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PreprintCited by 2Invertebrate Immune Response MechanismsMarine Invertebrate Physiology and EcologyDevelopmental Biology and Gene Regulation

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An ancient lysozyme in placozoans participates in acidic extracellular digestion

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Berndt, Henry, Duarte, Igor, Repnik, Urska, Struwe, Michel, Abukhalaf, Mohammad, Scheidig, Axel, Tholey, Andreas, Gruber-Vodicka, Harald, Leippe, Matthias·2024·Communications Biology
DOI 10.1101/2024.10.06.616844PMC PMC12855814

Preprint — published as 10.1038/s42003-025-09409-6

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Abstract

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and produced in gland cells of the ventral epithelium. PLys contains a protective and non-conserved cysteine-rich domain N-terminal of the conserved GH23 lysozyme domain. A truncation of this N-terminal domain in the maturation process of PLys leads to a drastic increase in enzymatic activity at the cost of stability. As the lysozyme is most active under acidic conditions, we investigated the pH trajectories during extracellular digestion in situ . Using a pH-senstive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied a structure-based phylogenetics approach to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla, including Porifera, Cnidaria, Placozoa and chordates, acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

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Fig. 1 PLys is an active lysozyme with a truncated mature proteoform. A Lysoplate assay with 8 μg and 12 μg total protein of raw Trichoplax sp. H2 protein extract, 20 μg hen egg white lysozyme (HEWL), and lysis buffer. B Structure model of PLys as ribbon diagram. The backbone of the mature proteoform of PLys is colored in light blue, while the cysteine-rich N-terminal domain is colored in dark blue. Cysteine residues forming disulphide bonds are highlighted and annotated. An arrow marks S100, the apparent N-terminus of the physiologically mature proteoform. C Representative mass spectrum of the top-down proteomics experiment with the identified mature PLys proteoform with charge states z = 19 and z = 20.
Fig. 1 PLys is an active lysozyme with a truncated mature proteoform. A Lysoplate assay with 8 μg and 12 μg total protein of raw Trichoplax sp. H2 protein extract, 20 μg hen egg white lysozyme (HEWL), and lysis buffer. B Structure model of PLys as ribbon diagram. The backbone of the mature proteoform of PLys is colored in light blue, while the cysteine-rich N-terminal domain is colored in dark blue. Cysteine residues forming disulphide bonds are highlighted and annotated. An arrow marks S100, the apparent N-terminus of the physiologically mature proteoform. C Representative mass spectrum of the top-down proteomics experiment with the identified mature PLys proteoform with charge states z = 19 and z = 20.CC BY · PubMed Central
Fig. 2 PLys is abundant primarily in cells of the ventral epithelium and detectable in secreted mucus. A Cross-section of a resin-embedded Trichoplax sp. H2 individual, highlighting dorsal (d) and ventral (v) epithelia as well as mucus-secreting gland cells (asterisks). DAPI = blue, Wheat-germ agglutinin (WGA) = orange. B Dorsal to ventral view on a maximum intensity projection of a Z-stack of a Trichoplax. sp. H2 fixed on a coverslip with its traces of mucus. The sample was stained with anti-PLys (green) and WGA (orange). Co-labeling of mucus and PLys in traces of the animal is indicated with an arrowhead. Partial orthogonal projections (C: XZ, D: YZ) of a Z-stack of Trichoplax sp. H2. Both projections cover a depth of 5 μm. Dorsal (d) and ventral (v) epithelia are annotated. DNA was stained with DAPI (blue), mucus with WGA (orange), and PLys with anti-PLys (green). PLys signal in the ventral epithelium is highlighted with closed arrowheads, occasional labeling in the dorsal epithelium is highlighted with open arrowheads. All presented colors are false colors applied to the specific channels. Scale bars correspond to 20 μm (A, B), or 10 μm (C, D).
Fig. 2 PLys is abundant primarily in cells of the ventral epithelium and detectable in secreted mucus. A Cross-section of a resin-embedded Trichoplax sp. H2 individual, highlighting dorsal (d) and ventral (v) epithelia as well as mucus-secreting gland cells (asterisks). DAPI = blue, Wheat-germ agglutinin (WGA) = orange. B Dorsal to ventral view on a maximum intensity projection of a Z-stack of a Trichoplax. sp. H2 fixed on a coverslip with its traces of mucus. The sample was stained with anti-PLys (green) and WGA (orange). Co-labeling of mucus and PLys in traces of the animal is indicated with an arrowhead. Partial orthogonal projections (C: XZ, D: YZ) of a Z-stack of Trichoplax sp. H2. Both projections cover a depth of 5 μm. Dorsal (d) and ventral (v) epithelia are annotated. DNA was stained with DAPI (blue), mucus with WGA (orange), and PLys with anti-PLys (green). PLys signal in the ventral epithelium is highlighted with closed arrowheads, occasional labeling in the dorsal epithelium is highlighted with open arrowheads. All presented colors are false colors applied to the specific channels. Scale bars correspond to 20 μm (A, B), or 10 μm (C, D).CC BY · PubMed Central
