A specific peptide inhibitor of the class B metallo-ß-lactamase L-1 from Stenotrophomonas maltophilia identified using phage display

François Sanschagrin and Roger C. Levesque*

Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4


* Corresponding author. Tel: +1-418-656-3070; Fax: +1-418-656-7176; Email: rclevesq{at}rsvs.ulaval.ca

Received 25 August 2004; returned 20 October 2004; revised 10 November 2004; accepted 24 November 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: In Gram-negative bacteria, resistance to ß-lactam antibiotics and to known inhibitors mediated by metallo-ß-lactamases is a major concern and a serious threat to public health. Since no clinically useful inhibitors are available against class B metallo-ß-lactamases, the aim of the study was to identify peptides as inhibitors.

Methods: The L-1 metalloenzyme from Stenotrophomonas maltophilia was cloned, over-expressed, purified to homogeneity and used in screening of peptide libraries by phage display with a selective and competitive biopanning assay. This was based upon the high affinity of L-1 for cefoxitin and its slow hydrolysis.

Results: From six peptides, the consensus sequence Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys was identified as a promising inhibitor of L-1 hydrolytic activity. This peptide showed a mixed inhibition of L-1 with a Ki competitive of 16 ± 4 µM and a Ki uncompetitive of 9 ± 1 µM. The same peptide was prepared without flanking Cys residues and demonstrated no detectable inhibition of L-1 hydrolytic activity with nitrocefin as a substrate. These data confirmed the importance of the peptide conformation for the inhibition of L-1 hydrolytic activity. Further analysis revealed rescue by Zn2+ ions. The mixed inhibition indicated peptide binding near the active site of L-1 and blocking of zinc atoms for optimal conformation in the pocket of the active site.

Conclusion: This is the first report of a peptide inhibitor for Class B metallo-ß-lactamases. It will be used as a lead to identify more potent small molecule inhibitors via peptidomimetics.

Keywords: L-1 ß-lactamase , selective biopanning , ß-lactamase inhibitors


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance against ß-lactam antibiotics is a major problem, mainly caused by the presence of ß-lactamases. Class A ß-lactamases are susceptible to mechanism-based inhibitors in clinical use but class B enzymes are not. Phage display is a powerful technique for the study of protein–ligand interactions, or to identify peptides as inhibitors of ß-lactamase activity.1 We have previously used this approach with success to identify peptide inhibitors of MurC ATPase and FtsZ GTPase activities.2,3

In this study, we used a phage-display technique coupled to a selective biopanning protocol for screening and for identification of peptides having significant inhibition of L-1 metallo-ß-lactamase hydrolytic activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, plasmids and DNA constructions

Escherichia coli Novablue (endA1 hsdR17(r12K m12K+) supE44 thi-1 recA1 gyrA96 relA1 lac [F'proA + B+ lacIqZ{Delta}M15::Tn10 (TcR)] (Novagen, Madison, WI, USA) was the recipient strain for construction and production of DNA for sequencing. E. coli BL21({lambda}DE3) (FompT hsdSB (rb mb) gal dcm (DE3) (Novagen) was the recipient strain for protein expression and purification. The plasmid pMON13 containing blaS coding for class B ß-lactamase L-1 from Stenotrophomonas maltophilia GN128734 was used to perform PCR cloning. PCR primers containing NdeI-XhoI restriction sites in plasmid pET30a were used to give the plasmid pMON19 coding for a fusion protein L-1 having six histidine residues at the C-terminus. Preparation of DNA and related techniques was by standard methods.5

Protein expression and purification

L-1 ß-lactamase expression and purification was carried out with a B-PER 6x His Fusion Protein Purification Kit, as recommended by the manufacturer (Pierce, Rockford, IL, USA), except that lysis of E. coli BL21 was performed by osmotic shock.6

