https://pmc.ncbi.nlm.nih.gov/articles/PMC8698798/
Lysozyme is a ~14 kDa protein present in many mucosal secretions (tears, saliva, and mucus) and tissues of animals and plants, and plays an important role in the innate immunity, providing protection against bacteria, viruses, and fungi. Three main different types of lysozymes are known: the c-type (chicken or conventional type), the g-type (goose type), and the i-type (invertebrate type). It has long been the subject of several applications due to its antimicrobial properties. The problem of antibiotic resistance has stimulated the search for new molecules or new applications of known compounds. The use of lysozyme as an alternative antibiotic is the subject of this review, which covers the results published over the past two decades. This review is focused on the applications of lysozyme in medicine, (the treatment of infectious diseases, wound healing, and anti-biofilm), veterinary, feed, food preservation, and crop protection. It is available from a wide range of sources, in addition to the well-known chicken egg white, and its synergism with other compounds, endowed with antimicrobial activity, are also summarized. An overview of the modified lysozyme applications is provided in the form of tables.
Keywords: innate immunity, peptidoglycan, Gram-positive, Gram-negative, antimicrobial, muramidase, lysozyme
1. Introduction
Lysozyme (or muramidase or N-acetylmuramic acid hydrolase E.C. 3.2.1.17) is a protein that exerts its enzymatic activity through the hydrolysis of the β-1,4-glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamide (NAG) in the polysaccharide backbone of the peptidoglycans of the Gram-positive bacterial cell wall.
Peptidoglycan is composed of polysaccharide chains cross-linked by short peptides. The polysaccharide chains contain alternate units of NAM and NAG.
The peptides are bound to the lactate moiety of NAM and usually consist of L-alanine, D-isoglutamic acid (D-isoglutamine in many Gram-positive bacteria), L-lysine or meso-diamino pimelic acid (in Gram-positive and Gram-negative bacteria, respectively), and two D-alanine residues. The side chain peptides of different polysaccharide chains are linked together with a direct link between meso-pimelic acid and the first D-alanine (Gram-negative), or through a cross-bridge (Gram-positive), a peptide that is characteristic of each species (Figure 1) [1].
Due to its capacity to disrupt the bacterial cell wall, lysozyme has been considered as an endogenous antibiotic, innately essential in the defense against microbes since its discovery by A. Fleming in 1921 [2]. Lysozyme is a small, monomeric protein stabilized by four disulfide linkages among the eight cysteine residues of its chain (Figure 2).
Lysozymes are present not only in human organs, tissues, and secretion, but also in the organs and secretions of various vertebrates, invertebrates, bacteria, and plants. They are classified into three main families: chicken type (c-type), goose type (g-type) and invertebrate type (i-type) [3]; also known are the phage type, bacterial type, and plant type lysozymes [4,5,6]. The chicken (cLys) and human (hLys) lysozymes are c-type Lys. The cLys is composed of 129 amino acid residues (14.3 kDa), whereas hLys is composed of 130 amino acid residues (14.7 kDa). There is a 59% identity between the sequence of human and chicken lysozymes but the antibacterial activity of hLys is three-fold greater than the antibacterial activity of cLys.
The antibacterial action of lysozyme is particularly efficient against Gram-positive bacteria because of its ability to hydrolyze the β-1,4-glycosidic bond present in the polysaccharide layer of these bacteria cell walls. The effect against the Gram-negative bacteria is significantly weaker due to the presence of a protective lipopolysaccharides layer on the outer membrane.
Many methods, physical and chemical, have been suggested and successfully developed, and aimed at enhancing the susceptibility of Gram-negative bacteria to lysozyme.
Taking into account the well-known antibacterial, antiviral, antifungal, anti-inflammatory, anticancer, and immunomodulatory activity [7], lysozyme has great potential, mainly in clinical, feed, and food applications, for treating pathogens of a different nature, and numerous examples have been reported in literature.
