HK1078883A - Nucleotide sequence coding for a tolc and a defined amino acid sequence - Google Patents

Description

Nucleotide sequence coding for TolC and defined amino acid sequence

Technical Field

The present invention relates to a nucleotide sequence encoding TolC, a plasmid comprising such a nucleotide sequence, a protein or peptide encoded by such a nucleotide sequence, a bacterium comprising such a nucleotide sequence and various uses of such a bacterium.

Background and Prior Art

In virulence attenuation, bacteria that multiply intracellularly can induce long-lasting immunity as live vaccines. Salmonella Typhi TY1a (Levine et al, Lancet 1: 1049-.

Variants of, for example, Listeria monocytogenes (Listeria monocytogenes), Salmonella enterica (Salmonella enterica), Salmonella typhimurium (Salmonella typhimurium), and Salmonella typhosa (Salmonella Typhi), as well as the BCG class, have been used as good digestible live vaccines against typhus and tuberculosis. These bacteria, including attenuated mutants thereof, are general immune stimulators, can elicit a good cellular immune response and are themselves useful as vaccine vectors.

The advantage of these bacteria as vaccine vectors is that they first induce a so-called Thl immune response (Hess and Kaufmann, FEMS Immunol. Med. Microbiol. 23: 165-173, 1999). These immune responses are characterized by the presence of cytotoxic lymphocytes (CTL) and specific IFN- γ secreting CD4+ T-cells (i.e., T-helper cells, Th) (Abbas et al, Nature 383: 787-.

For example, listeria monocytogenes (l.monocytogenes) stimulates THl cell activation and cytotoxic T-lymphocyte (CTL) proliferation to a specific extent. These bacteria provide secreted antigens which directly enter the cytoplasm of the antigen-providing cells (APC; macrophages and dendritic cells), which themselves inhibit costimulatory molecules and lead to effective stimulation of T cells. The listeria moiety reduces phage compatibility, so that antigens produced by these vector bacteria can be presented on the one hand in MHC class II molecules and thereby induce T-helper cells. On the other hand, listeria replicates in the cytosol of APCs upon release from the phage; the antigens produced and secreted by these bacteria are thus preferably presented via the MHC class I pathway and will therefore induce a CTL response against these antigens. Furthermore, it could be shown that the expression of these cytokines is induced by the interaction between Listeria with macrophages, natural killer cells (NK) and neutrophils (TNF-. alpha., IFN-. gamma., I1-2, IL-12; Unanuue, curr. Opin. Immunol., 9: 35-43, 1997; Mata and Paterson, J Immunol.163: 1449-.

Thus, recombinant bacteria are able to protect against heterologous tumors (Medina et al, Eur. J. Immunol.29: 693-699, 1999; Pan et al, Cancer Res.59: 5264-5269, 1999; Woodlock et al, J Immunol.22: 251-259, 1999; Paglia et al, Blood 92: 3172-3176, 1998; Paglia et al, Eur. J. Immunol.27: 1570-1575, 1997; Pan et al, nat. Med.1: 471-477, 1995; Pan et al, Cancer Res.55: 4776-4779, 1995).

Thus, by providing L.monocytogenes (L.monocytogens) that transduce expressed tumor antigens, it is possible to antigen-specifically inhibit the growth of experimental tumors (Pan et al, nat. Med.1: 471-477, 1995; Cancer Res.59: 5264-5269, 1999; Voest et al, Natl. Cancer Inst.87: 581-586, 1995; Beatty and Pater son, J.Immunol.165: 5502-5508, 2000).

Attenuated Salmonella enterica (Salmonella enterica) into which a nucleotide sequence encoding a tumor antigen has been introduced can be used as a bacterial vector for inhibiting tumor antigens after oral administration to result in specific protection against various experimental tumors (Medina et al, Eur. J. Immunol.30: 768-777, 2000; Zollerund Christ J. Immunol.166: 3440-34450, 2001; Xiaong et al, PNAS 97: 5492-5497, 2000).

Recombinant Salmonella bacteria are also effective as a prophylactic vaccine against viral infections (HPV; Benyacoub et al, infection. Immun.67: 3674-3679, 1999) and for the treatment of tumor virus (HPV) -immobilized mouse tumors (Revaz et al, Virology 279: 354-3679, 2001).

