Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host Marco Candela, 1 Elena Biagi, 1 Manuela Centanni, 1 Silvia Turroni, 1 Manuela Vici, 2 Francesco Musiani, 3 Beatrice Vitali, 1 Simone Bergmann, 4 Sven Hammerschmidt 5 and Patrizia Brigidi 1 Correspondence Patrizia Brigidi patrizia.brigidi@unibo.it 1 Department of Pharmaceutical Sciences, CIRB-centre for Biotechnology, University of Bologna, Italy 2 Department of Experimental Pathology, University of Bologna, Italy 3 Department of Agro Environmental Science and Technology, University of Bologna, Italy 4 Department of Microbial Pathogenicity, Helmholtz Centre for Infection Research GmbH, Braunschweig, Germany 5 Department Genetics of Microorganisms, Ernst Moritz Arndt University Greifswald, Greifswald, Germany Received 2 March 2009 Revised 29 May 2009 Accepted 30 June 2009 The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host. INTRODUCTION Bifidobacteria represent one of the most important health- promoting groups of human intestinal microbiota (Schell et al., 2002; Ventura et al., 2009). Even though numerous health-promoting activities have been related to the presence of bifidobacteria in the human gastrointestinal tract (Guarner & Malagelada, 2003), knowledge of the mechanisms of interaction with the host is still in its infancy. The understanding of the Bifidobacterium–host interaction process, as well as its impact on human health, could be clarified by the identification and characterization of the bacterial proteins involved. In particular, represent- ing the first line of contact with the intestinal epithelium, the proteins of the bacterial cell surface may play a critical role in the early interaction between microbes and the host (Klijn et al., 2005). Recently, a proteomic approach identified five highly conserved cytoplasmic proteins in the cell wall fraction of Bifidobacterium lactis BI07 as putative human plasminogen (Plg) receptors: DnaK, glutamine synthetase, enolase, bile salt hydrolase and phosphoglycerate mutase (Candela et al., 2007). Plg is the zymogen of plasmin, a trypsin-like serine protease with a broad substrate specificity. Plg is a single-chain glycopro- tein with a molecular mass of 92 kDa and comprises an N- terminal pre-activation peptide (~8 kDa), five consecutive disulfide-bonded triple-loop kringle domains (K1–5), and a serine-protease domain containing the catalytic triad (Vassalli et al., 1991). It is produced mainly by hepatocytes; however, other tissue sources for Plg synthesis have been identified, including the intestine (Zhang et al., 2002). The active form of plasmin is involved in fibrinolysis (Collen & Abbreviations: 2-PGE, 2-phosphoglycerate; EACA, e-aminocaproic acid; PEP, phosphoenolpyruvate; Plg, plasminogen. The GenBank/EMBL/DDBJ accession number for the enolase sequence of B. lactis BI07 is DQ117970. Microbiology (2009), 155, 3294–3303 DOI 10.1099/mic.0.028795-0 3294 028795 G 2009 SGM Printed in Great Britain