The continuing increase in bacteria resistance to antibiotics prompts the identification of new molecules or new applications of known compounds, such as the case of lysozyme. The chance of the emergence of resistance to peptidoglycan-degrading enzymes, including lysozyme, are described in an interesting 2020 review [1]. The authors explain that Gram-positive bacteria predominantly achieve resistance through peptidoglycan modifications; on the contrary, many Gram-negative bacteria tend to utilize inhibitor proteins that bind the active center of the peptidoglycan-degrading enzymes, blocking their activity. The development of resistance to these enzymes is defined by the authors as a rare event, at least in vitro, emerging not through de novo mutations, but through the horizontal transfer of resistance determinants. The clinical use of peptidoglycan-degrading enzymes is considered as being at less risk of resistance, due to their nature as recombinant proteins.
In the present review, the focus is the more relevant papers and patents published in the last 20 years. The present paper summarizes the main lysozyme source, its application as an alternative antibiotic, its synergism with other compounds, and the physical and chemical modifications aimed to improve its activity.
2. Sources
Lysozyme is a ubiquitous enzyme present in all living organisms and viruses with a wide variability in origin, quantity, structural, chemical, and enzymatic properties.
2.1. Lysozyme in Eggs
The chicken egg white is the richest source of this enzyme, and it is constituted by about 0.3% of lysozyme. This enzyme accounts for 3.4–5.8% of the total egg white proteins [8,9].
The wide availability makes the chicken egg white the main commercial source for this protein. Many efforts are aimed at obtaining lysozyme in a suitably pure form. In 2018, a review was published about the methods of the purification of lysozyme from egg whites [10]. The purification process of ultrafiltration, according to the authors of a 2009 article [11], extends the spectrum of its activity through the formation of lysozyme polymers. Indeed, it is known that the dimerization produces an enzyme that is active against both the Gram-positive and the Gram-negative bacteria (see the physical modifications Section).
Lysozyme was also found in the hen egg shell membrane, and a 2005 study [12] examined the effect of layer breed, bird age, membrane stabilization treatment, and storage time on the enzymatic and biological activity of lysozyme in egg shell membranes. In a later publication [13], it was observed that there was a lower antibacterial activity of purified lysozyme from egg shell membranes than that of purified lysozyme from egg whites.
Lysozyme from the egg white of quail was purified and characterized in 2014 [14], whereas the polymorphism of the egg white lysozyme from Japanese quail was investigated in 2012, and the authors demonstrated that two phenotypes of lysozyme were associated with significant differences in the antibacterial activity of the enzyme [15].
The g-type lysozyme can be isolated from goose egg whites [16]. It has a three-dimensional structure like the c-type and T4 phage lysozymes.
2.2. Lysozyme in Milk
Even though the egg white is the richest source of commercial lysozymes, milk from mammals also contains lysozyme molecules endowed with equally interesting properties. Lysozyme is present in mammals’ milk, either as a free soluble protein or within leucocytes and lysosomes. The lysozymes from milk belong to the c-type family with a wide variability from one species to another in terms of structure, physicochemical properties, and concentrations. Within the same species, the variation depends on many factors (breed, stage of lactation, parturition nutrition, and season of the year), as reported in a 2008 review [17]. A group of the sampled milks contains high levels of lysozyme (200–1330 mg/L) and another group has levels that are 3000 to 6000 times lower. Human, equine, and canine milks belong to the first group while bovine, ovine, and caprine milks represent the second group. In the same 2008 review [17], a table summarized the concentration of lysozyme in different mammals. According to the authors, the low level of lysozyme in milks of some mammals explains the conflicting literature data about the content, the presence, or the absence of lysozyme in the milk of some species (bovine, camel, and porcine).
Jenny milk is characterized by its high lysozyme content and has been used as an antimicrobial additive in dairy products; it is an alternative to the hen egg white lysozyme which can cause allergic reactions [18].
The buffalo milk lysozyme was purified and characterized in 2002 [19], showing a specific activity that was ten-fold that of the bovine milk lysozyme. The sequence of 23 amino acid residues of the N-terminal end was identified, and it showed a 56.5% homology with the bovine milk lysozyme and 30.4% with the equine milk lysozyme.
