Indian Patents. 223784:NUCLEOTIDE SEQUENCE CODING FOR A TOLC AND A DEFINED AMINO ACID SEQUENCE

Title of Invention	NUCLEOTIDE SEQUENCE CODING FOR A TOLC AND A DEFINED AMINO ACID SEQUENCE
Abstract	Nucleotide sequence coding for a TolC and a defined amino acid sequence The invention relates to a nucleotide sequence coding for a TolC, and a defined amlno acid sequence, said defined amino acid sequence being inserted in the permissive, membrane-external area of the TolC, and several uses thereof, particularly bacteria containing such a nucleotide sequence.

Full Text	Nucleotide sequence coding for a TolC and a defined amino acid sequence. Field of the invention. The invention relates to a nucleotide se- quence coding for a TolC, a plasmid containing such a nucleotide sequence, a protein or a pep- tide coded by such a nucleotide sequence, a bac- terium containing such a nucleotide sequence and several uses of such bacteria. Background of the invention and prior art. Viru1ence- attenuated, intrace11u1ar1y set- tling bacteria can induce a long-lasting immu- nity as live vaccines. Up to now, in particular Salmonella typhi TYla (Levine et al., Lancet 1:1049-1052, 1987), Mycobacterium bovis BCG (Fine and Rodrigues, Lancet 335:1016-1020, 1990) and Vibrio cholerae (Levine and Kaper, Vaccine 11: 20 7-212, 1993) were used as live vaccines. For instance, such variants of Listeria mono- cytogenes, Salmonella enterica sv. typhimurium and typhi, and BCG were already used as well- tolerated live vaccines against typhus and tu- berculosis. These bacteria, including their at- tenuated mutants are generally immune-stimulat- ing and can initiate a fair cellular immune re- sponse, and were therefore used a vaccine carri- ers. The advantage of these bacteria as vaccine carriers is that they mainly induce a so-called Th1 immune response (Hess and Kaufmann, FEMS Im- munol. Med. Microbiol. 23:165-173, 1999). This immune response is characterized by cytotoxic lymphocytes (CTL) and by the presence of spe- cific IFN-gamma- secreting CD4+ T cells (also T helper cells, Th) (Abbas et al., Nature 383:787- 793, 1996). For instance, L. monocytogenes stimulates to a special extent via the activation of TH1-cells the proliferation of cytotoxic T lymphocytes (CTL). These bacteria supply secreted antigens directly into the cytosol of antigen-presenting cells (APC; macrophages and dendritic cells), which in turn express the co-stimulating mole- cules and cause an efficient stimulation of T cells. The listeriae are in part degraded in phagosomal compartments, and the antigens pro- duced by these carrier bacteria can therefore on the one hand be presented by MHC class II mole- cules and thus lead to the induction of T helper cells. On the other hand, the listeriae repli- cate after release from the phagosome in the cy- tosol of APCs; antigens produced and these bacteria are therefore preferably pre- sented via the MHC class I pathway, thus CTL re- sponses against these antigens being induced. Further it could be shown that by the interac- tion of the listeriae with macrophages, natural killer cells (NK) and neutrophilic granulocytes, the expression of such cytokines (TNF-alpha, IFN-gamma, 11-2, IL-12; Unanue, Curr. Opin. Im- munol. 9:35-43, 19 9 7; Mata and Paterson, J. Im- munol. 163:14 4 9-144 56, 1999) is induced, for which an antitumoral effectiveness was detected. Recombinant bacteria were thus capable to protect against a heterologous tumor (Medina et al., Eur. J. Immunol. 29:693-699, 1999; Pan et al., Cancer Res. 59:5264-5269, 1999; Woodlock et al., J. Immunother. 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). By the administration of L. monocytogenes, which were transduced for the expression of tu- mor antigens, the growth of experimental tumors could be inhibited antigen-specifica11y (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 Paterson, J. Immunol. 16 5:5502-5508, 2000). Virulence-attenuated , Salmonella enterica strains, into which nucleotide sequences coding for tumor antigens had been introduced, as tumor antigen-expressing bacterial carriers, could provide after oral administration a specific protection against different experimental tumors (Medina et al., Eur. J. Immunol. 30:768-777, 2000; Zoller and Christ, J. Immunol. 166:3440- 34450, 2001; Xiang et al., PNAS 97:5492-5497, 2000) . Recombinant Salmonella strains were also ef- fective as prophylactic vaccines against virus infections (HPV; Benyacoub et al., Infect. Im- mun. 67:3674-367 9, 1999) and for the therapeutic treatment of a mouse tumor immortalized by a tu- mor virus (HPV) (Revaz et al., Virology 279:354- 360, 2001). For the use as a vaccine carrier, methods were developed to express expression products of nucleic acid sequences introduced into bacteria on the cell membrane of these bacteria or to have them secreted by these bacteria. The basis of these methods is the Escherichia coli hemo- lysin system hlyAs representing the prototype of a type I secretion system of gram-negative bac- teria. By means of the hlyAs, secretion vectors were developed that allow an efficient discharge of protein antigens in Salmonella enterica, Yersinia enterocolitica and Vibrio cholerae. Such secretion vectors contain the cDNA of an arbitrary protein antigen coupled to the nucleo- tide sequence for the hlyA signal peptide, for the hemolysin secretion apparatus, hlyB and hlyD and the hly-specific promoter. By means of this secretion vector, a protein can for instance be expressed on the surface of this bacterium. Such genetically modified bacteria induce as vaccines a considerably stronger immune protection than bacteria, wherein the protein expressed by the introduced nucleic acid remains inside the cell (Donner et al. , EP 1015023 A; Gentschev et al., Gene 179:133-140, 1996; Vaccine 19; 2621-2618, 2001; Hess et al., PNAS 9 3:1458-1463, 1996). The disadvantage of this system is however that by using the hly-specific promoter the amount of the protein expressed by the bacterium is small. Further transport systems in bacteria repre- sent for instance, i) the transport signal for the S-layer protein (Rsa A) of Caulobacter cres- centus, where - for the secretion and the mem- brane-bound expression - the C-terminal RsaA transport signal is to be used (Umelo-Njaka et al., Vaccine 19:1406-1415, 2001), and ii) the transport signal for the Internalin A of Lis- teria monocytogenes. For the secretion, the N - terminal transport signal is necessary, and for the membrane-bound expression, the N-terminal transport signal together with the C-terminal part containing the LPXTG motive responsible for the cell wall anchoring (Dhar et al., Biochemis- try 39 : 3725-3733, 2000) . In another context, the integral membrane protein TolC of E. coli is known. This is a mul- ti-functional pore-forming protein of the outer membrane of E. coli, which, in addition to func- tions, such as e.g. reception of Colicin El (Mo- rona et al., J. Bacteriol. 