Fig. 3 PLys is a potent lysozyme under acidic conditions. A SDS-PAGE gel of purified recombinant mPLys_M112 and pPLys. The molecular mass standard is indicated on the left and marked with the associated molecular masses in kDa. B Lysozyme assay of 32 pmol pPLys, 32 pmol mPLys_M112, and 32 pmol HEWL at pH 5.0. Reaction buffer consisted of 20 mM sodium acetate, pH 5.0 (I = 13 mM). Dependency of lysozyme activity of pPLys and mPLys_M112 on ionic strength (C) and pH (D) at room temperature. E Lysozyme activity of pPLys, mPLys_M112 and HEWL at different temperatures. All measurements were performed in triplicates. Bacterial lysis assay against B. subtilis (F) and E. coli D31 (G) using a membrane-impermeable dye (SYTOX Green). Fluorescence indicates lysis of cells. Sigmoidal functions were fitted to the dose response curves. mPLys_M112: open triangles, pPLys closed triangles, HEWL closed squares, Melittin closed circles, buffer control: crosses.
Fig. 3 PLys is a potent lysozyme under acidic conditions. A SDS-PAGE gel of purified recombinant mPLys_M112 and pPLys. The molecular mass standard is indicated on the left and marked with the associated molecular masses in kDa. B Lysozyme assay of 32 pmol pPLys, 32 pmol mPLys_M112, and 32 pmol HEWL at pH 5.0. Reaction buffer consisted of 20 mM sodium acetate, pH 5.0 (I = 13 mM). Dependency of lysozyme activity of pPLys and mPLys_M112 on ionic strength (C) and pH (D) at room temperature. E Lysozyme activity of pPLys, mPLys_M112 and HEWL at different temperatures. All measurements were performed in triplicates. Bacterial lysis assay against B. subtilis (F) and E. coli D31 (G) using a membrane-impermeable dye (SYTOX Green). Fluorescence indicates lysis of cells. Sigmoidal functions were fitted to the dose response curves. mPLys_M112: open triangles, pPLys closed triangles, HEWL closed squares, Melittin closed circles, buffer control: crosses.CC BY · PubMed Central
Fig. 4 Placozoa acidify their temporary feeding groove pulsatively. A Merged micrographs in brightfield and green-epifluorescence mode of Trichoplax sp. H2 feeding on zymosan particles coated with a pH-sensitive dye after 0 s and 15 s. Two feeding grooves are highlighted by dotted lines. The corresponding pH is presented next to the grooves. Scale bars: 50 μm. B Box plot of the minimum recorded pH (closed diamonds) of digestive events (n = 22). The open square shows the mean, the horizontal line represents the median.
Fig. 4 Placozoa acidify their temporary feeding groove pulsatively. A Merged micrographs in brightfield and green-epifluorescence mode of Trichoplax sp. H2 feeding on zymosan particles coated with a pH-sensitive dye after 0 s and 15 s. Two feeding grooves are highlighted by dotted lines. The corresponding pH is presented next to the grooves. Scale bars: 50 μm. B Box plot of the minimum recorded pH (closed diamonds) of digestive events (n = 22). The open square shows the mean, the horizontal line represents the median.CC BY · PubMed Central
Fig. 5 Metazoan g-type lysozymes cluster predominantly into a single clade and are present in six animal phyla. A Phylogenetic tree of 1428 glycoside hydrolase family 23 sequences from all domains of life and B expanded tree branch containing all metazoan sequences. Colors indicate host groups, orange dots show phylogenetic support (aLRT).
Fig. 5 Metazoan g-type lysozymes cluster predominantly into a single clade and are present in six animal phyla. A Phylogenetic tree of 1428 glycoside hydrolase family 23 sequences from all domains of life and B expanded tree branch containing all metazoan sequences. Colors indicate host groups, orange dots show phylogenetic support (aLRT).CC BY · PubMed Central
Fig. 6 G-type lysozymes are widely distributed over the animal tree of life and the only lysozyme of all early branching metazoans except ctenophores. Overview phylogram of metazoans according to Laumer et al.7 that shows the distribution of the most abundant lysozyme families (closed circles). The status of lysozyme abundance is based on Callewaert and Michiels2, Berndt et al.32 and this study. As only one GH22i lysozyme has been reported for poriferans33, the respective circle is dotted.
Fig. 6 G-type lysozymes are widely distributed over the animal tree of life and the only lysozyme of all early branching metazoans except ctenophores. Overview phylogram of metazoans according to Laumer et al.7 that shows the distribution of the most abundant lysozyme families (closed circles). The status of lysozyme abundance is based on Callewaert and Michiels2, Berndt et al.32 and this study. As only one GH22i lysozyme has been reported for poriferans33, the respective circle is dotted.CC BY · PubMed Central
Fig. 7 Structure-based phylogeny of GH families within the lysozyme superfamily. In A the whole tree is depicted, in B GH23 clades were collapsed and colored using the same color code as in (A). Two clades of myxobacterial lysozyme clades within the metazoan GH23 clade are labeled with “1” and “2”. Asterisks were added to collapsed clades to report minor occurrence of structures from different organismal groups in the respective attributed colors.
Fig. 7 Structure-based phylogeny of GH families within the lysozyme superfamily. In A the whole tree is depicted, in B GH23 clades were collapsed and colored using the same color code as in (A). Two clades of myxobacterial lysozyme clades within the metazoan GH23 clade are labeled with “1” and “2”. Asterisks were added to collapsed clades to report minor occurrence of structures from different organismal groups in the respective attributed colors.CC BY · PubMed Central

