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The Listeria monocytogenes Sortase-B Recognizes Varied Amino Acids at Position 2 of the Sorting Motif*

  • Javier F. Mariscotti
    Affiliations
    Departamento de Biología Molecular, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain

    Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain
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  • Francisco García-del Portillo
    Affiliations
    Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain
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  • M.Graciela Pucciarelli
    Correspondence
    An investigator of the Universidad Autónoma de Madrid and supported by a Ramón y Cajal contract of the Spanish Ministry of Science and Innovation. To whom correspondence should be addressed: Centro Nacional de Biotecnología-CSIC, Darwin 3, 28049 Madrid, Spain. Tel.: 34-91-5854551; Fax: 34-91-5854506
    Affiliations
    Departamento de Biología Molecular, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain

    Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain
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  • Author Footnotes
    2 The abbreviations used are: Srt, sortase; Inl, internalin-.
    * This work was supported by Grants BIO2006-14230 (to M. G. P.) and GEN2006-27774-C2-1-E/PAT (to F. G.-P.) from the Ministry of Science and Innovation of Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.
Open AccessPublished:January 07, 2009DOI:https://doi.org/10.1074/jbc.M807989200
      Sortases are bacterial enzymes that anchor surface proteins covalently to the peptidoglycan upon cleavage of a motif located at their C-terminal end. Motifs recognized by sortases of the class-B (SrtB) are defined by the consensus sequence NP(Q/K)(T/S)(N/G/S)(D/A). Evidence supporting this consensus is limited to IsdC of Staphylococcus aureus and Bacillus anthracis, cleaved at motifs NPQTN and NPKTG, respectively. In Listeria monocytogenes, StrB has two substrates, Lmo2185 and Lmo2186, containing NAKTN and NKVTN (or the overlapping sequence NPKSS) as putative sorting motifs. Some of these motifs do not match the consensus, because they lack either proline (P) at position 2 or glutamine/lysine (Q/K) at position 3. Here, we identified NPKSS as a sorting motif of Lmo2186 by monitoring anchoring to peptidoglycan of chimeras lacking each of its two predicted motifs. Motif-swapping experiments confirmed that NPKSS, but not NKVTN, could replace NAKTN for anchoring of an Lmo2185 chimera. Residue substitutions in the NPKSS sequence revealed the essentiality of proline at position 2 for recognition of this particular motif. Lysine at position 3 was however dispensable. Deletion of NAKTN, on the other hand, abrogated SrtB-mediated anchoring of the Lmo2185 chimera. NAKTN, therefore, represents an exception to the rule of a conserved proline in position 2 of the sorting motif. Taken together, our data indicate that proline is not absolutely required for substrate recognition by sortases of the class-B. In addition, they prove the capacity of a single sortase, as SrtB of L. monocytogenes, to recognize varied amino acids at position 2 of the sorting motif.
      The cell wall of Gram-positive bacteria is formed by a thick layer of peptidoglycan decorated with anionic polymers such as teichoic and lipoteichoic acids, and a myriad of surface proteins (
      • Pucciarelli M.G.
      • Bierne H.
      • García-del Portillo F.
      ,
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ,
      • Weidenmaier C.
      • Peschel A.
      ). Surface proteins can be partially embedded in the membrane via lipid modification in their N termini or hydrophobic C-terminal anchoring domains. Noncovalent association of certain surface proteins with outermost cell wall components such as teichoic acid, lipoteichoic acid, and peptidoglycan is also known (
      • Pucciarelli M.G.
      • Bierne H.
      • García-del Portillo F.
      ,
      • Weidenmaier C.
      • Peschel A.
      ). Another type of association is the covalent attachment of the surface protein to the peptidoglycan, exemplified by protein A of Staphylococcus aureus (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ,
      • Dramsi S.
      • Magnet S.
      • Davison S.
      • Arthur M.
      ,
      • DeDent A.C.
      • McAdow M.
      • Schneewind O.
      ).
      Sortases are enzymes conserved in Gram-positive bacteria that promote covalent anchoring of surface proteins to the peptidoglycan (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ). Sortases anchor to the peptidoglycan individual proteins and pili. In this latter case, a specialized pili-dedicated sortase together with the housekeeping sortase are involved in the anchoring of polymerized pilin (
      • Mandlik A.
      • Swierczynski A.
      • Das A.
      • Ton-That H.
      ). In all cases known, sortases catalyze a transpeptidation reaction that occurs upon cleavage at a specific motif present in the C-terminal sorting signal of the protein substrate (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ). The sorting signal is composed of this motif followed by a hydrophobic domain and a positively charged tail (
      • Schneewind O.
      • Model P.
      • Fischetti V.A.
      ,
      • Schneewind O.
      • Mihaylova-Petkov D.
      • Model P.
      ).
      Recent genomic analyses have unveiled the existence of sortase homologs, all sharing a conserved TLXTC motif and a histidine (H) involved in catalysis (
      • Comfort D.
      • Clubb R.T.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ). These sortases are classified in five classes or subfamilies, according to their homology in primary sequence, tridimensional structure, clustering with genes encoding protein substrates, location of the membrane anchor domain of the sortase, and sorting motifs predicted to be recognized by the enzyme (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ,
      • Comfort D.
