An internal thioester in a pathogen surface protein mediates covalent host binding
For commensal and pathogenic bacteria , adhesion to host surfaces is a pre-requisite for colonization and infection , and is mediated by surface-presented adhesins ( Pizarro-Cerdá and Cossart , 2006 ) . Through specific interactions , these proteins can define host and tissue tropism , providing niche environments and a competitive advantage in the search for nutrients . Bacterial adhesins bind either directly to integral host cell surface components , such as integrins or carbohydrates , or they interact with components of the extracellular matrix resulting in indirect binding to receptors on the host cell surface ( Kline et al . , 2009 ) . Such molecular interactions that define the host-microbe interface are generally non-covalent in nature and frequently involve extensive intermolecular interfaces and multivalent binding . The surprising discovery of internal thioester bonds in the pilus tip adhesin Cpa from the Gram-positive human pathogen Streptococcus pyogenes raised the possibility of pathogen-encoded covalent adhesion ( Pointon et al . , 2010; Linke-Winnebeck et al . , 2014 ) . Internal thioester bonds are formed between the side chains of Cys and Gln residues , most likely self-generated by a favorable environment during protein folding . Internal thioesters have previously only been observed in mammalian complement proteins C3 and C4 ( Law and Dodds , 1997 ) and related proteins ( Dodds and Law , 1998; Lin et al . , 2002; Cherry and Silverman , 2006; Wong and Dessen , 2014 ) . Complement thioester proteins are large , multi-domain constructs that upon proteolytic activation undergo a conformational change that exposes the reactive thioester ( Janssen et al . , 2006 ) . This is thought to react with nucleophiles on the surface of pathogens , thus mediating irreversible host-encoded covalent tagging of the pathogens for elimination by the host immune system . However , to the best of our knowledge , definitive evidence that internal thioesters in complement proteins deliver an intermolecular bond with a pathogen target is lacking , and it is not clear if target binding is specific or non-specific . Cpa and complement proteins share no identifiable sequence or structural relationship and therefore appear to be evolutionarily distinct . The thioester domains of Cpa are found in a much less complex structural context and do not require proteolytic activation . The only similarity to complement appears to be that the same strategy , a ‘chemical harpoon’ , has evolved independently in bacteria and complement-related proteins for the purpose of irreversible binding . Internal thioesters are one of the three unexpected self-generating cross-links between amino acid side chains found in subunits of pili and other adhesins of Gram-positive bacteria ( Schwarz-Linek and Banfield , 2014 ) , with the others being intramolecular isopeptide bonds ( Kang et al . , 2007; Kang and Baker , 2011 ) and ester bonds ( Kwon et al . , 2014 ) . Unlike intramolecular isopeptide or ester bonds , the role of thioesters does not appear to be protein stabilization ( Walden et al . , 2014 ) . Instead , by analogy to complement , thioesters presented on bacterial surfaces may react with nucleophilic groups on host tissue targets , thus establishing pathogen-encoded covalent adhesion . Indeed , binding of S . pyogenes to mammalian cells in vitro is severely impaired when an engineered Cpa variant lacking the thioester is expressed ( Pointon et al . , 2010 ) . While this does not confirm covalent attachment , it supports a role for the reactive bond in bacterial adhesion . Interestingly , a recent study showed covalent bond formation between a Cpa thioester domain and the small molecule nucleophile spermidine , confirming the accessibility and reactivity of the thioester ( Linke-Winnebeck et al . , 2014 ) . However , the molecular mechanisms of targeting and covalent binding to a relevant host receptor have yet to be discovered . Here we experimentally demonstrate that thioester domains are prevalent in Gram-positive surface proteins , and that they share a conserved three-dimensional structure despite being divergent in protein sequence . This suggests that thioester domains may target distinct receptors and have a widespread but currently unappreciated role in mediating pathogen adhesion to hosts . Further , we present the identification of host fibrinogen as a covalently bound target of the streptococcal surface protein SfbI . In a thioester-dependent mechanism , SfbI reacts with one specific lysine residue in fibrinogen , forming a very stable intermolecular amide bond . Using a combination of computational , biochemical , structural and cell-based assays we reveal a novel mechanism for host adhesion mediated by a pathogen-encoded covalent interaction .
The discovery of thioester-containing domains ( TEDs ) in Cpa prompted us to look for similar domains in other proteins . Using a TED of Cpa ( Cpa-TED2 ) as a template , extensive PSI-BLAST similarity searches ( Altschul et al . , 1997 ) yielded hundreds of potential hits , exclusively in Gram-positive bacteria . 54 sequences from 40 species were analyzed further ( Figure 1—figure supplement 1 ) . Putative TEDs appear in a variety of domain architectures ( Figure 1 ) , but are almost always located at the N-termini of proteins containing secretion signals and C-terminal LPXTG cell wall-anchoring motifs ( Schneewind and Missiakas , 2014 ) . Most TED-containing proteins are also predicted to contain intramolecular isopeptide and/or ester domains , usually present in tandem repeat arrays . Like isopeptide domains ( sIPDs ) in pilus assemblies ( Kang et al . , 2007 ) , these repeats probably act as ‘stalks’ presenting the TED away from the bacterial surface . We refer to these as TIE ( thioester , isopeptide , ester domain ) proteins . Other domains commonly associated with putative TEDs are fibronectin-binding repeats and proline-rich regions . 10 . 7554/eLife . 06638 . 003Figure 1 . Domain architectures of TED-containing proteins of clinically relevant bacteria . C , T and Q indicate positions of conserved motifs . The MEME signatures ( Bailey et al . , 2009 ) derived from 54 sequences ( Figure 1—figure supplement 1 ) are also shown with the thioester-forming residues in red; proline , hydrophobic , polar and small residues in yellow , green , blue and grey , respectively . TIE , thioester , isopeptide , ester domain protein; TEP , thioester domain containing protein . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 00310 . 7554/eLife . 06638 . 004Figure 1—figure supplement 1 . Sequence alignment of 54 TEDs from 40 species . Proteins with names shown in red were investigated in this study . Blue and green Uniprot accession codes belong to proteins of firmicutes and actinobacteria , respectively . Secondary structure elements of SfbI-A40-TED are displayed on top . The thioester-forming Cys and Gln residues are highlighted in red , partially conserved residues are shaded grey ( threshold for shading 85% ) . The following proteins are included in the alignment . UniProt accession codes and species names are given , also protein names in parentheses , where available: Q1JDZ6 ( SfbI-A40 ) , Streptococcus pyogenes; Q711F8 ( SfbI-A346 ) , S . pyogenes; Q01924 ( SfbI-A20 ) , S . pyogenes; Q6EWI8 ( GfbA ) , Streptococcus dysgalactiae subsp . equisimilis; Q8G9G1 ( FbaB ) , S . pyogenes; Q8GRA2 ( Cpa-TED2 ) , S . pyogenes; B1R775 ( CpTIE ) , Clostridium perfringens; H2G8R6 ( CodTIE ) , Corynebacterium diphtheriae; A5MCJ6/Q4K1R7 ( PnTIE ) , Streptococcus pneumoniae; C3PDP7 ( BaTIE ) , Bacillus anthracis; I3GYQ5/I3HJ59/I3HKL2 ( SaTIE ) , Staphylococcus aureus VRS11b; Q17ZZ0 ( CdTEP ) , Peptoclostridium difficile; F2ZZY0 , Gemella haemolysans; U2Q1B9 , Gemella bergeriae; E1JLX8 , Lactococcus salivarius; P72416 ( FnZ ) , Streptococcus equi; C0MAN5 ( FneC ) , Streptococcus equi subsp . equi; T1ZSL1 , Streptococcus constellatus; D0UFC3 ( Sbs13 ) , Streptococcus gallolyticus; G5JZC9 , Streptococcus ictaluri; S5FV19 ( Cpa-TED1 ) , S . pyogenes; G5KFG6 , Streptococcus urinalis; B1RSM5 , C . perfringens; H3NQ41 , Helcococcus kunzii; A8SJ15 , Parvimonas micra; G4D2J9 , Peptoniphilus indolicus; D3MRS9 , Peptostreptococcus anaerobius; K0ZKB8 , Actinomyces turicensis; D7BLE5 , Arcanobacterium haemolyticum; H2H5E2 , C . diphtheriae; H2GKP8 , C . diphtheriae; D9QDT9 , Corynebacterium pseudotuberculosis; G0CMN3 , Corynebacterium ulcerans; E6EBZ4 , Propionibacterium acnes; D2Q6N9 , Bifidobacterium dentium; S4GNG3 , Gardnerella vaginalis; D6L3Z6 , Parascardovia denticolens; J0D4V1 , Scardovia wiggsiae; B5UW14 , Bacillus cereus; C3AWK0 , Bacillus mycoides; E0WKP3 , Listeria grayi; Q8Y804 , Listeria monocytogenes; H7CR09 , L . monocytogenes; W7CRB3 , Listeria weihenstephanensis; Q833P7 , Enterococcus faecalis; R2PL19 , Enterococcus faecium; S4EXK4 , E . faecium; T4YK58 , P . difficile; X4QXD7 , Trueperella pyogenes; D4YNA9 , Brevibacterium mcbrellneri; D7AU59 , Nocardiopsis dassonvillei . