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Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

A NEW MECHANISM FOR SENSING PATHOGENS*
  • Bernd Bufe
    Correspondence
    To whom correspondence should be addressed: Dept. of Physiology, University of Saarland School of Medicine, Kirrbergerstr. Bldg. 58, 66424 Homburg, Germany. Tel.: 6841-16-26047; Fax: 6841-16-26655;
    Footnotes
    Affiliations
    From the Departments of Physiology, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Timo Schumann
    Footnotes
    Affiliations
    From the Departments of Physiology, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Reinhard Kappl
    Affiliations
    Departments of Biophysics, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Ivan Bogeski
    Affiliations
    Departments of Biophysics, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Carsten Kummerow
    Affiliations
    Departments of Biophysics, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Marta Podgórska
    Affiliations
    Departments of Virology, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Sigrun Smola
    Affiliations
    Departments of Virology, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Markus Hoth
    Affiliations
    Departments of Biophysics, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Frank Zufall
    Affiliations
    From the Departments of Physiology, University of Saarland School of Medicine, 66421 Homburg, Germany
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  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grants INST 256/271–1 FUGG (to F. Z. and B. B), Sonderforschungsbereich 894/A1 (to M. H.) and A17 (to F. Z.), Sonderforschungsbereich 1027/A2 and C4 (to M. H. and I. B.), and Schwerpunktprogramm 1392 (to F. Z.). This work was also supported by the University of Saarland through HOMFORexcellent Grants 2011 (to B. B.) and 2013 (to I. B.) and a graduate scholarship (GradUS) (to T. S.).
    This article contains supplemental Table S1 and Video 1.
    1 Both authors contributed equally to this work.
Open AccessPublished:January 20, 2015DOI:https://doi.org/10.1074/jbc.M114.626747
      Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

      Introduction

      The initial sensing of infection depends on innate pattern recognition receptors (PRRs)
      The abbreviations used are: PRR
      pattern recognition receptor
      FPR
      formyl peptide receptor
      GPCR
      G-protein-coupled receptor
      f-MLF
      N-formylmethionine-leucine-phenylalanine
      PAMP
      pathogen-associated molecular pattern
      ROS
      reactive oxygen species
      ND
      NADH-reductase subunit
      RANTES
      regulated on activation, normal T-cell expressed and secreted
      μPAR
      urokinase receptor
      HBSS
      Hanks' buffered saline solution
      HEK
      human embryonic kidney
      CMH
      1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine
      MMP
      matrix metallopeptidase.
      that recognize evolutionarily conserved structures of microorganisms known as pathogen-associated molecular patterns (PAMPs). Toll-like receptors represent a prime example of such PRRs (
      • Kawai T.
      • Akira S.
      The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.
      ,
      • Takeuchi O.
      • Akira S.
      Pattern recognition receptors and inflammation.
      ). Formyl peptide receptors (FPRs) belong to a class of G-protein-coupled receptors (GPCRs) involved in host defense against pathogens in the innate immune system (
      • Migeotte I.
      • Communi D.
      • Parmentier M.
      Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses.
      ,
      • Liu M.
      • Chen K.
      • Yoshimura T.
      • Liu Y.
      • Gong W.
      • Wang A.
      • Gao J.L.
      • Murphy P.M.
      • Wang J.M.
      Formylpeptide receptors are critical for rapid neutrophil mobilization in host defense against Listeria monocytogenes.
      • Ye R.D.
      • Boulay F.
      • Wang J.M.
      • Dahlgren C.
      • Gerard C.
      • Parmentier M.
      • Serhan C.N.
      • Murphy P.M.
      International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.
      ). FPR function is best known in phagocytic leukocytes (e.g. neutrophils and monocytes) that, in response to microbial chemoattractants, migrate and accumulate at sites of infection, where they release reactive oxygen species (ROS) and other factors to combat invading microorganisms (
      • Schiffmann E.
      • Corcoran B.A.
      • Wahl S.M.
      N-Formylmethionyl peptides as chemoattractants for leucocytes.
      ,
      • Fu H.
      • Karlsson J.
      • Bylund J.
      • Movitz C.
      • Karlsson A.
      • Dahlgren C.
      Ligand recognition and activation of formyl peptide receptors in neutrophils.
      • Le Y.
      • Gong W.
      • Tiffany H.L.
      • Tumanov A.