      • Clubb R.T.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ). Thus, sortases (Srt)
      The abbreviations used are: Srt, sortase; Inl, internalin-.
      of the class A (SrtA, subfamily 1) recognize motifs with the consensus sequence LPXTG and are encoded by genes often non-clustered with those encoding their substrates (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ). Proteomic analysis in srtA mutants of diverse Gram-positive bacteria, as well as in vitro assays with purified SrtA enzyme and peptides covering the predicted motifs, have unequivocally demonstrated the recognition of the LPXTG motif by SrtA and the cleavage of this motif between the threonine (T) and glycine (G) residues (
      • Ton-That H.
      • Liu G.
      • Mazmanian S.K.
      • Faull K.F.
      • Schneewind O.
      ,
      • Bierne H.
      • Mazmanian S.K.
      • Trost M.
      • Pucciarelli M.G.
      • Liu G.
      • Dehoux P.
      • Jansch L.
      • Garcia-del Portillo F.
      • Schneewind O.
      • Cossart P.
      ,
      • Lalioui L.
      • Pellegrini E.
      • Dramsi S.
      • Baptista M.
      • Bourgeois N.
      • Doucet-Populaire F.
      • Rusniok C.
      • Zouine M.
      • Glaser P.
      • Kunst F.
      • Poyart C.
      • Trieu-Cuot P.
      ,
      • Garandeau C.
      • Reglier-Poupet H.
      • Dubail I.
      • Beretti J.L.
      • Berche P.
      • Charbit A.
      ,
      • Kruger R.G.
      • Otvos B.
      • Frankel B.A.
      • Bentley M.
      • Dostal P.
      • McCafferty D.G.
      ,
      • Gaspar A.H.
      • Marraffini L.A.
      • Glass E.M.
      • Debord K.L.
      • Ton-That H.
      • Schneewind O.
      ). Sortases of the class B (SrtB, subfamily 2) are generally expressed in operons containing genes encoding their substrates and recognize motifs with the consensus sequence NP(Q/K)(T/S)(N/G/S)(D/A) (
      • Comfort D.
      • Clubb R.T.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ). Noteworthy, the sorting motif recognized by SrtB has been experimentally tested only with the surface protein IsdC involved in iron acquisition. IsdC of S. aureus and Bacillus anthracis are recognized by SrtB at NPQTN and NPKTG motifs, respectively (
      • Mazmanian S.K.
      • Ton-That H.
      • Su K.
      • Schneewind O.
      ,
      • Maresso A.W.
      • Chapa T.J.
      • Schneewind O.
      ). Bioinformatic analyses performed on available bacterial genome sequences have predicted different sorting motifs for sortases of other subfamilies and new putative sortase substrates (
      • Comfort D.
      • Clubb R.T.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ,
      • Litou Z.I.
      • Bagos P.G.
      • Tsirigos K.D.
      • Liakopoulos T.D.
      • Hamodrakas S.J.
      ,
      • Boekhorst J.
      • de Been M.W.
      • Kleerebezem M.
      • Siezen R.J.
      ). However, with the exceptions above noted for LPXTG, NPQTN, and NPKTG motifs, experimental data supporting the recognition of other sorting motifs are lacking.
      Listeria monocytogenes is a bacterial pathogen that contains two sortases, SrtA and SrtB (
      • Bierne H.
      • Mazmanian S.K.
      • Trost M.
      • Pucciarelli M.G.
      • Liu G.
      • Dehoux P.
      • Jansch L.
      • Garcia-del Portillo F.
      • Schneewind O.
      • Cossart P.
      ,
      • Bierne H.
      • Garandeau C.
      • Pucciarelli M.G.
      • Sabet C.
      • Newton S.
      • Garciadel Portillo F.
      • Cossart P.
      • Charbit A.
      ). L. monocytogenes, as well as other species of the genus Listeria, are among the Gram-positive bacteria containing the highest number of surface proteins, in the range of 40–45, with predicted covalent association to peptidoglycan (
      • Pucciarelli M.G.
      • Bierne H.
      • García-del Portillo F.
      ,
      • Boekhorst J.
      • de Been M.W.
      • Kleerebezem M.
      • Siezen R.J.
      ,
      • Glaser P.
      • Frangeul L.
      • Buchrieser C.
      • Rusniok C.
      • Amend A.
      • Baquero F.
      • Berche P.
      • Bloecker H.
      • Brandt P.
      • Chakraborty T.
      • Charbit A.
      • Chetouani F.
      • Couve E.
      • de Daruvar A.
      • Dehoux P.
      • Domann E.
      • Dominguez-Bernal G.
      • Duchaud E.
      • Durant L.
      • Dussurget O.
      • Entian K.D.
      • Fsihi H.
      • Garcia-del Portillo F.
      • Garrido P.
      • Gautier L.
      • Goebel W.
      • Gomez-Lopez N.
      • Hain T.
      • Hauf J.
      • Jackson D.
      • Jones L.M.
      • Kaerst U.
      • Kreft J.
      • Kuhn M.
      • Kunst F.
      • Kurapkat G.
      • Madueno E.
      • Maitournam A.
      • Vicente J.M.
      • Ng E.
      • Nedjari H.
      • Nordsiek G.
      • Novella S.
      • de Pablos B.
      • Perez-Diaz J.C.
      • Purcell R.
      • Remmel B.
      • Rose M.