153:693-699, 1983) and the secretion of Colicin V (Fath et al., J. Bacteriol. 173:7549-7556, 1991), also serves as a receptor for the U3 phage (Austin et al., J. Bacteriol. 17 2:5312-5325, 1990). This protein is not only found in E. coli, but also in a multi- tude of gram-negative bacteria (Wiener, Struc- ture Fold. Des. 8:171-5, 2000). The crystal structure of the TolC protein shows that it forms, as a homotrimer, a tunnel channel having a length of about 120 Angstroms, the biggest part of the homodimer, the tunnel domain, being localized in the periplasm and only two little loops (amino acids 52-61 and 257-279) being settled on the surface of the bacterium (Koronakis et al., Nature 405:914-919, 2000) . The tolC gene has the nucleotide sequence published by Niki et a 1., Nucleotide sequence of the tolC gene of Escherichia coli, Nucleic Acids Res. 18 (18), 5547 (1990). TolC is part of at least four different bacterial export systems by representing the membrane tunnel, through which the export of the bacterial protein is made pos- sible. For instance in the hlyA transport sys- tem, the connection between hlyD and the peri- plasmic end of the TolC permits the export of the hemolysin from the hlyD into the membrane tunnel of the TolC (Gentschev et al., Trends in Microbiology 10:39-45, 2002). Technical object of the invention. The invention is based on the technical ob- ject to specify a transport system, by means of which an expression product having a higher ef- ficiency can be presented on an outer cell mem- brane . Basic concept of the invention and preferred em- bodiment s . For achieving the above technical object, the invention teaches a nucleotide sequence coding for a TolC and a defined amino acid sequence, said defined amino acid sequence being inserted in the permissive, membrane-external area of the TolC . By the invention, a new transport system in gram-negative bacteria is achieved, by means of which larger amounts of a protein expressed by a gene within a bacterium can be transported on the outer cell membrane of the bacterium than was possible with prior art transport systems. Surprisingly, the transport system for the TolC protein of Escherichia coli permits a substan- tially stronger membrane-bound expression of a peptide or protein (arbitrary) than was known from prior art transport proteins, and that for a multitude of gram-negative bacteria. The mem- brane-bound expression of the defined amino acid sequence or gene product is exclusively achieved by the TolC. A defined amino acid sequence may be an arbi- trary qiven peptide or a protein, an arbitrary pharmaceutical active substance, an arbitrary antigen, an arbitrary antibody, or an arbitrary 1igand. The TolC may be a (wild-tvpe) TolC protein according to ACCESSION X54049, to which hereby explicitly reference is made, or a (preferably N-terminal) partial sequence thereof or a mutant of the protein or of the partial sequence, for the partial, s, eaue nee or the mutant the transport functionality being maintained. N-terminal par- tial sequences means any partial sequence begin- ning in the N-terminal area amino acids 1 to 50 of the TolC protein and ending at the C-terminal end of a loop, which is settled on the surface of the bacterium. Preferred is thus the N-termi- nal transport signal of TolC, but also the cen- tral part of the protein, which represents the extracellular areas of TolC. A mutant may com- prise an insertion, deletion or substitution, as long as the transport functionality is not dis- tinctly reduced thereby. For certain applications, it may be recom- mended that a defined amino acid sequence be in- serted on one side or both sides by a spacer se- quence. This will however only be helpful, if the defined amino acid sequence is to be pre- sented in a certain spatial structure, for in- stance in the case of an antigen, and this does however not take place by the defined amino acid sequence itself for steric or configurative rea- sons to a desired extent. Then a spacer sequence may be formed in particular by a sequence natu- rally following the defined amino acid sequence, thus the defined amino acid sequence being folded in the same way as in the natural anti- gen. The spacer sequence may however also be ar- tificial, if thus a desired presentation and/or folding of the defined amino acid sequence is obtained. This can easily be calculated by means of theoretical methods, under consideration of the spatial conditions at the position of inser- tion in the TolC . In detail it is preferred that the defined amino acid sequence is inserted in the N-termi- nal area of the TolC, in particular in the area of the amino acids 52 to 61 and/or 257 to 279 (each referred to the TolC protein) . Subject matter of the invention is further a plasmid containing a nucleotide sequence accord- ing to the invention and a protein or peptide coded by a nucleotide sequence according to the invention. The invention further teaches a bacterium containing a nucleotide sequence according to the invention, the TolC causing the transport of the defined amino acid sequence on the membrane of the bacterium. In other words, in the bacte- rium is caused the membrane-bound expression of a gene product by the TolC protein. Subject mat- ter of the invention is thus also a gram-nega- tive bacterium, which contains at least one nu- cleotide sequence coding for at least one de- fined amino acid sequence and for at least one E. coli TolC gene product. This E. coli TolC gene product preferably is wild-type. Subject matter of the invention are however also mutated E. coli TolC gene products, wherein the trans- port signal activity has been maintained. Pref- erably, the bacterium is selected from the group composed of "Salmonella spp., Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, Shig- ella spp. and