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Research works printed but not linked: 55

  1. 10.1073/pnas.1218525110 · DOI
  2. 10.1007/s12038-010-0015-5 · DOI
  3. 10.1007/s002390010136 · DOI
  4. Wohlkönig A , Huet J , Looze Y , Wintjens R. Structural relationships in the lysozyme superfamily: Significant evidence for glycoside hydrolase signature motifs. Uversky VN , editor. PLoS ONE. 2010 Nov 9;5(11):e15388. · DOI
  5. 10.1016/j.cell.2023.08.027 · DOI
  6. Laumer CE , Fernández R , Lemer S , Combosch D , Kocot KM , Riesgo A , et al. Revisiting metazoan phylogeny with genomic sampling of all phyla. Proc R Soc B Biol Sci. 2019 Jul 10;286(1906):20190831. · DOI · Paper page
  7. 10.1038/ncomms2424 · DOI
  8. DuBuc TQ , Ryan JF , Martindale MQ . “Dorsal–Ventral” genes are part of an ancient axial patterning system: Evidence from Trichoplax adhaerens (Placozoa). True J , editor. Mol Biol Evol. 2019 May 1;36(5):966–73. · DOI
  9. 10.1097/WNR.0000000000001436 · DOI
  10. 10.1038/s41564-019-0475-9 · DOI
  11. 10.1038/s41598-019-54037-w · DOI
  12. 10.1016/j.cub.2014.05.046 · DOI · Paper page
  13. 10.1038/s41598-021-02735-9 · DOI
  14. 15. Smith CL , Pivovarova N , Reese TS. Coordinated feeding behavior in Trichoplax, an animal without synapses. Steele RE , editor. PLOS ONE. 2015 Sep 2;10(9):e0136098. · DOI · Paper page
  15. Mayorova TD , Hammar K , Winters CA , Reese TS , Smith CL . The ventral epithelium of Trichoplax adhaerens deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides. Biol Open. 2019 Jan 1;bio.045674. · DOI
  16. 10.1186/s12864-018-5377-3 · DOI
  17. 10.1111/j.1742-4658.2008.06422.x · DOI
  18. 10.1110/ps.26801 · DOI
  19. 10.1186/s13100-019-0146-7 · DOI
  20. 10.1186/s12859-020-03599-y · DOI
  21. Schierwater B , Eitel M , Jakob W , Osigus HJ , Hadrys H , Dellaporta SL , et al. Concatenated Analysis Sheds Light on Early Metazoan Evolution and Fuels a Modern “Urmetazoon” Hypothesis. Penny D , editor. PLoS Biol. 2009 Jan 27;7(1):e1000020. · DOI · Paper page
  22. Mayorova TD , Koch TL , Kachar B , Jung JH , Reese TS , Smith CL. Placozoan secretory cell types implicated in feeding, innate immunity and regulation of behavior [Internet]. 2024 [cited 2025 Feb 1]. Available from: http://biorxiv.org/lookup/doi/10.1101/2024.09.18.613768 · DOI
  23. 10.1016/j.cub.2021.06.093 · DOI
  24. Weston A , Vladescu SC , Reddyhoff T , Griffiths A , Crouzier T , Fielden M , et al. The influence of ions on the lubricative abilities of mucin and the role of sialic acids. Colloids Surf B Biointerfaces. 2023 Jul;227:113327. · DOI
  25. 10.7554/eLife.36278 · DOI · Paper page
  26. Mukherjee K , Moroz LL . Evolution of g-type lysozymes in metazoa: insights into immunity and digestive adaptations. Front Cell Dev Biol. 2024 Nov 6;12:1487920. · DOI · Paper page
  27. 10.1186/s13568-022-01393-y · DOI
  28. Schultz DT , Haddock SHD , Bredeson JV , Green RE , Simakov O , Rokhsar DS . Ancient gene linkages support ctenophores as sister to other animals. Nature. 2023 Jun 1;618(7963):110– 7. · DOI · Paper page
  29. Berndt H , Fuchs S , Kraus-Stojanowic I , Pees B , Gelhaus C , Leippe M . Molecular and functional characterization of ILYS-5, a major invertebrate lysozyme of Caenorhabditis elegans . Dev Comp Immunol. 2024 Oct;159:105220. · DOI
  30. 10.1074/jbc.M504049200 · DOI
  31. 10.1002/prot.22458 · DOI
  32. 10.1016/j.str.2013.06.020 · DOI
  33. 10.1038/S41592-022-01488-1 · DOI · Paper page
  34. Moi D , Bernard C , Steinegger M , Nevers Y , Langleib M , Dessimoz C. Structural phylogenetics unravels the evolutionary diversification of communication systems in gram-positive bacteria and their viruses [Internet]. 2023 [cited 2024 May 31]. Available from: http://biorxiv.org/lookup/doi/10.1101/2023.09.19.558401 · DOI
  35. 10.1038/nsb0296-133 · DOI
  36. 10.1016/0003-2697(87)90587-2 · DOI
  37. 10.1084/jem.124.5.921 · DOI
  38. 10.7717/peerj.1860 · DOI
  39. 10.1038/s41587-019-0036-z · DOI
  40. 10.1016/0006-3002(52)90045-0 · DOI
  41. Ellis DA . A new universal buffer system. Nature. 1961 Sep;191(4793):1099–100. · DOI
  42. 10.1042/bj20030250 · DOI
  43. 10.1128/jb.121.2.455-464.1975 · DOI
  44. Huynh K , Partch CL . Analysis of protein stability and ligand interactions by thermal shift assay. Curr Protoc Protein Sci [Internet]. 2015 Feb [cited 2024 May 31];79(1). Available from: https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/0471140864.ps2809s79 · DOI
  45. 10.1093/nar/gkab1045 · DOI · Paper page
  46. 10.1093/bioinformatics/bts565 · DOI · Paper page
  47. 10.1038/s41467-022-34630-w · DOI
  48. 10.1093/bioinformatics/btu031 · DOI
  49. 10.1093/molbev/msu300 · DOI · Paper page
  50. 10.1038/nmeth.4285 · DOI · Paper page
  51. 10.1093/NAR/GKAB301 · DOI
  52. 10.1093/bioinformatics/bts578 · DOI
  53. 10.1093/sysbio/syy054 · DOI
  54. 10.1002/jcc.20084 · DOI
  55. 10.1038/nbt.2839 · DOI