      • Nedospasov S.
      • Shen W.
      • Dunlop N.M.
      • Gao J.L.
      • Murphy P.M.
      • Oppenheim J.J.
      • Wang J.M.
      Amyloid b42 activates a G-protein-coupled chemoattractant receptor, FPR-like-1.
      ). Activation of FPRs triggers classical GPCR signaling cascades involving G-protein-dependent phospholipase C stimulation, leading to intracellular Ca2+ mobilization (
      • Rabiet M.J.
      • Huet E.
      • Boulay F.
      Human mitochondria-derived N-formylated peptides are novel agonists equally active on FPR and FPRL1, while Listeria monocytogenes-derived peptides preferentially activate FPR.
      ,
      • Selvatici R.
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      Signal transduction pathways triggered by selective formylpeptide analogues in human neutrophils.
      • Panaro M.A.
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      • Sisto M.
      • Lisi S.
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      • Mitolo V.
      Biological role of the N-formyl peptide receptors.
      ). Humans are known to express three FPR genes, FPR1, FPR2, and FPR3, but this number varies across mammalian species (
      • Gao J.L.
      • Lee E.J.
      • Murphy P.M.
      Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor.
      ,
      • Liberles S.D.
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      • Kuang D.
      • Contos J.J.
      • Wilson K.L.
      • Siltberg-Liberles J.
      • Liberles D.A.
      • Buck L.B.
      Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ.
      ).
      FPRs have been proposed to function as PRRs (
      • Ye R.D.
      • Boulay F.
      • Wang J.M.
      • Dahlgren C.
      • Gerard C.
      • Parmentier M.
      • Serhan C.N.
      • Murphy P.M.
      International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.
      ,
      • Fu H.
      • Karlsson J.
      • Bylund J.
      • Movitz C.
      • Karlsson A.
      • Dahlgren C.
      Ligand recognition and activation of formyl peptide receptors in neutrophils.
      ,
      • Kretschmer D.
      • Nikola N.
      • Dürr M.
      • Otto M.
      • Peschel A.
      The virulence regulator Agr controls the staphylococcal capacity to activate human neutrophils via the formyl peptide receptor 2.
      ,
      • Kretschmer D.
      • Gleske A.K.
      • Rautenberg M.
      • Wang R.
      • Köberle M.
      • Bohn E.
      • Schöneberg T.
      • Rabiet M.J.
      • Boulay F.
      • Klebanoff S.J.
      • van Kessel K.A.
      • van Strijp J.A.
      • Otto M.
      • Peschel A.
      Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus.
      ), but their pathogen-associated molecular pattern remains unclear. In fact, one of the most puzzling features of the FPR family is its unusually high degree of molecular promiscuity (
      • Migeotte I.
      • Communi D.
      • Parmentier M.
      Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses.
      ,
      • Fu H.
      • Karlsson J.
      • Bylund J.
      • Movitz C.
      • Karlsson A.
      • Dahlgren C.
      Ligand recognition and activation of formyl peptide receptors in neutrophils.
      ,
      • Le Y.
      • Murphy P.M.
      • Wang J.M.
      Formyl-peptide receptors revisited.
      ). Although FPRs have been named according to their capability to detect formylated peptides (
      • Ye R.D.
      • Boulay F.
      • Wang J.M.
      • Dahlgren C.
      • Gerard C.
      • Parmentier M.
      • Serhan C.N.
      • Murphy P.M.
      International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.
      ,
      • Boulay F.
      • Tardif M.
      • Brouchon L.
      • Vignais P.
      Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNA.
      ), these receptors can recognize structurally diverse agonists with no obvious common pattern in amino acid sequence or natural origin (
      • Migeotte I.
      • Communi D.
      • Parmentier M.
      Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses.
      ,
      • Le Y.
      • Murphy P.M.
      • Wang J.M.
      Formyl-peptide receptors revisited.
      ). Such ligands include N-formylated, C-amidated, and unmodified peptides from bacterial and viral pathogens as well as host-endogenous mitochondrial peptides and several non-peptide agonists, such as resolvin D1 and lipoxin A4 (
      • Migeotte I.
      • Communi D.
      • Parmentier M.
      Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses.
      ,
      • Ye R.D.
      • Boulay F.
      • Wang J.M.
      • Dahlgren C.
      • Gerard C.
      • Parmentier M.
      • Serhan C.N.
