Lysozymes, first discovered by Alexander Fleming in 1922, are key antimicrobial enzymes within the innate immune system, widely known for their role in hydrolyzing the 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG), which are critical components of bacterial cell walls. This enzymatic activity is ubiquitous across various biological systems and tissues, including mammalian blood, tears, sweat, and milk, as well as in the egg whites of birds (Fleming, 1922; Rupley, 1967). Beyond their enzymatic role, lysozymes also exhibit potent antibacterial properties, as even their proteolytic fragments demonstrate antimicrobial effects (Ibrahim, Matsuzaki and Aoki, 2001).

Lysozymes are classified into several distinct types, each with evolutionary variations. These include the c-type (chicken-type), g-type (goose-type), i-type (invertebrate-type), and the less common ch-type (Chalaropsis-type). Additionally, lysozymes are found in bacteriophages (phage-type), bacteria (bacterial-type), and plants (plant-type), each contributing to breaking down bacterial cell walls and immune defense in different organisms. Despite differences in their tissue and organismal origins, and minimal sequence similarity, lysozymes exhibit conserved protein crystal structures, indicating convergent evolution across eukaryotic kingdoms from animals to plants and even prokaryotes (Beintema and Terwisscha van Scheltinga, 1996; Callewaert and Michiels, 2010; Fischetti, 2005; Grutter, Weaver and Matthews, 1983; Holtje, 1996; Monzingo, Marcotte, Hart and Robertus, 1996; Weaver et al., 1984).

In vertebrates, lysozymes are key components of the innate immune system, serving as a frontline defense against invading pathogens. These enzymes are typically associated with immune cells such as monocytes, macrophages, and neutrophils (Ragland and Criss, 2017; Rosowski, 2020). Neutrophils, in particular, are produced in the bone marrow and circulate in the bloodstream, mobilizing quickly during infections (Burgener and Schroder, 2020). These neutrophils are major phagocytic cells across vertebrates, including fish, amphibians, reptiles, birds, and mammals. During immune responses, neutrophils perform key functions such as phagocytosis, apoptosis, and degranulation, and can form neutrophil extracellular traps (NETs) to capture and neutralize pathogens (Brinkmann et al., 2004; Fingerhut, Dolz and de Buhr, 2020).

While vertebrate lysozymes are well-studied, little is known about highly diverse invertebrate groups, many of which rely entirely on innate immunity due to their lack of adaptive immune systems. In invertebrates, lysozymes serve both defense and digestive functions, particularly in filter-feeding species such as bivalves. These enzymes help protect against pathogens and can digest bacteria filtered through their gills. This dual role of lysozymes highlights a significant evolutionary adaptation in invertebrates, where digestive and immune functions have co-evolved together. Specifically, arrays of i-type lysozymes play critical roles in these processes, contrasting with the c-type lysozymes’ recruitment in vertebrate ruminants, where they facilitate similar digestive functions (Itoh et al., 2010; Itoh and Takahashi, 2009; Matsumoto et al., 2006).

The understanding of g-type lysozymes in invertebrates remains limited. These lysozymes were originally discovered in the egg whites of the Anser anser (Embden goose) and have since been identified in other chordates as well as various molluscs, such as scallops and mussels. This widespread presence across different phyla suggests that g-type lysozymes perform essential functions in both ecological and evolutionary contexts. Their functions range from aiding in digestion to providing defense against pathogens, highlighting their versatility and significance in the immune responses of these organisms (He et al., 2012; Hikima et al., 2001; Nilsen et al., 2003; Wang et al., 2012; Zhang et al., 2012; Zhao et al., 2007; Zou et al., 2005).

Lateral Gene Transfer (LGT) or Horizontal Gene Transfer (HGT) plays a significant role in biological innovations, shaping the evolutionary trajectories by cross-kingdom signaling and transfer novel genetic material across species. Recent studies have highlighted the importance of HGT in enabling organisms to acquire adaptive traits such as enhanced metabolism, versatile immunity, and reproductive functions (Li et al., 2022; Liu et al., 2023). The identification of g-type lysozymes in metazoans, which exhibit strong sequence similarities to bacterial lysozymes, particularly those of predatory myxobacteria, suggests that HGT may have contributed in their acquisition. This process may explain the mosaic distribution of g-type lysozymes across taxa, as observed in our study.