The N-terminal domain of the human milk lysozyme was treated with pepsin, and the N-terminal helix examined for its antimicrobial activity exhibited a potent bactericidal action to Gram-positive, Gram-negative bacteria, and the fungus Candida albicans, showing a potential use for the treatment of infectious diseases [20].
2.3. Saliva, Tears, Various Organs, and Tissues of Mammals
Lysozyme was discovered from a casual observation by A. Fleming [2,21,22,23] in 1921, when a few drops of his nasal discharge that contaminated an inoculated culture medium blocked the bacterial growth as observed a few days later. The presence of lysozyme in many forms of human secretion (such as saliva and tears) and tissue and organ secretion (such as placenta, sperm, leukocytes, blood) has been well known since then.
Numerous defense proteins are present in saliva and involved in innate and acquired immunity [24]. The same role is carried out by lysozyme in the lacrimal fluid, and its presence in the retinal pigment epithelial cells has been recently demonstrated [25]: the lysozyme expression is modulated, in this case, by pathogenic challenges.
Interestingly, in 2013 [26], authors from Saudi Arabia demonstrated that lysozyme purified from dromedary tears showed significant bactericidal activity against Listeria monocytogenes and Staphylococcus epidermidis, whereas the one purified from human tears was devoid of activity against these two strains.
Research about the c-type lysozyme genes in mice allowed the authors to hypothesize the role in mitochondrial functions of spermatozoon and its contribution to the innate immunity of the male genital tract [27].
Ruminant animals have been considered as lysozyme deficient. The expression of lysozyme in the tears, milk, and blood of cows is low. However, in the stomach, the antibacterial lysozymes were recruited as digestive enzymes useful to exploit plant material as a food resource [28]. In a work published in 2010 [29], the yak stomach lysozyme was compared with the cow stomach lysozyme. The result indicated that the yak stomach lysozyme was more closely related to the cow milk lysozyme than to the cow stomach lysozyme. The authors explained this result by concluding that there is a more recent common ancestor of at least one of the stomach lysozymes with milk lysozyme than with other stomach lysozymes. The interest in ruminant animals is also confirmed in a study [30] of a new c-type lysozyme from Lezhi black goat rumen (147 amino acid residues) sharing 70.27% of its identity with the capra hircus blood lysozyme that, likely, functions in host immunity and digestive systems.
2.4. Aquatic Organisms
2.4.1. Fish
Lysozyme is present in the mucus, lymphoid tissue, and serum of most fish species but not in cod and wolffish. It has been detected in the oocytes, fertilized eggs, and larval stages of fish species, including coho salmon, sea bass, and tilapia [31].
In aquatic environments, fish are in constant interaction with pathogenic and non-pathogenic microorganisms and, therefore, have developed mechanisms of defense aimed at their survival. The skin layer contains innate and adaptive immune factors that protect against infections. In the skin mucus innate immune factors are higher than in the serum. A study of these factors, comprising of lysozyme, was realized in the skin mucus of five marine teleost fish [32] and three freshwater fish [33], highlighting the variations in the considered fish in order to furnish important information for the aquaculture industry.
Several studies, aimed at identifying amino acid sequences, structure, and antimicrobial activity, focus on the fish lysozyme. The lysozyme from the rock bream (Oplegnathus fasciatus) was characterized in 2011 and classified as a g-type lysozyme [34]. In the same year, the lysozymes from kelp grouper (Epinephelus bruneus) [35] and from turbot (Scophthalmus maximus) [36] were identified and classified as c-type and g-type lysozymes, respectively. Two years later, a c-type lysozyme was isolated and characterized from the leukocytes of a nurse shark [37] and two lysozymes genes, and their recombinant proteins from Asian seabass (Lates calcarifer) were analyzed. In this case, either c-type and a g-type lysozymes were identified. The first was most abundant in the liver and the second was predominantly expressed in the intestine and weakly expressed in the muscle [38]. A g-type lysozyme was identified in 2016 [39] in seahorses (Hippocampus abdominalis), with the highest expression in the kidney and the least expression in the liver.