      • Murphy P.M.
      International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.
      ,
      • Bäck M.
      • Powell W.S.
      • Dahlén S.E.
      • Drazen J.M.
      • Evans J.F.
      • Serhan C.N.
      • Shimizu T.
      • Yokomizo T.
      • Rovati G.E.
      Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.
      ). FPRs detect a wide range of structurally diverse pro- and anti-inflammatory ligands associated with important human diseases, such as amyloidosis, Alzheimer disease, HIV, and inflammatory pain (
      • Li Y.
      • Ye D.
      Molecular biology for formyl peptide receptors in human diseases.
      ,
      • Mollica A.
      • Stefanucci A.
      • Costante R.
      • Pinnen F.
      Role of formyl peptide receptors (FPR) in abnormal inflammation responses involved in neurodegenerative diseases.
      ).
      Although FPR expression has been initially described in immune cells, it is becoming increasingly clear that FPRs are also expressed in other cell types and tissues, from the nervous system (
      • Liberles S.D.
      • Horowitz L.F.
      • Kuang D.
      • Contos J.J.
      • Wilson K.L.
      • Siltberg-Liberles J.
      • Liberles D.A.
      • Buck L.B.
      Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ.
      ,
      • Lacy M.
      • Jones J.
      • Whittemore S.R.
      • Haviland D.L.
      • Wetsel R.A.
      • Barnum S.R.
      Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia.
      ,
      • Chiu I.M.
      • Heesters B.A.
      • Ghasemlou N.
      • Von Hehn C.A.
      • Zhao F.
      • Tran J.
      • Wainger B.
      • Strominger A.
      • Muralidharan S.
      • Horswill A.R.
      • Bubeck Wardenburg J.
      • Hwang S.W.
      • Carroll M.C.
      • Woolf C.J.
      Bacteria activate sensory neurons that modulate pain and inflammation.
      • Rivière S.
      • Challet L.
      • Fluegge D.
      • Spehr M.
      • Rodriguez I.
      Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.
      ) to internal organs, including lung and gut (
      • Shao G.
      • Julian M.W.
      • Bao S.
      • McCullers M.K.
      • Lai J.P.
      • Knoell D.L.
      • Crouser E.D.
      Formyl peptide receptor ligands promote wound closure in lung epithelial cells.
      ,
      • Wentworth C.C.
      • Alam A.
      • Jones R.M.
      • Nusrat A.
      • Neish A.S.
      Enteric commensal bacteria induce extracellular signal-regulated kinase pathway signaling via formyl peptide receptor-dependent redox modulation of dual specific phosphatase 3.
      ), suggesting that FPRs could be generally involved in the sensing and management of the microbiome of the body. An important development has been the finding that a set of FPR-like proteins represents a distinct receptor family in chemosensory neurons of the mouse vomeronasal organ (
      • Liberles S.D.
      • Horowitz L.F.
      • Kuang D.
      • Contos J.J.
      • Wilson K.L.
      • Siltberg-Liberles J.
      • Liberles D.A.
      • Buck L.B.
      Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ.
      ,
      • Rivière S.
      • Challet L.
      • Fluegge D.
      • Spehr M.
      • Rodriguez I.
      Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.
      ) and that some of these neurons recognize formylated peptides (
      • Rivière S.
      • Challet L.
      • Fluegge D.
      • Spehr M.
      • Rodriguez I.
      Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.
      ,
      • Chamero P.
      • Katsoulidou V.
      • Hendrix P.
      • Bufe B.
      • Roberts R.
      • Matsunami H.
      • Abramowitz J.
      • Birnbaumer L.
      • Zufall F.
      • Leinders-Zufall T.
      G protein Gαo is essential for vomeronasal function and aggressive behavior in mice.
      ), suggesting an evolutionary link between recognition mechanisms in immune cells and subsets of sensory neurons of the vomeronasal organ (
      • Chamero P.
      • Leinders-Zufall T.
      • Zufall F.
      From genes to social communication: molecular sensing by the vomeronasal organ.
      ,
      • Leinders-Zufall T.
      • Ishii T.
      • Mombaerts P.
      • Zufall F.
      • Boehm T.
      Structural requirements for the activation of vomeronasal sensory neurons by MHC peptides.
      • Leinders-Zufall T.
      • Ishii T.
      • Chamero P.
      • Hendrix P.
      • Oboti L.
      • Schmid A.
