and females from the potato field indicated that the pre-imaginal stages (larvae and pupae) suffer the sublethal effects of exposure to the larvicide control action of lambda-cyhalothrin. Ultrastructural alterations recorded in Malpighian tubules at the level of plasma membrane, mitochondria and nucleus indicated the reduction of the detoxification capability. The basal phenoloxidase and lysozyme-like enzyme activities have measured as markers of immune competence. Spectrophometric analyses showed that the chronic exposure in field causes an increase of basal phenoloxidase enzyme activity, while the lytic activity of haemolymph was not affected. As a result, the use of larvicides and fungicides have a harmful effect on beneficial species such C. fuscipes living in the soil of potato fields. These morphological and physiological results recorded at the organismal level can provide useful information of effects at the population and community levels to preserve the biodiversity of agroecosystem.

How does temperature influences the development of lactococcosis? Transcriptomic and immunoproteomic invitro approaches.

Lactococcus garvieae is the aetiological agent of lactococcosis, a haemorrhagic septicaemia that affects marine and freshwater fish, with special incidence and economic relevance in farmed rainbow trout. Water temperature is one of the most important predisposing factors in the development of lactococcosis outbreaks. Lactococcosis in trout usually occur when water temperatures rise to about 18°C, while fish carriers remain asymptomatic at temperatures below 13°C. The aim of this work was to analyse the differences in the complete transcriptome response of L.garvieae grown at 18°C and at 13°C and to identify the immunogenic proteins expressed by this bacterium at 18°C. Our results show that water temperature influences the expression of L.garvieae genes involved in the lysis of part of the bacterial cell population and in the cold response bacterial adaptation. Moreover, the surface immunogenic protein profile at 18°C suggests an important role of the lysozyme-like enzyme, WxL surface proteins and some putative moonlighting proteins (proteins with more than one function, usually associated with different cellular locations) as virulence factors in L.garvieae. The results of this study could provide insights into the understanding of the virulence mechanisms of L.garvieae in fish.

Are immune responses gender-related in Carabus lefebvrei (Coleoptera: Carabidae)?

The “live hard, die young” theory predicts the evolution of gender differences in immunocompetence, with males having a weaker immune system than females. To test thishypothesis in Carabus lefebvrei, total and basal phenoloxidase (PO) activities and lysozyme-like enzyme activity were compared among males and females of different reproductive status. The sexual dimorphism occurred only in reproductively active adults and for total and basal PO levels, while no significant differences were recorded between sexes in virgin adults. Differences were not recorded for lytic activity between sexes. Basal PO and lytic activities decreased in both males and females after mating, while the total PO value increased in males and decreased in females. Thus, resources seem to be invested to increase the humoral response in pre-reproductive phase forming a barrier against pathogens and preserving the fecundity and longevity of both sexes. Males preserve their survivorship in reproductive phase by increasing enzymatic levels in hemolymph to avoid fitness reduction due to the increased exposure to pathogen as result of mating. Females shift resources from PO and lytic activity to other physiological systems involved in reproduction in order to maximize their fitness.

Response of the insect immune system to three different immune challenges

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD50 dose of LPS from Escherichia coli elicited a more rapid response than an LD50 dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2hpi, followed by an increase by 7dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system’s response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.

Maturation of the immune system of the male house cricket, Acheta domesticus

The immune system functions to counteract the wide range of pathogens an insect may encounter during its lifespan, ultimately maintaining fitness and increasing the likelihood of survival to reproductive maturity. In this study, we describe the maturation of the innate immune system of the male house cricket Acheta domesticus during the last two nymphal stages, and during early and late adulthood. Total hemolymph phenoloxidase enzyme activity, lysozyme-like enzyme activity, the number of circulating hemocytes, and encapsulation ability were all determined for each developmental stage or age examined. The number of circulating hemocytes and lysozyme-like enzyme activity were similar for all developmental stages examined. Nymphs and newly molted adult males, however, had significantly lower total phenoloxidase activity than later adult stages, yet nymphs were able to encapsulate a nylon thread just as well as adults. Encapsulation ability would thus appear to be independent of total phenoloxidase activity.

Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricketGryllus texensis

In many parts of the world, climate change is increasing the frequency and severity of heat waves. How do heat waves impact short-lived poikilotherms such as insects? In the cricket, Gryllus texensis, 6 days of elevated temperatures (i.e. 7°C above the average field temperature and 5°C above their preferred temperature) resulted in increased egg laying, faster egg development and greater mass gain. The increased temperature also increased activity of phenoloxidase and lysozyme-like enzymes, two immune-related enzymes, and enhanced resistance to the Gram-negative bacterium Serratia marcescens. When given a sublethal S. marcescens infection, G. texensis maintained increased reproductive output at the elevated temperature (33°C). These data suggest that heat waves could result in more numerous, disease resistant, crickets. However, resistance to the Gram-positive bacterium, Bacillus cereus was lower at temperatures above or below the average field temperature (26°C). A sublethal infection with B. cereus reduced egg laying at all temperatures and suppressed the increase in egg laying induced by higher temperatures. These results suggest that for some species-pathogen interactions, increased temperatures can induce trade-offs between reproduction and disease resistance. This result may partly explain why G. texensis prefers temperatures lower than those that produce maximal reproductive output and enhanced immune function.

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.

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage φ29 tail

The small bacteriophage phi29 must penetrate the approximately 250-A thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage phi29 is noncontractile and approximately 380 A long. A 1.8-A resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the phi29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13(-) mutants with the phi29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

Sex-specific differences in immunological costs of multiple mating in Gryllus vocalis field crickets

Although mating can be costly, most individuals must choose not whether to mate but how many times to mate. This study examined the immunological costs of additional matings once an individual has already mated. Gryllus vocalis field crickets were assigned to mate 5 or 10 times and their immunocompetence probed by measuring their ability to withstand a bacterial challenge by Serratia marcescens, assaying lysozyme-like enzyme activity and phenoloxidase activity, and measuring their success in encapsulating a monofilament implant. Although number of matings generally did not affect the strength of immune responses, females had superior immunity to males in most assays. Females that mated 10 times did, however, have lower lysozyme-like enzyme levels than females that mated 5 times, suggesting that mating can compromise at least one component of female standing immunity. When individuals were allowed to mate ad libitum and their lysozyme-like enzyme activity, phenoloxidase activity, and encapsulation responses measured, there was not a relationship between female mating frequency and lysozyme-like enzyme activity. This result suggests that females may avoid immunological costs of mating by differentially moderating their mating frequency.

The role of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra-peritoneal infection in mice

BackgroundMycobacterium tuberculosis can enter into a dormant state which has resulted in one third of the world's population being infected with latent tuberculosis making the study of latency and reactivation of utmost importance. M. tuberculosis encodes five resuscitation promoting factors (Rpfs) that bear strong similarity to a lysozyme-like enzyme previously implicated in reactivation of dormant bacteria in vitro.We have developed an intraperitoneal infection model in mice, with immune modulation, that models chronic infection with similar properties in mouse lungs as those observed in the murine aerosol infection model. We have assessed the behavior of mutants that lack two or three rpf genes in different combinations in our intraperitoneal model.MethodsC57Bl/6 mice were intraperitonealy infected with H37Rv wild type M. tuberculosis or mutant strains that lacked two or three rpf genes in different combinations. After 90 days of infection aminoguanidine (AG) or anti-TNFα antibodies were administrated. Organ bacillary loads were determined at various intervals post infection by plating serial dilutions of organ homogenates and enumerating bacteria.ResultsWe found that the rpf triple and double mutants tested were attenuated in their ability to disseminate to mouse lungs after intraperitoneal administration and were defective in their ability to re-grow after immunosuppression induced by administration of aminoguanidine and anti-TNFα antibodies.ConclusionRpf proteins may have a significant physiological role for development of chronic TB infection and its reactivation in vivo.