      • Schlueter T.
      • Simoes N.
      • Tierrez A.
      • Vazquez-Boland J.A.
      • Voss H.
      • Wehland J.
      • Cossart P.
      ,
      • Bierne H.
      • Cossart P.
      ). Proteomic analyses of cell wall material isolated from L monocytogenes srtA and srtB mutants revealed that SrtA anchors proteins bearing LPXTG motifs, while two surface proteins, Lmo2185 and Lmo2186, are anchored to the peptidoglycan in a StrB-dependent manner (
      • Bierne H.
      • Mazmanian S.K.
      • Trost M.
      • Pucciarelli M.G.
      • Liu G.
      • Dehoux P.
      • Jansch L.
      • Garcia-del Portillo F.
      • Schneewind O.
      • Cossart P.
      ,
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). Both lmo2185 and lmo2186 genes are clustered in the same operon containing srtB (
      • Bierne H.
      • Garandeau C.
      • Pucciarelli M.G.
      • Sabet C.
      • Newton S.
      • Garciadel Portillo F.
      • Cossart P.
      • Charbit A.
      ). This feature is also observed in S. aureus for srtB and isdC (
      • Mazmanian S.K.
      • Ton-That H.
      • Su K.
      • Schneewind O.
      ).
      Like IsdC of S. aureus and B. anthracis, Lmo2185 and Lmo2186 contain NEAT domains, implicated in iron metabolism, and are encoded by genes forming part of a large operon that encodes components of a putative iron transporter (
      • Maresso A.W.
      • Chapa T.J.
      • Schneewind O.
      ,
      • Bierne H.
      • Cossart P.
      ). This L. monocytogenes operon is induced under iron deprivation (
      • Newton S.M.
      • Klebba P.E.
      • Raynaud C.
      • Shao Y.
      • Jiang X.
      • Dubail I.
      • Archer C.
      • Frehel C.
      • Charbit A.
      ), although no evidence for a role of Lmo2185 or Lmo2186 in withstanding low iron stress has been found. Another aspect undefined is the exact motif recognized by L. monocytogenes SrtB in each of these two surface proteins. We have previously shown that SrtB anchors to the peptidoglycan chimeras consisting of an N-fragment from Internalin-B (lacking the GW domains that promote attachment to the cell envelope (
      • Braun L.
      • Dramsi S.
      • Dehoux P.
      • Bierne H.
      • Lindahl G.
      • Cossart P.
      )) fused in its C-end to the putative sorting signals of Lmo2185 or Lmo2186 (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). These sorting signals of Lmo2185 and Lmo2186 initiate with the NAKTN and NKVTNPKSS sequences, respectively. Given that the Lmo2186 sequence used in this work contained two putative SrtB motifs, NKVTN or NPKSS, our study did not formally identify the motif recognized by StrB in this surface protein. Of interest, neither the putative NAKTN motif of Lmo2185 nor NKVTN of Lmo2186 contain the invariant proline (P) at position 2 of the consensus NP(Q/K)(T/S)(N/G/S)(D/A) (
      • Comfort D.
      • Clubb R.T.
      ). Noteworthy, proteomic analyses reported the presence of a Lmo2186 peptide with the sequence SDSSNKVTNPK in peptidoglycan material of L. monocytogenes containing strongly associated mature proteins (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). This finding provided an indirect evidence for NPKSS as the motif recognized and cleaved by SrtB.
      Considering that the predicted motif of Lmo2185 lacks proline (P) at position 2 (NAKTN) and that two putative motifs can be assigned in Lmo2816, we were interested in defining the identity of the motifs recognized by SrtB of L. monocytogenes. Here, we provide the identity of these motifs and uncover the capacity of this sortase for naturally recognizing motifs with varied amino acid at position 2 of the sorting motif.

      EXPERIMENTAL PROCEDURES

      Bacterial Strains and Growth Conditions-The L. monocytogenes strains used in this study are isogenic derivates of the wild-type virulent strain EGD-e (serotype 1/2a), with the complete genome sequence known (
      • Glaser P.
      • Frangeul L.
      • Buchrieser C.
      • Rusniok C.
      • Amend A.
      • Baquero F.
      • Berche P.
      • Bloecker H.
      • Brandt P.
      • Chakraborty T.
      • Charbit A.
      • Chetouani F.
      • Couve E.
      • de Daruvar A.
      • Dehoux P.
      • Domann E.
      • Dominguez-Bernal G.
      • Duchaud E.
      • Durant L.
      • Dussurget O.
      • Entian K.D.
      • Fsihi H.
      • Garcia-del Portillo F.
      • Garrido P.
      • Gautier L.
      • Goebel W.
      • Gomez-Lopez N.
      • Hain T.
      • Hauf J.
      • Jackson D.
      • Jones L.M.
      • Kaerst U.
      • Kreft J.
      • Kuhn M.
      • Kunst F.
      • Kurapkat G.
      • Madueno E.
      • Maitournam A.
      • Vicente J.M.
      • Ng E.
      • Nedjari H.
      • Nordsiek G.
      • Novella S.
      • de Pablos B.
      • Perez-Diaz J.C.
      • Purcell R.
      • Remmel B.
      • Rose M.
      • Schlueter T.
      • Simoes N.
      • Tierrez A.