Phage display

Purified L-1 was used to screen for metallo-ß-lactamase peptide inhibitors by phage display using a PH.D.-C-7-C library (New England Biolabs, Mississauga, Ont., Canada) containing ~3.7 x 109 C-7-C mer random peptide sequences. Biopanning was carried out as described previously2 except for the following modifications: L-1 was coated on the bottom of a 96-well plate, four rounds of biopanning were performed instead of three and elution in rounds three and four was conducted in two steps—one elution of 5 min followed by a second elution of 30 min, both with 1 mM cefoxitin (Sigma, Oakville, Ont., Canada). Phage recovered after the second elution was used as input for the fourth biopanning. Phage titration, DNA preparation and sequencing of eluted phage after four rounds of biopanning were performed as described previously.2 Peptide sequences deduced were aligned and a consensus peptide was selected for synthesis.2 Peptides were synthesized on an ABI 433A Peptide Synthesizer using FastMoc chemistry and purified on a Vydak 22 x 250 mm C-18 reverse-phase HPLC column using a 0.1% TFA/acetonitrile gradient at 10 mL/min.

The dissociation constants (Ki competitive and Ki uncompetitive) for the consensus peptide were determined at 30 °C in 50 mM Tris–HCl buffer, pH 8.0, in a 1 mL cuvette reaction volume in a Cary 1 spectrophotometer (Varian, Mississauga, Ont., Canada). Hydrolysis for nitrocefin (Oxford, Mississauga, Ont., Canada) ({varepsilon}=17400 M–1 cm–1) was monitored at 485 nm. Kinetic constants Vmax and Km were determined by rates of hydrolysis calculated from the initial velocity in the linear portion, with the same cuvette and a least-squares calculation. The concentration of L-1 was 20 nM and the five concentrations of nitrocefin tested were 2, 10, 25, 50 and 100 µM with three concentrations of C-7-C peptide at 5, 20, 50 µM, respectively. Hydrolytic activity was measured immediately after mixing reagents. All experiments were carried out in triplicate. Analysis of enzyme kinetic data was carried out using the Leonora software for robust regression analysis of enzyme data and a biweighting regression system.7


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The S. maltophilia GN12873 L-1 metallo-ß-lactamase was purified in milligram quantities to homogeneity. Its six histidine residues were used for this purpose in a single chromatographic step on an affinity nickel column; the mature L-1 molecular mass was confirmed by LC-MS analysis (data not shown).

The purified L-1 enzyme was coated on a microtitre plate and incubated with a phage-display library expressing randomized Cys-(7 amino acids)-Cys peptides. Screening was conducted using four rounds of biopanning; each step included increasingly restrictive conditions of washing and limited time of contact. Competitive elution with cefoxitin was envisaged as a potential tool to identify specific peptides with high affinity for the L-1 active site. After a fourth round of competitive biopanning, DNA sequencing of 17 randomly selected phages and alignment of peptide sequences identified a consensus, repeated eight times, as depicted in Figure 1. We noted the specific motif—Ser-Pro-Asn—in the first two peptides, and in the third sequence in the reverse orientation for a total of 13 among 17 peptide sequences.



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Figure 1. Peptide sequences obtained from selected phage clones in phage display. Shown are the peptide sequences selected after four rounds of biopanning, including a competitive biopanning step using cefoxitin. The numbers to the left of the sequences indicate the six peptides selected; the fractions to the right indicate the number of times a specific sequence was identified out of a total of 17 clones selected. The conserved motif identified in 13 sequences is underlined.

 
The peptide Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys was synthesized and tested for inhibition of L-1 hydrolytic activity of the chromogenic substrate nitrocefin. The kinetic parameters obtained for L-1 with nitrocefin as substrate are a Km of 15 ± 3 µM, kcat of 24 ± 2 s–1 and with the consensus peptide a Ki competitive of 16 ± 4 µM and a Ki uncompetitive of 9 ± 1 µM. Activity revealed that the peptide inhibited the hydrolytic activity of L-1 in a mixed inhibition fashion (see Figure 2). To confirm specificity of L-1 inhibition, we tested a peptide obtained by phage display and known to bind to the capsid of herpes virus (data not shown). Nitrocefin hydrolysis by L-1 in the presence or absence of this peptide gave similar values. Additional testing with BSA (1000-fold) as a competitor did not affect the peptide inhibition observed. We determined the MIC of cefoxitin in the presence of the peptide inhibitor at 2 g/L using the strains of E. coli and S. maltophilia expressing L-1 ß-lactamase. No significant changes in MICs were observed.