2.4.2. Marine Invertebrates
a. Mollusks
The lysozyme from the viscera of scallops (Patinopecten yessoensis) was purified and characterized in 2008 [40]. With the same aim of identifying new, more active enzymes, the lysozymes of other bivalve mollusks, including Unio pictorum [41] (four-fold more active than the egg lysozyme in the inhibition of E. coli); Asian hard clams (Meretrix meretrix) [42]; freshwater mussels (Cristaria plicata) [43]; and Manila clams (Ruditapes philippinarum) [44], were isolated and identified.
From the mollusk abalone (Haliotis discus hannai Ino), a chicken-type lysozyme was obtained and characterized (147 amino acid residues, 15.64 kDa molecular mass, and pI 4.87). This lysozyme showed bacteriolytic activity against Gram-positive and Gram-negative bacteria [45].
b. Crustaceans
The lysozymes from white shrimp (Panaeus vannamei) [46,47], black shrimp (Panaeus monodon) [48], blue shrimp (Litopenaeus stylirostris) [49], and penaeid shrimp (Marsupenaeus japonicus) [50] were identified, characterized, and their antibacterial properties analyzed. In some cases, the c-type lysozyme was present while in others the i-type was identified.
The characterization of lysozyme from banana prawn (Fenneropenaeus merguiensis) [51] showed a 37–93% similarity with mouse, human, chicken, and tiger prawn counterparts (15 kDa), and a strong inhibition against shrimp pathogens.
c. Echinodermata
The lysozyme of sea cucumbers (Stichopus japonicus) was identified in 2009 [52] as an i-type, by cDNA isolation.
d. Anellida
The medicinal leech lives in muddy freshwater pools. In its secretions, from the salivary glands, a multifunctional i-type enzyme, the destabilase-lysozyme, is present. This enzyme is endowed with isopeptidase, muramidase, and antibacterial activity. It attracts interest because it also shows thrombolytic activity through the lysis of the bonds ε-(γ-Glu)-Lys present in fibrin. For these reasons, its recombinant isoforms [53] and antifungal activity [54] have been studied.
2.5. Insects
The first antibacterial factor purified from insect hemolymph was lysozyme. The insect hemolymph lysozymes have molecular weights and properties similar to those of the hen egg white lysozyme, but a higher enzymatic activity.
Cameraria ohridella is the most dangerous pest to the horse chestnut. In 2005, [55] the lysozyme-type activity of the pupae of this insect was identified against Micrococcus luteus and Bacillus megaterium. Additionally, the lysozyme c-1 of Anopheles gambiae, in the course of its characterization, inhibited the growth of M. luteus but not of E. coli [56]. The possibility of cloning and overexpressing the lysozyme of Spodoptera litura in E. coli offers a method for the production of the biologically active c-type lysozyme as a natural antibiotic [57,58]. The same authors applied a similar process to the overexpression of lysozyme from Agrius convolvuli obtaining a peptide active against B. megaterium and M. luteus [59].
The larvae of Galleria melonella, the honeycomb moth, parasitize the honeybees, and the economic loss caused by this species prompted numerous studies. The c-type lysozyme of G. melonella is endowed with antifungal activity against Candida albicans, and the mechanism of this action was investigated by a Polish group in 2016 [60].
The awareness of the presence of lysozyme in the hemolymph of honeybees has existed since 1968 [61], and the antimicrobial properties of honey alone [62] or in combination with milk [63] have been reviewed.
A new type of lysozyme from the Chinese oak silk moth (Antheraea pernyi) was investigated and the encouraging results obtained about the strong effectiveness against Gram-negative strains, according to the authors, laid the foundation for future improvement by protein engineering [64].
Moreover, the c-type lysozyme from the Asian corn borer (Ostrinia furnacalis) [65] showed to be active against Gram-positive and Gram-negative bacteria.
Recently, a c-type lysozyme from Coridius chinensis, a medicinal insect resource in China, was identified and analyzed [66].
2.6. Plants
A novel plant lysozyme was isolated in 2005 [67] from the mung bean (Phaseolus mungo), with a molecular weight of 14.4 kDa, and exhibited antifungal activity toward Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, and antibacterial action against Staphylococcus aureus. A similar antifungal and antibacterial activity was found in the case of lysozyme isolated from Canadian cranberry beans (Phaseolus vulgaris) [68].