      • Vazquez-Boland J.A.
      • Voss H.
      • Wehland J.
      • Cossart P.
      ). These strains are listed in Table 1. Bacteria were routinely grown in Brain-Heart-Infusion (BHI) medium (Difco) at 37 °C under shaking conditions (175 rpm). Erythromycin (5 μg/ml) was added to the medium to grow bacteria carrying chimera-expressing plasmids (see below).
      TABLE 1Listeria monocytogenes strains used in this study
      StrainRelevant genotype
      See Table 2 for definition of the pJM plasmids series.
      Source or Ref.
      EGD-eWild-type virulent strain(
      • Glaser P.
      • Frangeul L.
      • Buchrieser C.
      • Rusniok C.
      • Amend A.
      • Baquero F.
      • Berche P.
      • Bloecker H.
      • Brandt P.
      • Chakraborty T.
      • Charbit A.
      • Chetouani F.
      • Couve E.
      • de Daruvar A.
      • Dehoux P.
      • Domann E.
      • Dominguez-Bernal G.
      • Duchaud E.
      • Durant L.
      • Dussurget O.
      • Entian K.D.
      • Fsihi H.
      • Garcia-del Portillo F.
      • Garrido P.
      • Gautier L.
      • Goebel W.
      • Gomez-Lopez N.
      • Hain T.
      • Hauf J.
      • Jackson D.
      • Jones L.M.
      • Kaerst U.
      • Kreft J.
      • Kuhn M.
      • Kunst F.
      • Kurapkat G.
      • Madueno E.
      • Maitournam A.
      • Vicente J.M.
      • Ng E.
      • Nedjari H.
      • Nordsiek G.
      • Novella S.
      • de Pablos B.
      • Perez-Diaz J.C.
      • Purcell R.
      • Remmel B.
      • Rose M.
      • Schlueter T.
      • Simoes N.
      • Tierrez A.
      • Vazquez-Boland J.A.
      • Voss H.
      • Wehland J.
      • Cossart P.
      )
      BUG2049EGD-e/pInlB-CterLmo2185(
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      )
      BUG2052EGD-e/pInlB-CterLmo2186(
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      )
      BUG1777ΔsrtA(
      • Bierne H.
      • Mazmanian S.K.
      • Trost M.
      • Pucciarelli M.G.
      • Liu G.
      • Dehoux P.
      • Jansch L.
      • Garcia-del Portillo F.
      • Schneewind O.
      • Cossart P.
      )
      BUG1877ΔsrtB(
      • Bierne H.
      • Garandeau C.
      • Pucciarelli M.G.
      • Sabet C.
      • Newton S.
      • Garciadel Portillo F.
      • Cossart P.
      • Charbit A.
      )
      MD1231EGD-e/pJM021This work
      MD1232ΔsrtA/pJM021This work
      MD1233ΔsrtB/pJM021This work
      MD1259EGD-e/pJM022This work
      MD1260ΔsrtA/pJM022This work
      MD1261ΔsrtB/pJM022This work
      MD1265ΔsrtA/pInlB-CterLmo2186This work
      MD1266ΔsrtB/pInlB-CterLmo2186This work
      MD2011ΔsrtA/pInlB-CterLmo2185This work
      MD2012ΔsrtB/pInlB-CterLmo2185This work
      MD2013EGD-e/pJM023This work
      MD2014ΔsrtA/pJM023This work
      MD2015ΔsrtB/pJM023This work
      MD2016EGD-e/pJM025This work
      MD2017ΔsrtA/pJM025This work
      MD2018ΔsrtB/pJM025This work
      MD2019EGD-e/pJM024This work
      MD2020ΔsrtA/pJM024This work
      MD2021ΔsrtB/pJM024This work
      MD2030EGD-e/pJM026This work
      MD2033EGD-e/pJM027This work
      MD2036EGD-e/pJM028This work
      a See Table 2 for definition of the pJM plasmids series.
      Construction of Plasmids Expressing Variants of the InlB-CterLmo2185 and InlB-CterLmo2186 Chimeras-Plasmids expressing the InlB-CterLmo2185 and InlB-CterLmo2186 protein fusions have been previously described (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). Both fusions contain amino acids 1–398 of InlB (lacking GW domains) followed by residues 536–569 of Lmo2185 (InlB-CterLmo2185) or residues 170–207 of Lmo2186 (InlB-CterLmo2186). These plasmids were used as template to generate variants in which putative motifs were deleted or mutated at specific sites. Table 2 lists the complete set of plasmids constructed for this work. These variants were generated by amplification of two regions encompassing the InlB and C-terminal sorting signal sequences, respectively, followed by overlapping PCR and cloning of the resulting product in pP1 plasmid (
      • Trieu-Cuot P.
      • Carlier C.
      • Poyart-Salmeron C.
      • Courvalin P.
      ) using the SacI and SphI restriction sites. The ExSite PCR-based site-directed mutagenesis kit (Stratagene) was used in the case of the plasmid pJM021, expressing a InlB-CterLmo2186-variant bearing only the putative motif NKVTN (Table 2). The modification(s) introduced in every variant were verified by sequencing. Oligonucleotides used are listed in supplemental Table S1.