For use as a vaccine vector, the following methods are proposed: the expression product is inhibited by introducing a nucleic acid sequence into the bacteria on the cell membrane of these bacteria or secreted by these bacteria. These methods are based on the E.coli (Escherichia coli) hemolysin system HlyAs, which produces prototypes of the type I secretion system from gram-negative bacteria. The secretion vectors are produced by means of HlyAs, so that protein antigens in Salmonella enterica (Salmonella enterica), Yersinia enterocolitica (Yersinia enterocolitica) and Vibrio cholerae (Vibrio cholerae) can be efficiently carried out. Such secretion vectors comprise the cDNA of any protein antigen coupled to the nucleotide sequence of the HlyA-signal peptide, the hemolysin secreta, hlyB and hlyD, and the hly-specific promoter. Proteins on the surface of these bacteria can be inhibited, for example, by means of these secretion vectors. Such genetically modified bacteria as vaccines induce an increase in the immunoprotection better than bacteria retaining their cell lines by proteins inhibited by the introduction of nucleic acids (Donner et al, EP 1015023A; Gentschev et al, Gene 179: 133-. However, this system has the disadvantage that the number of proteins suppressed by the bacteria by using a Hly-specific promoter is small.

For example, other transport systems in bacteria are: i) transport signal of the S-layer protein (Rsa a) of corynebacterium crescentis (caulobactererchienceus), which-for secretion and membrane-bound expression-uses a C-terminal Rsa a-transport signal (Umelo-Njaka et al, Vaccine 19: 1406-1415, 2001) and ii) Internalin A transport signals of Listeria monocytogenes (Listeria monocytogenes). Secretion requires an N-terminal transit signal, whereas membrane-bound expression requires an N-terminal transit signal and a C-terminal part comprising an LPXTG-motif responsible for cell wall immobilization (Dhar et al, Biochemistry 39: 3725-3733, 2000).

Other relevant is the intact membrane protein TolC from e. This is a multifunctional, pore-forming protein of the outer membrane of E.coli (E.coli), which functions, for example, in addition to approximating the uptake of E1 (Morona et al, J.Bacteriol.153: 693-. These proteins are found not only in E.coli (E.coli) but also in most gram-negative bacteria (Wiener, Structure fold. Des., 8: 171-5, 2000).

The crystal structure of the TolC protein shows that it forms a tunnel of about 120 angstroms in length as a homotrimer, the largest part of the homodimer, the tunnel region is located in the periplasm, and there are only two small loops (amino acids 52-61 and 257-919) on the surface of the bacteria (Koronakis et al, Nature 405: 914-919, 2000). Niki et al, Nucleic acid sequence of the tolCgene of Escherichia coli, Nucleic Acids Res.18(18), 5547(1990) disclose that the tolC gene has this nucleotide sequence. TolC is a part of a system from at least four different bacterial exports whereby it forms a membrane tunnel through which bacterial proteins can be exported. For example, in this HlyA transport system, the linkage between HlyD and the periplasmic end of TolC allows export of hemolysin from HlyD in the TolC membrane tunnel (Gentschev et al Trends in Microbiology 10: 39-45, 2002).

Disclosure of Invention

Problem to be solved by the invention

The present invention is based on the technical object of describing a transport system by means of which expression products with a higher potency on the outer cell membrane can be provided.

Basic concept and preferred embodiments of the invention

To achieve this technical object, the present invention teaches a nucleotide sequence encoding TolC and a defined amino acid sequence inserted in the extra-membrane region of a permissive TolC.

The invention results in a novel transport system for gram-negative bacteria, by means of which a larger amount of proteins, which are inhibited by genes in the bacteria, can be transported to the outer cell membrane of the bacteria than in the prior art. Surprisingly, the transport system for the TolC-protein of E.coli (Escherichia coli) allows a much more powerful permanent expression of the membrane of (arbitrary) peptides or proteins, and this is for a large number of gram-negative bacteria, compared to the transport systems of the prior art. Thus, membrane permanent expression of the determined amino acid sequence or gene product is accomplished only by TolC.

The defined amino acid sequence may be any given peptide or protein, any pharmaceutically active substance, any antigen, any antibody or any ligand.

The TolC may be a (wild-type) TolC protein according to ACCESSION X54049 for the clear indication, or a (preferably N-terminal) partial sequence thereof or a mutant or partial sequence of the protein, wherein for the partial sequence or mutant the transport function is maintained. The N-terminal part sequence is referred to herein as any part sequence starting at amino acids 1 to 50 of the N-terminal region of the TolC protein and ending at the C-terminus of the loop, which is on the surface of the bacterium. Thus, the N-terminal transport signal of TolC and the central part of the protein, which forms the extracellular region of TolC, are preferred. The mutant may include insertions, deletions or substitutions as long as its transport function is not significantly reduced thereby.