Yersinia spp.". Nucleotide sequences and bacteria can be used for various applications. For instance, the in- vention also teaches a pharmaceutical composi- tion containing a bacterium according to the in- vention, and as an option at least one physio- logically tolerable carrier substance, wherein the defined amino acid sequence is selected ac- cording to a given substance to be bound in an organism. By means of such a pharmaceutical com- position, substances interfering with the normal cellular metabolism, for instance exogenous toxicants or mutation-caused endogenous sub- stances such as octagons can be bound and thus inhibited. Further, by binding certain cellular target substances, metabolism processes can be modulated by removal of normal complex partners or those being regulated-up because of a dis- ease. Thereby, for instance a defined associa- tion is inhibited, and the shuttle related thereto is regulated down. Such a process can in turn be used for regu1ating-up other related processes. Insofar, the defined amino acid se- quence needs only be selected according to the target molecule to be inhibited with high speci- ficity. Such a pharmaceutical composition thus serves at last for therapeutic purposes. A pharmaceutical composition suitable for vaccination purposes contains a bacterium ac- cording to the invention and as an option at least one physiologically tolerable carrier sub- stance, wherein the defined amino acid sequence is an immunization sequence. An immunization se- quence stimulates in an organism the generation of antibodies against a natural antigen, which contains as a partial sequence the immunization sequence or is composed thereof. For diagnostic purposes, the invention teaches a diagnostic kit containing a bacterium according to one of claims 7 to 9, wherein the defined amino acid sequence specifically binds a marker substance to be determined. If for in- stance a tissue or fluid sample is taken from an organism, and this sample, if applicable after a pre-treatment with separation of undesired sam- ple components, is incubated with the bacterium, the binding events at the defined amino acid se- quence can be detected, and in case of a binding event, it is detected that the substance spe- cifically binding to the defined amino acid se- quence is contained in the sample. The detection of binding events can be made in various ways well known to the average man skilled in the art . Finally, the invention teaches a preparative binding substance containing a bacterium accord- ing to the invention, wherein the defined amino acid sequence specifically binds a target sub- stance to be separated from a solution. By such a binding substance, undesired substances can on the one hand specifically be removed from a so- lution by that the solution is incubated with the bacterium, and the bacterium is discarded after separation. On the other hand, a separa- tion or an enrichment of a target substance may be performed in a corresponding manner, namely by that after the incubation the target sub- stance is eluted from the bacterium. In this context, too, the invention can be used for the separation and/or enrichment of antigens, of an- tibodies, peptides, proteins or ligands. Examples of execution. Example 1: preparation of the TolC vector. The tolC gene of E. coli including its wild- type promoter was amplified by means of PCR (1 min 94°C, 1 min 66°C, 1 min 30 s 72°C) with the oligonucleotides 5'TolC (5'-TAACGCCCTATGTCGAC- TAACGCCAACCTT-3 ' ) and 3'TolC ( 5 ' - AGAGGATGTCGAC- TCGAAATTGAAGCGAGA-3') from the plasmid pAX629 (C. Wandersman, Institute Pasteur, Paris). At both ends, an additional Sail interface was in- troduced. The purified PCR product (QIAquick PCR Purification Kit - Qiagen, Hilden, Germany) was digested with the restriction endonuclease SalI and cloned into the vector pBR322 pre-split with SalI. The vector thus constructed was designated pBR32 2toIC. The functionality of the cloned tolC gene was then investigated in several tests. Example 2: introduction of an antigen sequence into the sequence of the TolC pro- tein. In the tolC sequence coding for one of the extracellular loops, a KpnI interface was iden- tified. This was used for cloning antigenic pep- tide sequences of the p60 protein (iap gene) of Listeria monocytogenes and permitted an inser- tion of foreign antigens behind amino acid 271 of the mature TolC protein. The iap sequence coding for a B cell epitope (amino acids 291 - 301) and a C D4 - re s t r i nged T cell epitope (amino acids 301 - 312) of the p60 protein was cloned as a Kpnl fragment into the vector pBR322tolC pre-cut with Kpnl (Fig. 1) . The plasmid thus obtained was designated pBR322- tolC::LisTB. Fig. 1 shows the cloning strategy for the in- sertion of the p60 -specific epitope sequences into the wild-type pla smid-coded E. coli tolC gene on the vector pBR322. There are: bla - am- picillin resistance gene; Tc - tetracycline; T - L. monocytogenes p60 T cell epitope (AS 301 312); B - L . monocytogenes p60 B cell epitope (AS 291 - 301); PtolC - wild-type E. coli tolC p romote r. Example 3: expression of the antigen on the mem- brane of a gram-negative bacterium (Escherichia coli). The expression of the epitopes of the p60 protein from L. monocytogenes within the TolC protein was detected in a Western blot. For this purpose, cell lysate proteins of E. coli CC118 tolC, E. coli CC118tolC/pBR322tolC and E. coli CC118tolC/pBR322tolC::ListTB were isolated in the late logarithmic phase. The applied cell protein totals corresponded to approx. 100 mil- lions bacteria. The proteins were separated in a 15% SDS polyacrylamide gel, and the expression of the chimeric TolC proteins or of the inserted epitopes were detected on the one hand with a polyclonal serum against the TolC protein and on the other hand with the monoclonal antibody K317 (Rowan et al., J. Clin. Microbiol. 3 8:2643-2648, 2000) specifically directed against the B cell epitope from L. monocytogenes (Fig. 2B) . As expected, no TolC protein could be de- tected in the cell lysate of E. coli CC118 tolC, which can be explained by a mutation* in the chromosomal tolC gene in this strain (Schlor et al., Mol. Gen. Genet. 256:306-319, 1997). The complementation with pBR322toiC led in this strain to the expression of the 52 kDa large TolC protein. The insertion of the L. monocyto - genes epitopes into the TolC protein did not af- fect the expression of TolC and led to a slight modification of the size of the chimeric protein of approx. 