      TABLE 2InlB-CterLmo2185 and InlB-CterLmo2186 chimeras used in this work
      PlasmidModificationMotif testedC-ter (sorting signal)
      pInlB-CterLmo2186NKVTNPKSSLmo2186
      pJM021Δ(PKSS)NKVTNLmo2186
      pJM022Δ(NKVT)NPKSSLmo2186
      pInlB-CterLmo2185NAKTNLmo2185
      pJM023Δ(NAKTN)Lmo2185
      pJM024AK→KVNKVTNLmo2185
      pJM025A→P, TN→SSNPKSSLmo2185
      pJM026P→ANAKSSLmo2186 (plasmid pJM022)
      pJM027K→QNPQSSLmo2186 (plasmid pJM022)
      pJM028KSS→QTNNPQTNLmo2186 (plasmid pJM022)
      Bacterial Fractionation-The method described by Jonquieres et al. (
      • Jonquieres R.
      • Bierne H.
      • Fiedler F.
      • Gounon P.
      • Cossart P.
      ) was used to prepare cell wall extracts. Briefly, bacteria grown overnight in BHI medium (A600 = 3.0) and contained in 1 ml of culture were collected by centrifugation (5,000 × g, 10 min, 4 °C), washed once in phosphate-buffered saline, and once in TS buffer (10 mm Tris-HCl pH 6.9, 10 mm MgCl2, 0.5 m sucrose). The pellets were resuspended in lysis buffer (TS buffer containing 60 μg/ml mutanolysin (Sigma, catalog no. M9901), 250 μg/ml RNase, and complete EDTA-free protease inhibitor mixture (Roche Applied Science, catalog no. 1873580)) and incubated with slow-rotating agitation at 37 °C for 1 h. Protoplast formation was monitored using optical microscopy and plating. Protoplasts were recovered by centrifugation at 15,000 × g, 10 min, 4 °C, and resuspended in phosphate-buffered saline. The supernatants (cell wall fractions) were filtered and precipitated on ice in 16% trichloroacetic acid for 45 min. Proteins present in the cell wall fraction were recovered by centrifugation at 21,000 × g, 10 min, 4 °C, washed in cold-acetone and resuspended in phosphate-buffered saline. An appropriate volume of Laemmli sample buffer was added to both protoplast and cell wall fractions. The extracellular medium fraction was prepared from the supernatant collected after the centrifugation of the bacterial culture, which was passed through 0.22-μm pore size Millipore filters. Proteins were precipitated using a 10% trichloroacetic acid and acetone washing procedure as described (
      • Bierne H.
      • Garandeau C.
      • Pucciarelli M.G.
      • Sabet C.
      • Newton S.
      • Garciadel Portillo F.
      • Cossart P.
      • Charbit A.
      ). Unless otherwise indicated, the amount of each fraction loaded into the gels corresponded to a 6:4:1 ratio in the number of bacteria for the cell wall:extracellular medium:protoplast fractions, respectively. Proteins were resolved by Tris-glycine polyacrylamide electrophoresis, using 8% gels. The experiments were repeated a minimum of three times.
      Immunoblot Analyses-The different chimeras were detected with mouse monoclonal anti-InlB antibody (
      • Braun L.
      • Ohayon H.
      • Cossart P.
      ). Mouse monoclonal anti-InlA (
      • Mengaud J.
      • Ohayon H.
      • Gounon P.
      • Mege R.M.
      • Cossart P.
      ) was also used as control proteins for the cell wall fractions. Goat anti-mouse or anti-rabbit conjugated to horseradish peroxidase (Bio-Rad) was used as secondary antibodies. Proteins recognized by the antibodies were visualized by chemoluminescence using the luciferin-luminol reagents. In the case of requiring more sensitivity, the ECL Western blotting detection system was used (Amersham Biosciences, catalog: RPN2209). In some cases, the relative amount of protein anchored to the peptidoglycan was assessed by quantification of specific bands corresponding to the chimera obtained in cell wall versus protoplast fractions using the Quantity One 1-D Software, v. 4.6.5, from Bio-Rad.

      RESULTS

      NPKSS Is a Motif Recognized by Sortase-B (SrtB) of L. monocytogenes in Lmo2186-The surface protein Lmo2186 (207 amino acids) contains a C-terminal sorting region predicted to initiate with the motif NKVTNPKSS, followed by a stretch of 22 hydrophobic amino acids and a positively charged tail (Fig. 1). We have previously shown that an InlB-CterLmo2186 chimera containing this sorting signal is anchored to the peptidoglycan in a StrB-dependent manner (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). However, the exact identity of the motif was not investigated. To determine this, new InlB-Cter-Lmo2186 chimeras were constructed bearing either NKVTN or NPKSS as the only motif (Fig. 2A). These variants were expressed in wild-type (EGD-e) and isogenic ΔsrtA or ΔsrtB bacteria, which were grown to stationary phase in BHI nutrient medium. Upon mutanolysin digestion of peptidoglycan, bacteria were fractionated into cell wall extracts and protoplasts containing cytosol and membrane material. Proteins secreted to the growth medium were also examined as an additional fraction. InlB-CterLmo2186 chimeras containing as motifs NKVTNPKSS, NKTVN, or NPKSS were examined in these three fractions. Control assays with antibodies detecting Internalin-A (InlA), a SrtA substrate, verified the purity of the peptidoglycan fractions (Fig. 2B). Our results showed that the variants containing either NKVTNPKSS or NPKSS were efficiently anchored to the peptidoglycan (Fig. 2B). The NPKSS variant was however detected in the peptidoglycan at slightly lesser amounts than the one containing the entire NKVTNPKSS sequence (Fig. 2B). The presence of these two chimeras in the peptidoglycan was shown to require a functional SrtB and to be independent of SrtA (Fig. 2C). Interestingly, the variant bearing only the NKVTN sequence was not anchored to the peptidoglycan in any of the three genetic backgrounds tested (Fig. 2C). This variant was detected in large amounts in the growth medium (data not shown). Because NPKSS shares amino acids at positions 1 and 2 with the previously validated motifs NPQTN and NPKTG, our data support a prominent role of these two positions for substrate recognition by SrtB sortases.