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 00410 . 7554/eLife . 06638 . 005Figure 1—figure supplement 2 . 10 of the 12 purified TEDs used in this study . SDS-PAGE showing the final purity of 10 of the 12 TED samples . The second bands observed for CodTIE and BaTIE likely represent breakdown products of the TEDs . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 00510 . 7554/eLife . 06638 . 006Figure 1—figure supplement 3 . Identification of the thioester-forming Gln residues in class-II TEDs . MS of tryptic digests of TEDs following incubation with methylamine and blocking of the resulting free Cys thiol with iodoacetamide ( IAA ) . The thioester-forming Gln residues appear as methylated Gln ( Q ( Me ) ) . Precursor signals matching the methylated tryptic fragment are shown . The masses do not match any other tryptic fragments of the TEDs . Δ , difference between experimental and theoretical mass . The BaTIE-TED fragment contains two Gln residues , and MS/MS analysis was used to unambiguously identify Gln266 as the residue involved in thioester formation . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 006 Multiple sequence alignment suggests high diversity among putative TEDs , which can be divided into two classes ( class I and class II ) based on two indels ( Figure 1—figure supplement 1 ) . Secondary structure predictions suggest conservation of TED topology , with a central helical region comprising three core helices lying between predicted β-sheet regions . Sequence similarities are mainly limited to three short regions ( Figure 1 ) . The only fully conserved residues , the thioester-forming Cys and Gln , are found in a [YFL]CΦζ motif ( where Φ is any hydrophobic and ζ any hydrophilic residue ) and a weak ΦQζΦΦ motif , respectively . Both motifs are consistently predicted to reside in a β-sheet secondary structure context . A TQxxΦWΦxζ α-helical motif , where x is any residue ( previously TQxA ( I/V ) W [Linke-Winnebeck et al . , 2014] ) is conserved in the central region of most , though not all putative TEDs . Mutagenesis of a Cpa-TED demonstrated that neither the Gln nor Trp of this motif is essential for thioester formation ( Linke-Winnebeck et al . , 2014 ) , a notion supported by lack of full conservation of any TQxxΦWΦxζ motif residue ( Figure 1—figure supplement 1 ) . To obtain experimental evidence for the apparent abundance and high sequence diversity of TEDs , twelve domains from eight significant human pathogens ( Figure 1 ) were recombinantly expressed in Escherichia coli . In addition to Cpa-TED2 , three allelic variants of the S . pyogenes fibronectin-binding protein SfbI ( SfbI-A40 , SfbI-A346 and SfbI-A20 ) and its Streptococcus dysgalactiae ortholog GfbA were chosen , since their N-terminal domains , now annotated as TEDs , exert an unexplained differential effect on the uptake mechanism of streptococci by mammalian cells ( Rohde et al . , 2011 ) . Other class-I TEDs expressed originate from the fibronectin-binding protein FbaB of S . pyogenes , and from TIE proteins of Clostridium perfringens , Corynebacterium diphtheriae and Streptococcus pneumoniae . Finally , three class-II TEDs from Bacillus anthracis , vancomycin-resistant Staphylococcus aureus isolate VRS11b ( Kos et al . , 2012 ) and multidrug-resistant Peptoclostridium difficile CD630 ( formerly Clostridium difficile ) ( Sebaihia et al . , 2006 ) were also expressed . These twelve TEDs were purified to homogeneity ( Figure 1—figure supplement 2 ) . Each protein displayed an experimental molecular mass , as determined by liquid chromatography–mass spectrometry ( LC-MS ) , ∼17 Da lower than predicted ( Table 1 ) . This is consistent with loss of one molecule of ammonia , as would occur upon internal thioester formation . Seven TEDs were produced as Cys to Ala variants , targeting the Cys of the [YFL]CΦζ motif . For each of these variants , the experimentally determined molecular masses conformed to predicted values ( Table 1 ) , confirming that the −17 Da differences observed for the native proteins are attributable to internal thioesters . For class-II TEDs ( BaTIE , SaTIE and CdTEP ) the presence of internal thioesters and the identities of the thioester-forming Gln were established by tryptic-digest LC-MS/MS of the proteins after reacting the thioester bond with the small nucleophile methylamine ( Figure 1—figure supplement 3 ) . This analysis lends confidence to our definition of domain boundaries of class-II TEDs , which often lack an obvious ΦQζΦΦ motif to define the thioester-forming Gln from sequence alone ( Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 06638 . 007Table 1 . Protein intact mass MS analysisDOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 007ProteinVectorMolecular mass ( Da ) CalculatedObservedΔSfbI-A40-TEDpOPIN-F*24 , 156 . 924 , 139 . 6−17 . 3SfbI-A40-TED:Cys109AlapOPIN-F*24 , 124 . 924 , 124 . 7−0 . 2SfbI-A346-TEDpDEST†24 , 835 . 824 , 818 . 2−17 . 6SfbI-A346-TED:Cys103AlapOPIN-E‡25 , 626 . 625 , 626 . 3−0 . 3SfbI-A20-TEDpOPIN-E‡25 , 996 . 125 , 978 . 5−17 . 6SfbI-A20-TED:Cys97AlapOPIN-E‡25 , 964 . 125 , 963 . 6−0 . 5GfbA-TEDpDEST†24 , 382 . 424 , 364 . 7−17 . 7FbaB-TEDpOPIN-F*21 , 683 . 021 , 665 . 6−17 . 4FbaB-TED:Cys94AlapOPIN-E§22 , 447 . 722 , 447 . 5−0 . 2Cpa-TED2pOPIN-F*22 , 377 . 822 , 360 . 5−17 . 3CpTIE-TEDpOPIN-F*21 , 261 . 021 , 243 . 7−17 . 3CpTIE-TED:Cys138AlapOPIN-F*21 , 228 . 921 , 227 . 8−1 . 1CodTIE-TEDpOPIN-F*25 , 011 . 624 , 994 . 3−17 . 3CodTIE-TED:Cys157AlapOPIN-F*24 , 979 . 524 , 979 . 1−0 . 4PnTIE-TEDpDEST†25 , 779 . 025 , 761 . 7−17 . 3PnTIE-TED:Cys94AlapHisTEV†25 , 746 . 925 , 746 . 5−0 . 4BaTIE-TEDpHisTEV†28 , 541 . 128 , 523 . 7−17 . 4SaTIE-TEDpHisTEV†33 , 167 . 933 , 151 . 3−16 . 6CdTEP-TEDpHisTEV#46 , 363 . 746 , 345 . 7−18 . 0*Non-native residues remaining after 3C cleavage: N-terminal GP . †Non-native residues remaining after TEV cleavage: N-terminal GAM . ‡Non-native residues remaining: N-terminal M , C-terminal KHHHHHH . §Non-native residues remaining: C-terminal KHHHHH only . N-terminal M removed . #Non-native residue remaining after TEV cleavage: N-terminal M . Crystal structures of SfbI-A40-TED , PnTIE-TED , CpTIE-TED and CpTIE-TED:Cys138Ala were determined ( Figure 2 , Figure 2—figure supplement 1; Table 2 ) . Despite pairwise sequence identities as low as 12% ( Table 3 ) , implying no difference from chance , the overall structures of all TEDs solved to date are remarkably similar ( Figure 2—figure supplement 1A ) . The three native TED structures determined here show continuous electron density between the Cys and Gln side chains predicted to form the thioesters ( SfbI-A40-TED: Cys109-Gln261 , PnTIE-TED: Cys94-Gln247 , CpTIE-TED: Cys138-Gln267 , Figure 2B ) . Consistent with previous data ( Pointon et al . , 2010; Linke-Winnebeck et al . , 2014 ) , the thioesters are largely buried at the interface between α-helical and β-barrel subdomains . The thioester Cys backbone carbonyl and amide groups form hydrogen bonds to the Gln , and in some cases Trp , side chains of the TQxxΦWΦxζ motif of the central helix , but from the structures no obvious role of this motif for thioester bond formation is apparent . One notable difference between the TED structures is the position of the loop between the first two strands of the β-barrel , which lies adjacent to the thioester and results in very different protein surfaces around this region ( Figure 2A , C ) . Given the proximity to the thioester , we suggest that this loop may be involved in interactions that define thioester target specificity , although this has yet to be tested . Furthermore , the α-helical subdomain shows a larger degree of structural variation than the β-barrel subdomain . The structure of CpTIE-TED:Cys138Ala is essentially identical to the native protein ( Figure 2—figure supplement 1B , C ) , confirming that internal thioesters are not structural determinants ( Walden et al . , 2014 ) . 10 . 7554/eLife . 06638 . 