Botryllin, a Novel Antimicrobial Peptide from the Colonial Ascidian Botryllus schlosseri.

By mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, which we named botryllin. The gene is constitutively transcribed by circulating cytotoxic morula cells (MCs) as a pre-propeptide that is then cleaved to mature peptide. The synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity of bacterial and unicellular yeast cells, causing breakages of both the plasma membrane and the cell wall. Specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence of the propeptide and the mature peptide; in both cases, they label the MC granular content. Upon MC degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby MC remains. The obtained results suggest that the botryllin gene carries the information for the synthesis of an AMP involved in the protection of B. schlosseri from invading foreign cells.

[1]  A. Genevière,et al.  Biomaterials and Bioactive Natural Products from Marine Invertebrates: From Basic Research to Innovative Applications , 2022, Marine drugs.

[2]  L. Dufossé,et al.  Marine Natural Products from Tunicates and Their Associated Microbes , 2021, Marine drugs.

[3]  Q. Kong,et al.  Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields , 2020, Frontiers in Microbiology.

[4]  N. Franchi,et al.  Insights into the Complement System of Tunicates: C3a/C5aR of the Colonial Ascidian Botryllus schlosseri , 2020, Biology.

[5]  Marcello Casertano,et al.  The Ascidian-Derived Metabolites with Antimicrobial Properties , 2020, Antibiotics.

[6]  B. Lazzaro,et al.  Antimicrobial peptides: Application informed by evolution , 2020, Science.

[7]  N. Franchi,et al.  Complement system and phagocytosis in a colonial protochordate. , 2020, Developmental and comparative immunology.

[8]  A. Grimaldi,et al.  Functional amyloidogenesis in immunocytes from the colonial ascidian Botryllus schlosseri: Evolutionary perspective , 2019, Developmental and comparative immunology.

[9]  N. Franchi,et al.  Immunity in Protochordates: The Tunicate Perspective , 2017, Front. Immunol..

[10]  Franchi Nicola,et al.  Morula cells as key hemocytes of the lectin pathway of complement activation in the colonial tunicate Botryllus schlosseri , 2017, Fish & shellfish immunology.

[11]  B. Deslouches,et al.  Antimicrobial peptides with selective antitumor mechanisms: prospect for anticancer applications , 2017, Oncotarget.

[12]  S. Swarnakar,et al.  Bioactive Compounds from Marine Invertebrates for Potential Medicines - An Overview , 2015, International Letters of Natural Sciences.

[13]  T. Cullen,et al.  Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation , 2015, Science.

[14]  N. Franchi,et al.  Preliminary characterization of complement in a colonial tunicate: C3, Bf and inhibition of C3 opsonic activity by compstatin. , 2014, Developmental and comparative immunology.

[15]  A. Bahar,et al.  Antimicrobial Peptides , 2013, Pharmaceuticals.

[16]  Jun Wang,et al.  Distinct antimicrobial peptide expression determines host species-specific bacterial associations , 2013, Proceedings of the National Academy of Sciences.

[17]  Andrew W. Han,et al.  Genome streamlining and chemical defense in a coral reef symbiosis , 2012, Proceedings of the National Academy of Sciences.

[18]  Huajun Zheng,et al.  Bacterial biosynthesis and maturation of the didemnin anti-cancer agents. , 2012, Journal of the American Chemical Society.

[19]  J. Ravel,et al.  Origin and variation of tunicate secondary metabolites. , 2012, Journal of natural products.

[20]  J. Serôdio,et al.  Trends in the Discovery of New Marine Natural Products from Invertebrates over the Last Two Decades – Where and What Are We Bioprospecting? , 2012, PloS one.

[21]  L. Ballarin Ascidian cytotoxic cells: state of the art and research perspectives , 2012 .

[22]  G. Schneider,et al.  Designing antimicrobial peptides: form follows function , 2011, Nature Reviews Drug Discovery.

[23]  J. Fujimoto,et al.  Bacterial production of the tunicate-derived antitumor cyclic depsipeptide didemnin B. , 2011, Journal of natural products.

[24]  A. Heddi,et al.  Antimicrobial Peptides Keep Insect Endosymbionts Under Control , 2011, Science.

[25]  S. Tosatto,et al.  Immune roles of a rhamnose-binding lectin in the colonial ascidian Botryllus schlosseri. , 2011, Immunobiology.

[26]  N. Salzman,et al.  Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis , 2011, Nature Reviews Microbiology.

[27]  M. Leippe,et al.  The antimicrobial peptide Ci-MAM-A24 is highly active against multidrug-resistant and anaerobic bacteria pathogenic for humans. , 2010, International journal of antimicrobial agents.

[28]  A. Brandelli,et al.  Kinetics and thermodynamics of thermal inactivation of the antimicrobial peptide cerein 8A , 2009 .

[29]  M. Leippe,et al.  An exceptional salt-tolerant antimicrobial peptide derived from a novel gene family of haemocytes of the marine invertebrate Ciona intestinalis. , 2008, The Biochemical journal.

[30]  L. Ballarin,et al.  Immunomodulatory molecules in the compound ascidian Botryllus schlosseri: evidence from conditioned media. , 2008, Journal of invertebrate pathology.

