Highly Sensitive Quenched Fluorescent Substrate of Legionella

Supplemental Material can be found at: http://www.jbc.org/content/suppl/2012/04/23/M111.334334.DC1.html THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20221–20230, June 8, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis*□S Received for publication, December 22, 2011, and in revised form, April 12, 2012 Published, JBC Papers in Press, April 23, 2012, DOI 10.1074/jbc.M111.334334 Hervé Poras‡1, Sophie Duquesnoy‡, Emilie Dange‡, Anthony Pinon§, Michèle Vialette§, Marie-Claude Fournié-Zaluski‡, and Tanja Ouimet‡ From ‡Pharmaleads, Paris BioPark, 11 Rue Watt 75013 Paris and the §Institut Pasteur Lille, Unité de Sécurité Microbiologique, 1 Rue du Professeur Calmette, 59019 Lille Cedex, France Background: Legionella pneumophila secretes a protease without known specific substrate and three-dimensional structure. Results: Analysis of a quenched peptide library identified a lead substrate, ameliorated by rational design, using an Msp structural model obtained by the x-ray structure of pseudolysin. Conclusion: The study identifies the first selective substrate for Msp. Significance: This substrate could be useful for Legionella detection. Downloaded from Legionella pneumophila has been shown to secrete a protease (phosphatase, phospholipase C, proteases, and kinases), and termed major secretory protein (Msp). This protease belongs to the Mip gene product (1). the M4 family of metalloproteases and shares 62.9% sequence Among the proteases, one exotoxin, abundantly secreted by similarity with pseudolysin (EC 3.4.24.26). With the aim of different strains of L. pneumophila, was detected (2) and shown www.jbc.org developing a specific enzymatic assay for the detection and to possess a proteolytic activity toward some human serum quantification of Msp, the Fluofast substrate library was proteins (3), collagen, casein, gelatin, and hide powder but not screened using both enzymes in parallel. Moreover, based on the elastin (4). This extracellular protease, designated as major 2 crystal structure of pseudolysin, a model of the Msp structure secretory protein (Msp), has been purified from L. pneumo- at INSERM, on October 11, 2012 was built. Screening of the peptide library identified a lead sub- phila culture filtrates (5) and characterized as a neutral zinc- strate specifically cleaved by Msp that was subsequently opti- containing metalloprotease of 38 kDa, exhibiting hemolytic and mized by rational design. The proposed model for Msp is con- cytotoxic activities (6). sistent with the enzymatic characteristics of the studied peptide The protease has been cloned (7), and analysis of the gene has substrates and provides new structural information useful for shown that it encodes a large preproenzyme of 543 amino acids the characterization of the protease. This study leads to the (60,775 Da) transported to the periplasma before maturation identification of the first selective and high affinity substrate for into the 38-kDa active protein. The protease belongs to the M4 Msp that is able to detect picomolar concentrations of the puri- family of metalloproteases (clan MA/E) represented by its fied enzyme. The identified substrate could be useful for the archetypal member thermolysin. Within the M4 family, Msp development of a novel method for the rapid detection of shares the highest structural and functional homologies with Legionella. pseudolysin (Pseudomonas aeruginosa elastase, EC 3.4.24.26) (8, 9). Comparison of their amino acid sequences allowed the characterization of the main residues involved in the proteo- Legionella is a genus of Gram-negative bacteria, widely dis- lytic activity of Msp, i.e. the consensus sequences 377HEVSH381 tributed in freshwater environments, where it survives as intra- and 401ESFSD405 containing the three zinc ligands His377, cellular parasites of protozoa. Among the different species of His381, Glu401, and the catalytic glutamate Glu378. As expected, Legionella, the most frequently associated with human disease the mutation of E378Q leads to an inactive protease deprived of is Legionella pneumophila, causing acute pneumonia, referred cytotoxic activity (9), thus probing the involvement of the to as legionellosis, and Pontiac fever, a severe influenza-like hydrolytic properties of the protease in L. pneumophila illness. In the human lung, the bacteria are able to replicate pathogenesis. within alveolar macrophages and epithelial cells, causing tissue A chromogenic substrate MeO-succinimide-Arg-Pro-Tyr- damage. Several potential virulence factors have been identified p-nitroanilide (S-2586), initially developed for ␣-chymotrypsin in L. pneumophila, including a peptide toxin, various enzymes (10), has been used to detect Msp in different strains of Legio- nella (11, 12). Despite the lack of specificity of this substrate, species-discriminating responses were obtained. All the sero- * This work was supported by a grant from Agence Nationale pour la Recher- che (Programme de Recherche sur les ECOtechnologies et le Développe- ment Durable, Ralf project). 