For specific applications, it is advisable to insert the defined amino acid sequence on one or both sides of the spacer sequence. However, it is only advantageous if a defined amino acid sequence is provided in a specific spatial structure, for example in the case of an antigen, which is not produced to the desired extent by the defined amino acid sequence itself, for spatial or conformational reasons. In particular, it is therefore possible to construct the spacer sequence by naturally joining the sequence following the defined amino acid sequence, so that the defined amino acid sequence is folded like the natural antigen. However, the spacer sequence may also be artificial, as long as the presentation and/or folding of the desired defined amino acid sequence is obtained thereby. This is easily calculated using theoretical methods and considering the spatial conditions of the insertion position of the TolC.

In particular, it is particularly preferred that the defined amino acid sequence is inserted in particular in the N-terminal region of TolC, in particular in the region of amino acids 52 to 61 and/or 257-279 (respectively TolC protein).

Furthermore, the subject of the invention is plasmids comprising the nucleotide sequences of the invention and proteins or peptides encoded by the nucleotide sequences of the invention.

Furthermore, the present invention teaches a bacterium comprising a nucleotide sequence of the present invention, wherein TolC causes transport of the defined amino acid sequence to the bacterial membrane. In other words, the TolC protein causes membrane-permanent expression of the gene product to occur in bacteria. The subject of the invention is therefore also a gram-negative bacterium which comprises at least one nucleotide sequence which codes for at least one defined amino acid sequence and codes for at least one E.coli (E.coli) TolC-gene product. The E.coli (E.coli) TolC-gene product is preferably the wild type. The subject of the invention is also a mutated E.coli (E.coli) TolC gene product in which the transport signal activity is maintained. Preferably the bacteria are selected from the following: "Salmonella spp.", Escherichia coli (Escherichia coli), Vibrio cholerae (Vibrio cholerae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Shigella spp. ", and Yersinia spp.".

The nucleotide sequences and bacteria of the present invention may be used in a variety of applications. For example, the present invention also teaches a pharmaceutical composition comprising a bacterium of the present invention and optionally at least one physiologically acceptable carrier substance, wherein the defined amino acid sequence is selected according to the given substance to be bound in vivo. By means of such pharmaceutical compositions, substances which interfere with normal cellular metabolism, for example exogenous poisons or endogenous substances caused by mutations, such as (Octagons), can be bound and thus inhibited. In addition, by binding to certain cellular targets, metabolic processes can be regulated by eliminating normal complex participants or complex participants that are upregulated due to pathological conditions. Thus, the shuttle associated therewith is down-regulated, for example by suppressing a determined linkage. This method can also be used to adjust other related methods. Accordingly, the defined amino acid sequence need only be selected with respect to the target molecule to be inhibited with high specificity. Such pharmaceutical compositions are therefore ultimately intended for therapeutic purposes.

Pharmaceutical compositions suitable for vaccination purposes comprise the bacterium of the invention and optionally at least one physiologically acceptable carrier substance, wherein the defined amino acid sequence is an immunological sequence. The immunological sequence stimulates the production of antibodies against the natural antigen in the organism, which comprises or consists of the immunological sequence as a partial sequence.

For diagnostic purposes, the present invention teaches a diagnostic kit comprising a bacterium according to one of claims 7 to 9, wherein the determined amino acid sequence specifically binds to the marker to be determined. If, for example, tissue or liquid samples are taken from the organism and, if necessary after a pretreatment for separating undesired sample components, these samples are incubated with bacteria so that binding events at a defined amino acid sequence can be detected and, in the case of binding events, substances which bind specifically to a defined amino acid sequence are detected in the samples. The detection of binding events can therefore be a variety of methods known to those of ordinary skill in the art.

Finally, the present invention teaches a preparative binding substance comprising a bacterium of the present invention, wherein the determined amino acid sequence specifically binds to a target substance isolated from a solution. By means of such a combination, undesired substances can be removed from the solution in particular, on the one hand, by incubating the solution with the bacteria and removing the bacteria after isolation. On the other hand, the isolation or enrichment of the target substance can be carried out in a corresponding manner, i.e.the target substance is eluted from the bacteria after the cultivation. For this purpose, the invention can also be used for the isolation and/or enrichment of antigens, antibodies, peptides, proteins or ligands.

Drawings

FIG. 1: source pBR 322;

a Tc resistance gene;

cloning the tolC gene from e.coli;

cloning of the p60 epitope sequence from Listeria monocytogenes (L.monocytogenes).