3 kDa. The expression of the p60-specific epitopes in E. coli CC118 to 1C/pBR322to1C: :ListB could be confirmed with the monoclonal p60 antibody K317. Example 4: detection of the exposed localization of the L. monocytogenes p60 epitopes in Salmonella enteritidis SM6T ( tolC) . Since the insertion position of the two lis- terial p60 epitopes was in an extracellular loop of TolC behind amino acid 271 of the mature pro- tein, they should be present in an exposed man- ner at the surface of S. enteritidis SM6T (Stone et al., Mol. Microbiol. 17:701-712, 1995). The definitive extracellular localization of the p60-specific epitopes in S. enteritidis SM6T was tested by indirect immuno fluorescenee . 25 µl each of an overnight culture of S. enteritidis SM6T/pBR322tolC and S. enteritidis SM6T/pBR322 tcolC::LisTB were dropped onto object carriers and air-dried. The cells were stained with the monoclonal p60 antibody K317 (1:200), and bound antibodies were then detected with an FITC-la- beled secondary anti-mouse serum (Dianova, Ger- many, working titer: 1:40) . The f1uorescence-microscopic analysis con- firmed the extracellular localization of the L. monocytogenes-specific epitopes in the strain S. enteritidis SM6T/pBR322tolC::LisTB. Example 5: immunization tests with the gram-neg- ative bacterium and analysis of the protective immune responses after in- fection with wild-type L. monocyto- gene s. In order to find out whether the exposed ex- pression of the T cell epitope from the p60 pro- tein of L. monocy togenes in the murine listerio- sis model leads to a protection, 8 female balb/c mice (Charles River, Sulzfeld, Germany) having an age of six weeks were orally immunized with a dose of 1x107 S. enteritidis SM6T/pBR322tolC: :LisTB. For control purposes, 5 female mice were orally immunized with S. enteritidis SM6T. The animals were immunized a second time three weeks later with the same dose of bacteria. The immunization success was tested five weeks after the first immunization in an immune blot, on which were applied supernatant proteins of Listeria monocytogenes. Anti-p60 -specific an- tibodies could be detected in the serum of the mice immunized with S. ente ri t idis SM6T/pBR322- tolC::LisTB. Three weeks after the second immunization, the animals were intravenously infected with 5x104 L. monocytogene s EGD, the five-fold LD50. Whilst the survival rate for the balb/c mice im- munized with S. enteritidis SM6T/pBR322tolC: - : LisTB after intravenous infection with L. mono- cytogene s EGD was 88%, the survival rate in the control group was only 20%. Thus the expression of the p60-specific epi- topes within an extracellular loop of TolC in the attenuated S. enteritidis carrier strain SM6T led to the induction of L i s te r ia mono cyto- genes-specific immune responses, which were ca- pable to protect balb/c mice against a usually lethal infection. Since the induction of anti- bodies against the B cell epitope from the p60 protein could be detected in a Western blot, it is obvious that an immune reaction under par- ticipation of the antibodies has caused the ob- served protection of the mice against an other- wise lethal infection with L. monocytogenes. 1. A nucleotide sequence coding for a TolC and a defined amino acid sequence, wherein the defined amino acid sequence is inserted in the permissive, membrane-external area of the TolC. 2. A nucleotide sequence according to claim 1, wherein the TolC is a TolC protein according to ACCESSION X54049 or a preferably N-terminal partial sequence thereof or a mutant of the pro- tein or of the partial sequence, and wherein for the N-terminal partial sequence or the mutant the transport functionality is maintained. 3. A nucleotide sequence according to claim 1 or 2, wherein the defined amino acid sequence is inserted on one side or both sides by a spacer sequence. 4 . A nucleotide sequence according to one of claims 1 to 3, wherein the defined amino acid sequence is inserted in the N-terminal area of the TolC, in particular in the area of the amino acids 52 to 61 and/or 257 to 279 (each referred to the TolC protein). 5. A plasmid containing a nucleotide se- quence according to one of claims 1 to 4. 6. A protein or a peptide coded by a nucleo- tide sequence according to one of claims 1 to 4. 7. A bacterium containing a nucleotide se- quence according to one of claims 1 to 4, where- in the TolC causes the transport of the defined amino acid sequence on the membrane of the bac- terium . 8. A bacterium according to claim 7, wherein the /defined amino acid sequence represents a peptide, a protein, an active substance, an an- tigen, an antibody or a ligand. 9. A bacterium according to one of claims 7 or 8, selected from the group composed of "Sal- monella spp., Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, Shigella spp. and Yer- sinia spp.". 10. A pharmaceutical composition containing a bacterium according to one of claims 7 to 9, and as an option at least one physiologically toler- able carrier substance, wherein the defined amino acid sequence is selected according to "a given substance to be bound in an organism. 11. A pharmaceutical composition containing a 'bacterium according to one of claims 7 to 9 and as an option at least one physiologically toler- able carrier substance, wherein the defined amino acid sequence is an immunization sequence. 12. A diagnostic kit containing a bacterium according to one of claims 7 to 9, wherein the defined amino acid sequence specifically binds a marker substance to be determined. 13. A preparative binding substance contain- ing a bacterium according to one of claims 7 to 9, wherein the defined amino acid sequence spe- cifically binds a target substance to be sepa- rated from a solution. Legend of the figures. Fig. 1 origin pBR322 Tc resistance gene cloning of the tolC gene from E. coli cloning of the p60 epitope sequences from L. monocyto- genes Nucleotide sequence coding for a TolC and a defined amino acid sequence The invention relates to a nucleotide sequence coding for a TolC, and a defined amlno acid sequence, said defined amino acid sequence being inserted in the permissive, membrane-external area of the TolC, and several uses thereof, particularly bacteria containing such a nucleotide sequence.