008Figure 2 . Crystal structures of TEDs . ( A ) Cartoon representations of crystal structures of SfbI-A40-TED ( blue ) , CpTIE-TED ( burgundy ) and PnTIE-TED ( yellow ) with thioesters shown as sticks . Variable loops adjacent to the thioester are colored red , purple and green , respectively . ( B ) Close-up views of the thioesters . Residues forming the thioesters are shown overlaid with electron density ( 2mFobs − DFcalc contoured at 1 . 0σ ) . The ‘Q’ , ‘W’ and ‘ζ’ residues of the TQxxΦWΦxζ motif are also shown . ( C ) Surface representations with variable loops colored as in A . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 00810 . 7554/eLife . 06638 . 009Figure 2—figure supplement 1 . Comparison of TED structures . ( A ) Overlay of SfbI-A40-TED ( blue ) , CpTIE-TED ( burgundy ) , Pn-TIE-TED ( yellow ) , Cpa-TED2 ( pale blue , PDB:2XI9 ) and Cpa-TED1 ( grey , PDB:4C0Z ) . Thioester and TQxxΦWΦxζ motif residues are shown as sticks . ( B ) Crystal structure of CpTIE-TED:Cys138Ala with adjacent variable loop colored cyan . ( C ) Overlay of CpTIE-TED ( burgundy ) and CpTIE-TED-Cys138Ala ( orange ) . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 00910 . 7554/eLife . 06638 . 010Table 2 . X-ray data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 010 ( A ) CpTIE-TED and CpTIE-TED:Cys138AlaCpTIE-TEDCpTIE-TED:Cys138AlaIodideNativeNativeData collection Space groupP1P21212P2 Cell dimensions a , b , c ( Å ) 70 . 82 , 74 . 36 , 82 . 8198 . 42 , 110 . 72 , 68 . 7797 . 91 , 110 . 63 , 68 . 45 α , β , γ ( ° ) 107 . 32 , 104 . 32 , 98 . 6390 , 90 , 9090 , 90 , 90 Resolution ( Å ) *44 . 88–2 . 62 ( 2 . 69–2 . 62 ) 44 . 97–1 . 60 ( 1 . 64–1 . 60 ) 58 . 21–2 . 83 ( 2 . 90–2 . 83 ) Rmerge13 . 3 ( 76 . 4 ) 5 . 4 ( 74 . 4 ) 19 . 5 ( 101 . 8 ) I/σI7 . 5 ( 1 . 8 ) 31 . 6 ( 4 . 3 ) 22 . 7 ( 4 . 4 ) Completeness ( % ) Overall98 . 0 ( 97 . 3 ) 100 ( 99 . 9 ) 98 . 2 ( 97 . 3 ) Anomalous98 . 1 ( 96 . 2 ) Redundancy Overall4 . 7 ( 4 . 9 ) 18 . 1 ( 18 . 1 ) 33 . 3 ( 33 . 0 ) Anomalous17 . 5 ( 16 . 8 ) CC ( 1/2 ) ( % ) 99 . 3 ( 58 . 9 ) 100 ( 90 . 9 ) 99 . 8 ( 86 . 0 ) Refinement Resolution ( Å ) 44 . 8–2 . 62 ( 2 . 69–2 . 62 ) 44 . 97–1 . 60 ( 1 . 64–1 . 60 ) No . reflections42 , 485 ( 3115 ) 94 , 622 ( 6864 ) Rwork/Rfree19 . 6/22 . 6 ( 35 . 1/33 . 7 ) 17 . 7/21 . 0 ( 20 . 5/22 . 0 ) No . atoms Protein82606310 Ligand/ion/water36403 B-factors Protein50 . 924 . 5 Ligand/ion/water37 . 828 . 5 R . m . s deviations Bond lengths ( Å ) 0 . 0140 . 013 Bond angles ( ° ) 1 . 601 . 57 MolProbity Score1 . 11 ( 100th percentile ) 1 . 13 ( 99th percentile ) ( B ) SfbI-A40-TED and PnTIE-TEDSfbI-A40-TEDPnTIE-TEDNative/ZincNativeIodideData collection Space groupI41P41212P41212 Cell dimensions a , b , c ( Å ) 165 . 12 , 165 . 12 , 42 . 5259 . 86 , 59 . 86 , 121 . 7059 . 37 , 59 . 37 , 122 . 4 α , β , γ ( ° ) 90 , 90 , 9090 , 90 , 9090 , 90 , 90 Resolution ( Å ) †52 . 22–1 . 35 ( 1 . 39–1 . 35 ) 42 . 67–1 . 30 ( 1 . 32–1 . 30 ) 31 . 22–2 . 80 ( 2 . 95–2 . 80 ) Rmerge5 . 3 ( 75 . 4 ) 5 . 4 ( 30 . 4 ) 13 . 3 ( 35 . 7 ) I/σI18 . 7 ( 2 . 7 ) 33 . 8 ( 7 . 4 ) 24 . 9 ( 13 . 0 ) Completeness ( % ) Overall99 . 9 ( 99 . 1 ) 93 . 6 ( 62 . 7 ) 100 ( 100 ) Anomalous99 . 6 ( 97 . 5 ) 100 ( 100 ) Redundancy Overall9 . 2 ( 9 . 1 ) 23 . 9 ( 11 . 9 ) 32 . 8 ( 31 . 9 ) Anomalous4 . 6 ( 4 . 6 ) 18 . 1 ( 16 . 6 ) CC ( 1/2 ) ( % ) 99 . 9 ( 85 . 2 ) 100 ( 96 . 2 ) 99 . 6 ( 99 . 2 ) Refinement Resolution ( Å ) 52 . 22–1 . 35 ( 1 . 39–1 . 35 ) 53 . 71–1 . 30 ( 1 . 33–1 . 30 ) No . reflections119869 ( 8679 ) 48 , 978 ( 2374 ) Rwork/Rfree13 . 1/15 . 3 ( 22 . 6/22 . 9 ) 11 . 9/15 . 1 ( 12 . 6/15 . 9 ) No . atoms Protein33891848 Ligand/ion/water470252 B-factors Protein23 . 012 . 5 Ligand/ion/water39 . 426 . 9 R . m . s deviations Bond lengths ( Å ) 0 . 0120 . 012 Bond angles ( ° ) 1 . 531 . 50 MolProbity Score1 . 06 ( 99th percentile ) 1 . 11 ( 98th percentile ) *The highest resolution shell is shown in parenthesis . †The highest resolution shell is shown in parenthesis . 10 . 7554/eLife . 06638 . 011Table 3 . Pairwise sequence identities of TEDsDOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 011SfbI-A40SfbI-A346SfbI-A20GfbAFbaBCpa-TED2CpTIECodTIEPnTIEBaTIESaTIECdTIECpa-TED1SfbI-A4054 . 254 . 252 . 327 . 125 . 827 . 424 . 312 . 07 . 67 . 67 . 250 . 0SfbI-A34654 . 249 . 164 . 322 . 721 . 431 . 023 . 49 . 96 . 96 . 78 . 046 . 0SfbI-A2054 . 249 . 156 . 426 . 223 . 727 . 721 . 38 . 78 . 25 . 07 . 652 . 2GfbA52 . 364 . 356 . 422 . 721 . 029 . 726 . 09 . 56 . 66 . 77 . 550 . 2FbaB27 . 122 . 726 . 222 . 719 . 121 . 917 . 719 . 57 . 88 . 05 . 524 . 8Cpa-TED225 . 821 . 423 . 721 . 019 . 127 . 132 . 616 . 99 . 55 . 06 . 220 . 3CpTIE27 . 431 . 027 . 729 . 721 . 927 . 124 . 420 . 18 . 46 . 77 . 226 . 8CodTIE24 . 323 . 421 . 326 . 017 . 732 . 624 . 419 . 69 . 04 . 76 . 625 . 4PnTIE12 . 09 . 98 . 79 . 519 . 516 . 920 . 119 . 65 . 25 . 16 . 021 . 6BaTIE7 . 66 . 98 . 26 . 67 . 89 . 58 . 49 . 05 . 29 . 311 . 89 . 1SaTIE7 . 66 . 75 . 06 . 78 . 05 . 06 . 74 . 75 . 19 . 39 . 27 . 0CdTIE7 . 28 . 07 . 67 . 55 . 56 . 27 . 26 . 66 . 011 . 89 . 28 . 0Cpa-TED150 . 046 . 052 . 250 . 224 . 820 . 326 . 825 . 421 . 69 . 17 . 08 . 0Bold values correspond to pairs of TEDs with known structures . Values highlighted in grey indicate that a pairwise alignment was not meaningful; the values given correspond to pairwise identities as calculated from the alignment of 54 TEDs ( Figure 1—figure supplement 1 ) . Alignments of randomized sequences commonly resulted in pairwise identities of 10–20% . Pairwise alignments were produced with BioEdit using a GONNET similarity matrix . We next tested the hypothesis that TEDs can target host proteins , forming intermolecular covalent bonds in a thioester-dependent manner . Of our panel of twelve TED proteins , the streptococcal protein SfbI is the most thoroughly characterized . SfbI mediates internalization of bacteria by host cells through binding to the extracellular protein fibronectin ( Schwarz-Linek et al . , 2006 ) . The N-terminal domain of SfbI ( revealed here to be a TED ) is not strictly required for this process , but defines the uptake mechanism and is a determinant of intracellular bacterial survival ( Rohde et al . , 2011 ) . It has also been reported to interact with fibrinogen ( Katerov et al . , 1998 ) . We investigated if this interaction involves thioester-dependent covalent bond formation by mixing purified TEDs with fibrinogen and analyzing the resulting samples by SDS-PAGE . For all three SfbI-TED variants ( SfbI-A40-TED , SfbI-A346-TED , SfbI-A20-TED ) , and FbaB-TED , a new band is present at a molecular mass consistent with an adduct of each TED with one fibrinogen subunit ( Figure 3A ) . Interestingly , a group of protein bands corresponding to the heterogeneous fibrinogen Aα chain ( Mosesson et al . , 1972 ) show depletion in the adduct samples , which is clearest for SfbI-A20-TED with the conditions used here . This suggested that SfbI-TED and FbaB-TED were covalently binding to the Aα chain of fibrinogen . When fibrinogen is incubated with the Cys/Ala variants of the SfbI-TEDs and FbaB-TED , no adduct bands are observed ( Figure 3B ) , suggesting their formation critically depends on the thioesters . Other TEDs did not form a fibrinogen adduct under the conditions of the assay , indicating this activity is not a generic , non-specific property of TEDs . 10 . 7554/eLife . 06638 . 012Figure 3 . SDS-PAGE analysis of TED:fibrinogen ( Fg ) adduct formation . ( A ) Stable adducts between fibrinogen and TEDs revealed by SDS-PAGE . Red asterisks , adduct bands . ( B ) Cys-to-Ala mutants of TEDs do not result in adduct formation after incubation with Fg . ( C ) Schematic representation of the intermolecular isopeptide bond formed between a Lys residue of fibrinogen Aα and the Gln residue of the TED . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 01210 . 7554/eLife . 06638 . 013Figure 3—figure supplement 1 . Effect of acetylation of Lys side chains on fibrinogen ( Fg ) :TED adduct formation . The hash symbol indicates protein pre-incubated with sulfo-NHS acetate . Red asterisks highlight the positions of adduct bands . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 013 Tryptic-digest nanoLC-MSE of excised adduct gel bands revealed the respective TEDs and the fibrinogen Aα chain as top hits following database searches ( Supplementary file 1 ) . Peptides from the fibrinogen γ ( but not the Bβ ) chain were observed at comparably low scores . This can be explained by the presence of a fibrinogen γ dimer , commonly observed in fibrinogen samples , which has a mass similar to the TED-Aα cross-linked protein ( Figure 4—figure supplement 1 ) . The MS results implicate a site in the fibrinogen Aα chain as the target of SfbI- and FbaB-TEDs . Acetylation of primary amines in fibrinogen abrogates TED/fibrinogen adduct formation ( Figure 3—figure supplement 1 ) , suggesting Lys side chains as the likely nucleophiles to react with the thioester . This would result in formation of an intermolecular isopeptide bond to the side chain carbonyl group of the TED Gln in the ΦQζΦΦ motif ( Figure 3C ) . To assess the specificity of SfbI-TED and FbaB-TED binding to fibrinogen , pull-down assays using human blood plasma were performed . TEDs were immobilized using isopeptide domain ( IPD ) complementation; a strategy that allowed us to use a toolbox approach for many constructs that were used in both pull-down and cell binding ( described below ) experiments . IPD complementation relies on the spontaneous amide bond formation between a truncated , or split , IPD of the streptococcal FbaB protein that lacks the C-terminal β-strand ( sIPD ) , and a peptide representing this missing strand ( Zakeri et al . , 2012 ) . The peptide , named here isopep-tag ( iPT ) is used as an expression tag fused as bait to the C-termini of TEDs ( TED-iPT ) while the sIPD is immobilized on sepharose beads ( Figure 4A ) . The advantage of this covalent pull-down strategy is the ability to greatly reduce the amount of non-specific binding . Each of the SfbI-TEDs pulled down fibrinogen from this complex biologically relevant sample by forming an adduct with fibrinogen Aα , although to differing degrees ( Figure 4B; Figure 4—figure supplement 1 ) . For FbaB-TED , little fibrinogen was detectable and the protein appeared to bind non-covalently to albumin . All TED Cys/Ala variants failed to pull down fibrinogen from plasma . There is no evidence for covalent binding of TEDs to any other protein in plasma pull-downs . Gel bands present in samples eluted from the beads were analyzed by MS and contained sequences matching fibrinogen , or the TED-sIPD pull-down constructs . For FbaB , the albumin band contained no TED peptides . 10 . 7554/eLife . 06638 . 014Figure 4 . TED:fibrinogen ( Fg ) adduct formation in plasma pull down assays . ( A ) Schematic representation of the isopeptide domain ( green ) complementation used in the pull-down assays . Pink triangle , tobacco etch virus protease cleavage site . ( B ) SfbI and FbaB-TEDs pull down fibrinogen from blood plasma , forming covalent adducts ( red asterisks ) . ‘a’ denotes bands corresponding to human serum albumin . Bands below 31 kDa are breakdown products of tagged TEDs . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 01410 . 7554/eLife . 06638 . 015Figure 4—figure supplement 1 . TED:fibrinogen ( Fg ) adduct formation in plasma pull down assays . An overloaded gel of the plasma pull-down better shows adduct formation between fibrinogen and TEDs , and its dependence on the thioester . P = plasma depleted of the majority of albumin by blue sepharose chromatography . Red asterisks , adduct bands , ‘a’ and ‘d’ denote human serum albumin and Fg γ dimer , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 015 The fibrinogen Aα Lys residue involved in covalent bond formation was identified by searching both low and high trap collision energy nanoLC-MSE spectra for precursor and fragment ion masses consistent with calculated values for theoretical cross-links . This analysis was carried out for gel bands obtained from experiments using isolated fibrinogen and , for SfbI-A40-TED and SfbI-A20-TED , also for plasma pull-down experiments . Multiple precursor ions and peptide fragments were recovered , all of which were consistent with a single candidate nucleophilic residue , fibrinogen Aα-Lys100 ( Lys81 in the mature protein ) ( Figure 5; Supplementary file 1 ) , which formed covalent links with the TED Gln residues of the ΦQζΦΦ motifs . 10 . 7554/eLife . 06638 . 016Figure 5 . Mass spectrometric identification of the SfbI-TED and FbaB-TED target residue in fibrinogen ( Fg ) . ( A ) Precursor ion masses identified in tryptic-digest nanoLC-MSE of excised adduct gel bands for different samples . ( B ) Several charged states are observed in nanoLC-MSE of cross-linked precursor peptides obtained by tryptic digestion of adducts formed by TEDs with fibrinogen or in plasma pull-downs . ( C ) Fragmentation nanoLC-MSE spectra of the cross-linked precursor obtained for SfbI-A40-TED in a plasma pull-down . The low and high mass range spectrum is shown on the left and right , respectively . Fragmentations observed in nanoLC-MSE are indicated in the schematic drawings above the spectra by hooks , y-series on top of sequences , b-series below sequences . Numbers correspond to positions in the cross-linked fragments; red , SfbI-A40-TED; blue , fibrinogen Aα . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 01610 . 7554/eLife . 06638 . 017Figure 5—figure supplement 1 . Acetylation of Lys residues in the fibrinogen Aα subunit in absence and presence of SfbI-A40-TED . Sequence coverage by peptide mass fingerprinting is indicated by colors mapped onto the sequence of human fibrinogen subunit Aα . Grey , signal sequence ( not present in mature fibrinogen Aα ) ; red , trypsin coverage; blue and green , additional coverage obtained by endoproteinase GluC and chymotrypsin digestion , respectively; black , no coverage . All Lys residues are highlighted in boldface . Underlined K indicates that the residue was observed in its acetylated form; non-underlined K were observed only in the non-acetylated form . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 01710 . 7554/eLife . 06638 . 018Figure 5—figure supplement 2 . Structure of fibrinogen with important sites labeled . The positions of Lys100 ( magenta ) and RGDF platelet-binding motif ( cyan ) are highlighted on the crystal structure of Fg ( PDB:3GHG ) . α , β and γ chains are colored blue , green and red , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 018 To further support specific targeting of Aα-Lys100 by SfbI-A40-TED , we subjected fibrinogen to acetylation in the absence or presence of SfbI-A40-TED . Acetylation results in specific modification of solvent-accessible Lys ε-amine groups . Following proteolytic digests of bands corresponding to fibrinogen Aα and the SfbI-A40-TED:fibrinogen Aα covalent adduct , we observed near-complete coverage of the Aα sequence ( Figure 5—figure supplement 1 ) . Of all Aα-Lys residues , only Lys100 was acetylated in the free , but not the bound form of fibrinogen . One other Lys residue , Lys446 , was not covered by the analyses of either free or bound fibrinogen . nanoLC-MSE spectra were scrutinized for potential cross-links between the tryptic peptide containing this Lys residue and TEDs , but no matching precursor peptide masses could be found . All available evidence supports reaction of SfbI and FbaB TEDs with fibrinogen Aα-Lys100 exclusively . If binding were not specific for a single Lys , we would expect to observe multiple binding of TEDs to different Lys residues on a single fibrinogen molecule . We do not see evidence for such higher-order complexes in SDS-PAGE gels . Further , if only one TED can covalently bind to fibrinogen , but through different Lys residues , this would result in a mixed population of cross-linked species with the same molecular mass , but different acetylation and tryptic digest patterns . There is no evidence to support such events in our exhaustive MS analyses , which only support modification of Aα-Lys100 . Aα-Lys100 is presented on the surface of the α-helical coiled-coil region of fibrinogen ( Figure 5—figure supplement 2 ) . It does not participate in the cross-linking of fibrinogen that results in fibrin formation ( Sobel and Gawinowicz , 1996 ) , but is a plasmin cleavage site ( Kirschbaum and Budzynski , 1990 ) . This suggests SfbI-TEDs may also bind to fibrin with implications for fibrinolysis . Aα-Lys100 also lies in the vicinity of the integrin-binding RGDF motif of fibrinogen involved in platelet interactions ( Ugarova et al . , 1993 ) . To determine a role for thioester-mediated binding to fibrinogen by bacteria , strains of the model Gram-positive bacterium Lactococcus lactis expressing either SfbI-A40 or the corresponding SfbI-A40:Cys109Ala variant were produced . Immunogold labeling confirmed the presence of wildtype and variant SfbI at similar levels on the surface of L . lactis ( Figure 6—figure supplement 1 ) . Since fibrinogen Aα-Lys100 is not involved in fibrin formation , it was possible to use a fibrin-based assay for visualization of bacterial binding by electron microscopy . L . lactis expressing SfbI-A40 , but not bacteria expressing the Cys109Ala variant , show intimate adherence to both single fibrin fibrils ( Figure 6 ) and fibrin clots , with the latter reminiscent of biofilms ( Figure 6—figure supplement 2 ) . Similar results were obtained for L . lactis expressing SfbI-A20 and SfbI-A20:Cys97Ala ( Figure 6—figure supplement 2 ) . 