The multifarious lysozyme arsenal of Dictyostelium discoideum

Developmental and Comparative Immunology 107 (2020) 103645

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The multifarious lysozyme arsenal of Dictyostelium discoideum a,∗

a

a

b

Otmane Lamrabet , Tania Jauslin , Wanessa Cristina Lima , Matthias Leippe , Pierre Cosson a b

T a

Faculty of Medicine, University of Geneva, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211, Geneva 4, Switzerland Zoological Institute, Comparative Immunobiology, University of Kiel, Kiel, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Amoeba Dictyostelium discoideum Lysozymes Phylogenetic distribution Protozoa

Dictyostelium discoideum is a free-living soil amoeba which feeds upon bacteria. To bind, ingest, and kill bacteria, D. discoideum uses molecular mechanisms analogous to those found in professional phagocytic cells of multicellular organisms. D. discoideum is equipped with a large arsenal of antimicrobial peptides and proteins including amoebapore-like peptides and lysozymes. This review describes the family of lysozymes in D. discoideum. We identified 22 genes potentially encoding four different types of lysozymes in the D. discoideum genome. Although most of these genes are also present in the genomes of other amoebal species, no other organism is as well-equipped with lysozyme genes as D. discoideum.

1. Introduction Dictyostelium discoideum belongs to dictyostelids, a group of heterotrophic amoebae also known as social amoebae (Eichinger et al., 2005; Schilde and Schaap, 2013; Sheikh et al., 2018). Dictyostelids are characterized by a life cycle alternating unicellularity and multicellularity: while unicellular amoebae grow and divide in good trophic conditions, they aggregate and form multicellular structures when starved (Raper, 1984; Dunn et al., 2018). Around 150 species of dictyostelids have been described (Sheikh et al., 2018). Molecular phylogenies based on small subunit ribosomal RNA and alpha-tubulin datasets divide dictyostelid species into four major groups (1–4) (Schaap et al., 2006) and three minor groups named the violaceum, polycephalum, and polycarpum complexes (Singh et al., 2016; Sheikh et al., 2018). D. discoideum is a member of group 4 (Schaap et al., 2006). D. discoideum feeds upon bacteria in the soil (Raper K.B, 1935; Raper K.B, 1936). To bind, ingest, and kill bacteria, D. discoideum uses molecular mechanisms analogous to those found in specialized phagocytic cells of multicellular organisms, such as neutrophilic granulocytes and macrophages (Cosson and Soldati, 2008; Dunn et al., 2018). Consequently, D. discoideum has been used as a model organism to study interactions between phagocytic eukaryotic cells and pathogenic or non-pathogenic bacteria (Cosson and Lima, 2014; Dunn et al., 2018). Analysis of the D. discoideum genome reveals that it is equipped with a large arsenal of antimicrobial peptides, notably lysozymes (Chisholm et al., 2006). Since their discovery by Fleming in 1922 (Fleming, 1922), lysozymes have been extensively studied. They were one of the first

proteins to be completely sequenced and one of the first enzymes for which the X-ray structure was determined (Wohlkönig et al., 2010). Lysozymes are defined exclusively by their enzymatic activity: they are enzymes that degrade bacterial peptidoglycan. The peptidoglycan is a component of the bacterial cell wall which maintains the rigidity of bacteria. It is a heteropolymer of alternating β-1,4-linked N-acetylglucosamine (NAG) and N-acetyl muramic acid (NAM), crosslinked with short peptides (Schleifer and Kandler, 1972). Lysozymes catalyze the hydrolysis of the β-1,4-linkages between the NAM and NAG residues. Lysozymes (E.C. 3.2.1.17) are ubiquitous enzymes, occurring in all major taxa of living organisms. They are used by animals and plants as a defense against bacterial invasion although their exact role and importance in this process and in others, such as digestion, is not fully established (Jollès and Jollès, 1984; Callewaert and Michiels, 2010). Several classes of lysozymes have been identified and their biochemical and enzymatic properties as well as their protein structure have been determined to different degrees: the C-(chicken-), the G-(goose-), the V(viral-), the I-(invertebrate-), the Entamoeba- and the Aly-type (Canfield, 1963; Jollès and Jollès, 1984; Nickel et al., 1998; Müller et al., 2005; Callewaert and Michiels, 2010; Wohlkönig et al., 2010; Van Herreweghe and Michiels, 2012). Chicken-type lysozymes are the most prevalent, found in a large number of vertebrates, from fish to mammals (Prager and Jollès, 1996), as well as in insects (Hultmark, 1996). Although all lysozymes share the same enzymatic activity, their sequences and structures are highly divergent, and alignments of protein sequences can only be achieved reliably within each type. Similarly,

