Background The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPα, EspPβ, EspPγ, and EspPδ), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown. Methodology/Principal Findings The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the β-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His127, Asp156 and Ser263 in Domain 1 form the catalytic triad of EspP. Conclusions/Significance Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPβ and EspPδ.
References
[1]
Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692–744.
[2]
Dautin N, Bernstein HD (2007) Protein secretion in gram-negative Bacteria via the autotransporter pathway. Annu Rev Microbiol 61: 89–112.
[3]
Kajava AV, Steven AC (2006) Beta-rolls, beta-helices, and other beta-solenoid proteins. Adv Protein Chem 73: 55–96.
[4]
Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, et al. (2004) Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol 186: 5432–5441.
[5]
Henderson IR, Nataro JP (2001) Virulence functions of autotransporter proteins. Infect Immun 69: 1231–1243.
Brunder W, Schmidt H, Karch H (1997) EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. Mol Microbiol 24: 767–778.
[8]
Karch H, Tarr PI, Bielaszewska M (2005) Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 295: 405–418.
[9]
Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365: 1073–1086.
[10]
Aldick T, Bielaszewska M, Zhang W, Brockmeyer J, Schmidt H, et al. (2007) Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Microbes Infect 9: 282–290.
[11]
Bielaszewska M, Sinha B, Kuczius T, Karch H (2005) Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells. Infect Immun 73: 552–562.
[12]
Bielaszewska M, Karch H (2005) Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thromb Haemost 94: 312–318.
[13]
Dautin N, Barnard TJ, Anderson DE, Bernstein HD (2007) Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism. EMBO J 26: 1942–1952.
[14]
Barnard TJ, Dautin N, Lukacik P, Bernstein HD, Buchanan SK (2007) Autotransporter structure reveals intra-barrel cleavage followed by conformational changes. Nat Struct Mol Biol 14: 1214–1220.
[15]
Ieva R, Skillman KM, Bernstein HD (2008) Incorporation of a polypeptide segment into the beta-domain pore during the assembly of a bacterial autotransporter. Mol Microbiol 67: 188–201.
[16]
Brockmeyer J, Bielaszewska M, Fruth A, Bonn ML, Mellmann A, et al. (2007) Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity. Appl Environ Microbiol 73: 6351–6359.
[17]
Khan AB, Naim A, Orth D, Grif K, Mohsin M, et al. (2008) Serine protease espP subtype α, but not β or γ, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups. Int J Med Microbiol 299: 247–254.
[18]
Kraut J (1977) Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem 46: 331–358.
[19]
Siezen RJ, Leunissen JA (1997) Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6: 501–523.
[20]
Fink DL, Cope LD, Hansen EJ, Geme JW3 St (2001) The Hemophilus influenzae Hap autotransporter is a chymotrypsin clan serine protease and undergoes autoproteolysis via an intermolecular mechanism. J Biol Chem 276: 39492–39500.
[21]
Baker D, Sali A (2001) Protein structure prediction and structural genomics. Science 294: 93–96.
[22]
Martí-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, et al. (2000) Comparative protein structure modelling of genes and genomes. Annu Rev Biophys Biomol Struct 29: 291–325.
[23]
Emsley P, Charles IG, Fairweather NF, Isaacs NW (1996) Structure of Bordetella pertussis virulence factor P.69 pertactin. Nature 381: 90–92.
[24]
Nummelin H, Merckel MC, Leo JC, Lankinen H, Skurnik M, et al. (2004) The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll. EMBO J 23: 701–711.
[25]
Yeo H, Cotter SE, Laarmann S, Juehne T, St. Geme JW 3rd, et al. (2004) Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter. EMBO J 23: 1245–1256.
[26]
Szczesny P, Linke D, Ursinus A, B？r K, Schwarz H, et al. (2008) Structure of the head of the Bartonella adhesin BadA. PLoS Pathogens 4: e1000119.
[27]
Otto BR, Sijbrandi R, Luirink J, Oudega B, Heddle JG, et al. (2005) Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli. J Biol Chem 280: 17339–17345.
[28]
Fernandez-Fuentes N, Madrid-Aliste CJ, Rai BK, Fajardo JE, Fiser A (2007) M4T: a comparative protein structure modelling server. Nucleic Acids Res 35: W363–368.
[29]
Dutta PR, Sui BQ, Nataro JP (2003) Structure-function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis. J Biol Chem 278: 39912–39920.
[30]
Oliver DC, Huang G, Nodel E, Pleasance S, Fernandez RC (2003) A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain. Mol Microbiol 47: 1367–1383.
[31]
Ohnishi Y, Nishiyama M, Horinouchi S, Beppu T (1994) Involvement of the COOH-terminal pro-sequence of Serratia marcescens serine protease in the folding of the mature enzyme. J Biol Chem 269: 32800–32806.
[32]
Velarde JJ, Nataro JP (2004) Hydrophobic residues of the autotransporter EspP linker domain are important for outer membrane translocation of its passenger. J Biol Chem 279: 31495–31504.
[33]
Jong WSP, ten Hagen-Jongman CM, den Blaauwen T, Slotboom DJ, Tame JRH, et al. (2007) Limited tolerance towards folded elements during secretion of the autotransporter Hbp. Mol Microbiol 63: 1524–1536.
[34]
Skillman KM, Barnard TJ, Peterson JH, Ghirlando R, Bernstein HD (2005) Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter. Mol Microbiol 58: 945–958.
[35]
Voulhoux R, Bos MP, Geurtsen J, Mols M, Tommassen J (2003) Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299: 262–265.
[36]
Kim S, Malinverni JC, Sliz P, Silhavy TJ, Harrison SC, et al. (2007) Structure and function of an essential component of the outer membrane protein assembly machine. Science 317: 961–964.
[37]
Bernstein HD (2007) Are bacterial ‘autotransporters’ really transporters? Trends Microbiol 15: 441–447.
[38]
Robert V, Volokhina EB, Senf F, Bos MP, van Gelder P, et al. (2006) Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol 4: e377.
[39]
Klauser T, Kramer J, Otzelberger K, Pohlner J, Meyer TF (1993) Characterization of the Neisseria Iga beta-core. The essential unit for outer membrane targeting and extracellular protein secretion. J Mol Biol 234: 579–593.
[40]
Maurer J, Jose J, Meyer TF (1999) Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions. J Bacteriol 181: 14–20.
[41]
Deber CM, Therien AG (2002) Putting the β-breaks on membrane protein misfolding. Nat Struct Biol 9: 318–319.
[42]
Chou PY, Fasman GD (1978) Empirical predictions of Protein conformation. Annu Rev Biochem 47: 251–276.
[43]
Clantin B, Delattre A, Rucktooa P, Saint N, Meli AC, et al. (2007) Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily. Science 317: 957–961.
[44]
Holm L, Kaariainen S, Wilton C, Plewczynski D (2006) Using Dali for structural comparison of proteins. Curr Protoc Bioinformatics Chapter 5: Unit 5.5.
[45]
Fiser A, Sali A (2003) Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol 374: 461–491.
[46]
Eslava C, Navarro-Garcia F, Czeczulin JR, Henderson IR, Cravioto A, et al. (1998) Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Inf Immun 66: 3155–3163.
[47]
Melo F, Feytmans E (1998) Assessing protein structures with a non-local atomic interaction energy. J Mol Biol 277: 1141–1152.
[48]
Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17: 355–362.
[49]
Laskowski RA, MacArthur MW, Moss D, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26: 283–291.
[50]
Benkert P, Tosatto SCE, Schomburg D (2008) QMEAN: A comprehensive scoring function for model quality assessment. Proteins 71: 261–277.