      • Kircher S.
      • Pyrski M.
      • Akiyoshi S.
      • Khan M.
      • Vaes E.
      • Zufall F.
      • Mombaerts P.
      A family of nonclassical class I MHC genes contributes to ultrasensitive chemodetection by mouse vomeronasal sensory neurons.
      ).
      To gain new insight into the function and recognition capabilities of FPRs and to better understand the molecular promiscuity of these receptors, we investigated conserved features in the structure of disparate peptide agonists required for activation of human and mouse FPRs. Through this approach, we generated critical knowledge leading to the discovery of an unsuspected, extremely large family of natural FPR agonists with common structural properties: bacterial signal peptides and their short breakdown products. Signal peptides are N-terminal protein signatures that are required for directing the transfer of bacterial proteins through the plasma membrane, during which they are cleaved off to give rise to the native form of membrane-associated or secreted proteins (
      • Dalbey R.E.
      • Wang P.
      • van Dijl J.M.
      Membrane proteases in the bacterial protein secretion and quality control pathway.
      ,
      • Hegde R.S.
      • Bernstein H.D.
      The surprising complexity of signal sequences.
      ). We observed a remarkably high degree of sensitivity, selectivity, and functional conservation of signal peptide recognition across species and receptor subtypes for FPR1 and FPR2, strongly arguing for an important role of this novel agonist family during evolution of mammalian FPR function. Dynamic measurements in human and mouse innate immune cells demonstrate that all tested signal peptides are recognized by these cells and trigger classical innate immune responses, such as intracellular Ca2+ mobilization, generation of reactive oxygen species, release of metallopeptidase, and chemotactic cell migration. These observations argue that mammalian FPRs may have evolved originally as germ line-encoded pattern recognition receptors that recognize structurally conserved export motifs of bacterial signal sequences as their cognate, pathogen-associated molecular pattern.

      DISCUSSION

      FPRs are a unique family of GPCRs with an exceptionally broad and promiscuous agonist spectrum showing no obvious common pattern in primary structure or natural origin (
      • Migeotte I.
      • Communi D.
      • Parmentier M.
      Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses.
      ,
      • Fu H.
      • Karlsson J.
      • Bylund J.
      • Movitz C.
      • Karlsson A.
      • Dahlgren C.
      Ligand recognition and activation of formyl peptide receptors in neutrophils.
      ,
      • Le Y.
      • Gong W.
      • Tiffany H.L.
      • Tumanov A.
      • Nedospasov S.
      • Shen W.
      • Dunlop N.M.
      • Gao J.L.
      • Murphy P.M.
      • Oppenheim J.J.
      • Wang J.M.
      Amyloid b42 activates a G-protein-coupled chemoattractant receptor, FPR-like-1.
      ). Because of this molecular promiscuity, there is little understanding of what the biological targets of these receptors are and why FPRs detect a range of structurally dissimilar molecules. Here, we report the ability of FPRs to recognize bacterial signal peptides, a vast family of novel natural FPR agonists that differ extensively in their primary structure but exhibit a well conserved secondary structure. Detailed structural variation analysis of peptide agonists defined the key features that enable both promiscuity and high specificity of FPR molecular detection. Finally, we argue that these results identify a common structural basis or molecular signature for FPR activation that is used to detect natural pathogens. These results provide new insight into the function of these receptors and their molecular promiscuity and make specific inferences about their biological roles and evolution.
      Our results indicate that bacterial signal peptides provide an exceptionally large pool of sequence-divergent FPR agonists that all contain a conserved secondary structure. The f-MLF motif alone exists at the N terminus of several hundred signal peptides, whereas the core agonist motif of all 21 signal peptides used in this study occurs at the N terminus of a total of 4,293 peptides (Table 3). Moreover, our results suggest that FPR1 and FPR2 together may recognize far more than 100,000 distinct signal peptides. In fact, there is currently no reason for ruling out that FPR1 and FPR2 could detect the entire set of bacterial signal peptides. As such, bacterial signal peptides represent the largest family of natural FPR agonists and one of the largest families of GPCR ligands known to date. They are also one of the most complex classes of natural activators of the innate immune system discovered thus far.