Abbreviations: NAG, N-acetylglucosamine; NAM, N-acetyl muramic acid; GH, Glycoside Hydrolase Family ∗ Corresponding author. E-mail address: [email protected] (O. Lamrabet). https://doi.org/10.1016/j.dci.2020.103645 Received 9 January 2020; Received in revised form 12 February 2020; Accepted 12 February 2020 Available online 13 February 2020 0145-305X/ © 2020 Elsevier Ltd. All rights reserved.

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Table 1 Lysozymes genes present in Dictyostelium discoideum. Gene Name DDB_G0288143 DDB_G0291137 DDB_G0278563 DDB_G0274551 DDB_G0272494 DDB_G0274181 DDB_G0293492 DDB_G0293566 DDB_G0276439 DDB_G0273175 DDB_G0273875 DDB_G0269248 DDB_G0275123 DDB_G0275119 DDB_G0275121 DDB_G0273197 DDB_G0273731 DDB_G0286229 DDB_G0282905 DDB_G0282893 DDB_G0274831 DDB_G0274291

Annotation dictybase

lyC2 rcdBB

cf50-1 cf50-2 cf45-1 alyA alyB alyC alyD-1 alyD-2 alyL

lyT2-4

Synonyms

Lysozyme type

Size (aa)

Chromosome (position)

Reference

DdLyC1 DdLyC2 DdLyC3 DdLyC4 DdLyC5

Chicken Chicken Chicken Chicken Chicken

187 185 199 202 369

5 5 3 2 2

(1,139,463–1140,026) (5,040,435–5041,278) (1,076,940–1077,754) (4,193,351–4194,293) (1,799,833–1801,293)

Muller et al. (2005) Muller et al. (2005) Muller et al. (2005) This work This work

DdLyEh1 DdLyEh2 DdLyEh3 DdLyEh4 cf50-1 cf50-2 cf45-1

Entamoeba Entamoeba Entamoeba Entamoeba Entamoeba Entamoeba Entamoeba

212 213 216 480 303 303 297

2 6 6 2 2 2 1

(4,831,162–4832,097) (2,951,996–2953,178) (3,051,866–3052,770) (6,954,075–6955,794) (2,522,303–2523,214) (3,508,572–3509,483) (3,267,307–3268,805)

Muller et al. (2005) Muller et al. (2005) This work This work This work This work This work

AlyA AlyB AlyC AlyD-1 AyD-2 AlyL AlyTM1 AlyTM2

Aly Aly Aly Aly Aly Aly Aly Aly

181 181 181 260 260 572 356 356

2 2 2 2 2 4 3 3

(5,002,742–5003861) (5,005,709–5006809) (5,004,280–5005342) (2,696,096–2697,063) (3,334,723–3335,690) (4,218,365–4220,358) (6,351,923–6353,597) (6,349,872–6351,423)

Muller et al. Muller et al. Muller et al. Muller et al. Muller et al. Nasser et al. This work This work

LyT1-4 LyT2-4

Phage Phage

170 170

2 (4,061,568–4062,080) 2 (4,062,693–4063,205)

(2005) (2005) (2005) (2005) (2005) (2013); This work

Muller et al. (2005) Muller et al. (2005)

