BETA-LACTAMASE VARIANTS

Abstract

The present invention relates to an isolated polypeptide having beta-lactamase activity and nucleic acid sequences encoding the polypeptide. The isolated polypeptide of the invention is a VIM-2 variant with improved properties such as improved protease stability.

Claims

1. An isolated polypeptide having beta-lactamase activity, which comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1, said polypeptide further comprising a substitution at a position corresponding to residue 34 in SEQ ID NO: 1 and a C-terminal truncation of residues 237-240 of SEQ ID NO:1.

2. The polypeptide according to claim 1, which comprises: a C-terminal truncation of residues 236-240 of SEQ ID NO:1; or a C-terminal truncation of residues 235-240 of SEQ ID NO:1.

3. The polypeptide according to claim 1, wherein said substitution is Q34R.

4. An isolated polypeptide having beta-lactamase activity, which comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1, and which comprises a C-terminal truncation of exactly five residues from position 236 to 240 of SEQ ID NO:1.

5. The polypeptide according to claim 1, wherein the first residue of SEQ ID NO:1 is replaced with a methionine residue.

6. The polypeptide according to claim 1, further comprising a signal peptide at its N-terminal end having the sequence of SEQ ID NO:2.

7. The polypeptide according to claim 1, comprising a truncation at its N-terminal end as compared to the sequence of SEQ ID NO:1.

8. The polypeptide according to claim 1, comprising or consisting of the amino acid sequence of any one of SEQ ID NO:3 to 6.

9. An isolated nucleic acid sequence comprising a nucleic acid sequence encoding the polypeptide according to claim 1.

10. A nucleic acid construct comprising the nucleic acid sequence of claim 9, operably linked to one or more control sequences that direct the expression of the polypeptide in a suitable expression host.

11. A recombinant host cell comprising the nucleic acid construct of claim 10.

12. A composition comprising the polypeptide of claim 1.

13. The composition of claim 12, which is orally administrable and is able to release the polypeptide in the intestine.

14. A kit-of-parts comprising (a) the composition of claim 13; and (b) a beta-lactam antibiotic which is sensitive to said polypeptide contained in the composition of (a); for separate, sequential or simultaneous administration.

15. A method of treatment comprising administering the polypeptide of claim 1 to a patient.

16. The method of claim 15, wherein the patient has a bacterial infection which is caused by a bacteria which is susceptible to a beta-lactam antibiotic.

17. The method of claim 15, comprising administering the polypeptide in combination with a beta-lactam antibiotic which is sensitive to said polypeptide.

18. The composition of claim 12, which is orally administrable and is able to release the polypeptide in the jejunum, the ileum, the caecum or the colon.

Description

LEGEND OF THE FIGURES

[0077] FIG. 1 is a graph representing the specific activity of wild-type VIM-2, VIM-2 DCT236 and VIM-2.sub.[Q34R] DCT236 variants after 0, 30, 60, 90, 120 and 240 minutes in piglets ileal medium.

EXAMPLES

Example 1: Determination of the Positions of Interest in the Sequence of VIM-2 Enzyme

[0078] In order to identify amino acid positions relevant for either the enzyme activity, its substrate profile, its stability or its level of production in the host cell, random mutagenesis was used to create a library of bla.sub.VIM-2 genes carrying up to 12 mutations. To this end, bla.sub.VIM-2 mutants were introduced using an error-prone polymerase chain reaction, in the presence of nucleotide analogues. The bla.sub.VIM-2-derived nucleotide sequences were then cloned in a suitable Escherichia coli plasmid vector and the properties of the enzyme carrying various substitutions (thus a laboratory variants deriving from the introduction of mutations in the bla.sub.VIM-2 nucleotide sequence) were analyzed using the evaluation of the beta-lactam susceptibility profiles of the strains producing the variants, the determination of the specific-hydrolyzing activity for the degradation of various beta-lactams and the stability of the variant enzyme.

[0079] From the experiments described above performed on wild-type VIM-2, the following positions were discovered as positions of interest in the VIM-2 sequence wherein the positions correspond to the positions of the beta-lactamase represented in SEQ ID NO:1: positions 34 and 236.

Example 2: Production and Purification of One VIM-2 Variant at One Position

[0080] The VIM-2 variant was produced in Escherichia coli using either a P.sub.lac-promoter-based system (using pLB-II high copy number plasmid, as described in Borgianni et al., Antimicrob. Agents Chemother.; 2010; 54:3197-3204) or a T7 promoter-based expression system (using the pET-9a expression plasmid). Briefly, the mutant bla.sub.VIM-2 gene was cloned in the plasmid vector pLB-II or pET-9a using the NdeI and BamHI restriction sites, and the resulting plasmid introduced in E. coli DH5 or BL21(DE3) cells by electroporation. The resulting host cell was grown in either Luria-Bertani medium or the rich auto-inducing cell culture medium ZYP-5052 (Studier, F. W. 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41:207-234.) for 24 h, and the culture supernatant clarified by centrifugation. The resulting sample was then concentrated by ultrafiltration and loaded on an anion exchange chromatography column. Proteins were eluted using a linear NaCl gradient and the active fractions pooled and concentrated. The protein sample was then loaded on a gel filtration column and the proteins eluted with 50 mM HEPES (pH 7.5) supplemented with 50 M ZnSO.sub.4. The purified protein was then concentrated to 1-2 mg/ml and stored at 20 C.