      Not only do our findings demonstrate that bacterial signal peptides are recognized by human and mouse FPR1 and FPR2 in vitro; they also provide extensive evidence that this recognition process occurs in primary leukocytes of the human and mouse innate immune systems. These cells are equipped with native FPRs, and we provide clear evidence that bacterial signal peptides are detected in a sensitive manner by monocytes and granulocytes and that they trigger a range of classical innate immune defense mechanisms, including chemotactic migration, ROS production, and matrix metalloproteinase release. Pharmacologic and genetic manipulation indicates that this recognition process is indeed FPR-dependent.
      Our experiments establish that FPR1 and FPR2 function as broad signal peptide receptors. A goal of future experiments will be to determine whether this finding applies to other FPR subtypes as well. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 seem to be much more narrowly tuned in the detection of signal peptides. Compared with FPR1 and FPR2, these receptors reacted to a relatively small subset of signal peptides and required higher concentrations. Possibly, hFPR3 and mFpr-rs1 focus on the detection of a specific set of bacterial pathogens, whereas FPR1 and FPR2 function as more general sensors of bacteria.
      FPRs were originally discovered as receptors for formylated peptides, such as f-MLF, present in the supernatant of bacterial cultures (
      • Schiffmann E.
      • Corcoran B.A.
      • Wahl S.M.
      N-Formylmethionyl peptides as chemoattractants for leucocytes.
      ,
      • Marasco W.A.
      • Phan S.H.
      • Krutzsch H.
      • Showell H.J.
      • Feltner D.E.
      • Nairn R.
      • Becker E.L.
      • Ward P.A.
      Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.
      ). However, the precise source and release pathway of such peptides has never been identified (
      • Ye R.D.
      • Boulay F.
      • Wang J.M.
      • Dahlgren C.
      • Gerard C.
      • Parmentier M.
      • Serhan C.N.
      • Murphy P.M.
      International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.
      ). Our finding that the sequences of these ligands are contained within bacterial signal peptides and that these signal peptides and their N-terminal breakdown products function as potent activators of FPRs identifies a likely natural source for these molecules. Signal peptides are cleaved off the maturing protein by extracellular proteases during membrane targeting and subsequently digested into short fragments (
      • Dalbey R.E.
      • Wang P.
      • van Dijl J.M.
      Membrane proteases in the bacterial protein secretion and quality control pathway.
      ). Although the fate of bacterial signal peptides after their cleavage is currently not well understood (
      • Dalbey R.E.
      • Wang P.
      • van Dijl J.M.
      Membrane proteases in the bacterial protein secretion and quality control pathway.
      ), recent mass spectrometry studies of bacterial secretomes found complete signal peptides as well as N-terminal fragments in the extracellular medium of bacteria cultures, demonstrating that these molecules indeed can be secreted by bacteria (
      • de Souza G.A.
      • Leversen N.A.
      • Målen H.
      • Wiker H.G.
      Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway.
      ,
      • Ravipaty S.
      • Reilly J.P.
      Comprehensive characterization of methicillin-resistant Staphylococcus aureus subsp. aureus COL secretome by two-dimensional liquid chromatography and mass spectrometry.
      • Schwartz K.
      • Sekedat M.D.
      • Syed A.K.
      • O'Hara B.
      • Payne D.E.
      • Lamb A.
      • Boles B.R.
      The AgrD N-terminal leader peptide of Staphylococcus aureus has cytolytic and amyloidogenic properties.
      ). One possibility of how this happens is through lysis, either through autolysis during bacterial growth (
      • Yarmolinsky M.B.
      Programmed cell death in bacterial populations.
      ) or through immune cell-mediated lysis during an infection (
      • Martner A.
      • Dahlgren C.
      • Paton J.C.
      • Wold A.E.
      Pneumolysin released during Streptococcus pneumoniae autolysis is a potent activator of intracellular oxygen radical production in neutrophils.
      ). Both mechanisms are well established and, therefore, represent likely pathways by which signal peptides can be made available for the recognition by FPRs. Determination of whether other more specific release mechanisms exist for bacterial signal peptides remains a goal of future research. Compared with eukaryotes, bacteria employ highly complex secretion mechanisms, and their secretory pathways are less well examined (
      • Pugsley A.P.
      The complete general secretory pathway in Gram-negative bacteria.
      ,
      • Tseng T.T.
      • Tyler B.M.
      • Setubal J.C.
      Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology.
      ).