[31]  M. Leippe,et al.  A reverse search for antimicrobial peptides in Ciona intestinalis: identification of a gene family expressed in hemocytes and evaluation of activity. , 2008, Developmental and comparative immunology.

[32]  B. Spolaore,et al.  Novel rhamnose-binding lectins from the colonial ascidian Botryllus schlosseri. , 2008, Developmental and comparative immunology.

[33]  H. S. Ramaswamy,et al.  Thermal processing and quality: Principles and overview , 2007 .

[34]  G. Pirri,et al.  Antimicrobial peptides: an overview of a promising class of therapeutics , 2007, Central European Journal of Biology.

[35]  R. Hancock,et al.  Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies , 2006, Nature Biotechnology.

[36]  R. Hancock,et al.  Peptide Antimicrobial Agents , 2006, Clinical Microbiology Reviews.

[37]  F. Delsuc,et al.  Tunicates and not cephalochordates are the closest living relatives of vertebrates , 2006, Nature.

[38]  L. Ballarin,et al.  Release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian , 2005 .

[39]  L. Ballarin,et al.  Cytochemical properties of Botryllus schlosseri haemocytes: indications for morpho-functional characterisation. , 2005, European journal of histochemistry : EJH.

[40]  Steven W. Taylor,et al.  Antimicrobial Peptides from Marine Invertebrates , 2004, Antimicrobial Agents and Chemotherapy.

[41]  L. Ballarin,et al.  Cellular aspects of allorecognition in the compound ascidian Botryllus schlosseri. , 2004, Developmental and comparative immunology.

[42]  L. Otvos,et al.  Primary Structure and in Vitro Antibacterial Properties of the Drosophila melanogaster Attacin C Pro-domain* , 2004, Journal of Biological Chemistry.

[43]  H. Sahl,et al.  Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes , 1985, Archives of Microbiology.

[44]  Steven W. Taylor,et al.  Plicatamide, an Antimicrobial Octapeptide from Styela plicata Hemocytes* , 2003, The Journal of Biological Chemistry.

[45]  Steven W. Taylor,et al.  Natural Peptide Antibiotics from Tunicates: Structures, Functions and Potential Uses1 , 2003, Integrative and comparative biology.

[46]  M. Floreani,et al.  Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri. , 2002, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[47]  Michel Salzet,et al.  Antimicrobial peptides from animals: focus on invertebrates. , 2002, Trends in pharmacological sciences.

[48]  R. Lehrer,et al.  Immunolocalization of clavanins in Styela clava hemocytes. , 2002, Developmental and comparative immunology.

[49]  M. Zasloff Antimicrobial peptides of multicellular organisms , 2002, Nature.

[50]  A. Waring,et al.  Clavaspirin, an antibacterial and haemolytic peptide from Styela clava. , 2001, The journal of peptide research : official journal of the American Peptide Society.

[51]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[52]  E. Ottaviani,et al.  Morula Cells as the Major Immunomodulatory Hemocytes in Ascidians: Evidences From the Colonial Species Botryllus schlosseri , 2001, The Biological Bulletin.

[53]  S. Lovas,et al.  The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. , 2001, Biochemistry.

[54]  Steven W. Taylor,et al.  Styelin D, an Extensively Modified Antimicrobial Peptide from Ascidian Hemocytes* , 2000, The Journal of Biological Chemistry.

[55]  R I Lehrer,et al.  Crystallization of antimicrobial pores in membranes: magainin and protegrin. , 2000, Biophysical journal.

[56]  K. Matsuzaki Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. , 1999, Biochimica et biophysica acta.

[57]  Y. Shai,et al.  Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. , 1999, Biochimica et biophysica acta.

[58]  L. Ballarin,et al.  Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri , 1999 .

[59]  I. Gould Stewardship of antibiotic use and resistance surveillance: the international scene. , 1999, The Journal of hospital infection.

[60]  L. Ballarin,et al.  Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. , 1998, Developmental and comparative immunology.

[61]  R I Lehrer,et al.  Styelins, broad-spectrum antimicrobial peptides from the solitary tunicate, Styela clava. , 1997, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[62]  R. Lehrer,et al.  cDNA cloning of three cecropin‐like antimicrobial peptides (Styelins) from the tunicate, Styela clava , 1997, FEBS letters.

[63]  James M. Wilson,et al.  Human β-Defensin-1 Is a Salt-Sensitive Antibiotic in Lung That Is Inactivated in Cystic Fibrosis , 1997, Cell.

[64]  L. Ballarin,et al.  Morula Cells and Histocompatibility in the Colonial Ascidian Botryllus schlosseri , 1995 .

[65]  L. Ballarin,et al.  Phenoloxidase in the colonial ascidian Botryllus schlosseri (Urochordata: Ascidiacea) , 1994 .

[66]  L. Ballarin,et al.  Histoenzymatic staining and characterization of the colonial ascidian Botryllus schlösseri hemocytes , 1993 .

[67]  G. Zaniolo,et al.  Genetic and cytological aspects of histocompatibility in ascidians , 1992 .

[68]  H. Yokosawa,et al.  Halocyamines: novel antimicrobial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian Halocynthia roretzi. , 1990, Biochemistry.

[69]  H V Westerhoff,et al.  Magainins and the disruption of membrane-linked free-energy transduction. , 1989, Proceedings of the National Academy of Sciences of the United States of America.