2 The abbreviations used are: Msp, major secretory protein; HPI, N-(1-carboxy- □ S This article contains supplemental Fig. 1. 3-phenylpropyl)-phenylalanyl-␣-asparagine; Nop, (p-nitro)-L-phenylala- 1 To whom correspondence should be addressed. Tel.: 33144066095; Fax: nine; Pya, L-pyrenylalanine; Fmoc, N-(9-fluorenyl)methoxycarbonyl; Orn, 33144066099; E-mail: [email protected]. ornithine; hSer, homo-serine; hArg, homo-arginine. JUNE 8, 2012•VOLUME 287•NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 20221 Fluorescently Quenched Substrate of Legionella Endopeptidase groups of L. pneumophila cleaved this substrate, whereas other continuous shaking. The final volume of each culture was 3 Legionella species (2 of 5), Pseudomonas strains (4 of 8), or liters. A sample was removed for enumeration on BCYE␣, and Enterobacteriaceae strains (0 of 11) were cleaved poorly or were the remaining culture medium was centrifuged at 5000 rpm for not responsive. These results suggested a relatively high speci- 10 min at 20 °C to remove suspended matter. The supernatant ficity of L. pneumophila Msp as compared with analogous was filter-sterilized on a 0.2-␮m membrane (Millipore). secreted activities of other bacteria. Msp has interesting prop- Purification—Msp was purified based on the protocol from erties, i.e. its abundant secretion, its possible implication in L. Dreyfus and Iglewski (5). Proteins from the sterilized culture pneumophila pathogenicity, and its specificity toward other media were precipitated overnight at 4 °C under constant stir- pathogens. Taking into account these properties, the aim of this ring with 65% ammonium sulfate. The media were then centri- study was to develop a highly sensitive and selective substrate of fuged at 14,000 rpm for 30 min at 4 °C, and the protein pellets Msp as a marker of L. pneumophila. were resuspended using 25 mM Tris-HCl, pH 7.8, 25 mM NaCl, Consequently, based on the crystal structure of pseudolysin, 0.01% Triton X-100 (buffer A), loaded on a HiPrep 26/10 desalt- obtained with 1.5-Å resolution (13), a three-dimensional model ing column (GE Healthcare, Akta Systems), and eluted in the of Msp was constructed and refined. The secreted Msp protease same buffer at a flow rate of 10 ml/min. The fractions contain- was purified from culture media of L. pneumophila Philadel- ing Msp activity (identified by SDS-PAGE or enzymatic activ- phia 1 (CIP 103854T-ATCC 33152). The Fluofast library of ity) were pooled (120 ml) and concentrated (ϳ10 ml at 4 °C intramolecularly quenched fluorescent substrates containing using Amicon Ultra-15 (cutoff of 10 kDa) (Millipore)). The con- the Pya/Nop fluorophore/quencher pair (14, 15) was screened centrated fraction was loaded onto a HiPrep DEAE FF 16/10 in parallel with purified Msp and pseudolysin. Results of this column (GE Healthcare, Akta Systems) equilibrated with buffer Downloaded from screening provided a lead selective substrate. Docking of this A, and the protein was eluted according to a multistep gradient substrate in the three-dimensional model of Msp and sequence buffer B (25 mM Tris-HCl, pH 7.8, 1 M NaCl, 0.01% Triton refinement based on the observed substrate-enzyme interac- X-100). The first step consisted of 15% of buffer B (6 column tions allowed the synthesis of highly efficient and selective sub- volumes), the second of 60% of buffer B (6 column volumes), strates of L. pneumophila Msp. and the last elution step was set at 100% of the same (5 column volumes). The fractions containing Msp activity, eluting at 60% www.jbc.org EXPERIMENTAL PROCEDURES buffer B, were pooled and concentrated to a volume of 200 ␮lat Reagents—Fmoc-protected amino acids, piperidine, N-methyl- 4 °C. This concentrated fraction was loaded onto a HiLoad pyrrolidone, dichloromethane, dicyclohexylcarbodiimide, 16/60 Superdex 75 preparation grade (GE Healthcare, Akta at INSERM, on October 11, 2012 1-hydroxybenzotriazole, and O-(7-azabenzotriazol-1-yl)- Systems) and eluted in 500-␮l fractions with 25 mM Tris-HCl, 1,1,3,3-tetramethyluronium were purchased from Applied pH 7.2, 0.15 M NaCl, 0.01% Triton X-100 at a flow rate of 0.25 Biosystems (Courtaboeuf, France). Fmoc-(p-Nitro)-L-phenyl- ml/min across 1.5 bed volumes. The pure fractions containing alanine and methylbenzhydrylamine resin (0.73 mmol/g) were Msp activity were pooled and concentrated using Amicon Ultra from Novabiochem. Fmoc-L-pyrenylalanine (Pya), prepared as 10-kDa ultrafiltration. Proteins were quantified using BCA described previously (16), were from Polypeptide Laboratories (Pierce). (Strasbourg, France). Trifluoroacetic acid was from SDS-Carlo The purified preparation was electrophoresed on a 12% Erba (France). Triisopropylsilane was from Sigma. BCYE␣ acrylamide denaturing gel, silver-stained, and quantified by (agar-solidified Buffered Charcoal Yeast Extract supplemented densitometry using a BSA scale and the Quantity One program with 0.1% ␣-ketoglutarate) was obtained from Oxoid (Basing- of Bio-Rad. Purity of the preparation was also evaluated using stoke, UK).

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