      The ability of FPRs to recognize thousands of peptides with distinct amino acid sequences yet maintaining selectivity requires a compromise between broad specificity and high affinity. Our results suggest a remarkable solution to this problem. We provide evidence for an FPR detection mechanism that seems to focus on the recognition of a conserved three-dimensional motif rather than the linear peptide sequences. Several critical features of this mechanism can now be defined. For example, our data predict that FPR peptide agonists possess a well defined secondary structure, probably containing an α-turn. Such agonists can vary considerably in their length, but a minimal size of three amino acids is required. The first (or last) amino acid residue of a given peptide represents a key element for agonist potency because this residue has the most stringent spatial and chemical limitations. The N-terminal residue should consist of a formylated methionine, or, alternatively, the C-terminal residue should be an amidated methionine. Rather than site-specific hydrogen donor/acceptor or ionic bonds, flexible shape-oriented van der Waals interactions of most other amino acid side chains determine agonist potency. Especially the second and/or third residue next to this methionine should comprise amino acids that preferentially permit van der Waals interactions, although a certain amount of polarity can be tolerated. We define a core motif that comprises three key residues forming a hydrophobic, tripartite clawlike structure with an α-turn oriented around a carbonyl group. The symmetrical organization of this agonist motif, together with the observation that a similar carbonyl group exists in both the N-terminal formyl and the C-terminal amide group (Fig. 3F), provides an explanation for the finding that FPRs recognize in very similar ways both N-terminally formylated and C-terminally amidated peptides. We predict that a C-terminally amidated peptide interacts first with the receptor binding pocket via its C-terminal methionine, whereas an N-terminally formylated peptide binds first through its N terminus. Experimental evidence for the validity of this mechanism is provided by our demonstration that C-terminally amidated peptides are equally potent FPR agonists as corresponding peptides in which the amino acid sequence has been reversed and now comprises an N-terminal formylation.
      The fact that this mechanistic concept enabled us to identify bacterial signal peptides as a novel class of FPR agonists provides direct support for its usefulness. Bacterial signal peptides have structural features that fit particularly well to our model predictions, including a highly variable primary structure that contains a largely hydrophobic α-helical domain close to a conserved N terminus starting with a formylated methionine (
      • von Heijne G.
      Signal sequences. The limits of variation.
      ). Importantly, key features derived from our analyses can also be found in other previously identified FPR agonists, such as mitochondrial peptides, suggesting that these results could be of general significance. N-terminally formylated mitochondrial peptides from membrane proteins, such as ND1, ND4, ND6, and CO1, are well known to function as FPR agonists (
      • Rabiet M.J.
      • Huet E.
      • Boulay F.
      Human mitochondria-derived N-formylated peptides are novel agonists equally active on FPR and FPRL1, while Listeria monocytogenes-derived peptides preferentially activate FPR.
      ). Mitochondria are of ancient bacterial origin (
      • Sagan L.
      On the origin of mitosing cells.
      ), and we predict that these mitochondrial peptides served originally as signal peptides enabling protein translocation through the mitochondrial membrane. Consistent with this idea, the ND1 N terminus reveals clear structural similarities to our agonist model (Fig. 3I). Further structural comparisons demonstrate that other known FPR ligands, such as humanin and f-MLF, also display striking similarities with our agonist model (Fig. 3I). Independent support for this concept comes from a recent study using non-peptide agonists to identify a potential binding motif in FPR2 (
      • Schepetkin I.A.
      • Kirpotina L.N.
      • Khlebnikov A.I.
      • Leopoldo M.
      • Lucente E.
      • Lacivita E.
      • De Giorgio P.
      • Quinn M.T.
      3-(1H-indol-3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide enantiomers with human formyl-peptide receptor agonist activity: molecular modeling of chiral recognition by FPR2.
      ). Moreover, other FPR agonists, such as phenol-soluble modulin peptide toxins (
      • Kretschmer D.
      • Gleske A.K.
      • Rautenberg M.
      • Wang R.
      • Köberle M.
      • Bohn E.
      • Schöneberg T.
      • Rabiet M.J.
      • Boulay F.
      • Klebanoff S.J.
      • van Kessel K.A.
      • van Strijp J.A.
      • Otto M.
      • Peschel A.
      Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus.
      ), amyloid-β(1–42) (
      • Le Y.
      • Gong W.
      • Tiffany H.L.
      • Tumanov A.
      • Nedospasov S.
      • Shen W.
      • Dunlop N.M.