      Figure thumbnail gr1
      FIGURE 1Sorting signals of peptidoglycan-anchored proteins in which experimental evidence has been demonstrated to be recognized by sortases of the class B (SrtB):IsdC of S. aureus; IsdC of B. anthracis; and, Lmo2185-Lmo2186 of L. monocytogenes. Indicated are the different regions of the protein substrate, including the N-terminal signal peptide and the three regions that compose the C-terminal sorting signal: the sorting motif, followed by the hydrophobic retention domain, and the positively charged tail. Highlighted are the sorting motifs predicted to be recognized by SrtB in each substrate protein. In the case of Lmo2186, the two putative motifs are overlapped (NKVTNPKSS). Asterisks denote residues in each sorting motif that are conserved with NPQTN, the first SrtB motif demonstrated experimentally. The length of the hydrophobic domain is also indicated for each substrate.
      Figure thumbnail gr2
      FIGURE 2The NPKSS sequence of Lmo2186 is recognized as sorting motif by the sortase SrtB. A, map of the InlB-CterLmo2186 chimera harboring the NKVTNPKSS sequence and variants containing only the first or second putative sorting motifs. The plasmids expressing these chimeras are also indicated. B, immunodetection of the InlB-CterLmo2186 chimera and variants in subcellular fractions and the extracellular medium using anti-InlB antibodies. Note that only two of the three chimeras tested were detected in the cell wall material (material released upon mutanolysin treatment). Control experiments demonstrated that anchoring of InlA to the cell wall was similar in all strains used. C, cell wall fractions of wild-type, ΔsrtA, and ΔsrtB strains expressing the InlB-CterLmo2186 chimera and variants. Chimeras were detected with anti-InlB antibodies. Immunodetection of InlA was also made as control. Note that NPKSS is recognized as a sorting motif in an SrtB-dependent manner.
      NPKSS, but Not NKVTN, Can Mediate SrtB-dependent Anchoring of an InlB-Cter-Lmo2185 Fusion-To unequivocally demonstrate the identity of the sorting motif recognized by SrtB in Lmo2186, we generated a new series of InlB-Cter-Lmo2185 chimeras in which the endogenous NAKTN motif was exchanged to either NKVTN or NPKSS, the two putative motifs of Lmo2186 (Fig. 3A). These chimeras were expressed in wild-type, ΔsrtA, and ΔsrtB strains. Analysis of peptidoglycan material obtained from these strains showed that NPKSS, but not NKVTN, was recognized by SrtB to promote anchoring of the InlB-Cter-Lmo2185 chimera (Fig. 3B). An InlB-Cter-Lmo2185 variant lacking the NAKTN motif was also generated for these assays. Importantly, the analysis of protoplast, cell wall, and extracellular medium fractions showed that the lack of NAKTN totally abrogated the recognition and anchoring to peptidoglycan of the chimera by SrtB, resulting in its secretion to the extracellular medium (Fig. 3B). Taken together, these results confirmed the recognition of NPKSS by SrtB, even when placed in the context of the heterologous sorting signal of Lmo2185. Likewise, the data unveiled the unique capacity of L. monocytogenes SrtB for recognizing a sorting motif lacking the invariant proline at position (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ), exemplified by NAKTN.
      Figure thumbnail gr3
      FIGURE 3NPKSS, but not NKVTN, is recognized by SrtB in the heterologous sorting signal of Lmo2185. A, map of the InlB-Cter-Lmo2185 and variants that either lack the NAKTN motif or have this motif exchanged by one of the putative motifs predicted in Lmo2186 (NPKSS, NKVTN). The substituted residues are marked with asterisks. The plasmids expressing these chimeras are also indicated. B, immunodetection of the distinct InlB-CterLmo2185 chimeras in protoplast, cell wall, and extracellular medium extracts using anti-InlB antibodies. Note that the chimera lacking the NAKTN motif (marked as Δmotif) is not anchored to the peptidoglycan and, instead, secreted to the medium. The amount of protein loaded for each fraction corresponded to the following bacteria: 1 × 108 (protoplast), 4.2 × 108 (extracellular medium), and 6.2 × 108 (cell wall).