Full Text

Nucleotide sequence coding for a TolC and a
defined amino acid sequence.
Field of the invention.
The invention relates to a nucleotide se-
quence coding for a TolC, a plasmid containing
such a nucleotide sequence, a protein or a pep-
tide coded by such a nucleotide sequence, a bac-
terium containing such a nucleotide sequence and
several uses of such bacteria.
Background of the invention and prior art.
Viru1ence- attenuated, intrace11u1ar1y set-
tling bacteria can induce a long-lasting immu-
nity as live vaccines. Up to now, in particular
Salmonella typhi TYla (Levine et al., Lancet
1:1049-1052, 1987), Mycobacterium bovis BCG
(Fine and Rodrigues, Lancet 335:1016-1020, 1990)
and Vibrio cholerae (Levine and Kaper, Vaccine
11: 20 7-212, 1993) were used as live vaccines.
For instance, such variants of Listeria mono-
cytogenes, Salmonella enterica sv. typhimurium
and typhi, and BCG were already used as well-
tolerated live vaccines against typhus and tu-
berculosis. These bacteria, including their at-
tenuated mutants are generally immune-stimulat-
ing and can initiate a fair cellular immune re-
sponse, and were therefore used a vaccine carri-
ers.
The advantage of these bacteria as vaccine
carriers is that they mainly induce a so-called
Th1 immune response (Hess and Kaufmann, FEMS Im-
munol. Med. Microbiol. 23:165-173, 1999). This
immune response is characterized by cytotoxic
lymphocytes (CTL) and by the presence of spe-
cific IFN-gamma- secreting CD4+ T cells (also T
helper cells, Th) (Abbas et al., Nature 383:787-
793, 1996).
For instance, L. monocytogenes stimulates to
a special extent via the activation of TH1-cells
the proliferation of cytotoxic T lymphocytes
(CTL). These bacteria supply secreted antigens
directly into the cytosol of antigen-presenting
cells (APC; macrophages and dendritic cells),
which in turn express the co-stimulating mole-
cules and cause an efficient stimulation of T
cells. The listeriae are in part degraded in
phagosomal compartments, and the antigens pro-
duced by these carrier bacteria can therefore on
the one hand be presented by MHC class II mole-
cules and thus lead to the induction of T helper
cells. On the other hand, the listeriae repli-
cate after release from the phagosome in the cy-
tosol of APCs; antigens produced and
these bacteria are therefore preferably pre-
sented via the MHC class I pathway, thus CTL re-
sponses against these antigens being induced.
Further it could be shown that by the interac-
tion of the listeriae with macrophages, natural
killer cells (NK) and neutrophilic granulocytes,
the expression of such cytokines (TNF-alpha,
IFN-gamma, 11-2, IL-12; Unanue, Curr. Opin. Im-
munol. 9:35-43, 19 9 7; Mata and Paterson, J. Im-
munol. 163:14 4 9-144 56, 1999) is induced, for
which an antitumoral effectiveness was detected.
Recombinant bacteria were thus capable to
protect against a heterologous tumor (Medina et
al., Eur. J. Immunol. 29:693-699, 1999; Pan et
al., Cancer Res. 59:5264-5269, 1999; Woodlock et
al., J. Immunother. 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).
By the administration of L. monocytogenes,
which were transduced for the expression of tu-
mor antigens, the growth of experimental tumors
could be inhibited antigen-specifica11y (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 Paterson, J.
Immunol. 16 5:5502-5508, 2000).
Virulence-attenuated , Salmonella enterica
strains, into which nucleotide sequences coding
for tumor antigens had been introduced, as tumor
antigen-expressing bacterial carriers, could
provide after oral administration a specific
protection against different experimental tumors
(Medina et al., Eur. J. Immunol. 30:768-777,
2000; Zoller and Christ, J. Immunol. 166:3440-
34450, 2001; Xiang et al., PNAS 97:5492-5497,
2000) .
Recombinant Salmonella strains were also ef-
fective as prophylactic vaccines against virus
infections (HPV; Benyacoub et al., Infect. Im-
mun. 67:3674-367 9, 1999) and for the therapeutic
treatment of a mouse tumor immortalized by a tu-
mor virus (HPV) (Revaz et al., Virology 279:354-
360, 2001).
For the use as a vaccine carrier, methods
were developed to express expression products of
nucleic acid sequences introduced into bacteria
on the cell membrane of these bacteria or to
have them secreted by these bacteria. The basis
of these methods is the Escherichia coli hemo-
lysin system hlyAs representing the prototype of
a type I secretion system of gram-negative bac-
teria. By means of the hlyAs, secretion vectors
were developed that allow an efficient discharge
of protein antigens in Salmonella enterica,
Yersinia enterocolitica and Vibrio cholerae.
Such secretion vectors contain the cDNA of an
arbitrary protein antigen coupled to the nucleo-
tide sequence for the hlyA signal peptide, for
the hemolysin secretion apparatus, hlyB and hlyD
and the hly-specific promoter. By means of this
secretion vector, a protein can for instance be
expressed on the surface of this bacterium. Such
genetically modified bacteria induce as vaccines
a considerably stronger immune protection than
bacteria, wherein the protein expressed by the
introduced nucleic acid remains inside the cell
(Donner et al. , EP 1015023 A; Gentschev et al.,
Gene 179:133-140, 1996; Vaccine 19; 2621-2618,
2001; Hess et al., PNAS 9 3:1458-1463, 1996). The
disadvantage of this system is however that by
using the hly-specific promoter the amount of
the protein expressed by the bacterium is small.
Further transport systems in bacteria repre-
sent for instance, i) the transport signal for
the S-layer protein (Rsa A) of Caulobacter cres-
centus, where - for the secretion and the mem-
brane-bound expression - the C-terminal RsaA
transport signal is to be used (Umelo-Njaka et
al., Vaccine 19:1406-1415, 2001), and ii) the
transport signal for the Internalin A of Lis-
teria monocytogenes. For the secretion, the N -
terminal transport signal is necessary, and for
the membrane-bound expression, the N-terminal
transport signal together with the C-terminal
part containing the LPXTG motive responsible for
the cell wall anchoring (Dhar et al., Biochemis-
try 39 : 3725-3733, 2000) .
In another context, the integral membrane
protein TolC of E. coli is known. This is a mul-
ti-functional pore-forming protein of the outer
membrane of E. coli, which, in addition to func-
tions, such as e.g. reception of Colicin El (Mo-
rona et al., J. Bacteriol. 153:693-699, 1983)
and the secretion of Colicin V (Fath et al., J.