[0081] All the mutants characterized in the following examples have been produced with a similar method. In particular, this production protocol was successfully used to produce the following variant VIM-2 enzymes: VIM-2 DCT236 (with a C-terminal deletion from position 236, i.e. the variant does not comprise amino acid 236-240 of wild-type VIM-2 of SEQ ID NO:1), VIM-2.sub.[Q34R], VIM-2.sub.[Q34R] DCT236.

Example 3: Determination of the Increased Resistance to Protease for Variants of VIM-2 Enzyme

[0082] To measure the sensitivity of purified enzyme preparations (e. g. VIM-2, VIM-2 DCT236 and variants thereof, prepared as described in Example 2) to proteolytic degradation by commercially-available purified proteases, the enzyme (final concentration, 50 g/mL) was incubated, at 35 C., for up to 150 min in a buffer (50 mM HEPES, 50 M ZnSO4, pH 7.5) containing up to 2.5 mg/mL of trypsin (from bovine pancreas, Type III, Sigma-Aldrich Cat. No. T-8253). The residual activity of the resulting sample was determined by means of a spectrophotometric assay, in which the hydrolysis rate of a 150 M imipenem solution (in 50 mM HEPES, 50 M ZnSO4, pH 7.5) was measured and compared to that of the sample at the beginning of the incubation (time, 0 min) or in the absence of protease.

[0083] In these conditions and in the absence of trypsin in the buffer, both tested enzymes (VIM-2 and VIM-2 DCT236) incubated for up to 150 min show a residual activity of 10015%, indicated a good intrinsic stability of the enzymes in the buffer system used in these experiments. In the presence of 2.5 mg/mL trypsin, the VIM-2 only retains 70 and 6610% of its initial activity after 30 or 90 min of incubation, respectively. By contrast, the VIM-2 DCT236 shows a residual activity of 9810% in the presence of 2.5 mg/mL trypsin, even after 150 min of incubation. These data show that the VIM-2 DCT236 enzyme is less susceptible to trypsin-mediated proteolytic degradation than the VIM-2 enzyme.

Example 4: Determination of Increased Stability of Several Variants of VIM-2

[0084] To measure the stability of VIM-2 variants in intestinal samples (i. e. where the enzyme is for example intended to exert its hydrolytic activity), the following procedure is applied: the imipenem-hydrolyzing activity of purified enzymes was determined after incubation within ileal medium collected from piglets. The specific activity (Sp. Act.) for the variants was measured at different time points (0, 30, 60, 90, 120 and 240 min) during the incubation. The specific activity at time t was compared with the specific activity at time t=0 to assess the loss of activity of the variant in the intestinal medium. The residual activity at different time points could be compared to evaluate the greater stability in intestinal medium of some variants compared to the wild-type enzyme.

[0085] The specific activity of wild-type VIM-2 enzyme and VIM-2 variants VIM-2 DCT236 and VIM-2.sub.[Q34R] DCT236 over time when incubated in ileal medium are presented in FIG. 1.

[0086] As shown in FIG. 1, the wild-type enzyme lost 88% of its activity after 240 minutes incubation in ileal medium. The VIM-2.sub.[Q34R] DCT236 variant lost only 45% of its activity in the same conditions. The VIM-2 DCT236 variant lost only 80% of its activity in the same conditions.

Example 5: Determination of the Specific Activity of Given VIM-2 Variants Against Beta-Lactams

[0087] For the mutant enzymes produced as mentioned in example 3, it is possible to measure the specific activity against specific beta-lactam compounds such as specific beta-lactam antibiotics. The specific activity assessment is performed as described hereafter: the hydrolysis of beta-lactams in the presence of crude extracts, prepared from bacterial cultures in the log or stationary growth phase as previously described (Docquier J.-D. et al., J. Antimicrob. Chemother.; 2003; 51:257-266), containing a VIM-2 variant and/or purified VIM-2 variants was monitored spectrophotometrically at a suitable wavelength. The specific activity may be expressed in nmol of beta-lactam substrate hydrolyzed by min and per mg of total protein in the sample.

[0088] In the following, all the specific activities will be expressed relatively to the specific activity of the wild-type enzyme measured in the same conditions.

[0089] For VIM-2 variants, the measured specific activities are:

TABLE-US-00005 (Specific activity of the enzyme/ Specific activity of the wild- Enzyme type enzyme) for ampicillin VIM-2.sub.[WT] 1 VIM-2.sub.[Q34R] 10.06

TABLE-US-00006 (Specific activity of the enzyme/ Specific activity of the wild- Enzyme type enzyme) for piperacillin VIM-2.sub.[WT] 1 VIM-2.sub.[Q34R] 4.14

TABLE-US-00007 (Specific activity of the enzyme/ Specific activity of the wild- Enzyme type enzyme) for ceftazidime VIM-2.sub.[WT] 1 VIM-2.sub.[Q34R] 17

TABLE-US-00008 (Specific activity of the enzyme/ Specific activity of the wild- Enzyme type enzyme) for imipenem VIM-2.sub.[WT] 1 VIM-2 DCT236 1.63 VIM-2.sub.[Q34R] 1.47

TABLE-US-00009 (Specific activity of the enzyme/ Specific activity of the wild- Enzyme type enzyme) for meropenem VIM-2.sub.[WT] 1 VIM-2.sub.[Q34R] 8.72