10 . 7554/eLife . 06638 . 019Figure 6 . Thioester-dependent binding of bacteria to fibrin . ( A ) and ( B ) fibrin binding by L . lactis expressing SfbI-A40 . ( C ) SfbI-A40:Cys109Ala does not confer fibrin-binding activity to L . lactis . ( D ) Control of L . lactis transformed with empty pOri23 plasmid . All scale bars 2 μm , except in the insert in panel B , 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 01910 . 7554/eLife . 06638 . 020Figure 6—figure supplement 1 . Immunogold labeling of SfbI-A40 and SfbI-A40:Cys109Ala on L . lactis surfaces . A and B L . lactis expressing SfbI-A40 . ( C ) L . lactis expressing SfbI-A40:Cys109Ala . ( D ) L . lactis transformed with empty pOri23 plasmid shows essentially no cross-reactivity of the antibody with the bacterial surface . All scale bars 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 02010 . 7554/eLife . 06638 . 021Figure 6—figure supplement 2 . Fibrin clot binding of L . lactis expressing SfbI variants . A control of L . lactis transformed with empty pOri23 plasmid is also shown . All scale bars 10 μm except in the right column , 2 μm . White arrows point to bacteria in the experiments with the Cys-to-Ala mutants and the empty plasmid . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 021 Fibrinogen is an abundant plasma protein produced mainly by hepatocytes and required for hemostasis . It is however also produced by extrahepatic epithelial cells , and is present in the extracellular matrix ( Pereira et al . , 2002 ) , the provisional matrix in wound healing ( Clark et al . , 1982 ) and in inflamed tissues ( Lawrence and Simpson-Haidaris , 2004 ) . Fibrinogen expression is upregulated during inflammatory acute phase response in hepatocytes , but also in epithelial cells by the synergistic action of corticosteroids and interleukin-6 ( Snyers et al . , 1990; Haidaris , 1997 ) . This inflammatory response can be induced in cell culture by incubation with dexamethasone and interleukin-6 . Under these conditions human A549 lung epithelial cells express and secrete fibrinogen , which remains associated with cell surfaces ( Guadiz et al . , 1997 ) . We investigated if SfbI-TEDs interact with such induced A549 cells . SfbI-TED binding to unfixed , viable , adherent cells was visualized by conjugating TED-iPT constructs via IPD complementation to GFP fused to sIPD ( Zakeri et al . , 2012 ) ( Figure 7A ) . SfbI-A40-TED bound to A549 cells only after they had been pre-incubated with dexamethasone and interleukin-6 ( Figure 7B ) . SfbI-A40-TED:Cys109Ala binding to A549 cells was not detectable with or without induction . To obtain direct evidence for fibrinogen binding , Western blot analyses of cell homogenates using an anti-fibrinogen α antibody were performed . These confirmed upregulation of fibrinogen expression following exposure of cells to dexamethasone/interleukin-6 , in agreement with published data ( Snyers et al . , 1990; Haidaris , 1997 ) . Critically , in homogenates from induced cells incubated with SfbI-A40-TED , a second band is detected of a molecular mass consistent with an SfbI-A40-TED:fibrinogen Aα adduct ( Figure 7C ) . The lack of this band in homogenates from induced cells incubated with SfbI-A40-TED:Cys109Ala confirms formation of this adduct is dependent on the thioester . These data show that SfbI-A40-TED adherence to A549 cells under conditions mimicking inflammation depends on thioester-mediated covalent binding to cell surface-associated fibrinogen . 10 . 7554/eLife . 06638 . 022Figure 7 . Thioester-dependent binding of SfbI-A40-TED to A549 cells . ( A ) Schematic representation of the covalent cell labeling experiment . After incubation with cells , isopep-tag ( iPT ) labeled TED is detected by GFP fused to sIPD . ( B ) SfbI-A40-TED and SfbI-A40-TED:Cys109Ala binding to A549 cells before and after incubation with interleukin-6 ( IL6 ) and dexamethasone ( dex ) . No TED , sIPD-GFP controls . Cell nuclei appear in blue . Scale bars , 200 μm . ( C ) Western blot of whole-cell extracts of induced and non-induced A549 cells , probed with an anti-fibrinogen α antibody . The same membrane was re-probed with an anti-β-actin antibody ( shown in separate panel ) . MW , molecular weight markers; TED , cells incubated with SfbI-A40-TED-iPT; TED C-A , cells incubated with SfbI-A40-TED:Cys109Ala-iPT . DOI: http://dx . doi . org/10 . 7554/eLife . 06638 . 022 Pathogenic bacteria disseminated in the blood stream directly or indirectly target fibrinogen and fibrin , resulting in immune evasion , induction of inflammatory response , or platelet activation ( Sun , 2006; Rivera et al . , 2007 ) . Two well-studied examples for bacterial fibrinogen-binding proteins involved in invasive infections are clumping factor A of S . aureus ( Ganesh et al . , 2008 ) and the M1 protein of S . pyogenes ( Macheboeuf et al . , 2011 ) . In this study we have not investigated the role of SfbI binding to fibrinogen during bacterial infection . However , S . pyogenes is known to target epithelial and endothelial cells for adhesion and invasion , and to cause invasive infections involving dissemination in the blood stream . Therefore our experiments using fibrin and epithelial cells represent meaningful biological models to probe the role of thioester-dependent covalent interaction in infections . Perhaps most significantly our data suggest that fibrinogen may have an unappreciated role as a host cell-surface associated adhesion target for bacteria . Potentially SfbI through its fibrinogen-binding activity may confer a tropism for inflamed tissue or the provisional matrix of healing wounds . To mediate strong attachment , bacterial adhesion complexes typically present extensive , multivalent binding interfaces . Our results show that a wide and diverse range of Gram-positive bacteria have evolved a mechanism for covalent bond formation with specific host factors via a pathogen-encoded reactive internal thioester . Thioester proteins could form a continuous covalent bridge between the bacterial cell envelope and host adhesion targets . This would provide rapid , mechanically very strong attachment that may be particularly advantageous under conditions of shear stress . Lack of similarity in the amino acid sequences of TEDs , together with the observation that only a small subset of those tested here bind fibrinogen , suggests there are undiscovered targets of these proteins . Thioester-dependent covalent adhesion may be a common molecular mechanism in host-microbe interactions , playing a key role in host colonization by both commensal and pathogenic Gram-positive bacteria . As such , TEDs are potentially attractive targets for the design of small molecules to inhibit infection , but they also present an opportunity to engineer beneficial interactions . TEDs hold promise as reactive protein fusion tags for use in tissue engineering , diagnostics or as tools for cell and molecular biology . While we think it is likely that many thioester-containing bacterial proteins will play a role in host-microbe interactions , it is also possible that they function in other processes such as bacteria–bacteria interactions and biofilm formation .