to the complete genomes of seven amoeba species [Naegleria fowleri ATCC (GCA_000499105.1), Entamoeba histolytica HM-1:IMSS (AAFB00000000.2), Acanthameoba castellanii str. Neff (AHJI00000000.1), Acytostelium subglobosum (BAUZ00000000.1), Polysphondylium pallidum (ADBJ01000000), Dictyostelium fasciculatum (NC_010,653), and Dictyostelium purpureum (NZ_ADID00000000.1)] using the BLASTp program as implemented on the NCBI, DictyBase and AmoebaDB (Aurrecoechea et al., 2011) servers. Inclusion criteria were again e-value < 10−4, coverage ≥80% and similarity ≥30%. The identity of analyzed genes was confirmed by BLASTp and DELTABLAST (inclusion criteria e-value < 10−4, coverage ≥40% and similarity ≥25%) against manually annotated UniProt entries and against PDB structures (Supplementary Table S1). The conserved domains of selected ORFs were annotated with SMART (Letunic et al., 2015) and InterPro (Mitchell et al., 2019). Alignments of lysozymes from each class were done with PROMALS3D (Pei et al., 2008) and Espresso (Armougom et al., 2006), two methods which consider secondary structure predictions and homologous 3D structures to build alignments.

searching genomic databases with the sequence of a given lysozyme retrieves sequences of putative lysozymes of the same type only. Functionally, lysozymes very often exhibit an antibacterial activity (Callewaert and Michiels, 2010). Generally, Gram-positive bacteria are more sensitive to lysozymes than Gram-negative bacteria (Masschalck and Michiels, 2003). This probably reflects the fact that Gram-positive bacterial cell walls are composed of 30%–70% peptidoglycan, while Gram-negative bacterial cell walls are composed of less than 10% peptidoglycan and are additionally surrounded by an outer membrane containing lipopolysaccharides (Schleifer and Kandler, 1972; Masschalck and Michiels, 2003). Lysozymes have been implicated in a variety of biological processes, such as defense of multicellular organisms against bacterial infections (animals and plants), digestion of bacteria as food (animals and protozoa), bacterial cell wall synthesis and remodeling, and lysis of bacteria at the end of the phage replication cycle (Callewaert and Michiels, 2010). In this review, we describe and classify the putative lysozyme genes found in the D. discoideum genome, we analyze their genomic organization, and we discuss the molecular evolution of lysozymes in dictyostelids and other amoeba species.

2.2. Multiple sequence alignment and phylogenetic tree of lysozymes

2. Methods

For phylogenetic reconstructions, the amino acid sequences of lysozymes were aligned using the MUSCLE algorithm (Larkin et al., 2007). The alignments were manually refined, in order to remove regions containing gaps or highly divergent sequences, using the BioEdit program v7.0.9 (Hall, 1999). Phylogenetic trees were generated using MEGA 7.0 (Kumar et al., 2016). Genetic distances were computed using the Neighbor-Joining (NJ) algorithm, and Maximum-likelihood (ML) phylogenetic trees were obtained with the JTT matrix-based model (Jones et al., 1992). Bootstrap assessment of tree topology with 100 and 1′000 replicates was performed to find the support for the inferred clades. Bootstrap support of > 70% and posterior probability of > 90% were considered to identify supported nodes.

2.1. Identification of lysozyme genes in dictyostelids A first survey of the genome of D. discoideum AX4 (NC_007087–92) was done by searching for the“Lysozyme” keyword on the DictyBase server (Fey et al., 2013) or in published articles on PubMed. Twelve genes were originally identified in this manner. New putative lysozymelike gene products were identified by similarity searches using the twelve previously described Dictyostelium lysozyme genes as seeds, the V-type lysozyme gene from Escherichia coli phage RB14 (Uniprot #C3V1I9) (Lukacik et al., 2012), the I-type lysozyme gene from the bivalve Ruditapes philippinarum (Uniprot #C8CBP0) (Ito et al., 1999) and the G-type lysozyme gene from the goose Anser anser (Uniprot #P00718) (Simpson and Morgan, 1983). The BLASTp program was used (inclusion criteria: e-value < 10−4, coverage ≥80% and similarity ≥30%) as implemented on the NCBI and DictyBase servers. In addition, each lysozyme gene of D. discoideum AX4 was compared 2

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Fig. 1. Schematic representation of the four lysozyme types present in D. discoideum. Schematic alignment of protein sequences of: (A) the two phage-like lysozymes from D. discoideum with one from Escherichia coli virus RB14; (B) the five C-type lysozymes from D. discoideum with one from chicken (Gallus gallus); (C) the seven Entamoeba-type lysozymes with one from E. histolytica; and (D) the eight Aly-type lysozymes. The putative critical residues of the active sites are indicated with asterisks.