The seed oil from Carthamus tinctorius safflower [69] and the milky juice of papaya fruits [70] are rich sources of proteolytic enzymes, including lysozyme. For this reason, they are applied in the treatment of various skin injuries.
The lysozyme isolated from Momordica charantia L. was found to exhibit antifungal activity toward Mucor racemosus and Rhizoctonia solani, in addition to the antibacterial action against E. coli and S. aureus [71].
2.7. Microorganisms
The research of new antibacterial drugs able to overcome the antibiotic-resistant bacteria problem, also focused the attention toward bacteria, bacteriophages, and yeast as sources of new lysozymes.
Recently [72], a Chinese patent reported the identification of a bacteriophage lysozyme and its gene, as well as its use for preventing and treating bacterial infections.
Pichia pastoris is a methylotrophic yeast which has proven to be an efficient system for the expression of many heterologous proteins. The DNA of an unstable mutant in the hen egg lysozyme was integrated in P. pastoris, and the amount of secreted enzyme was 422-fold greater than what was observed with Saccharomyces cerevisiae [73]. The same yeast was employed to insert the T4 lysozyme gene, and the obtained protein inhibited the growth of S. aureus and Streptococcus pneumoniae [74]. In another example, P. pastoris was modified with the gene encoding the lysozyme. The produced lysozyme, in the presence of silicic acid, mediated the encapsulation of yeast cells within silica, paving a novel way for the preparation of composites, finalized to biotechnological applications [75].
The lysozyme of Bacillus licheniformis from soil was cloned and expressed in E. coli. The produced lysozyme was resistant to pepsin and trypsin, to some extent at, 40 °C, and efficiently active in the pH range between 3 and 9 and from 20° to 60 °C, respectively. The promising properties of this preparation [76] as a food or feed additives was also found for the fungal lysozyme isolated from Trichoderma reesei, showing an antimicrobial activity improved at the acidic pH (<6.5) [77].
According to the authors [78], another fungal lysozyme, produced by the Chalaropsis species, could be utilized in a variety of settings where bacterial infections proliferate, such as hospital settings (S. aureus) or in veterinary applications (mastitis from S. aureus in cows), and as a means of combating bioterror agents (such as Staphylococcal Enterotoxin B and Clostridium botulinum).
Chitinases are the enzymes which hydrolyze chitin, the β-1,4-linear polymer of N-acetylglucosamine, one of the most abundant natural polysaccharides. A bifunctional chitinase/lysozyme from Bacillus pumilus, capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls of many kinds of bacteria (Xanthomonas translucens, Xanthomonas axonopodis, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa, and Pseudomonas putida), was cloned and expressed in the E. coli strain M15 [79]. The chitinases from plants and animals are frequently endowed with lysozyme activity, whereas the bifunctionality of microbial chitinases is rare.
2.8. Recombinant Human Lysozyme (rhLys)
The great antimicrobial activity of lysozyme makes it interesting in medicine, cosmetics, and the food industry. The chicken egg white lysozyme is commonly used for these purposes but individuals sensitive to chicken eggs have also demonstrated an allergic reaction to the lysozyme isolated from egg whites. Many investigations have been carried out to produce the human lysozyme, considering its limited source, in bacteria, yeast, plants, and other organisms. In a 2006 patent [80], the preparation and the purification of rhLys with a transgenic organism, E. coli, was described. The obtained protein exhibited enzymatically active bacteriolytic properties, showing that E. coli can produce a functionally active human lysozyme. A bioreactor, with plastic composite support, was used to optimize the growth parameters of Kluyveromyces lactis K7, a genetically modified organism that expresses the human lysozyme [81].
A novel human c-type lysozyme was produced in recombinant Pichia pastoris using a fed-batch strategy; the obtained lysozyme showed a specific activity toward Micrococcus lysodeikticus of 7069 U/mg (Micrococcus lysodeikticus is a Gram-positive organism, isolated by A. Fleming when he discovered lysozyme. It is the standard microorganism utilized for the evaluation of muramidase activity. The current name for M. lysodeikticus is M. luteus [82]; in this review, we have used the former name or the new one, depending on the choice by the authors of the cited articles), suggesting a possible industrial application [83]. The expression of rhLys in Pichia pastoris, fused with the peptide tachyplesin I, was reviewed in 2013 [84].