Bacteriol. 173:7549-7556, 1991), also serves as
a receptor for the U3 phage (Austin et al., J.
Bacteriol. 17 2:5312-5325, 1990). This protein is
not only found in E. coli, but also in a multi-
tude of gram-negative bacteria (Wiener, Struc-
ture Fold. Des. 8:171-5, 2000).
The crystal structure of the TolC protein
shows that it forms, as a homotrimer, a tunnel
channel having a length of about 120 Angstroms,
the biggest part of the homodimer, the tunnel
domain, being localized in the periplasm and
only two little loops (amino acids 52-61 and
257-279) being settled on the surface of the
bacterium (Koronakis et al., Nature 405:914-919,
2000) . The tolC gene has the nucleotide sequence
published by Niki et a 1., Nucleotide sequence of
the tolC gene of Escherichia coli, Nucleic Acids
Res. 18 (18), 5547 (1990). TolC is part of at
least four different bacterial export systems by
representing the membrane tunnel, through which
the export of the bacterial protein is made pos-
sible. For instance in the hlyA transport sys-
tem, the connection between hlyD and the peri-
plasmic end of the TolC permits the export of
the hemolysin from the hlyD into the membrane
tunnel of the TolC (Gentschev et al., Trends in
Microbiology 10:39-45, 2002).
Technical object of the invention.
The invention is based on the technical ob-
ject to specify a transport system, by means of
which an expression product having a higher ef-
ficiency can be presented on an outer cell mem-
brane .
Basic concept of the invention and preferred em-
bodiment s .
For achieving the above technical object, the
invention teaches a nucleotide sequence coding
for a TolC and a defined amino acid sequence,
said defined amino acid sequence being inserted
in the permissive, membrane-external area of the
TolC .
By the invention, a new transport system in
gram-negative bacteria is achieved, by means of
which larger amounts of a protein expressed by a
gene within a bacterium can be transported on
the outer cell membrane of the bacterium than
was possible with prior art transport systems.
Surprisingly, the transport system for the TolC
protein of Escherichia coli permits a substan-
tially stronger membrane-bound expression of a
peptide or protein (arbitrary) than was known
from prior art transport proteins, and that for
a multitude of gram-negative bacteria. The mem-
brane-bound expression of the defined amino acid
sequence or gene product is exclusively achieved
by the TolC.
A defined amino acid sequence may be an arbi-
trary qiven peptide or a protein, an arbitrary
pharmaceutical active substance, an arbitrary
antigen, an arbitrary antibody, or an arbitrary
1igand.
The TolC may be a (wild-tvpe) TolC protein
according to ACCESSION X54049, to which hereby
explicitly reference is made, or a (preferably
N-terminal) partial sequence thereof or a mutant
of the protein or of the partial sequence, for
the partial, s, eaue nee or the mutant the transport
functionality being maintained. N-terminal par-
tial sequences means any partial sequence begin-
ning in the N-terminal area amino acids 1 to 50
of the TolC protein and ending at the C-terminal
end of a loop, which is settled on the surface
of the bacterium. Preferred is thus the N-termi-
nal transport signal of TolC, but also the cen-
tral part of the protein, which represents the
extracellular areas of TolC. A mutant may com-
prise an insertion, deletion or substitution, as
long as the transport functionality is not dis-
tinctly reduced thereby.
For certain applications, it may be recom-
mended that a defined amino acid sequence be in-
serted on one side or both sides by a spacer se-
quence. This will however only be helpful, if
the defined amino acid sequence is to be pre-
sented in a certain spatial structure, for in-
stance in the case of an antigen, and this does
however not take place by the defined amino acid
sequence itself for steric or configurative rea-
sons to a desired extent. Then a spacer sequence
may be formed in particular by a sequence natu-
rally following the defined amino acid sequence,
thus the defined amino acid sequence being
folded in the same way as in the natural anti-
gen. The spacer sequence may however also be ar-
tificial, if thus a desired presentation and/or
folding of the defined amino acid sequence is
obtained. This can easily be calculated by means
of theoretical methods, under consideration of
the spatial conditions at the position of inser-
tion in the TolC .
In detail it is preferred that the defined
amino acid sequence is inserted in the N-termi-
nal area of the TolC, in particular in the area
of the amino acids 52 to 61 and/or 257 to 279
(each referred to the TolC protein) .
Subject matter of the invention is further a
plasmid containing a nucleotide sequence accord-
ing to the invention and a protein or peptide
coded by a nucleotide sequence according to the
invention.
The invention further teaches a bacterium
containing a nucleotide sequence according to
the invention, the TolC causing the transport of
the defined amino acid sequence on the membrane
of the bacterium. In other words, in the bacte-
rium is caused the membrane-bound expression of
a gene product by the TolC protein. Subject mat-
ter of the invention is thus also a gram-nega-
tive bacterium, which contains at least one nu-
cleotide sequence coding for at least one de-
fined amino acid sequence and for at least one
E. coli TolC gene product. This E. coli TolC
gene product preferably is wild-type. Subject
matter of the invention are however also mutated
E. coli TolC gene products, wherein the trans-
port signal activity has been maintained. Pref-
erably, the bacterium is selected from the group
composed of "Salmonella spp., Escherichia coli,
Vibrio cholerae, Pseudomonas aeruginosa, Shig-
ella spp. and Yersinia spp.".
Nucleotide sequences and bacteria can be used
for various applications. For instance, the in-
vention also teaches a pharmaceutical composi-
tion containing a bacterium according to the in-
vention, and as an option at least one physio-
logically tolerable carrier substance, wherein
the defined amino acid sequence is selected ac-
cording to a given substance to be bound in an
organism. By means of such a pharmaceutical com-
position, substances interfering with the normal
cellular metabolism, for instance exogenous
toxicants or mutation-caused endogenous sub-
stances such as octagons can be bound and thus
inhibited. Further, by binding certain cellular