      • Gao J.L.
      • Murphy P.M.
      • Oppenheim J.J.
      • Wang J.M.
      Amyloid b42 activates a G-protein-coupled chemoattractant receptor, FPR-like-1.
      ), or μPAR (
      • Resnati M.
      • Pallavicini I.
      • Wang J.M.
      • Oppenheim J.
      • Serhan C.N.
      • Romano M.
      • Blasi F.
      The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R.
      ), may also fit into this scheme. For example, a recent study demonstrated that the signal peptide of the Staphylococcus aureus quorum-sensing signal, AgrD, shares clear structural and functional similarities with the phenol-soluble modulin family (
      • Schwartz K.
      • Sekedat M.D.
      • Syed A.K.
      • O'Hara B.
      • Payne D.E.
      • Lamb A.
      • Boles B.R.
      The AgrD N-terminal leader peptide of Staphylococcus aureus has cytolytic and amyloidogenic properties.
      ). The fact that critical agonist features as described here extend to a number of distinct FPR agonists suggests that our results should prove useful in future developments, such as the identification of the FPR ligand binding site and the discovery of subtype-selective FPR antagonists.
      The mammalian innate immune system as the first line of host defense has evolved multiple strategies to detect pathogen-associated molecules to subsequently eliminate infective pathogens in the body. A major challenge for the innate immune system is the recognition of a multitude of rapidly adapting microorganisms via a limited number of germ line-encoded PRRs. Therefore, PRRs focus on the recognition of highly conserved microbial components, so-called PAMPs, which are difficult to alter because they are essential for the survival of an invading pathogen. Bacterial signal peptides show a number of specific molecular properties that are typical hallmarks of PAMPs. First, export processes initiated by signal peptides represent an evolutionarily conserved mechanism that is essential for bacterial survival (
      • Dalbey R.E.
      • Wang P.
      • van Dijl J.M.
      Membrane proteases in the bacterial protein secretion and quality control pathway.
      ). Second, bacteria use an N-terminally formylated methionine to initiate translation, whereas this methionine in eukaryotic cells is unformylated (
      • Benelli D.
      • Londei P.
      Begin at the beginning: evolution of translational initiation.
      ). Because the formylation is critical for FPR recognition, this difference provides an elegant solution to enable discrimination between host-endogenous and microbial signal peptides. Third, our studies using human and mouse leukocytes provide clear evidence that signal peptides are indeed capable of triggering classical innate immune responses mediated by FPRs. Fourth, we observed a remarkably high degree of conservation in signal peptide recognition between human and mouse FPR1 and FPR2, thus arguing for an important role of bacterial signal peptides during evolution of FPR function. Consistent with this concept, human and mouse FPR1 and FPR2 both show relatively broad tuning and respond to a large number of signal peptides, and these receptors display striking similarities in their sensitivity and selectivity toward structurally divergent peptides. In summary, the recognition capabilities that mammalian FPRs have evolved are perfect for sensing the molecular properties of bacterial signal peptides, which combine an exceptionally high sequence variability with a conserved secondary structure. Hence, we propose that mammalian FPRs may have evolved originally as germ line-encoded PRRs that recognize structurally conserved export motifs of bacterial signal sequences as their cognate, pathogen-associated molecular pattern.
      Both FPRs and the Toll-like receptors focus on the recognition of invading bacteria. However, these receptors trigger distinct signal transduction cascades. It is tempting to speculate that there could be an interaction between both systems. A critical feature of FPRs is their ability to detect not only molecular patterns associated with pathogens, such as bacterial signal peptides, but also host-endogenous mitochondrial peptides. This specific property enables the FPRs to function not only as PAMP receptors but also as sensors of danger-associated molecular patterns (known as DAMPs), for example in the case of tissue damage. This aspect could be useful in the general management of the microbiome of the body, consistent with the large distribution of FPRs in multiple tissues and organs. Taken together, our findings provide an essential foundation for understanding the function of FPRs, with far reaching consequences for their biological roles.

      Acknowledgments

      We thank S. Plant, C. Hässig, and R. Bender-Omlor for expert technical and G. Mörschbächer for editorial assistance. R. Zimmermann, M. Pyrski, M. Bischoff, and T. Boehm provided critical advice on the manuscript. We thank H. Eichler for providing human blood samples and S. Saul for technical advice regarding monocyte culture.

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