Lysozyme is highly expressed in human milk but is found only in trace amounts in cow’s milk. In a work conducted in 2011, 17 healthy cloned cattle expressing the recombinant human lysozyme were produced. The transgenic cattle milk offered similar nutritional benefits as human milk, and the described techniques are appliable for the production of the active human lysozyme on a large scale [85]. More recently, recombinant human lactoferrin and lysozyme were produced and characterized in a bi-transgenic cow. The enzymatic activity of the lysozyme in the transgenic milk was comparable to that of human milk, which is 6 and 10 times higher than that of the bovine lysozyme present in milk [86].
A bovine mammary gland expression vector, expressing the human lysozyme gene, was constructed and tested on lactation rabbits [87] and in mouse mammary epithelial cells [88]. Transgenic mice were also developed for the expression of large amounts (18.5–35 g/L) of rhLys in milk [89], representing a model system for the cost-effective production of hLys. I previous studies, significantly lower amounts of lysozyme (1.20–1.76 g/L) were obtained using similar approaches [90,91,92].
Transgenic swine expressing rhLys were generated by a somatic cell transfer, with the aim to feed piglets with the human lysozyme to avoid pathogenic infections and, hence, a negative impact on neonatal survival. One of the 3 cloned female pigs expressed rhLys at 0.32 μg/mL in milk, 50-fold higher than the native pig lysozyme [93].
Transgenic dairy goats that expressed the human lysozyme in their milk, at 68% of the level normally found in human milk, were developed, in order to extend the beneficial protective properties of human milk into livestock milk and make it readily available for people of all ages [94,95].
Both the chicken egg lysozyme and human lysozyme belong to the c-type. Although the chicken egg lysozyme is easily obtained from egg whites, hLys displays a 3-fold higher antibacterial activity and is more thermal stable. For these reasons, transgenic chickens suitable for the production of active rhLys, were generated and the analysis of the obtained rhLys showed physicochemical and biological properties similar to commercial hLys. Moreover, the transgene of rhLys was genetically stable across the different generations [96].
Th breast feeding of fresh human milk has traditionally been considered the best means for providing nutrition to infants; indeed, it has been demonstrated that lysozyme and other milk proteins are immune factors that compensate for the undeveloped defense mechanism of the gut of infants [97]. For the situations in which the mother’s milk is not available, synthetic infant milk formulas are used in the place of breast feeding. In this context, the expression of human milk proteins (including lysozyme) in transgenic plants was developed.
A synthetic gene of hLys was introduced into the calli of rice [98]. The obtained rhLys was purified and the amino acid sequence verified, showing the promising potential for using a rice-derived lysozyme as a food supplement for infant formula and baby foods. The transgenic rice expressing lactoferrin and lysozyme was fed to chicks, showing antibiotic-like properties similar to subtherapeutic doses of baritrocin + roxarsone in the protection of the intestinal tract [99].
3. Applications of Lysozyme
The large number of investigated sources of different types of lysozymes can be explained by the many applications in medicine, cosmetics, the food industry, and agriculture. The wide spectrum of applications depends not only on its antibacterial activity, but also on the inactivation of certain viruses and fungi.