Here, we conducted an extensive screening of g-type lysozymes across metazoans, uncovering a complex and intermittent distribution pattern, particularly within the lophotrochozoan/spiralian superclade. Our findings suggest that this distribution may be driven by lateral gene transfer (LGT) events from bacteria. We also identified two major g-type lysozyme groups in gastropods, using Aplysia californica as the reference species. Notably, our analysis suggests that A. californica and kin lacks both c-type and i-type lysozymes, indicating its reliance on three unique g-type lysozymes. Of these, two are highly expressed in the hepatopancreas, a key digestive organ, aligning with findings from other mollusks. The third g-type lysozyme shows variable expression across organs associated with both feeding and immune defense, highlighting the gene’s functional diversity.

These targeted expression patterns point to evolutionary adaptation through convergent evolution and lateral gene transfer to meet specialized dietary and defense challenges and needs. Additionally, we observed high sequence similarity between metazoan g-type lysozymes and those in myxobacteria, suggesting horizontal gene transfer from bacteria to animals. These findings provide insights into the evolutionary dynamics of lysozymes, shedding light on their mosaic functional diversification across metazoans.

Figure 2A presents a multiple sequence alignment (MSA) of g-type lysozyme genes, including one from humans (LYG2), Anthozoan D. gigantea, the more ancestral A. californica gene (LYZg3), as well as the Trichoplax g-type lysozyme. Accompanying this alignment, secondary structure predictions obtained from the JPRED4 server underscore the preservation of key structural elements across these diverse taxa (Drozdetskiy et al., 2015). Notably, the catalytic residues (aspartic acid and glutamic acid) are highlighted with stars on the MSA, illustrating their surprising conservation across broad phylogenetic distances. These catalytic residues are also conserved across all lysozyme types, including c-type and i-type lysozymes (Kuwano et al., 2013; Taylor et al., 2019).

Further, 3D structural analyses were performed using AlphaFold2 (Jumper et al., 2021) (Figure 2B). Predictions from AlphaFold2 demonstrated that the g-type lysozyme genes across a broad evolutionary spectrum fold into a structural motif observed in other lysozyme types, such as c-type or i-type (Kuwano et al., 2013). Despite lacking sequence similarity with these types, g-type lysozymes adopt similar structural conformations. Thus, the 3D structures predicted by AlphaFold2 reveal a remarkable consistency in the structural architecture of these enzymes across metazoans, again highlighting the deep evolutionary conservation of essential structural features critical for antimicrobial defense.

In order to validate the structural models of the g-type lysozyme genes across metazoans, we employed AlphaFold for structure prediction. The predicted accuracy, represented by the pLDDT (predicted Local Distance Difference Test) and pTM (predicted Template Modeling score), was calculated for each lysozyme model. As summarized in Table 1, the highest-ranked models for each gene showed consistent and high accuracy, with pLDDT values ranging from 87.4 to 97.8 and pTM values from 0.815 to 0.940, indicating reliable predictions. These results further illuminate the evolutionary preservation of g-type lysozyme architectures.

In addition to the predicted accuracy, we compared the Root Mean Square Deviation (RMSD) between different model pairs for each gene, as shown in Table 2. The RMSD values across model comparisons ranged from 0.100 to 0.183, indicating close structural similarity between different AlphaFold predictions for each gene. Furthermore, the structural alignments between the AlphaFold2-predicted models and experimentally determined structures yielded the following RMSD values: 0.717 Å for Hs_LYG2, 0.566 Å for Dg_27517, and 0.503 Å for Ta_63166 (all aligned with PDB ID: 4G9S). Additionally, the alignment between the AlphaFold2-predicted model for Ac_LYZg3 and the experimentally determined structure (PDB ID: 4G9S) produced an RMSD of 0.644 Å, demonstrating a high degree of similarity between the predicted and experimentally derived conformations.

Figure 2C presents a multiple sequence alignment of g-type lysozymes from various representative species, focusing on the amino acids surrounding the critical catalytic residues, glutamic and aspartic acids. These essential residues are highlighted with stars above the alignment to underscore their importance in the enzyme’s catalytic mechanism. Previous research has demonstrated that site-directed mutagenesis of aspartic acid (D52N) and glutamic acid (E35Q) in the chicken egg white lysozyme significantly impairs enzymatic activity. The mutant enzyme D52N retains approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, whereas E35Q exhibits virtually no measurable activity (0.1% ± 0.1%) (Malcolm et al., 1989). These findings affirm the pivotal role these residues play in the enzyme’s catalytic function.