Estimating disease resistance in insects: phenoloxidase and lysozyme-like activity and disease resistance in the cricket Gryllus texensis

An animal’s ability to resist disease is usually estimated by measuring one or more components of the immune system. There is an assumption that these assays of immunity measure an animal’s ability to mount an effective immune response. This paper tests this assumption by examining the relationship between two common estimates of insect immunocompetence, phenoloxidase and lysozyme-like enzyme activity, and resistance to three common insect bacterial pathogens: Serratia marcescens, Serratia liquefaciens, and Bacillus cereus. There was a correlation (Spearman’s r s=0.33, p<0.001, n=190 pairs) between total phenoloxidase and baseline lysozyme-like activity within individuals. However, total phenoloxidase and baseline lysozyme-like activity levels did not predict which male crickets would survive any of the three bacterial challenges. Lysozyme-like activity increased after an immune challenge (Friedman, 33.72, p<0.001), and the greater the increase, the greater the chance that the cricket would survive S. marcescens (slope=0.15, χ 2=8.2, p=0.005) or B. cereus (slope=0.8, χ 2=6.4, p=0.01). The crickets with a greater total hemolymph protein concentration were also more likely to survive a challenge with any of the three bacterial pathogens than the crickets with lower total hemolymph protein concentrations ( S. liquefaciens: slope=0.02, χ 2=9.2, p=0.002; B. cereus: slope=0.02, χ 2=6.5, p=0.01; S. marcescens: slope=0.03, χ 2=7.8, p=0.005). Because of the complexity of the immune system, empirical tests of the relationship between assays of immunity and resistance to a range of actual pathogens are important for correctly interpreting these measures.

Biochemical and bacteriological properties of the lysozyme of the clam Tivela stultorum

A lysozyme-like enzyme from the crystalline style of the clam Tivela stultorum was purified by chromatography of cationic exchange in carboxymethyl cellulose. The product had a specific activity of 25,407 units per milligram of protein, which represents a purification of 100 times. The specific activity observed with this enzyme was almost six times greater than the activity observed in the hen egg-white lysozyme under the same conditions. Using the isoelectric focusing technique, a component was observed that indicates an isoelectric point of 7.7 (IEF-PAGE, Phast Gel). A one-dimensional electrophoresis (SDS-PAGE) revealed an apparent molecular weight of 17,000 daltons. The enzyme was optimally active at pH 5.6–5.8, in 0.1 M phosphate-citrate buffer, with maximum activity at a temperature of 40ºC, and an ionic strength of 0.1043. At 4ºC, the lysozyme presented 26.7% of remnant activity, and was highly stable at elevated temperatures (90ºC) and extreme pHs, from 2.8 to 11.8, with activities above 75%. This enzyme proved to have antimicrobial properties with or without EDTA in the lysis of three bacteria (Streptococcus alfa, Micrococcus lisodeikticus and Escherichia coli), whereas hen egg lysozyme showed lysis in the presence of EDTA on S. alfa and E. coli. The results were also compared against the lysozyme of the scallop Chlamys islandica.

The question of whether gut microflora of the millipede Ommatoiulus sabulosus could function as a threshold to food infections

Summary The specific composition of bacterial flora in the intestines of O. sabulosus cannot be explained simply by antibiotic inhibition of contaminating microflora since the predominant types of bacteria present are unable to produce any bacteriolytic activity of lysozyme-like enzymes, bacteriocins or other antimicrobial molecules. The lack of microbial contaminators could rather result from the unfavourable biochemical niche in the midgut enzymes and little or no competition for the enteric bacteria group predominant in the millipede alimentary tract. In other words, the rapid colonization and overgrowth of the Ommatoiulus intestines eliminate adventitious bacteria, yeasts and moulds ingested with food.

Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity

An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5–6.2) and at low temperature (4–35°C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70°C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.

Lysozyme-like enzyme from the scallop Chlamys islandica

A lysozyme-like enzyme isolated from the crystalline style of the scallop Chlamys islandica, was purified by cation exchange chromatography on carboxymethyl cellulose and Mono S columns. The enzyme was optimally active at pH 4.8, similar to that observed for the lysozyme of Mytilis edulis. The present enzyme has an unusual 67%, remainder activity at 4ºC when cell walls of Micrococcus lysodeikticus were used as substrate. The enzyme had a specific activity of 486,090 U/mg, corresponding to ten times higher than 1ha1 of hen egg while lysozyme.