      Proline (P) at Position 2, and Not Lysine (K) at Position 3, Is Essential for the Recognition of NPKSS by SrtB-The above experiments demonstrated that NAKTN (Lmo2185) and NPKSS (Lmo2186), but not NKVTN, are recognized as sorting motifs by L. monocytogenes SrtB. These data suggested that lysine (K) at position 3 could play an important role for SrtB recognition. To test this hypothesis, we generated new variants using the NPKSS sequence as template and the C-terminal sorting signal of Lmo2186. These NPKSS variants carried changes at position 2 (NAKSS); 3 (NPQSS); and 3-4-5 (NPQTN) (Fig. 4A). The latter variant was designed to recreate the NPQTN motif recognized in IsdC by the S. aureus SrtB, which lacks lysine (K) at position 3. Analysis of cell wall, protoplast, and extracellular fractions revealed that NPKSS and NPQSS were recognized as sorting motifs since the respective chimeras were detected in the cell wall (Fig. 4B). Although the chimera harboring the NPQTN motif was expressed at lower levels than the others chimeras used, it was also detected in cell wall extracts. A densitometry analysis confirmed the recognition of the NPQTN motif to an extent comparable to those of the motif NPKSS and the NAKTNPKSS sequence present in Lmo2186 (Fig. 4, C and D). Therefore, we concluded that NPQTN is recognized by the L. monocytogenes sortase SrtB. On the other hand, a P → A change in the position 2 of the NPKSS motif of Lmo2186 (NAKSS variant) abrogated anchoring to the peptidoglycan (Fig. 4, B and C). Control experiments using a ΔsrtB strain revealed that in those cases in which the anchoring to peptidoglycan was detected for a particular chimera, this was SrtB-dependent (data not shown). Taken together, these results demonstrated that, unlike lysine (K) at position 3, proline (P) at position 2 is essential for recognition of the NPKSS sorting motif by the L. monocytogenes SrtB sortase.
      Figure thumbnail gr4
      FIGURE 4Substitution of proline (P) at position 2, but not lysine (K) at position 3, impairs recognition of the NPKSS sorting motif by the L. monocytogenes SrtB sortase. A, map of the different mutations generated at positions 2, 3, or 3-4-5 of the NPKSS motif, which are indicated with asterisks. The plasmids expressing these chimeras are also indicated; B, distribution of the chimeras InlB-CterLmo2186 harboring distinct variants of the NPKSS motif in protoplast, cell wall, and extracellular medium extracts. Chimeras were immunodetected with anti-InlB antibodies. Note the lower production of the chimera with the NPQTN variant respect the rest of variants tested. The amount of protein loaded for each fraction corresponded to the following bacteria: 1 × 108 (protoplast), 4.2 × 108 (extracellular medium), and 6.2 × 108 (cell-wall). C, samples as shown in panel B in which the extracts from the strain producing the chimera with the NPQTN variant were loaded 4-fold more concentrated than for the rest of strains (see as reference the signal of endogenous InlB in protoplasts). D, densitometry analysis of samples of panel C, showing the ratio between the signal corresponding to the chimera obtained in cell wall versus protoplast extracts.

      DISCUSSION

      In this work, we have identified the sorting motifs of Lmo2185 and Lmo2186, two L. monocytogenes surface proteins that are anchored to the peptidoglycan in an SrtB-dependent manner (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). Our previous proteomic study identified the SDSSNKVTNPK peptide, corresponding to Lmo2186, in material obtained from cell wall extracts (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). The detection of such peptide in the cell wall-derived extract suggested, although not proved, that NPKSS could be the sorting motif recognized by SrtB in Lmo2186. These proteomic analyses also provided evidence for a marked presence of peptides from Lmo2185 and Lmo2186 within the pool of peptides obtained upon trypsin digestion of the cell wall material (
      • Pucciarelli M.G.
      • Calvo E.
      • Sabet C.
      • Bierne H.
      • Cossart P.
      • Garciadel Portillo F.
      ). In fact, Lmo2185 and Lmo2186 peptides were identified at a higher rate that those of other surface proteins with similar molecular weight. Lmo2185 and Lmo2186 seem therefore to be abundant among the set of proteins covalently bound to the peptidoglycan, which implies that SrtB of L. monocytogenes must be a sortase displaying high activity in vivo.
      Taking in account these observations, we focused on the unequivocal identification of sorting motifs recognized by SrtB in Lmo2185 and Lmo2186. To this aim, chimeras harboring natural and recombinant sequences expected to be recognized by this sortase were analyzed. This experimental design relied on predictions considering that the sequences NAKTN in Lmo2185, and NKVTN (or the overlapping NPKSS) in Lmo2186, could be recognized as sorting motifs (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ,
      • Comfort D.
      • Clubb R.T.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ,
      • Bierne H.
      • Garandeau C.
      • Pucciarelli M.G.
      • Sabet C.
      • Newton S.
      • Garciadel Portillo F.
      • Cossart P.
      • Charbit A.
      ). Our experiments showed that NPKSS, but not the upstream sequence NKVTN, is absolutely required for SrtB-mediated anchoring of a chimera containing the C-terminal end of Lmo2186. This result was in agreement with the consensus motif NP(Q/K)(T/S)(N/G/S)(D/A), proposed by Comfort and Clubb (
      • Comfort D.
      • Clubb R.T.
      ) for the class-B sortases (StrB) upon comparison of multiple bacterial genome sequences.