target substances, metabolism processes can be
modulated by removal of normal complex partners
or those being regulated-up because of a dis-
ease. Thereby, for instance a defined associa-
tion is inhibited, and the shuttle related
thereto is regulated down. Such a process can in
turn be used for regu1ating-up other related
processes. Insofar, the defined amino acid se-
quence needs only be selected according to the
target molecule to be inhibited with high speci-
ficity. Such a pharmaceutical composition thus
serves at last for therapeutic purposes.
A pharmaceutical composition suitable for
vaccination purposes contains a bacterium ac-
cording to the invention and as an option at
least one physiologically tolerable carrier sub-
stance, wherein the defined amino acid sequence
is an immunization sequence. An immunization se-
quence stimulates in an organism the generation
of antibodies against a natural antigen, which
contains as a partial sequence the immunization
sequence or is composed thereof.
For diagnostic purposes, the invention
teaches a diagnostic kit containing a bacterium
according to one of claims 7 to 9, wherein the
defined amino acid sequence specifically binds a
marker substance to be determined. If for in-
stance a tissue or fluid sample is taken from an
organism, and this sample, if applicable after a
pre-treatment with separation of undesired sam-
ple components, is incubated with the bacterium,
the binding events at the defined amino acid se-
quence can be detected, and in case of a binding
event, it is detected that the substance spe-
cifically binding to the defined amino acid se-
quence is contained in the sample. The detection
of binding events can be made in various ways
well known to the average man skilled in the
art .
Finally, the invention teaches a preparative
binding substance containing a bacterium accord-
ing to the invention, wherein the defined amino
acid sequence specifically binds a target sub-
stance to be separated from a solution. By such
a binding substance, undesired substances can on
the one hand specifically be removed from a so-
lution by that the solution is incubated with
the bacterium, and the bacterium is discarded
after separation. On the other hand, a separa-
tion or an enrichment of a target substance may
be performed in a corresponding manner, namely
by that after the incubation the target sub-
stance is eluted from the bacterium. In this
context, too, the invention can be used for the
separation and/or enrichment of antigens, of an-
tibodies, peptides, proteins or ligands.
Examples of execution.
Example 1: preparation of the TolC vector.
The tolC gene of E. coli including its wild-
type promoter was amplified by means of PCR (1
min 94°C, 1 min 66°C, 1 min 30 s 72°C) with the
oligonucleotides 5'TolC (5'-TAACGCCCTATGTCGAC-
TAACGCCAACCTT-3 ' ) and 3'TolC ( 5 ' - AGAGGATGTCGAC-
TCGAAATTGAAGCGAGA-3') from the plasmid pAX629
(C. Wandersman, Institute Pasteur, Paris). At
both ends, an additional Sail interface was in-
troduced. The purified PCR product (QIAquick PCR
Purification Kit - Qiagen, Hilden, Germany) was
digested with the restriction endonuclease SalI
and cloned into the vector pBR322 pre-split with
SalI. The vector thus constructed was designated
pBR32 2toIC. The functionality of the cloned tolC
gene was then investigated in several tests.
Example 2: introduction of an antigen sequence
into the sequence of the TolC pro-
tein.
In the tolC sequence coding for one of the
extracellular loops, a KpnI interface was iden-
tified. This was used for cloning antigenic pep-
tide sequences of the p60 protein (iap gene) of
Listeria monocytogenes and permitted an inser-
tion of foreign antigens behind amino acid 271
of the mature TolC protein.
The iap sequence coding for a B cell epitope
(amino acids 291 - 301) and a C D4 - re s t r i nged T
cell epitope (amino acids 301 - 312) of the p60
protein was cloned as a Kpnl fragment into the
vector pBR322tolC pre-cut with Kpnl (Fig. 1) .
The plasmid thus obtained was designated pBR322-
tolC::LisTB.
Fig. 1 shows the cloning strategy for the in-
sertion of the p60 -specific epitope sequences
into the wild-type pla smid-coded E. coli tolC
gene on the vector pBR322. There are: bla - am-
picillin resistance gene; Tc - tetracycline; T -
L. monocytogenes p60 T cell epitope (AS 301
312); B - L . monocytogenes p60 B cell epitope
(AS 291 - 301); PtolC - wild-type E. coli tolC
p romote r.
Example 3: expression of the antigen on the mem-
brane of a gram-negative bacterium
(Escherichia coli).
The expression of the epitopes of the p60
protein from L. monocytogenes within the TolC
protein was detected in a Western blot. For this
purpose, cell lysate proteins of E. coli CC118
tolC, E. coli CC118tolC/pBR322tolC and E. coli
CC118tolC/pBR322tolC::ListTB were isolated in
the late logarithmic phase. The applied cell
protein totals corresponded to approx. 100 mil-
lions bacteria. The proteins were separated in a
15% SDS polyacrylamide gel, and the expression
of the chimeric TolC proteins or of the inserted
epitopes were detected on the one hand with a
polyclonal serum against the TolC protein and on
the other hand with the monoclonal antibody K317
(Rowan et al., J. Clin. Microbiol. 3 8:2643-2648,
2000) specifically directed against the B cell
epitope from L. monocytogenes (Fig. 2B) .
As expected, no TolC protein could be de-
tected in the cell lysate of E. coli CC118 tolC,
which can be explained by a mutation* in the
chromosomal tolC gene in this strain (Schlor et
al., Mol. Gen. Genet. 256:306-319, 1997). The
complementation with pBR322toiC led in this
strain to the expression of the 52 kDa large
TolC protein. The insertion of the L. monocyto -
genes epitopes into the TolC protein did not af-
fect the expression of TolC and led to a slight
modification of the size of the chimeric protein
of approx. 3 kDa.
The expression of the p60-specific epitopes
in E. coli CC118 to 1C/pBR322to1C: :ListB could be
confirmed with the monoclonal p60 antibody K317.
Example 4: detection of the exposed localization
of the L. monocytogenes p60 epitopes
in Salmonella enteritidis SM6T
( tolC) .
Since the insertion position of the two lis-
terial p60 epitopes was in an extracellular loop
of TolC behind amino acid 271 of the mature pro-
tein, they should be present in an exposed man-
ner at the surface of S. enteritidis SM6T (Stone
et al., Mol. Microbiol. 17:701-712, 1995). The