Detailed Description

Examples

Example 1: construction of TolC vector

The tolC genes from E.coli (E.coli), including their wild-type promoters, were used by PCR (1 min 94 ℃, 1 min 66 ℃, 1 min 30s 72 ℃) with plasmid pAX629 (C.Wand)The 5 'TolC (5' -TAACGCCCTAT) oligonucleotide of ersman, Institute Pasteur, ParisGTCGACTAACGCCAACCTT-3) and 3' TolC (5-AGAG-GAT)GTCGACTCGAAATTGAAGCGAGA-3') was amplified. Thereby introducing an additional SalI-interface at both ends. The purified PCR product was digested with the restriction endonuclease SalI (QIA Rapid PCR purification kit-Qiagen, Hilden, Germany) and cloned into the vector pBR322 previously isolated with SalI-. The vector thus constructed was designated pBR322 tolC. The cloned tolC gene was then studied for function in more relevant experiments.

Example 2: insertion of an antigenic sequence into a TolC protein sequence

In the tolC sequence, which codes for one of the extracellular loops, a KpnI interface is defined. This was used to clone the antigenic peptide sequence (iap gene) of the p 60-protein of Listeria monocytogenes (Listeria monocytogenes) and to allow insertion of foreign antigens after amino acid 271 of the mature TolC-protein.

The iap-sequences coding for the B-cell epitope (amino acids 291-301) and the CD 4-restricted T-cell epitope (amino acids 301-312) of the p 60-protein were cloned as KpnI fragment into the vector pBR322tolC from which KpnI had been removed beforehand (FIG. 1). The plasmid thus formed was designated pBR322 tolC:LisTB.

FIG. 1 shows the cloning strategy for inserting a p 60-specific epitope sequence into the wild-type, plasmid-encoded E.coli (E.coli) tolC gene on vector pBR 322. They are: bla-ampicillin resistance gene; tc-tetracycline; T-Listeria monocytogenes (L.monocytogenes) p 60-T-cell epitope (AS 301-312); B-Listeria monocytogenes (L.monocytogenes) p 60B cell epitope (AS 291-301); PtolC-wild type e.coli (e.coli) tolC-promoter.

Example 3: expression of antigens on gram-negative bacteria (E.coli) membranes

Expression of an epitope of p 60-protein from Listeria monocytogenes (L.monocytogenes) within the TolC-protein was detected by Western blotting. For this purpose, cell lysate proteins from E.coli (E.coli) CC118tolC, E.coli (E.coli) CC118tolC/pBR322tolC and E.coli (E.coli) CC118tolC/pBR322tolC:: LisTB were isolated in the subsequent log phase. The total cellular protein for this application is equivalent to about 100 million bacteria. The proteins were isolated in a 15% SDS-polyacrylamide gel and the expression of the TolC-protein or of the inserted epitope of the chimera was detected, on the one hand, with polyclonal serum against TolC-protein and, on the other hand, with the monoclonal antibody K317(Rowan et al, J.Clin.Microbiol.38: 2643-2648, 2000), which specifically directed against B-cell epitopes from Listeria monocytogenes (L.monocytogenes) (FIG. 2B).

As can be expected, no TolC-protein could be detected in the cell lysate of E.coli CC118tolC, which could be attributed to a mutation of the chromosomal tolC gene in this strain (Schlor et al, mol. cen. Genet.256: 306-319, 1997). Complementation of pBR322tolC resulted in the expression of the 52kDa large TolC-protein in these strains. Insertion of a listeria monocytogenes (l.monocytogenes) epitope in the TolC-protein did not affect the expression of TolC and resulted in a slight modification of the size of the chimeric protein of approximately 3 kDa.

Coli (E.coli) CC118tolC/pBR322 tolC:expressionof the p 60-specific epitope on LisTB can be demonstrated with monoclonal p 60-antibody K317.

Example 4: detection of the exposed localization of the L.monocytogenes (L.monocytogenes) p 60-epitope in Salmonella enteritidis (Salmonella enteritidis) SM6T (tolC)

Since the two Listeria p60 epitopes in the extracellular loop of TolC are inserted after amino acid 271 of the mature protein, they should be present on the surface of Salmonella enteritidis (S. enteritidis) SM6T in an exposed manner (Stone et al, mol. Microbiol. 17: 701-712, 1995). The defined extracellular localization of this p 60-specific epitope in salmonella enteritidis (s.enteritidis) SM6T was examined by indirect immunofluorescence. Mu.l each of an overnight culture of Salmonella enteritidis (S.enteritidis) SM6T/pBR322tolC and Salmonella enteritidis (Salmonella enteritidis) SM6T/pBR322tolC:: LisTB was dropped onto the target vector and air-dried. Monoclonal p 60-antibody K317 (1: 200) stained the cells, and bound antibodies bound to FITC-labeled secondary anti-mouse serum were detected (Dianova, Germany, Albeitster: 1: 40).