      Next, we further explored the essentiality of the residues composing the NPKSS sorting motif of Lmo2186. The P → A substitution was shown to abrogate anchoring of the corresponding chimera bearing the NAKSS variant. This result, to our knowledge, represents the first evidence supporting the essentiality of proline (P) at position 2 of the motif for recognition by sortases of the class B (StrB). It is important to note that the relevance of proline (P) at position 2 for substrate recognition was previously shown for sortases of the class A (SrtA). In this case, a P → N substitution at position 2 of the LPETG motif of protein A resulted in impaired anchoring of this surface protein to the S. aureus cell wall (
      • Schneewind O.
      • Model P.
      • Fischetti V.A.
      ). The key role of proline (P) for sortase recognition is also evident in most substrates predicted for other sortases, as those of subfamilies 3 and 4 (
      • Comfort D.
      • Clubb R.T.
      ).
      Interestingly, genes encoding StrB homologs have been found in several bacterial genera as Bacillus, Staphylococcus, Listeria, Clostridium, and Streptococcus (
      • Marraffini L.A.
      • Dedent A.C.
      • Schneewind O.
      ,
      • Comfort D.
      • Clubb R.T.
      ,
      • Dramsi S.
      • Trieu-Cuot P.
      • Bierne H.
      ). Noteworthy, while every StrB substrate predicted in staphylococci has proline (P) at position 2, SrtB substrates with varied amino acids at position 2 have been predicted in both Bacillus and Listeria species. Thus, Bacillus spp. have substrates with either proline (P) or serine (S) at position 2, whereas those of Listeria spp. contain either proline (P) or alanine (A) at the same position. These observations, together with the data obtained for the case of the Lmo2185 motif (see below), show that proline (P) at position 2 is not an absolute requirement for substrate recognition by SrtB sortases.
      Similarly, the mutational analysis performed on the NPKSS motif of Lmo2186 led us also to conclude that lysine (K) at position 3 is not essential for SrtB recognition. Thus, a chimera bearing the NPQSS variant was efficiently anchored to the peptidoglycan. This result was unexpected considering the rather different chemical character of lysine (K), a charged residue, with respect to glutamine (Q), a hydrophilic uncharged residue. The apparent dispensability of lysine (K) at position 3 of the NPKSS motif of Lmo2186 contrasts with its conservation in the NAKTN motif of Lmo2185. This lysine is also conserved in most of the StrB substrates predicted by bioinformatic analyses (
      • Boekhorst J.
      • de Been M.W.
      • Kleerebezem M.
      • Siezen R.J.
      ) and in the NPKTG motif of IsdC, validated in B. anthracis (
      • Maresso A.W.
      • Chapa T.J.
      • Schneewind O.
      ). The dispensability of the lysine (K) in position 3 of the NPKSS motif could be explained in the context of the flanking sequences. Our experiments with Lmo2186 chimeras lacking each of the two probable overlapping sorting motifs revealed that the wild-type sequence NKVTNPKSS was recognized with a slightly higher efficiently than the construction bearing only NPKSS (see Fig. 2C). Although indirectly, this observation supports the idea of residues located upstream of NPKSS contributing to the recognition of Lmo2186 by SrtB. Mutational analysis of these flanking residues might provide insights on the molecular features that ultimately determine the extent of interaction and recognition by the sortase. The concept of highly conserved amino acids that are apparently dispensable for recognition is not limited to the lysine (K) in position 3 of the NPKSS motif. Thus, the pioneering work of Schneewind et al. (
      • Schneewind O.
      • Model P.
      • Fischetti V.A.
      ) revealed that, despite its high conservation, the threonine (T) present in the LPETG motif of S. aureus protein A was dispensable for anchoring of this surface protein to the peptidoglycan.
      Our study has also uncovered NAKTN as the genuine sorting motif of Lmo2185 recognized by SrtB of L. monocytogenes. This finding reinforces the idea of a single sortase capable of recognizing in its natural substrates distinct amino acids at position 2 of their respective motifs. Interestingly, sortases classified in the subfamily 5 (class-D) are predicted to recognize motifs with a consensus sequence NA(E/A/S/H)TG (
      • Comfort D.
      • Clubb R.T.
      ), which contains an invariant alanine (A) at position 2. Sortases of subfamily 5 are found in Streptomyces spp., Corynebacterium spp., Tropheryma whipplei, Thermobifida fusca, and Bifidobacterium longum. None of these Gram-positive bacteria have either SrtA or SrtB homologs. In fact, it has been proposed that sortases of the subfamily 5 could play a housekeeping role equivalent to that assigned to SrtA (
      • Comfort D.
      • Clubb R.T.
      ). Based on these observations, it is tempting to speculate that SrtB could have evolved by acquiring some properties inherent to sortases of the subfamily 5 and the class-A (SrtA), which recognize alanine (A) or proline (P) at position 2 of the sorting motif, respectively. Such assumption could explain the flexibility of the L. monocytogenes SrtB for recognizing varied amino acids at the position 2 of the sorting motif. To our knowledge, this property had not been shown for any sortase before this study. Data validating SrtB substrates of Bacillus species containing serine (S) at position 2 of the motif could be of high value to further support the capacity of sortases of the class B for recognizing varied amino acids at this position.

      Acknowledgments

      We thank Pascale Cossart and Hélène Bierne for the anti-InlB and anti-InlA antibodies, and the original InlB-Cter-Lmo2185 and InlB-CterLmo2186 chimeras.

      Supplementary Material

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