Metadata

Authors
Berndt, H., Duarte, I., Repnik, U., Struwe, M., Abukhalaf, M., Scheidig, A., Tholey, A., Gruber-Vodicka, H., & Leippe, M.
Affiliations
Christian-Albrechts-Universität zu Kiel (DE)
Corresponding author
Leippe, Matthias
ORCID
Venue
Communications Biology
Publisher
openRxiv
Published
2024-10-06
Volume
9
Issue
1
Pages
130-130
ISSN
2399-3642 (ISSN-L)
DOI
10.1101/2024.10.06.616844
PMC
PMC12855814
OpenAlex
W7119052418
Cited by
2
References
1
Citations by year
2025: 2
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Deutsche Forschungsgemeinschaft (777‐1 FUGG, 152/777-1, 5028/1-1, INST 152/772-1, INST 257/650-1 FUGG, INST 152/772-1 | 152/774-1 | 152/775-1 | 152/776-1 | 152/777-1 FUGG, 152/777 -1 FUGG, 152/775-1, 152/772-1, INST 152/774-1 FUGG, INST 152/772-1|152/774-1|152/775-1|152/776-1, 774-1, 261376515, 152/774-1, INST 152/776-1 FUGG, 776-1, 775-1, 152/776-1, INST 152/772-1 FUGG)
  • Universität Hamburg (INST 152/772-1, 774-1)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Invertebrate Immune Response Mechanisms
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Marine Invertebrate Physiology and Ecology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
Keywords
placozoans, Trichoplax, extracellular digestion, pH-dependent fluorescence, horizontal gene transfer, antibacterial defense, peptidoglycan hydrolysis, cysteine-rich domain
MeSH terms
Amino Acid Sequence, Animals, Hydrogen-Ion Concentration, Muramidase, Phylogeny, Evolution, Molecular, Placozoa

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Berndt, H., Duarte, I., Repnik, U., Struwe, M., Abukhalaf, M., Scheidig, A., Tholey, A., Gruber-Vodicka, H., & Leippe, M. (2024). An ancient lysozyme in placozoans participates in acidic extracellular digestion. Communications Biology. https://doi.org/10.1101/2024.10.06.616844