3. Characterization of distinct types of lysozymes in D. discoideum

lysozyme-like protein belonging to the Aly-type, named AlyL (for amoeba-lysozyme-Like), has been described (Nasser et al., 2013). We searched for new putative gene products with lysozyme-like features in D. discoideum using different strategies. BLAST searches with the AlyA amino acid sequence did not reveal any new lysozyme genes other than the four previously identified in the original study (Müller et al., 2005). However, similarity searches with the other 11 previously described genes allowed us to find 9 new putative lysozyme genes, belonging to the same three types (Table 1). DDB_G0282905 and DDB_G0282893 (named alyTM1 and alyTM2, for amoeba-lysozymeTransmembrane Domain 1 and 2) belong to the Aly-type. RcdBB (DDB_G0274551) and DDB_G0272494, here re-named DdlyC4 and DdlyC5, belong to the C-type family. DDB_G0293566 and DDB_G0276439, here annotated DdlyEh3 and DdlyEh4, belong to the Entamoeba-type family (Table 1). In addition, three other genes, cf50-1 (DDB_G0273175), cf50-2 (DDB_G0273875) and cf45-1 (DDB_G0269248), contain a GH25 (Glycoside Hydrolase Family 25) lysozyme domain (the characteristic domain observed in Entamoebatype lysozymes, see below) (Table 1 and Supplementary Table S1). No

3.1. D. discoideum lysozyme repertoire A formerly published survey of the D. discoideum genome revealed the existence of seven D. discoideum genes that potentially code for lysozymes belonging to previously identified types (Müller et al., 2005) (Table 1). Two (DDB_G0274181 and DDB_G0293492) encode Entamoeba-type lysozymes first described in E. histolytica (Jacobs and Leippe, 1995; Nickel et al., 1998); two (DDB_G0274831 and DDB_G0274291) encode bacteriophage T4 type (V-type) lysozymes (Fastrez, 1996); and three (DDB_G0291137, DDB_G0278563, and DDB_G0288143) are homologous to C-type lysozymes (Müller et al., 2005) (Table 1). In addition, purification and molecular cloning of a Dictyostelium protein with lysozyme activity revealed the existence of a new type of lysozymes, named Aly (for Amoeba LYsozymes), containing four members (AlyA-D: DDB_G0273197, DDB_G0275123, DDB_G0275119 and DDB_G0275121; in the AX4 strain, alyD is present in a duplicated region on chromosome 2) (Table 1). More recently, a 3

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gene transfer (possibly from a bacteriophage inhabiting a bacterium ingested by the amoeba) followed by duplication in the D. discoideum genome.

additional T4 phage-like lysozyme was detected. Our analysis also confirmed the absence of G- and I-type lysozymes. Lysozyme activity has also been described in plants, including in the original Fleming experiments in 1922 (Fleming, 1922). However, plant lysozymes also have an additional chitinase activity (E.C. 3.2.1.14) (Beintema and Terwisscha van Scheltinga, 1996). Chitinase enzymes catalyze the breakdown of chitin, a linear polymer found in insects, crustaceans, and fungal cell walls consisting of NAG (Chipman et al., 1967). Some lysozymes can hydrolyze chitin, but less efficiently than their natural substrate (Boller et al., 1983; Jekel et al., 1991; Terwisscha van Scheltinga et al., 1994; Ito et al., 1999). However, there is no obvious amino acid sequence similarity found between these lysozymes and chitinases (Monzingo et al., 1996; Wohlkönig et al., 2010). The genome of D. discoideum presents six genes encoding chitinase-related proteins (Funkhouser and Aronson, 2007). This category represents another class of polysaccharide-hydrolyzing enzymes, and is not discussed further in this review. The fact that amoeba cells possess different classes of lysozymes is remarkable, as organisms usually have only one specific type of lysozyme (Callewaert and Michiels, 2010). As D. discoideum lives in the soil where it may encounter a wide variety of microorganisms, the large number of lysozymes found in its genome may indicate that each of them is useful to kill and degrade a specific subset of microorganisms. The full list of D. discoideum lysozymes is detailed below.