Fluorescence microscopy analysis confirmed the extracellular localization of specific epitopes of Listeria monocytogenes (L.monocytogenes) in the strain Salmonella enteritidis SM6T/pBR322tolC:: LisTB.

Example 5: immunoassays with gram-negative bacteria and analysis of protective immune responses following infection with wild-type listeria monocytogenes (l

To determine whether L.monocytogenes (L.monocytogenes) leads to protection by the emerging expression of T-cell epitopes from the p 60-protein in a mouse model of Listeria disease, the oral dose was 1X 10⁷8 six-week-old female Balb/c-mice (Charles River, Sulzfeld, Germany) were immunized by LisTB. For control, 5 female mice were immunized by oral administration of salmonella enteritidis (s.enteritidis) SM 6T. The animals were immunized a second time with the same bacterial dose after 3 weeks.

The immunization results were checked 5 weeks after the first immunization with immunoblotting, on which supernatant proteins from Listeria monocytogenes (Listeria monocytogenes) were applied. Antibodies specific for p60 can thus be detected in the serum of mice immunized with Salmonella enteritidis SM6T/pBR322tolC:: LisTB.

3 weeks after the second immunization, 5X 10⁴Listeria monocytogenes (L.monocytogenes) EGD, 5-folded LD₅₀Animals were infected intravenously. During this period, after intravenous infection with Listeria monocytogenes (L.monocytogenes) EGD, the survival rate of Balb/c-mice immunized with Salmonella enteritidis SM6T/pBR322tolC:: LisTB was 88%, which was only 20% lower than the control group.

Thus expression of the p 60-specific epitope within the extracellular loop of TolC in the attenuated salmonella enteritidis (s.enteritidis) vector strain SM6T resulted in the induction of a Listeria monocytogenes (Listeria monocytogenes) -specific immune response that could be used to protect Balb/c mice against normally lethal infections. The induction of antibodies against B-cell epitopes from p60 protein can therefore be detected by Westen blotting. Apparently, the immune response in which the antibody is involved has led to the protection observed here to protect mice from other lethal infections with listeria monocytogenes (l.

Claims (13)

1. A nucleotide sequence encoding TolC and a defined amino acid sequence, wherein the defined amino acid sequence is inserted in the extracellular region of a permissive TolC.

2. Nucleotide sequence according to claim 1, wherein TolC is a protein according to acessionx 54049 or preferably an N-terminal part sequence thereof or a mutant of said protein or part sequence, and wherein the transport function is maintained for said N-terminal part sequence or mutant.

3. A nucleotide sequence according to claim 1 or 2, wherein the defined amino acid sequence is inserted on one or both sides of the spacer sequence.

4. Nucleotide sequence according to one of claims 1 to 3, in which the defined amino acid sequence is inserted in the N-terminal region of TolC, in particular in the region of amino acids 52 to 61 and/or 257 to 279 (each in connection with TolC protein).

5. A plasmid comprising a nucleotide sequence according to any one of claims 1 to 4.

6. A protein or peptide encoded by a nucleotide sequence according to any one of claims 1 to 4.

7. Bacterium comprising a nucleotide sequence according to one of claims 1 to 4, wherein TolC causes transport of a defined amino acid sequence on the bacterial membrane.

8. The bacterium according to claim 7, wherein the determined amino acid sequence represents a peptide, protein, active substance, antigen, antibody or ligand.

9. The bacterium according to one of claims 7 or 8, which is selected from the group consisting of "Salmonella spp.", Escherichia coli (Escherichia coli), Vibrio cholerae (Vibrio cholerae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Shigella spp. ", and Yersinia spp.".

10. A pharmaceutical composition comprising a bacterium according to one of claims 7 to 9 and optionally at least one physiologically acceptable carrier substance, wherein said defined amino acid sequence is selected according to the given substance to be bound in the organism.

11. A pharmaceutical composition comprising a bacterium according to one of claims 7 to 9 and optionally at least one physiologically acceptable carrier substance, wherein the determined amino acid sequence is an immune sequence.

12. A diagnostic kit comprising a bacterium according to any one of claims 7 to 9, wherein the determined amino acid sequence specifically binds to a label to be determined.

13. Preparative binding substance comprising a bacterium according to one of claims 7 to 9, wherein the determined amino acid sequence specifically binds to a target substance isolated from a solution.