definitive extracellular localization of the
p60-specific epitopes in S. enteritidis SM6T was
tested by indirect immuno fluorescenee . 25 µl
each of an overnight culture of S. enteritidis
SM6T/pBR322tolC and S. enteritidis SM6T/pBR322
tcolC::LisTB were dropped onto object carriers
and air-dried. The cells were stained with the
monoclonal p60 antibody K317 (1:200), and bound
antibodies were then detected with an FITC-la-
beled secondary anti-mouse serum (Dianova, Ger-
many, working titer: 1:40) .
The f1uorescence-microscopic analysis con-
firmed the extracellular localization of the L.
monocytogenes-specific epitopes in the strain S.
enteritidis SM6T/pBR322tolC::LisTB.
Example 5: immunization tests with the gram-neg-
ative bacterium and analysis of the
protective immune responses after in-
fection with wild-type L. monocyto-
gene s.
In order to find out whether the exposed ex-
pression of the T cell epitope from the p60 pro-
tein of L. monocy togenes in the murine listerio-
sis model leads to a protection, 8 female balb/c
mice (Charles River, Sulzfeld, Germany) having
an age of six weeks were orally immunized with a
dose of 1x107 S. enteritidis SM6T/pBR322tolC:
:LisTB. For control purposes, 5 female mice were
orally immunized with S. enteritidis SM6T. The
animals were immunized a second time three weeks
later with the same dose of bacteria.
The immunization success was tested five
weeks after the first immunization in an immune
blot, on which were applied supernatant proteins
of Listeria monocytogenes. Anti-p60 -specific an-
tibodies could be detected in the serum of the
mice immunized with S. ente ri t idis SM6T/pBR322-
tolC::LisTB.
Three weeks after the second immunization,
the animals were intravenously infected with
5x104 L. monocytogene s EGD, the five-fold LD50.
Whilst the survival rate for the balb/c mice im-
munized with S. enteritidis SM6T/pBR322tolC: -
: LisTB after intravenous infection with L. mono-
cytogene s EGD was 88%, the survival rate in the
control group was only 20%.
Thus the expression of the p60-specific epi-
topes within an extracellular loop of TolC in
the attenuated S. enteritidis carrier strain
SM6T led to the induction of L i s te r ia mono cyto-
genes-specific immune responses, which were ca-
pable to protect balb/c mice against a usually
lethal infection. Since the induction of anti-
bodies against the B cell epitope from the p60
protein could be detected in a Western blot, it
is obvious that an immune reaction under par-
ticipation of the antibodies has caused the ob-
served protection of the mice against an other-
wise lethal infection with L. monocytogenes.
1. A nucleotide sequence coding for a TolC
and a defined amino acid sequence, wherein the
defined amino acid sequence is inserted in the
permissive, membrane-external area of the TolC.
2. A nucleotide sequence according to claim
1, wherein the TolC is a TolC protein according
to ACCESSION X54049 or a preferably N-terminal
partial sequence thereof or a mutant of the pro-
tein or of the partial sequence, and wherein for
the N-terminal partial sequence or the mutant
the transport functionality is maintained.
3. A nucleotide sequence according to claim
1 or 2, wherein the defined amino acid sequence
is inserted on one side or both sides by a
spacer sequence.
4 . A nucleotide sequence according to one of
claims 1 to 3, wherein the defined amino acid
sequence is inserted in the N-terminal area of
the TolC, in particular in the area of the amino
acids 52 to 61 and/or 257 to 279 (each referred
to the TolC protein).
5. A plasmid containing a nucleotide se-
quence according to one of claims 1 to 4.
6. A protein or a peptide coded by a nucleo-
tide sequence according to one of claims 1 to 4.
7. A bacterium containing a nucleotide se-
quence according to one of claims 1 to 4, where-
in the TolC causes the transport of the defined
amino acid sequence on the membrane of the bac-
terium .
8. A bacterium according to claim 7, wherein
the /defined amino acid sequence represents a
peptide, a protein, an active substance, an an-
tigen, an antibody or a ligand.
9. A bacterium according to one of claims 7
or 8, selected from the group composed of "Sal-
monella spp., Escherichia coli, Vibrio cholerae,
Pseudomonas aeruginosa, Shigella spp. and Yer-
sinia spp.".
10. A pharmaceutical composition containing a
bacterium according to one of claims 7 to 9, and
as an option at least one physiologically toler-
able carrier substance, wherein the defined
amino acid sequence is selected according to "a
given substance to be bound in an organism.
11. A pharmaceutical composition containing a
'bacterium according to one of claims 7 to 9 and
as an option at least one physiologically toler-
able carrier substance, wherein the defined
amino acid sequence is an immunization sequence.
12. A diagnostic kit containing a bacterium
according to one of claims 7 to 9, wherein the
defined amino acid sequence specifically binds a
marker substance to be determined.
13. A preparative binding substance contain-
ing a bacterium according to one of claims 7 to
9, wherein the defined amino acid sequence spe-
cifically binds a target substance to be sepa-
rated from a solution.
Legend of the figures.
Fig. 1
origin
pBR322
Tc resistance gene
cloning of the tolC gene from E. coli
cloning of the p60 epitope sequences from L. monocyto-
genes
Nucleotide sequence coding for a TolC
and a defined amino acid sequence
The invention relates to a nucleotide sequence coding for a TolC,
and a defined amlno acid sequence, said defined amino acid
sequence being inserted in the permissive, membrane-external
area of the TolC, and several uses thereof, particularly bacteria
containing such a nucleotide sequence.

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Patent Number

223784

Indian Patent Application Number

01390/KOLNP/2004

PG Journal Number

39/2008

Publication Date

26-Sep-2008

Grant Date

23-Sep-2008

Date of Filing

20-Sep-2004

Name of Patentee

ZENTARIS GMBH

Applicant Address

WEISMULLERSTRASSE 45 D 60314 FRABKFURT

Inventors:

#	Inventor's Name	Inventor's Address
1	GOEBEL, WERNER	AM HAPPACH 12, 97218 GERBRUNN
2	GENTSCHEV IVAYLO	BAUWEG 5, 97270 KIST
3	SPRENG, SIMONE	BRUNNERSTRASSE 59, CH-3018 BERN

PCT International Classification Number

C12R 1/42, 1/19

PCT International Application Number

PCT/DE03/00469

PCT International Filing date

2003-02-13

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10208175.1	2002-02-20	Germany