3.1.2. Chicken-type lysozymes The D. discoideum genome contains five genes encoding putative Ctype lysozymes: DdlyC1, DdlyC2, DdlyC3, DdlyC4, and DdlyC5 (Table 1). The five C-type lysozyme genes are not clustered in the genome, but rather distributed over three different chromosomes. They all exhibit a signal peptide, a GH22 lysozyme domain in the N-terminal region (the characteristic domain of C-type lysozymes) and low complexity regions at the C terminus (Fig. 1B). DdLyC5 also has two repeat regions at the C terminus (Fig. 1B). In 2001, Vocadlo et al. 2001 published experimental evidence describing the enzymatic activity of C-type lysozymes at the atomic level. The hydrolysis of the β-(1,4)-glycosidic bond between NAM and NAG occurs through a double displacement reaction. The enzymatic activity in hen egg white lysozyme is ensured by Glu-53 and Asp-70 residues (Callewaert and Michiels, 2010). Alignment of the amino acid sequences of D. discoideum C-type lysozymes with the hen egg white lysozyme showed that only one (Glu-53) of these two amino acids considered essential for the catalytic activity shows positional identity in D. discoideum (Fig. 1B and Supplementary Fig. S1). The second critical acidic residue (Asp-70), although conserved in four D. discoideum genes, is positioned differently when compared to the chicken lysozyme (Fig. 1B and Supplementary Fig. S1B). Despite the presence of a GH22 lysozyme domain in the DdLyC5 protein, we were not able to localize its putative catalytic site.

3.1.1. Viral-type lysozymes The D. discoideum genome has two genes coding for T4 phage-like type lysozymes (V-type), lyT1-4 and lyT2-4 (Table 1). These two adjacent genes are probably the result of a recent gene duplication. Their high level of sequence identity (76% at the amino acid level) reinforces this hypothesis. Both genes have 46% of sequence identity with the prototypical T4 bacteriophage enzyme (Fig. 1A and Supplementary Fig. S1). The T4 lysozyme is an archetypal lysin, and members of the T4 lysozyme family present an essential catalytic triad composed of Glu11, Asp-20, and Thr-26 (Kuroki et al., 1993). The sequence alignment of D. discoideum LyT1-4 and LyT2-4 with the endolysin of the E. coli T4 bacteriophage shows a perfect conservation of these residues (Fig. 1A and Supplementary Fig. S1A). A signal peptide has not been detected in all these phage-like lysozymes (Fig. 1A and Supplementary Fig. S1A) suggesting that they are located in the cytosol. It has been proposed that phage lysozyme genes were horizontally transferred from bacteria to fungi, insects, plants, and archaea (Metcalf et al., 2014) and to the bivalve Manila clam (Ruditapes philippinarum) (Ding et al., 2014). The horizontally transferred lysozymes could be used as antibacterial proteins by the recipient organisms, complementing their antibacterial arsenal (Metcalf et al., 2014). It has also been described that coopted lysozyme genes undergo dramatic structural changes and gene duplication events (Ren et al., 2017). Although there are two V-type lysozymes in D. discoideum, we did not find orthologous genes coding for V-type lysozymes in the genomes of other very closely related amoeba species (Table 2). This observation strongly suggests that D. discoideum acquired a V-type lysozyme via horizontal

3.1.3. Entamoeba-type lysozymes In the D. discoideum genome four genes encoding Entamoeba-type lysozymes (DdLyEh) can be found: DdlyEh1 to DdlyEh4 (Table 1). They are distributed on three different chromosomes (Table 1). All primary translation products possess a putative signal peptide and a GH25 lysozyme domain (the characteristic domain of Entamoeba-type lysozymes). In addition, three other genes (cf45-1 and the tandem duplicated genes cf50-1 and cf50-2) also harbor a GH25 lysozyme domain. However, previous studies have shown that the products of these genes are components of the counting factor, a protein complex which regulates the series of processes that result in multicellularity during the developmental cycle and exhibits only a low, if any, enzymatic activity in standard lysozyme assays (Brock et al., 2002, 2003). Interestingly, knockout of cf50-1 impairs cell growth in the presence of the Gramnegative bacteria Klebsiella pneumoniae and the Gram-positive Bacillus subtilis (Žitnik et al., 2015). Two lysozymes have been isolated from extracts of the human-pathogenic protozoan E. histolytica (Jacobs and Leippe, 1995; Nickel et al., 1998). The primary structures of these E. histolytica-type lysozymes are similar to those of lysozymes found in the fungus Chalaropsis (Jacobs and Leippe, 1995), in the bacteria Streptomyces coelicolor (Rau et al., 2001) and in Caenorhabditis elegans (Nickel et al., 1998; Boehnisch et al., 2011). It has been reported that only one of the two acidic amino acids considered essential for the catalytic activity of Chalaropsis