Interestingly, we observed variations in the conservation of these residues across different species. In the human Hs_LYG1 gene, both glutamic acid and aspartic acid are not conserved, as indicated by the black arrow next to the gene name. Hs_LYG1 is highly expressed in the kidney, which plays a key role in detoxification and filtration, with lower expression levels noted in the liver and testes. A similar pattern of non-conservation is found in two of the three g-type lysozyme genes from the poriferan C. candelabrum (genes Cc_98523 and Cc_98591), which also lack conservation of these catalytic residues, marked by black arrows next to the gene names. In contrast, these catalytic residues are generally conserved in most other g-type lysozymes across other taxa. An exception to this trend is the gene Lg_LYZg3, where the aspartic acid (D) is replaced by tyrosine (Y), again indicated by a black arrow. While the specific effects of these mutations on enzyme activity have not yet been experimentally validated, it is hypothesized that these changes may result in reduced or altered enzymatic function, as seen with the chicken lysozyme mutants (D52N and E35Q).

In summary, the broad preservation of these catalytic residues highlights their evolutionary significance in maintaining enzymatic functions, with a few notable exceptions. These exceptions likely represent lineage-specific adaptations that have altered the canonical amino acids involved in the catalytic mechanism.

In various organisms, lysozymes play dual roles: they protect against microbial invasions and facilitate essential digestive processes (Dobson et al., 1984; Itoh and Takahashi, 2007). This study reveals a highly mosaic distribution of g-type lysozyme genes across metazoan phyla, with the apparent absence of these enzymes in ctenophores and most sponge classes (except the homoscleromorph C. candelabrum). The potential absence of g-type lysozymes in basal groups like ctenophores and sponges may represent either their primary absence in the common metazoan ancestor or early divergence of lysozyme subtypes and gene loss in many lineages, as opposed to lateral gene transfer in bilaterians, for example, illuminated in insects (Li et al., 2022; Liu et al., 2023).

Notably, the demosponge Suberites domuncula, possesses a different lysozyme subtype (i-type) and employs it for immune defense and digestive processes (Wiens et al., 2005). Sponges, being bacterial feeders, utilize specialized cells, choanocytes, for a filter mechanism similar to unicellular and colonial choanoflagellates. Both sponges and choanoflagellates share a critical structural feature for feeding–a collar: an apical cilium that generates water currents to draw bacteria surrounded by microvilli, which capture bacteria for intracellular digestion by phagocytosis (Mah et al., 2014). Although we did not identify a g-type lysozyme in the sequenced choanoflagellate genomes, we discovered geneologically unrelated families of genes encoding ch-type (Chalaropsis-type) and other unconventional lysozyme-like proteins and their potential involvement in digestive and defensive functions.

A broader look at the distribution of g-type lysozymes across bilaterians further suggests the same two alternative hypotheses: first, these genes may be conserved across the Tree of Life, playing fundamental roles across bacteria and animals, which we think is unlikely due to the absence of these enzymes in many other eukaryotic lineages. Alternatively, the observed distribution in Metazoa could be explained by lateral gene transfer (LGT) followed by differential gene loss in some lineages, too. Additional studies exploring the phylogenetic relationships and functional similarities between bacterial and metazoan lysozymes will be necessary to pinpoint whether the pattern is a result of gene conservation or LGT events.

Placozoans, the simplest metazoans with the primary absence of neurons and muscles, have developed extracellular digestion without a mouth and gut (Romanova and Moroz, 2024). G-type lysozyme gene in Trichoplax, with no evidence of other lysozyme types, might likely contribute to both immunity and digestion as in soft coral species like Dendronephthya and Xenia sp., with possible functional specialization for both diet and defense. While specific physiological reports on lysozymes in anthozoans are lacking, lysozyme-like activity has been observed in the mucus of Actinia equina (Stabili et al., 2015). Furthermore, our findings suggest that cnidarians, including Nematostella vectensis, may possess non-conventional types of lysozyme (unpublished data). The hemichordate Saccoglossus kowalevskii appears to encode genealogically different i-type and other non-conventional lysozyme as examples of convergent evolutionary adaptation, likely for both defensive and digestive roles.

Lysozymes broadly exhibit multifunctionality in marine bivalves, including scallops, clams, oysters, and mussels. These enzymes are utilized to degrade bacteria ensnared within the mucus of their gills, a process vital for nutrient uptake and enhancing digestive efficacy (Van Herreweghe and Michiels, 2012). Similarly, terrestrial invertebrates such as worms and flies, which feed on decomposing organic matter, depend on other families of lysozymes for both defensive measures against potential pathogens and the efficient extraction of nutrients from their microbial food sources. In flies, specifically in Drosophila melanogaster, lysozymes are part of the immune response system and are known to target bacterial peptidoglycans, as well as perform digestive roles in the midgut (Hultmark, 1996). Other arthropods, such as crustaceans, also utilize lysozymes for immune defense, breaking down bacterial cell walls to protect against infections (Bachali et al., 2002). In nematodes like Caenorhabditis elegans, lysozyme genes are expressed in the intestine, playing roles in pathogen defense and digestion (Boehnisch et al., 2011). Genealogically different i-type lysozymes also perform digestive roles akin to the function of c-type lysozymes in the foreguts of ruminants, highlighting a striking example of convergent evolution spanning diverse biological groups (Bachali et al., 2002; Olsen et al., 2003).