The multiple sequence alignment ( Figure 1—figure supplement 1 ) was produced with MAFFT/L-INS-i ( Katoh et al . , 2002 ) , refined and formatted using BioEdit ( Hall , 1999 ) taking into account structural data and secondary structure predictions by JPred ( Cole et al . , 2008 ) . Five E . coli expression vectors were used to encode TED proteins: pOPIN-F , pOPIN-E , pHisTEV , pDEST and pDEST-iPT . Expression from pOPIN-F results in an N-terminal His6-tag cleavable by 3C protease and from pOPIN-E , a non-cleavable C-terminal His6-tag ( Berrow et al . , 2007 ) . pHisTEV is derived from pET30a ( Liu and Naismith , 2009 ) and pDEST from pDEST14 . Both incorporate an N-terminal His6-tag cleavable by TEV protease . The pDEST-iPT vector is derived from pDEST with a C-terminal extension consisting of a trypsin cleavage site followed by the iPT sequence AHIVMVDAYK , representing the C-terminal β-strand of the FbaB CnaB2 domain ( Zakeri et al . , 2012 ) . DNA encoding the TEDs of SfbI-A40 ( Asp63-Pro270 ) , SfbI-A20 ( Asp51-Pro262 ) , GfbA ( Asp51-Pro256 ) , PnTIE ( Gly42-Pro258 ) and CdTEP ( Glu171-Asp578 ) were amplified from genomic DNA . The SaTIE ORF is incorrectly annotated in the published genome ( Kos et al . , 2012 ) , appearing fragmented into three ORFs , likely due to short identical sequence repeats . The complete ORF comprises 1223 residues . ORFs I3GYQ5 , I3HJ59 and I3HKL2 represent residues 1–179 , 172–232 , and 281–1223 , respectively . Missing residues and the correct length of the repeat region were obtained through cloning and DNA sequencing . The SaTIE-TED construct encompassed Gln254-Ala514 ( bases 48 , 874–49 , 658 in supercontig 1 . 1 , GenBank AHBV01000001 . 1 ) . Constructs of SfbI-A346-TED ( Asp51-Pro260 ) and FbaB ( Gly35-Asp365 ) in pDEST were created by the SPoRT laboratory ( University of St Andrews ) ( Oke et al . , 2010 ) . The FbaB construct was truncated to FbaB-TED ( Gly35-Pro247 ) by mutation of Trp248 to a stop codon . DNA encoding the TEDs of CpTIE and CpTIE:Cys138Ala ( Ser92-Pro277 ) , CodTIE and CodTIE:Cys157Ala ( Ser110-Pro327 ) , Cpa-TED2 ( Ser390-Pro272 ) and FbaB:Cys94Ala ( Ser56-Pro241 ) were synthesized and supplied in pUC57 by GenScript ( USA ) . BaTIE-TED ( Glu35-Glu288 ) was amplified from a synthetic gene created by Biomatik ( Canada ) . Where required , NcoI and BamHI sites were incorporated at the 5′ and 3′ ends respectively . Amplified DNA was inserted into pHisTEV ( BaTIE-TED , SaTIE-TED and CdTEP ) or pDEST ( all others ) . Cys to Ala point mutants for the SfbI-A40-TED , SfbI-A346-TED , SfbI-A20-TED , FbaB-TED ( Gly35-Pro247 ) and PnTIE-TED were created by site-directed mutagenesis . A selection of the TEDs were sub-cloned into pOPIN-F or pOPIN-E by In-Fusion ( Clontech , France ) cloning ( Berrow et al . , 2007 ) . A truncated version of FbaB-TED ( Ser56-Pro241 ) was also sub-cloned into pOPIN-F and pOPIN-E . The three SfbI-TEDs and FbaB-TED ( Gly35-Pro247 ) , and their mutants , were also cloned into pDEST-iPT . For mass spec identification of fibrinogen cross-links , a T231K mutant of FbaB-TED ( Ser56-Pro241 ) in pOPIN-E was synthesised by GenScript ( Piscataway , NJ , USA ) . This allowed generation of a shorter C-terminal Gln234-containing tryptic peptide ( NLQAVISVEP ) , more amenable for mass spec analysis . The sIPD construct was derived from pDEST-Cna114 ( residues 1–113 of the FbaB CnaB2 in pDEST14 ) ( Zakeri et al . , 2012 ) . This was shortened to residues 22–107 by deletion of residues 1–21 and mutation of Lys108 to a stop codon . A Cys residue was added N-terminal to the His6-tag , allowing immobilisation on a solid support . The sIPD-GFP construct was generated by insertion of a BamHI site between the His6-tag and sIPD , and cloning of GFP ( residues 2–238 ) between BamHI and NcoI . All primers used for this study are detailed in the Supplementary file 2 . TED proteins were produced in E . coli BL21 ( DE3 ) grown in Luria Broth at 37°C until A600 0 . 5–0 . 8 . Cultures were induced with 0 . 5–1 mM isopropyl 1-thio-β-d-galactopyranoside and incubated at 18°C for 16–20 hr . TED proteins for crystallization , MS and fibrinogen binding assays were expressed from pDEST , pHisTEV , pOPIN-E or pOPIN-F . Cell pellets were re-suspended in 50 mM Tris–HCl or 50 mM bis-tris ( pH 7 . 5 or 6 . 8 respectively ) , 500 mM NaCl , 50 mM glycine , 5% ( vol/vol ) glycerol , 20 mM imidazole , with one EDTA-free protease inhibitor cocktail tablet ( Roche ) per 40 ml of buffer . Cells were lysed by sonication . For proteins expressed with a cleavable N-terminal His6-tag ( pDEST , pHisTEV , pOPIN-F ) , clarified lysate was applied to a Ni2+-IMAC column ( GE Healthcare , UK ) and bound proteins were step eluted with buffer ( as above ) supplemented with 500 mM imidazole . The eluate was loaded onto a Hi-Load 26/60 Superdex 75 gel filtration column ( GE Healthcare ) pre-equilibrated with 20 mM HEPES ( pH 7 . 5 ) , 150 mM NaCl . Fractions containing TED proteins were incubated with appropriate protease ( 1:50 , wt/wt ) at 4°C for 16–20 hr . Imidazole was added to 40 mM and the sample applied to a Ni2+-immobilized column . Cleaved TED protein was collected in the flow through , which was concentrated and injected onto the Superdex 75 column pre-equilibrated with either 20 mM HEPES ( pH 7 . 5 ) , 150 mM NaCl , or 20 mM bis-tris ( pH 6 . 0 ) , 150 mM NaCl . Fractions containing purified TED proteins were concentrated to ∼5–10 mg/ml for binding assays and to ∼10–30 mg/ml for crystallization . Protein concentration was determined by A280 . For proteins expressed with a non-cleavable C-terminal His6-tag ( pOPIN-E ) , only the initial two-step purification by Ni2+-IMAC , and gel filtration chromatography was carried out . Gel filtration buffer was 20 mM bis-tris ( pH 6 . 0 ) , 150 mM NaCl . TED proteins for cell binding assays and pulldowns from plasma were expressed from pDEST-iPT . Cell pellets were resuspended in phosphate-buffered saline ( PBS ) ( pH 6 . 0 ) , 10 mM imidazole , with one EDTA-free protease inhibitor cocktail tablet ( Roche , UK ) per 50 ml of buffer . Cells were lysed by sonication . Clarified lysate was applied to a Ni2+-IMAC column and bound proteins eluted with PBS ( pH 6 . 0 ) , 250 mM imidazole . The eluate was dialyzed against PBS ( pH 6 . 0 ) at 4°C to remove imidazole . The His6-tag was cleaved with His6-tagged TEV protease ( 1:50 , wt/wt ) for 16 hr at 4°C . Imidazole was added to 20 mM and the sample applied to a Ni2+-immobilized column . Purified TED proteins were collected in the flow through and concentrated to 2–4 mg/ml as determined by A280 . The sIPD and sIPD-GFP proteins were expressed from pDEST and purified as above , except that PBS ( pH 7 . 2 ) buffer was used throughout , and the His6-tag was not removed . Protein intact masses were determined by LC-MS on a Synapt G2 mass spectrometer coupled to an Acquity UPLC system ( Waters , Milford , MA , USA ) . 50–100 pmol of protein were injected onto an Aeris WIDEPORE 3 . 6μ C4 column ( Phenomenex , UK ) and eluted with a 10–90% acetonitrile gradient over 13 min ( 0 . 4 ml/min ) . The spectrometer was controlled by the Masslynx 4 . 1 software ( Waters ) and operated in positive MS-TOF and resolution mode with capillary voltage of 2 kV , cone voltage , 40 V . Leu-enkephalin peptide ( 2 ng/ml , Waters ) was infused at 10 µl/min as a lock mass and measured every 30 s . Spectra were generated in Masslynx 4 . 1 by combining scans and deconvoluted using the MaxEnt1 tool ( Waters ) . BaTIE-TED , SaTIE-TED and CdTEP-TED ( 40 μl at 1 mg/ml in PBS ) were incubated with 6 μl 2 M methylamine ( in PBS ) , pH 7 . 6–8 . 0 at 25°C for 15 min . The pH was adjusted to 7 . 0 , 4 μl 200 mM iodoacetamide was added , and the mixture incubated for 60 min at 25°C in darkness . 4 μl 200 mM DTT was added to quench excess IAA . Protein solutions ( 5 µl ) were dialyzed into 50 mM ammonium bicarbonate , trypsin added and the samples incubated at 37°C overnight . The acidified peptides were separated on an Acclaim PepMap 100 C18 trap and RSLC C18 column ( Thermo Fisher Scientific , UK ) , using a nanoLC Ultra 2D plus loading pump and nanoLC as-2 autosampler ( Eskigent , UK ) . The peptides were eluted with a gradient of increasing acetonitrile . The eluent was sprayed into a TripleTOF 5600 electrospray tandem mass spectrometer ( ABSciex , UK ) and analyzed in Information Dependent Acquisition mode , performing 250 ms of MS followed by 100 ms MS/MS analyses on the 20 most intense peaks . The MS/MS data file generated in PeakView ( ABSciex ) was analyzed using the Mascot algorithm ( Matrix Science , UK ) , against an internal database containing the TED sequences as an error-tolerant search , which considers all modifications in Unimod . CpTIE-TED:Cys138Ala crystals were obtained from pOPIN-F-expressed protein , purified at pH 7 . 5 , at ∼28 mg/ml ( iodide dataset ) or ∼14 mg/ml ( native dataset ) . Crystals grown in 0 . 2 M sodium iodide , 0 . 1 M bis-tris propane ( pH 6 . 5 ) , 20% ( wt/vol ) PEG 3350 , were cryoprotected with mother liquor plus 15% ( vol/vol ) ethylene glycol . These were used for SAD X-ray data collection and phasing . Crystals grown in 0 . 2 M potassium thiocyanate , 0 . 1 M bis-tris propane ( pH 6 . 5 ) , 20% ( wt/vol ) PEG 3350 , were cryoprotected with mother liquor plus 15% ( vol/vol ) ethylene glycol . These were used for native X-ray data collection . CpTIE-TED crystals were obtained from pOPIN-F-expressed protein , purified at pH 6 . 0 , at ∼26 mg/ml . Crystals grown in 0 . 2 M tri-potassium citrate , 20% ( wt/vol ) PEG 3350 were cryoprotected with mother liquor plus 30% ( vol/vol ) ethylene glycol . PnTIE-TED crystals were obtained from pDEST-expressed protein , purified at pH 7 . 5 , at ∼6 . 