Table 2 Lysozymes genes in free-living amoebae. Amoeba species

Chicken-type

Viral-type

Aly-type

E. histolytica-type

Total genes

Dictyostelium discoideum AX4 Dictyostelium fasciculatum Dictyostelium purpureum Naegleria fowleri ATCC Entamoeba histolytica HM-1:IMSS Acanthamoeba castellanii str. Neff Acytostelium subglobosum Polysphondylium pallidum

5 3 6 0 0 3 3 1

2 0 0 0 0 0 0 0

8 1 4 0 0 0 2 3

7 12 7 1 6 3 6 8

22 16 17 1 6 6 11 12

4

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Fig. 2. Phylogeny of the lysozyme family. The unrooted maximum likelihood tree was obtained using the amino acid dataset. Branch lengths are proportional to the number of amino acid substitutions per site. Numbers at the nodes represent the percentage of bootstrap support (only values > 70% are shown).

alyTM2 (Table 1). The genes are distributed over three different chromosomes (Table 1). AlyA, AlyB, and AlyC are positioned next to each other, with very high levels of sequence identity (87%–93% at the amino acid level). This configuration is typical of recent gene duplications in D. discoideum. In addition, alyTM1 and alyTM2 are highly similar genes arranged in a tandem repeat, presumably also resulting from a recent gene duplication. AlyD (AlyD-1 and AlyD-2) is located on a region of chromosome 2 duplicated in certain axenic strains. All aly gene products, except AlyTM1 and AlyTM2, are equipped with a signal peptide. AlyD and AlyL present an extended low complexity region within their primary structure. In addition, AlyTM1 and AlyTM2 exhibit in their N-terminal region one and two putative transmembrane domains, respectively. Nothing is known about the catalytic site of Aly-type lysozymes. Alignment of the eight genes allowed the identification of one conserved aspartic acid residue and one glutamic acid residue in the Cterminal portion (Fig. 1D and Supplementary Fig. S1). These two amino acid residues may be essential for the catalytic activity of these lysozymes but this hypothesis remains to be tested. AlyA and AlyL have previously been characterized by functional genetics: (i) deletion of the alyA gene reduces the total cellular lysozyme activity by half, and also decreases the ability of cells to feed upon bacteria (Müller et al., 2005); (ii) deletion of alyL reduces the ability of

lysozyme (the most N-terminal aspartic acid residue, Asp-18) (Fouche and Hash, 1978) is found at the same position in E. histolytica (Jacobs and Leippe, 1995). A second acidic amino acid residue may not be required for the catalytic activity, as has been described for goose-type lysozymes (Weaver et al., 1995), or it may be positioned differently. In the Streptomyces cellosyl protein, which belongs to the Chalaropsis lysozyme family, the catalytic site has been determined by X-ray crystallography, and comprises the acidic residues Asp-9, Asp-98, and Glu100 (Rau et al., 2001). To identify these critical amino acid residues, we aligned the amino acid sequences of D. discoideum Entamoeba-type lysozymes with two lysozymes present in the genome of E. histolytica (Loftus et al., 2005) and one lysozyme present in the genome of A. castellanii (Rosenthal et al., 1969). We observed that the first Asp-18 residue is conserved in all DdlyEh genes; the second critical residue (Glu-108) could also be identified, as well as the second conserved Asp residue (position 106) from the bacterial catalytic site (Fig. 1C and Supplementary Fig. S1C). In summary, all three residues critical for enzymatic activity of Entamoeba-type lysozymes are apparently conserved in these D. discoideum lysozymes. 3.1.4. Aly-type lysozymes The D. discoideum AX4 genome contains eight genes coding for Alytype lysozymes: alyA, alyB, alyC, alyD-1, alyD-2, alyL, alyTM1, and 5

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present in free-living amoebae. The D. discoideum lysozymes are even more numerous than previously anticipated, with 22 genes encoding four different types of lysozymes. In addition, we observed that many lysozyme genes are found in the genomes of other amoeba species. To the best of our current knowledge, no other organism is as wellequipped with lysozyme genes as D. discoideum. Further studies are needed for the vast majority of the various lysozyme proteins to identify their enzymatic activities (if any) and their biological function(s). D. discoideum still has much to teach us.