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Figure 2. Lineweaver–Burk plot showing inhibition of nitrocefin hydrolysis activity by peptide Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys. Diamonds, hydrolysis without inhibitor; open squares, 5 µM peptide; open triangles, 20 µM peptide; open circles, 50 µM peptide. The position where lines intercept indicates the type of inhibition, in this case a mixed inhibition. The equilibria describing this system are shown above. Abbreviations: E, enzyme; S, substrate; I, inhibitor; P, product; Ks dissociation constant of complex enzyme-substrate; Ki competitive (Ki), dissociation constant of complex enzyme-inhibitor; Ki uncompetitive (Ki'), dissociation constant of complex enzyme-substrate-inhibitor.

 
The L-1 peptide inhibitor was tested for inhibition of Class A TEM-1 and PSE-4, the Class C P99 and three metallo-ß-lactamase—BcII, CphA and FEZ-1. No significant inhibition of the hydrolytic activity of Class A or Class C enzymes was found with nitrocefin as a substrate. In contrast, the L-1 peptide inhibitor diminished the hydrolytic activity of metallo-ß-lactamases, such as BcII, by 17% and the CphA enzyme by 25%; the FEZ-1 enzyme was completely inhibited at 100% (data not shown). Curiously, we noted that L-1 and FEZ-1 are in the same subclass—BBL B3—whereas BcII is in BBL B1 and CphA is classified in BBL B in a recent standard numbering scheme.8

To assess the importance of peptide conformation, the peptide Val-His-Ser-Pro-Asn-Arg-Glu was synthesized without Cys residues and had no significant inhibition of nitrocefin hydrolysis by L-1, even in 1000-fold excess. Hydrolysis of nitrocefin with 20 µM of peptide incubated with L-1 is not affected by the addition of 20 µM ZnCl2; however, we noted that 1000 µM of ZnCl2 rescued the hydrolytic activity from the peptide inhibitor.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we identified a peptide inhibitor of Class B ß-lactamase L-1 and FEZ-1. Using phage display combined with selective biopanning with cefoxitin, we isolated six different peptides. Among the aligned peptide sequences, the Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys was found in ~50% and the motif Ser-Pro-Asn was present in 75% of the peptides. Huang and collaborators1 have used phage display to identify a peptide inhibitor of class A ß-lactamase TEM-1; even when using a different ß-lactamase, we also obtained a peptide consensus in half the phage isolated. Even though the peptide of Huang et al.1 is a broad-spectrum inhibitor of ß-lactamases, no homology was found between their sequence and those reported here.1 Our biopanning strategies differed mostly by the use of cefoxitin in the elution step in round four. Cefoxitin was chosen for its high affinity for L-1 with a Km of 3.3 µM and its relatively ‘slow’ hydrolysis with a kcat of 2.2 s–1, as previously determined.9 In our selective strategy for biopanning, we expected that cefoxitin would probably compete with phage-displaying peptides competing for the active site of L-1, presumably selecting a peptide inhibitor of hydrolytic activity. Kinetic values with nitrocefin and our recombinant L-1 from S. maltophilia GN12873 were compared with those from L-1 from strain ULA-511 and are similar: a Km of 15 ± 3 µM versus 12 ± 1 µM and a kcat of 24 ± 2 s–1 and 31 ± 2 s–1, respectively.9 We noted that the presence or absence of Zn2+ in the buffer did not change the kinetic constants for hydrolysis of nitrocefin.

The Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys peptide gave a mixed inhibition. The apparent affinity of the peptide is almost equal for the enzyme and the enzyme–substrate complex (Ki competitive {approx} Ki uncompetitive) at µM concentrations indicating that the behaviour of inhibition is almost purely non-competitive.10 The peptide Val-His-Ser-Pro-Asn-Arg-Glu had no inhibitory activity suggesting that flanking Cys residues were essential. The TEM-1 consensus peptide, His-Ser-Ala-Cys-Asp-Thr-Arg-Arg-Gly-Asp-Cys-Gly, obtained by phage display was initially found to inhibit very weakly TEM-1 ß-lactamase with a Km of ~3.5 mM.1 Compared with the L-1 peptide inhibitor, this two-log difference can be explained by differences in biopanning where we used a competitive elution with cefoxitin. We entertain the possibility that competitive biopanning, such as using cefoxitin, is a more promising approach for finding ß-lactamase inhibitors. Huang and collaborators1 suggested that the disulphide bond consensus peptide constrained its conformation and was not optimal for binding and inhibition of TEM-1. For the L-1 peptide inhibitor, we noted that the disulphide bond was essential for its binding and blocking hydrolytic activity. Specific inhibition of Class B L-1 versus Class A TEM-1 and PSE-4, Class C P-99, is not surprising because these enzymes share low sequence identity and have major differences in their active site. Metallo-ß-lactamases require zinc, whereas other ß-lactamases have a serine protease mechanism. The kinetic data suggested binding of the enzyme and enzyme–substrate complex. The consensus peptide probably binds in the active site region, presumably displacing one of the two Zn atoms essential in the active site for maintaining hydrolytic activity.11 The peptide inhibitor described here will now be used as a lead compound in peptidomimetics to screen, identify and develop small molecules as inhibitors of class B metallo-ß-lactamases.


    Acknowledgements
 
We thank André Darveau, Département de Biochimie, Université Laval, for suggestions and comments using phage display, Moreno Gallinei, Centre d'Ingénieries des Protéines, Institut de Chimie, Université de Liège, Liège, Belgium for the gifts of enzymes and plasmids expressing the BCII, CphA and FEZ-1 enzymes and Le Service de Séquences de Peptides de l'Est du Québec for their excellent support. This work was supported by a Fonds de la Recherche en Santé du Québec team grant to R.C.L. and a Centres of Excellence operating grant. R.C.L. is a scholar of exceptional merit from the FRSQ.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Huang, W., Beharry, Z., Zhang, Z. et al. (2003). A broad-spectrum peptide inhibitor of ß-lactamase identified using phage display and peptide arrays. Protein Engineering 16, 853–60.[Abstract/Free Full Text]

2 . El Zoeiby, A., Sanschagrin, F., Darveau, A. et al. (2003). Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries. Journal of Antimicrobial Chemotherapy 51, 531–43.[Abstract/Free Full Text]

3 . Paradis-Bleau, C., Sanschagrin, F. & Levesque, R. C. (2004). Identification of Pseudomonas aeruginosa FtsZ peptide inhibitors as a tool for development of novel antimicrobials. Journal of Antimicrobial Chemotherapy 54, 278–80.[Free Full Text]

4 . Sanschagrin, F., Dufresne, J. & Levesque, R. C. (1998). Molecular heterogeneity of the L-1 metallo-ß-lactamase family from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 42, 1245–8.[Abstract/Free Full Text]

5 . Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

6 . Neu, H. C. & Heppel, L. A. (1965). The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. Journal of Biological Chemistry 240, 3685–92.[Free Full Text]

7 . Cornish-Bowden, A. (1995). Analysis of Kinetic Data. Oxford Science Publication, Oxford, UK.

8 . Garau, G., Garcia-Saez, I., Bebrone, C. et al. (2004). Update of the standard numbering scheme for class B ß-lactamases. Antimicrobial Agents and Chemotherapy 48, 2347–9.[Free Full Text]

9 . Crowder, M. W., Walsh, T. R., Banovic, L. et al. (1998). Overexpression, purification, and characterization of the cloned metallo-ß-lactamase L1 from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 42, 921–6.[Abstract/Free Full Text]

10 . Engel, P. C. (1996). Enzymology Labfax. BIOS Scientific, Oxford, UK.

11 . Ullah, J. H., Walsh, T. R., Taylor, I. A. et al. (1998). The crystal structure of the L1 metallo-ß-lactamase from Stenotrophomonas maltophilia at 1.7Å resolution. Journal of Molecular Biology 284, 125–36.[CrossRef][ISI][Medline]