5 mg/ml ( iodide dataset ) or ∼13 mg/ml ( native dataset ) . Crystals grown in 0 . 1 M sodium acetate ( pH 4 . 6 ) , 25% ( wt/vol ) PEG 3000 , were cryoprotected with mother liquor plus 0 . 8 M potassium iodide and 25% ( vol/vol ) glycerol . These were used for SAD data collection and phasing . Crystals grown in the Morpheus Screen ( Molecular Dimensions , UK ) condition H3 , ( 0 . 02 M sodium l-glutamate , 0 . 02 M dl-alanine , 0 . 02 M glycine , 0 . 02 m dl-lysine , 0 . 02 M dl-serine , 0 . 1 M MES/imidazole [pH 6 . 5] , 10% [wt/vol] PEG 4000 , 20% [vol/vol] glycerol ) , which served as the cryoprotectant also , were used for native data collection . SfbI-A40-TED crystals were obtained from pOPIN-F-expressed protein , purified at pH 6 . 0 , at ∼20 mg/ml . Crystals grown in 0 . 1 M zinc acetate , 0 . 1 M HEPES ( pH 7 . 5 ) , 14% ( wt/vol ) PEG 8000 , were cryoprotected with mother liquor plus 20% ( vol/vol ) glycerol . Diffraction data were collected at the Diamond Light Source ( UK ) using beamlines i02 ( CpTIE-TED , CpTIE-TED:Cys138Ala , PnTIE-TED ) and i04 ( SfbI-A40-TED ) . Data were processed using Xia2 ( CpTIE-TED WT , Cys138Ala , SfbI-A40-TED ) or iMOSFLM and Aimless in CCP4 ( PnTIE-TED ) ( Winter , 2010; Winn et al . , 2011; Evans and Murshudov , 2013 ) . Structures of CpTIE-TED:Cys138Ala , PnTIE-TED and SfbI-A40-TED were solved by SAD phasing using Phenix AutoSol , which also built initial models ( Adams et al . , 2010 ) . For CpTIE-TED:Cys138Ala and PnTIE-TED , the calculated phases were applied and extended to the native data using PARROT ( Cowtan , 2010 ) and a model built in the high resolution data using BUCCANEER ( Cowtan , 2006 ) . For CpTIE-TED , the structure was determined by molecular replacement using PHASER ( McCoy et al . , 2007 ) with a single chain of the CpTIE-TED:Cys138Ala structure as a search model . Final models were produced through iterative rounds of refinement using REFMAC5 ( Murshudov et al . , 2011 ) and manual rebuilding with COOT ( Emsley et al . , 2010 ) . Translation-Liberation-Screw ( TLS ) and non-crystallographic symmetry restraints were used for CpTIE-TED , whilst only TLS was used for CpTIE-TED:Cys138Ala . TLS groups were based on the α-helical and β-strand subdomains . The β-strand domain comprises Ser100-Gly162 and Pro251-Thr276; the α-helical domain Ser163-Ile250 . In Chain C of the CpTIE-TED:Cys138Ala model ( PDB:5a0d ) , it was clear from the electron density that the α-helical domain is somewhat flexible and can be modeled in two distinct positions . For SfbI-A40-TED and PnTIE-TED data , anisotropic B-factor refinement was used . Data collection , phasing and refinement statistics are shown in Table 2 . Structure validation was performed using MOLPROBITY ( Chen et al . , 2010 ) and COOT . Analyses indicated that 98/98/99/97% and 0 . 19/0 . 13/0/0% of the residues were in the favored and non-favored Ramachandran regions for the CpTIE-TED , CpTIE-TED:Cys138Ala , SfbI-A40-TED and PnTIE-TED models respectively . Lyophilized human fibrinogen ( Sigma , UK ) was reconstituted to 2 mg/ml in reaction buffer ( 20 mM HEPES [pH 7 . 5] , 150 mM NaCl ) . TED protein purified in 20 mM bis-tris ( pH 6 . 0 ) , 150 mM NaCl was diluted to 10 µM in reaction buffer . Equal volumes of fibrinogen and TED were mixed and left at RT for 60 min . Controls were performed with either fibrinogen or TED replaced with reaction buffer . Samples were analyzed by SDS-PAGE . Bands corresponding to the TED-fibrinogen Aα adduct were excised for analysis . sIPD protein was immobilized on SulfoLink Coupling Resin ( Thermo Fisher Scientific ) equilibrated in 50 mM Tris , 5 mM EDTA , pH 8 . 5 by reaction at 37°C for 45 min . Unreacted iodoacetyl groups were quenched with 50 mM L-cysteine in PBS ( pH 7 . 2 ) at 37°C for 30 min . After washing ( PBS , pH 7 . 2 ) the resin was incubated with iPT-TED at 37°C for 30 min . Excess iPT-TED was removed by washing ( PBS ) . The resin was incubated with 1 ml plasma ( TCS Biosciences , UK , human plasma mixed pool , citrated ) at 37°C for 60 min . For SfbI-A20-TED , prior to this step the plasma was partially depleted of albumin by applying it to a 1 ml HiTrap Blue Sepharose column ( GE Healthcare ) pre-equilibrated in PBS ( pH 7 . 2 ) . Non-covalently bound proteins were removed by washing ( PBS ) . Protein complexes were cleaved from the resin using TEV protease in PBS ( pH 7 . 2 ) , 1 mM DTT , 0 . 5 mM EDTA , at 37°C for 60 min . Samples were analyzed by SDS-PAGE . Bands corresponding to the TED-fibrinogen Aα adduct were excised for analysis . Excised bands from TED-fibrinogen and TED-plasma binding reactions were digested with trypsin according to standard procedures . Peptides were extracted and analyzed by nanoLC-MSE on a Synapt G2 mass spectrometer coupled to a nanoAcquity UPLC system . Peptides were trapped using a pre-column ( Symmetry C18 , 5 µm , 180 µm × 20 mm , Waters ) , which was switched in-line to an analytical column ( BEH C18 , 1 . 7 µm , 75 µm × 250 mm , Waters ) for separation . Peptides were eluted with an 8–50% acetonitrile gradient in water/0 . 1% formic acid at 0 . 75% per minute ( 250 nl/min ) . The column was connected to a 10 µm SilicaTip nanospray emitter ( New Objective , Woburn , MA , USA ) for infusion into the mass spectrometer . [Glu1]-fibrinopeptide B ( 1 pmol/µl , Sigma ) was infused at 0 . 5 µl/min as a lock mass and measured every 30 s . The spectrometer was controlled by the Masslynx 4 . 1 software ( Waters ) and operated in positive MSE and sensitivity mode with capillary voltage of 3 kV , cone voltage , 40 V . Scan time was 1 s over the range 50–2000 m/z range . For the low energy scan the trap collision was off , and for the high energy scan the trap collision energy ( CE ) was ramped from 20–60 V . For de novo identification of proteins , raw files were processed in Protein Lynx Global Server 2 . 5 . 2 ( Waters ) , including a search on a Homo sapiens protein database to which the TED protein sequences had been added . Acquisition in MSE mode uses alternating low and high CE scans and generates 2 separate chromatograms . To detect the TED-fibrinogen Aα cross-linked peptides , extracted ion chromatograms were generated from the high CE trace in Masslynx 4 . 1 . and inspected for characteristic y-ions expected from C-terminal TED-peptide fragments starting with proline . Peptides are known to fragment to the N-terminus of prolines , and the sequences searched for here were Pro ( 116 . 071 Da , SfbI-A40-TED-fibrinogen ) , Pro–Lys ( 244 . 166 Da , SfbI-A346-TED- and SfbI-A20-TED-fibrinogen ) and Pro-Gly-Ser-Arg ( 416 . 226 Da , SfbI-A40-TED- and SfbI-A20-TED-plasma ) . Detected peaks for those y-ions were aligned with the corresponding low CE traces , which reliably lead to the detection of masses consistent with the cross-linked precursor peptides . In some cases we also searched the low CE trace for masses consistent with all possible combinations of tryptic fibrinogen Aα peptides , cross-linked with the relevant TED peptides , but we could find no evidence for additional combinations . Precursor spectra and high CE fragment spectra were inspected for the presence of characteristic fragment ions of the identified cross-linked peptides . The lysine-acetylated TED-fibrinogen complex was generated by incubation of 20 µM fibrinogen with a fivefold molar excess of SfbI-A40-TED at 37°C for 1 hr . Sulfo-NHS-acetate ( Thermo Fisher Scientific ) was then added to a concentration of 10 mM and the reaction mixture incubated at 37°C for 2 hr . The sample components were separated by SDS-PAGE , and the TED-fibrinogen Aα adduct bands excised for analysis by MS . For comparison lysine-acetylated fibrinogen bands were produced by the same protocol , with the omission of the TED reaction step . Excised gel bands were digested with either trypsin or endoproteinase GluC or , in the case of the TED-fibrinogen adduct , chymotrypsin . The extracted peptides were analyzed by nanoLC-MSMS by the same protocol used for identification of Gln residues involved in thioester bond formation . Peptides with charges +2 to +5 with an ion count over 150 were selected for MSMS , and then excluded for further analysis for 15 s . A rolling CE was applied to fragment the peptides . The data was searched using the Mascot algorithm against an in-house database containing fibrinogen sequences . Carboamidomethyl modification of cysteines was set as a fixed modification , acetylation on lysine and oxidation of methionine were set as variable modifications . MS tolerance was ± 20 ppm and MSMS tolerance ± 0 . 1 Da . L . lactis was grown at 30°C in M17 medium plus 0 . 