D. discoideum to grow on some Gram-negative bacteria (Nasser et al., 2013). In addition, it was observed that AlyA displayed antibacterial activity against some viable Gram-positive bacteria (Müller et al., 2005). No study has been conducted to date assessing the bacteriolytic activity of AlyL lysozyme. 4. Lysozyme genes in amoebal species Due to the presence of peptidoglycans in bacterial walls, lysozymes are commonly found in organisms that feed on bacteria, including protists and other heterotrophic organisms. To determine how common these enzymes are in amoebae, we searched for similar sequences of the four different classes of lysozymes in the available genomes of five different species of free-living amoeba: A. castellanii, A. subglobosum, E. histolytica, N. fowleri, and P. pallidum. The number of lysozyme genes ranged from 1 in N. fowleri to 12 in P. pallidum (Table 2 and Supplementary Table S2). Aly-type and Entamoeba-type lysozymes appear to be present among these amoeboid protozoa in Dictyostelid species only (Table 2 and Supplementary Table S2). None of these five genomes contain genes coding for V-type lysozymes, which strengthens the hypothesis that in D. discoideum this type of lysozyme originates from a horizontal gene transfer. Also, neither G-type lysozymes nor Itype lysozymes were identified in the amoebal species analyzed here. In addition, we searched for genes similar to those coding for all 22 D. discoideum lysozymes in the available genomes of two other species of the genus Dictyostelium, namely D. fasciculatum and D. purpureum (Table 2). The D. fasciculatum and D. purpureum genomes encode a total number of 16 and 17 lysozymes, respectively (Table 2). No V-type lysozymes were identified in the genome of these species.

Author contributions OL and PC conceived and designed the study. OL, WCL and TJ analyzed the data. OL, WCL, TJ, ML and PC wrote the manuscript. All authors read and contributed to the manuscript. Funding This research was supported by Swiss National Science Foundation [31003A- 172951 to P.C.]. Declaration of competing interest The authors declare no competing or financial interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dci.2020.103645.

5. Molecular evolution of amoeba lysozymes

References

Phylogenetic analyses of the D. discoideum lysozymes confirmed the expected distribution in different classes (Entamoeba-type, Aly-type, Ctype, and V-type) (Fig. 2). Aly-type lysozymes form a group with homologous gene products from other dictyostelid species. D. discoideum V-type lysozymes cluster together with typical phage-like lysozymes. Likewise, D. discoideum Entamoeba-type lysozymes are present in a clade with lysozymes detected in other amoebae such as E. histolytica and A. castellanii. D. discoideum C-type lysozymes cluster together in a group with high bootstrap support, but not in the same clade as other metazoan C-type lysozymes; this might indicate a high degree of divergence from a putative common ancestor. It is well-known that the sequence similarity between members from different lysozyme families is very low. It has been argued - although it remains to be empirically proven - that different lysozymes function at very different pH, and that the different isoelectric points needed could account for the high diversity in amino acid sequences (Callewaert and Michiels, 2010). The fact that D. discoideum C-type lysozymes do not cluster together with those from vertebrates might reflect such high divergence in primary structures. Despite having very divergent primary structures, all lysozymes share a surprisingly conserved secondary structure, with two domains (one mostly an α-helix, the other a β-sheet) separated by a binding cleft (Wohlkönig et al., 2010). Notably, the Aly-type lysozymes were identified only because the protein AlyA of Dictyostelium was purified from amoebic extracts and subsequently the primary structure was solved. Standard BLAST searches, based on sequence identity, did not allow the detection of similar proteins outside the amoeba group. Alignments that consider information about secondary and tertiary structures suggest that the lysozymes in each Dictyostelium family have the same overall structure, and identify putative active residues (Supplementary Fig. S1).

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6. Conclusion This study provides an updated review of the lysozyme arsenal 6

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