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 Verona integron-encoded metallo-β-lactamase (VIM-2) variant with improved properties such as improved protease stability, stability in intestinal medium, improved activity against one or more antibiotics, improved specific activity and/or improved production in a host cell.

Claims

1. An isolated polypeptide having beta-lactamase activity, which comprises an amino acid sequence having beta-lactamase activity, wherein said amino acid sequence has at least 90% sequence identity to SEQ ID NO:1, said amino acid sequence comprising substitutions in position 34 and in at least one position selected from positions 22 and 130, wherein the positions correspond to the positions in SEQ ID NO:1.

2. The polypeptide according to claim 1, wherein the polypeptide comprises: a substitution at each of positions 22 and 34; a substitution at each of positions 34 and 130; or a substitution at each of positions 22, 34 and 130.

3. The polypeptide of claim 1, which comprises at least one modification selected from the following: Q22H or Q22N; Q34R; and E130D.

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

5. The polypeptide according to claim 1, further comprising a signal peptide at its N-terminal end.

6. The polypeptide according to claim 1, wherein the amino acid sequence comprises a truncation at its N-terminal or C-terminal end as compared to the sequence shown in SEQ ID NO:1.

7. The polypeptide according to claim 6, which comprises: a C-terminal truncation at residues corresponding to 237-240 of SEQ ID NO:1; a C-terminal truncation at residues corresponding to 236-240 of SEQ ID NO: 1; or a C-terminal truncation at residues corresponding to 235-240 of SEQ ID NO: 1.

8. The polypeptide according to claim 1, consisting of the amino acid sequence of any one of SEQ ID NO:3 to 22 or a fragment thereof having beta-lactamase activity.

9. An isolated nucleic acid sequence comprising a 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.

19. The polypeptide according to claim 1, wherein the amino acid sequence comprises any one of SEQ ID NO: 3 to 22.

20. The polypeptide according to claim 1, wherein the amino acid sequence comprises a fragment of any one of SEQ ID NO: 3 to 22, said fragment having beta-lactamase activity.

Description

LEGEND OF THE FIGURES

(1) FIG. 1 is a graph representing the specific activity of wild-type VIM-2 and VIM-2.sub.[Q22H,Q34R,E130D] variant 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

(2) In order to identify amino acid position 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 (deriving from the introduction of mutations in the bla.sub.VIM-2 nucleotide sequence) were analyzed using the determination of the MIC of beta-lactams for 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.

(3) 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: 22, 34 and 130. A further possible modification includes the truncation of the C-terminal part of VIM-2 starting from, and including, position 236, resulting in a variant ending with the residue in position 235 of SEQ ID NO: 1.

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

(4) 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 DH5α 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 μM ZnSO.sub.4. The purified protein was then concentrated to 1-2 mg/mL and stored at −20° C. All the variants 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.sub.[Q34R], VIM-2.sub.[Q22H,Q34R,E130D].

(5) Another variant that may be produced thanks to a similar method is DCT236 (with a C-terminal deletion from position 236), and variants thereof such as VIM-2.sub.[Q34R] DCT236 and VIM-2.sub.[Q22H,Q34R,E130D] DCT236.

Example 3: Determination of Increased Stability of a Variant of VIM-2 in Intestinal Medium

(6) To measure the stability of VIM-2 variants, the following procedure is applied: the imipenem-hydrolyzing activity of purified protein samples was determined after incubation in ileal, jejunal and caecal media collected from piglets or adult pigs. 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 change over time was expressed as the ratio between the initial activity (at t=0) and the activity measured later in time ((Sp. Act. Variant)t/(Sp. Act. Variant)t=0. A value lower than one indicates a loss of activity when incubated in the intestinal medium. The residual activity at different time points are compared to evaluate the greater stability in intestinal medium of some variants compared to the wild-type enzyme.

(7) The specific activity of wild-type VIM-2 enzyme and VIM-2 variant VIM-2.sub.[Q22H,Q34R,E130D] over time when incubated in ileal medium are presented in FIG. 1.

(8) The loss of activity over time is also summarized in the following table:

(9) TABLE-US-00005 Residual activity after x minutes in ileal medium 0 30 60 90 120 240 Wild-type VIM-2 100% 66% 46% 33% 23% 13% VIM-2.sub.[Q22H, Q34R, E130D] 100% 88% 80% 73% 60% 56% variant

(10) As shown in the table or FIG. 1, the wild-type enzyme lost 87% of its activity after 240 minutes incubation in ileal medium. The VIM-2.sub.[Q22H,Q34R,E130D] variant lost only 44% of its activity in the same conditions.

(11) The combination of substitutions Q22G, Q34D, E130D and truncation at C-terminal end starting from amino acid position 236 also results in variants with dramatically improved properties in an industrial perspective.

Example 4: Determination of the Specific Enzymatic and or Catalytic Activity of Given VIM-2 Variants Towards Various Beta-Lactams

(12) For the variant enzymes produced as mentioned in example 3, it is possible to measure the specific activity towards specific beta-lactam compounds such as 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.

(13) 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.

(14) For VIM-2.sub.[Q22H,Q34R,E130D] variant, the measured specific activities are:

(15) TABLE-US-00006 (Specific activity of the enzyme/ Specific activity of the wild-type Enzyme enzyme) for ampicillin VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D] 8.57

(16) TABLE-US-00007 (Specific activity of the enzyme/ Specific activity of the wild-type Enzyme enzyme) for piperacillin VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D] 5.86

(17) TABLE-US-00008 (Specific activity of the enzyme/ Specific activity of the wild-type Enzyme enzyme) for ceftazidime VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D] 9.97

(18) TABLE-US-00009 (Specific activity of the enzyme/ Specific activity of the wild-type enzyme) for imipenem VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D] 6.15

(19) TABLE-US-00010 (Specific activity of the enzyme/ Specific activity of the wild-type Enzyme enzyme) for meropenem VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D] 5.98

(20) The above results illustrate that crude extracts containing the VIM-2.sub.[Q22H,Q34R,E130D] exhibited a strongly improved activity against several beta-lactams of interest compared with the wild-type enzyme.

(21) In addition, variants with a truncated C-terminal end starting from amino acid position 236 present improved characteristics. The combination of substitutions Q22G, Q34D, E130D and said truncation therefore result in a variant with dramatically improved properties in an industrial perspective.

(22) In particular, the variant VIM-2.sub.[Q22H,Q34R,E130D] DCT236 exhibits improved catalytic properties when considering imipenem as a substrate:

(23) TABLE-US-00011 k.sub.cat K.sub.M k.sub.cat/K.sub.M VIM-2 WT 34 9 3.8 VIM-2.sub.[Q22H, Q34R, E130D] DCT236 85 13.5 6.3