The antibacterial activity against Gram-positive bacteria has been explained by the lysozyme enzymatic action on the peptidoglycans present in the cell wall. The peptidoglycans present in the inner membrane of Gram-negative bacteria are shielded by a lipidic outer membrane, but the lysozyme shows, even if weakly, to be active. Some authors explained this activity, proposing that the antibacterial mechanism of action is independent of its enzymatic activity, also in the case of Gram-positive bacteria. The role of the lysozyme, according to this hypothesis, is the removal of the cell wall of the bacteria previously killed by antimicrobial polypeptides. Ibrahim et al., in 2001 [100], showed that the catalytically inactive mutant of the hen egg white lysozyme was as bactericidal as the wild -type lysozyme against S. aureus and B. subtilis. An opposite opinion was suggested in the same year by Masschlck et al. [101], who observed that a high pressure treatment, in the presence of lysozyme, sensitized a series of Gram-negative bacteria; the denaturation of lysozyme, by heat treatment, fully eliminated the bactericidal effect observed under high pressure conditions, while a partially denatured lysozyme maintained its activity. The bactericidal effect, due to the high-pressure treatment, was observed also in the case of two peptides, devoid of enzymatic activity, obtained from lysozyme: the authors ascribed these results to the cationic nature and the increased hydrophobicity of the chains.
The role of cationic peptide chains (the depolarization and permeabilization of membranes) was discussed in an article published in 2004 [102], together with the hypothesis of the indirect bactericidal action of lysozyme: the cationic peptide can behave as antibacterial by activating an autolytic wall muramidase of bacteria (a phenomenon defined as “Trojan horse”), resulting in bacteriolysis.
The action mechanism of lysozyme was studied also in vivo to verify if its activity depends or not on the muramidase action. From the observed results in transgenic mice deficient in lysozyme or expressing a muramidase-deficient lysozyme transgene, the authors concluded that lysozyme kills bacteria independently of its muramidase activity [103].
The cationic nature of the lysozyme chain was hypothesized to be the cause of its fungicidal activity. The ionic interactions between the cationic peptide and the anionic structures in the microbial cell wall can result in the damage to the cell wall, which is disrupted by a subsequent event, such as the exposure to salt and detergent by the effect of osmotic pressure. [104].
In an article published in 1999, the presence of a protein with the N-terminal 15 amino acids sequence identical to the human urinary lysozyme C in preparations of the β-subunit of human chorionic gonadotropin, was reported. The antiviral activity of this protein and of the lysozymes from chicken egg whites, from human milk, and from human neutrophils against HIV-1, was explained by the authors as being due to the degradation of viral polysaccharides [105].
The number of suggested potential mechanisms of action is as wide as the field of lysozyme applications against microorganisms, which differ greatly from one another.
3.1. Medical Applications
3.1.1. Skin Diseases
The milky juice of papaya fruits is a source of proteolytic enzymes, including lysozyme, and it is applied in surgery for the treatment of fistulas, cleaning wounds from necrotized tissues, and for skin grafting [70].
High antibacterial effects produced by both bacteriostatic and bactericidal pathways, including lysozyme activity, was demonstrated for the seed oil from Carthamus tinctorius (safflower) [69], in the management of skin injuries.
Staphylococcus aureus is the most common cause of primary and post-operative skin infections, and it has become increasingly resistant to antibiotics, such as methicillin and vancomycin. The use of lysozyme from egg whites and its dextran conjugate was investigated as an alternative topical ointment for the treatment of the infected skin of mice [106]. The two preparations were tested in vitro against S. aureus and E. coli. The results showed that both the lysozyme and lysozyme conjugate exhibited antibacterial activity against S. aureus, but only the lysozyme conjugate was active against E. coli. The activity of the conjugated lysozyme was explained by the authors by the strong surface activity which can enhance the lytic action of the enzyme toward the peptidoglycan layer in the inner membrane. The studies on mice also confirmed the improvement of the antibacterial activity of the lysozyme in wound healing, due to the conjugation with dextran. The activity of the dextran conjugated lysozyme was comparable with tetracycline, suggesting that lysozyme is a natural antimicrobial agent and a suitable replacement for synthetic antibiotic.
In a 2017 publication [107], a smart antimicrobial system, activated in the case of infection, based on elevated lysozyme activity, was presented. A synthesized N-acetyl chitosan was subjected to the lysozyme hydrolysis in artificial wound fluid, presenting N-acetylated chitooligosaccharides (COS). COS, by action of cellobiose dehydrogenase, afforded antimicrobial hydrogen peroxide (1 mM), which is able to inhibit the growth of E. coli and S. aureus (Figure 3).
Figure 3.