The observed high sequence similarity between g-type lysozymes in animals and those found in myxobacteria such as Corallococcus, Myxococcus, Pyxidicoccus, Archangium, Stigmatella, and Hyalangium presents a compelling case of both molecular convergence and horizontal gene transfer across diverse domains of life. This striking homology not only suggests that similar selective pressures may have independently shaped these enzymes in vastly different organisms but also highlights the essential functional role of lysozymes in both predation and immune defense.

In myxobacteria, these lysozyme-like proteins are likely integral to their predatory mechanisms, facilitating the breakdown of bacterial cell walls, similar to the role of g-type lysozymes in the immune systems of animals. These conserved enzymatic functions across evolutionary distances underscore the adaptive significance of lysozymes and may reflect ancient evolutionary connections (LGT) that have preserved these critical activities. This finding contributes to our broader understanding of how essential functions can be recruited or arise independently across phyla, thus offering new insights into the evolutionary dynamics of molecular function and adaptation.

The phylogenetic analysis of g-type lysozyme genes in Aplysia californica illuminate functional divergence and suggests distinct evolutionary pressures resulting in division of labor between digestive and likely immune functions. The characteristic distribution of g-type1 and g-type2 lysozymes across marine and freshwater gastropods further supports functional divergence associated with different habitats. G-type1 lysozymes are exclusively found in marine gastropods in more stable marine environments. In contrast, g-type2 lysozymes, present in both marine and freshwater species, likely reflect phenotypic plasticity to cope with fluctuating salinity, temperature, and microbial diversity in freshwater habitats. This observation highlights shaping the evolutionary trajectories of differential recruitments of these lysozyme families, closely tied to the ecological niches. The positioning of bivalve g-type lysozyme genes between the two gastropod groups (g-type1 and g-type2) in the phylogenetic tree could reflect divergence from a common ancestral lineage shared by gastropods and bivalves or LGT events or both. This finding underscores the complexity of lysozyme evolution in molluscs.

In this context, A. californica further demonstrates the molecular division of labor for three g-type lysozymes, showing distinct expression patterns across cells and tissues. Similarly, among scallops and abalone, g-type lysozymes are primarily located in organs vital for nutrient processing and environmental pathogen management, such as the hepatopancreas and mantle (Bathige et al., 2013). In A. californica, LYZg1 and LYZg3 are predominantly associated with components of the digestive system, also implying that A. californica leverages these enzymes to decompose bacterial cells, thus pursuing an evolutionary path distinct from that of mammals (Mackie, 2002). On the other hand, the pronounced expression of LYZg2 in the skin, mantle, foot, chemosensory organs, and reproductive organs likely represents A. californica -specific adaptation, with a highly reduced shell, boosting its defenses against pathogens.

In summary, our findings highlight the multifunctional and evolutionary significance of lysozymes across Metazoa. The roles of these enzymes extend beyond immune defense, showcasing their involvement in critical digestive processes, and are shaped by lineage-specific adaptations. The observed mosaic distribution of g-type lysozymes and their varying conservation patterns point to complex evolutionary trajectories influenced by ecological and physiological pressures.

1. In this study, we explored the evolutionary diversification and functional roles of g-type lysozymes across a broad range of metazoans, revealing a mosaic pattern in their distribution. Certain phyla, such as ctenophores and most sponge classes, lack these enzymes entirely, while others, like C. candelabrum and Trichoplax, retain or acquire g-type lysozymes that play critical roles in both immune defense and digestion. Additionally, the identification of non-conventional lysozyme-like proteins in choanoflagellates suggests that digestive and defensive functions may have evolved in parallel in these organisms despite the absence of g-type lysozymes.

This study provides new insights into the evolutionary dynamics of g-type lysozymes and their recruitment for diverse functional roles across metazoans. Future studies, including experimental validation of lysozyme activity and further genomic analysis, will be crucial to understanding the full scope of their contributions to both immune defense and digestion.