5% glucose ( GM17 ) . S . pyogenes was grown at 37°C in Todd-Hewitt broth plus 0 . 5% yeast extract . Where appropriate , antibiotics were added: erythromycin at 3 mg/l for L . lactis and 400 mg/l for E . coli . Chromosomal DNA from S . pyogenes A40 was prepared as described previously ( Bergmann et al . , 2014 ) as a template for amplification of the sfbI-A40 gene . Amplified DNA was inserted into the shuttle vector pOri23 ( Que et al . , 2000 ) and transformed into E . coli XL1-Blue . Site-directed mutagenesis was used to create SfbI-A40:Cys109Ala plasmid DNA . 1 µg of plasmid was used to transform L . lactis by electroporation ( Holo and Nes , 1989 ) . L . lactis constructs SfbI-A40 , SfbI-A40:Cys109Ala and pOri23 were grown in GM17 and fixed in the growth medium with 2% formaldehyde . After quenching free aldehydes with 10 mM glycine , samples were incubated with purified polyclonal anti-SfbI IgG-antibodies for 60 min at 37°C , washed and incubated with protein A-gold nanoparticles ( 15 nm ) for 30 min at 37°C . After washing , samples were fixed in 2% glutaraldehyde , absorbed onto butvar-coated 300 mesh grids , washed in distilled water , air-dried and examined in a Zeiss Merlin scanning electron microscope at an acceleration voltage of 10 kV using the high efficiency Everhart-Thornley SE-detector . Fibrin was fibrillized on cover slips by incubating 1 mg plasminogen-depleted human fibrinogen ( Calbiochem , Merck Millipore , Billerica , MA , USA ) with 10 U of thrombin ( from bovine plasma , Sigma ) for 12 hr at 37°C in 100 µl PBS . PBS washed L . lactis strains , harboring the pOri23 , SfbI-A40 , SfbI-A40:Cys109Ala plasmids were incubated for 2 hr at 37°C on the fibrin clots . PBS washed cover slips were fixed for scanning electron microscopy with 2% glutaraldehyde and 5% formaldehyde , dehydrated with a graded series of acetone ( 10 , 30 , 50 , 70 , 90 , 100% ) , critical-point dried and sputter-coated with gold-palladium before examination in a Zeiss Merlin field emission scanning electron microscope ( Oberkochen , Germany ) at an acceleration voltage of 5 kV . Human alveolar basal epithelial ( A549 ) cells were propagated in Dulbecco's modified Eagle medium ( DMEM; Life Technologies , Thermo Fisher Scientific ) supplemented with 10% ( vol/vol ) fetal bovine serum ( FBS , Thermo Fisher Scientific ) , 100 U/ml penicillin and 50 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2 . Cells were seeded at a density of 4 × 104 cells/well and grown to approximately 60% confluency . Inflammatory response was induced in half of the cell cultures by incubating cell monolayers with 0 . 39 mg/ml dexamethasone ( Sigma ) and 50 ng/ml human recombinant interleukin-6 ( Cambridge Bioscience , UK ) in DMEM supplemented with 10% FBS . After 48 hr , cells were chilled at 4°C for 30 min and then incubated with 1 mg/ml SfbI-A40-TED-iPT , or SfbI-A40-TED:Cys109Ala-iPT in PBS , or PBS for 30 min at 4°C . Cells were fixed with pre-chilled ( −20°C ) methanol for 20 min at −20°C , then stained with 1 mg/ml sIPD-GFP in PBS for 30 min at 25°C . Nuclei were stained with 0 . 5 µg/ml DAPI ( 15 min , 25°C ) . Between each incubation cell monolayers were washed extensively with sterile PBS . After 24 hr at 4°C , cells were imaged on an EVOS Digital Inverted Microscope ( Life Technologies , Thermo Fisher Scientific ) . A549 cells were grown , induced and incubated with wildtype or Cys109Ala mutant TEDs as described above . Cells were homogenized in lysis buffer ( 8 M urea , 5% SDS , 10% β-mercaptoethanol ) by sonication in an ultrasonic water bath at 4°C for 45 min . 10 μl of cell homogenate were separated by SDS-PAGE on a 12% acrylamide gel ( Bio-Rad ) in Tris-glycine buffer ( 2 . 5 mM Tris , 19 . 2 mM glycine , 0 . 01% SDS , pH 8 . 3 ) . The proteins were then transferred to a BioTrace PVDF membrane ( PALL Gelman Laboratory ) at 100 V for 1 hr in Tris-glycine buffer containing 20% ( vol/vol ) methanol . The membrane was subsequently washed with PBS containing 0 . 1% ( vol/vol ) Tween20 ( Sigma ) , blocked for 1 hr with 5% ( wt/vol ) BSA in PBS-Tween , and incubated for 12 hr at 4°C with 1:10 , 000 diluted primary anti-fibrinogen α antibody ( C-7 , mouse IgG ) ( Santa Cruz Biotechnology ) . The membrane was washed 3 times for 10 min with PBS-Tween , and incubated with 1:20 , 000 diluted secondary IRDye 800CW goat anti-mouse IgG ( LI-COR Biosciences ) , for 1 hr at room temperature . After three 10 min washes with PBS-Tween , the membrane was imaged ( Odyssey CLx imaging system , LI-COR Biosciences ) . The same membrane was subsequently incubated for 1 hr at room temperature with 1:10 , 000 diluted primary anti-β-actin mouse antibody ( Sigma ) , washed 3 times for 10 min with PBS-Tween , incubated with 1:20 , 000 diluted secondary IRDye 800CW goat anti-mouse IgG for 1 hr and re-imaged . Protein structures , and the data used to derive these , have been deposited at the PDB with accession numbers 5a0l ( SfbI-A40-TED ) , 5a0n ( PnTIE-TED ) , 5a0g ( CpTIE-TED ) and 5a0d ( CpTIE-TED:Cys138Ala ) .
To cause disease and persist in a host , pathogenic and commensal microbes must adhere to tissues . Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins , or adhesins . To date , adhesins are only known to bind to host receptors non-covalently . Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’ . This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation . Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria , including many clinically relevant pathogens . Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions . This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions .
The human body is home to many trillions of microbes; most are harmless , but some may cause disease . To live inside a host , microbes must first attach to host tissues . This process involves multiple proteins on each microbe's surface , called adhesins , which interact with the molecules that make up these tissues . Like all proteins , adhesins are long chains of simpler building blocks called amino acids , and each amino acid is connected to the next via a strong ‘covalent’ bond . Adhesins , however , typically attach bacteria to host molecules through the combined strength of many weak ‘non-covalent’ interactions . It was recently discovered that one adhesin from a bacterium called Streptococcus pyogenes contains a rare , extra covalent bond—called a thioester—in an unusual location between two of its amino acids . S . pyogenes is a common cause of throat infections in humans , and can also cause the life-threatening ‘flesh-eating disease’ . Walden , Edwards et al . have now used a range of computational , biochemical , structural biology and cell-based techniques to study other adhesins that have thioester bonds in more detail . Computational searches identified hundreds of bacterial proteins containing similar bonds . These included many from bacteria that infect humans: such as Streptococcus pneumoniae , which is the most common cause of pneumonia in adults; and Clostridium difficile , which is notorious for causing severe gut infections in hospital patients . Closer examination of the three-dimensional structures of three of these proteins—including one called SfbI from S . pyogenes—revealed that each had a clear thioester bond . Biochemical tests of an additional nine of the identified proteins strongly suggested they too contained thioester bonds . Walden , Edwards et al . then showed that SfbI was able to not only attach to tissues like conventional adhesins , but also chemically react with fibrinogen: a human protein that is essential for blood clotting and commonly found in inflamed tissues and healing wounds . This chemical reaction results in the formation of a covalent bond between SfbI and fibrinogen , which is as stable as the bonds that link the amino acids in a protein chain . Further experiments revealed that SfbI strongly binds to human cells grown in the lab under conditions that mimic tissue inflammation . Finally , Walden , Edwards et al . made a mutant version of SfbI that did not contain a thioester , and found that it could not interact with fibrinogen nor bind to human cells . Together , these findings suggest that thioesters in bacterial adhesins act like ‘chemical harpoons’ , which microbes can use to irreversibly attach themselves to molecules within their host's tissues . This attachment mechanism has not been seen before in host-microbe interactions , and further research is now needed to explore whether interfering with this process could represent a new way to treat bacterial infections .