A T4 lysozyme fused with a cellulose binding module was prepared and immobilized to a wound dressing gauze. The immobilized protein retained the bacterial activity against Gram-positive and Gram-negative bacteria. The unmodified T4 lysozyme could not bind to the gauze. The immobilized lysozyme can constitute an innovative strategy for producing antimicrobial wound dressing materials [108].
Acticoat, an antibacterial silver nanoparticle-loaded dressing, is a commonplace for the prevention of infection in burns and with open wound patients. The efficacy of this dressing against methicillin-resistant S. aureus (MRSA) was evaluated, investigating additives that can improve its activity [109]. The greatest reduction in bacterial survival was observed when Acticoat was soaked with a combination of 10% glycerol, lysozyme (1 mg/mL), and an antimicrobial peptide (bac8c, a truncated and modified bovine neutrophile peptide).
A promising preparation for the development of antibacterial wound dressing was obtained in 2018 [110], starting from hairy steric stabilized nanocrystalline cellulose (SNCC) functionalized with aldehyde groups; by the reaction of these groups, lysozyme or nisin was immobilized on cellulose. Lysozyme and nisin in free and immobilized forms were tested against B. subtilis and S. aureus. S. aureus is the bacterial species more commonly detected in infected wounds and B. subtilis is closely related to several animal pathogens, including B. cereus, which is associated with wound infections. Immobilized nisin showed to be active against S. aureus, whereas free nisin became ineffective against the growth of S. aureus after 24 h. Lysozyme was not effective against S. aureus, but the immobilized lysozyme was active against B. subtilis. The authors of the study suggest that the combination of antimicrobial agents immobilized onto SNCC can offer an effective broad spectrum antibacterial wound dressing.
Recently [111], the effect of wound moisture on wound healing was studied considering the moisture balance of a polyurethane foam dressing. A moisture balanced antibacterial dressing was constructed by loading lysozyme onto a polyurethane foam dressing, by means of dopamine adsorption. The prepared dressing experiment in wound healing in infected mice provided the appropriate wound moisture and at the same time prevented bacterial infections.
The most common skin disorder is the acne vulgaris caused by Propionobacterium acnes. The use of lysozyme-shelled microbubbles (MBs) and ultrasound-mediated Lys-MBs cavitation against P. acnes, in vitro and in vivo, aimed to reduce the dose and the duration of antibiotic therapy, was investigated [112]. The results of the study showed that the combined Lys-MBs and ultrasound significantly reduced the treatment duration and inhibited P. acnes-induced skin diseases.
A different approach to the control of P. acnes by lysozyme was proposed in 2018. Bacteriocin AS-48 is a 70-amino acid residue circular peptide produced by different Enterococcus species, endowed with bactericidal activity on many Gram-positive and Gram-negative bacteria. The effectiveness against P. acnes by AS-48 alone, and in combination with lysozyme, was examined using a range of microscopy and bioassay techniques. The improvements of the action of AS-48 through the combination with lysozyme showed that these two natural compounds are promising candidates against dermatological diseases, such as acne vulgaris [113].
The use of a lysozyme gel formulation in the disinfection of the skin, during pre- and post-surgery, for facial care and the care of hands, feet, and nails, was reported in a 2013 U.S. Patent [114]. The gelled lysozyme was prepared by the addition of water to a suspension of lysozyme in alcohol, without the addition of other gelling substances. The formulation, retaining the enzymatic activity, was successfully used for local applications in the pre- and post-operative therapy of phlebopathic patients.
A polyethylene-based material loaded with an antibiotic is often used as a surgical sealant, but the developed drug resistance prompted the development of a variety of bioactive molecules modified by a PEG-based hydrogel. The antibacterial activity of lysozyme prompted the development of a PEG-lysozyme injectable sealant. A four-arm PEG suitably functionalized at each terminal arm was linked to lysozyme through its amine groups. It was observed that the hydrogel sealed gas or blood leakage in a rabbit trachea, and it could close the transmural left ventricular wall defect. The bacteriostatic activity was demonstrated against S. aureus and E. coli [115].
3.1.2. Medical Devices
Antimicrobial system involving lysozyme.
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