EP0797669A1

EP0797669A1 - Screening model

Info

Publication number: EP0797669A1
Application number: EP95941056A
Authority: EP
Inventors: Klaus P. SCHÄFER; Thomas Weitzenegger; Wolfram Steinhilber; Klaus Melchers
Original assignee: Byk Gulden Lomberg Chemische Fabrik GmbH
Current assignee: Takeda GmbH
Priority date: 1994-12-02
Filing date: 1995-11-30
Publication date: 1997-10-01
Also published as: US5981184A; AU4259096A; WO1996017066A1; JPH10509875A

Abstract

A screening-model is disclosed for determining the inhibiting effect of substances on the P-type ATP-ase activity of Helicobacter. The screening model in question comprises: (a) a recombinant organism consisting of host cells transformed with at least one P-type ATP-ase gene which can be controlled via a promoter; (b) an inductor for gene activation of the P-type ATP-ase; (c) cations which inhibit the metabolic activity of the recombinant organism only in the presence of Helicobacter P-type ATP-ase; and (d) a measurement device for determining the metabolic activity of the recombinant organism.

Description

Screening model

Tftf.hnisι'h <ι.ς Ohigf

The invention relates to a screening model for determining the activity of substances which inhibit the P-ATPase activity of Helicobacter.

Stand Arr technology

Helicobacter pylori (H. pylori) is a gastric bacterium that is pathogenic to humans and whose eradication from the stomach is now an essential prerequisite for the permanent cure of H. pylori-associated diseases. The germ is found in the stomach, preferably in and under the mucous membrane, sometimes also between the epithelial cells. Due to the uniqueness of the milieu in which the germ is located, H. pylori must have developed physiological strategies for metabolic changes that enable its survival in the gastrointestinal area of humans.

It is known today that the appearance of the germ is often causally associated with the development of gastritis, ulcers and with certain forms of gastric cancer. The current forms of therapy are based on the administration of substances that inhibit the activity of the gastric proton pump, the H + / K + -ATPase, together with antibiotic substances, which on the one hand are not always understood mechanically and on the other hand the risk of developing resistance include. Due to the widespread use of the germ and the unsatisfactory therapy strategies, the need for new therapy strategies is great.

The primary screening for helicobactericidal substances today is largely based on in vitro methods such as the agar dilution test, which can be used to determine the minimum inhibitory concentration with regard to H. pylori growth. In addition, helicobacter-infected animals are often used as in vivo models for the screening of helicobactericidal substances. In both cases, the relatively complex handling of the germ is necessary, which is characterized on the one hand by a relatively slow growth, which is why longer breeding or cultivation times are required, and on the other hand by demanding media conditions (microaerophilia, serum addition is necessary). In addition, H. pylori is human pathogenic (classified as S2 according to the German Federal Disease Law) and its handling is therefore associated with appropriate safety measures and requirements. In the development of helicobactericidal medicinal products, there is therefore a great need for simple in vitro primary screening models which can also be used to reduce the use of experimental animals in the primary screening phase. Such Screening models can be the basis for the development of suitable, safe and efficient forms of therapy for the eradication of this human pathogenic bacterium. The targets of future eradication strategies should be able to be described in their molecular structure and biochemical-physiological significance for the germ in order to ensure a safe and helicobacter-specific effect. On the basis of recombinant screening models, on which the effect of drugs on cloned Helicobacter functions can be investigated, the development and improvement of drugs taking into account drug-target interactions down to the molecular range is possible.

idiing

An object of the invention is to be seen in the provision of a screening model which allows the primary screening of substances with Helicobacter inhibitory activity without special safety requirements and without the use of experimental animals.

It has now been found that the measurement of the metabolic activity of a recombinant organism which has been transformed with at least one helicobacter-specific P-ATPase gene which can be controlled via a promoter, in the presence of cations which only together with Helicobacter P-ATPase, the metabolic activity of the recombinant organism inhibit, can be used for the effective and highly selective determination of the helicobacter-inhibiting effect of substances to be investigated.

The invention therefore relates to a screening model for determining the activity of substances which inhibits the P-ATPase activity of Helicobacter, comprising a) a recombinant organism consisting of at least one host cell which has been transformed via a promoter and can be controlled by a promoter , b) an inducer for gene activation of the P-ATPase, c) cations which only impair the metabolic activity of the recombinant organism in the presence of Helicobacter-P-ATPase, and d) a measuring device for determining the metabolic activity of the recombinant organism.

Further objects result from the subclaims.

From the H. pylori genome, DNA fragments were isolated which encode P-type ATPases associated with plasma membrane, hereinafter called P-ATPases or Helicobacter-P-ATPases. ATPases 439 and 948 in particular were examined in more detail. The Helicobacter P-ATPase genes were purified, cloned, sequenced and functionally expressed in transformed E. coli. The function or activity of the heterologous gene products interferes with the metabolic activity of the recombinant microorganism, such as with the proton concentration in the immediate vicinity of the cells, and can therefore be measured. The recombinant organisms that express the specific P-ATPase gene can be used to preferably detect (screen) biological and / or chemical compounds which preferably inhibit the activity of the HP-P-ATPase and for the action on Helicobacter to opume.

The host vector system allows screening for substances which interact with the cloned ATPase without having to work primarily with the H. pylori, which is relatively complex to cultivate, and without germs classified according to security level S2.

In addition, the molecular interaction between protein (target) and inhibitor can be analyzed in a targeted manner on this basis (expression of the mechanism of action or binding) via the expression of specifically mutated ATPase sequences. The system thus also provides the basis for the targeted design of inhibitors (drug development).

The isolation of the ATPase genes from the H. pylori genoin and their insertion into bacterial expression vectors and cloning are carried out according to methods known per se.

Descendants of E. coli K12 were preferably used as host cells, E. coli MM294 being preferred.

The usual, commercially available plasmids for bacterial expression come into question as bacterial expression vectors. The hybrid Tac promoter from the lae / trp system is preferred, but other promoters such as trc, lac or trp can also be used. PT12-1 is also particularly suitable as an expression plasmid, which is described in detail in Chapter 3.1.2.

The model according to the invention for screening for substances which inhibit the P-ATPase activity of Helicobacter consists of a recombinant organism, transformed with at least one Helicobacter-P-ATPase gene, which is expressed from a promoter. The synthesis of the P-ATPase leads in the host cell to sensitivity to media which, in addition to C or N sources, contain a number of ions, including NH ions. This P-ATPase-mediated sensitivity of the host cell is expressed phenotypically by an immediate and significant reduction or change in cellular metabolic activity, which can be measured. This effect on the cellular activity of the recombinant Organism that is found only with expression of the Helicobacter P-ATPase (s) can be prevented, for example, by ortho-vanadate, an inhibitor of P-ATPases. The model is therefore suitable for the screening and improvement of specific inhibitors of Helicobacter P-ATPase (s) that specifically interact with the Helicobacter P-ATPase activity.

The function of the model is shown below using the example of a recombinant organism which consists of an E. coli K12 host cell and a recombinant expression plasmid. A P-ATPase was inserted into the plasmid, which was isolated from H. pylori (HP). The derived amino acid sequence of the P-ATPase gene shows in the computer-assisted homology analysis significant similarity with already known bacterial and eukaryotic P-ATPases, in particular with those whose function lies in the transport of divalent cations. The gene or gene product represents a possible target for differential drug therapy of H. pylori-associated diseases. The gene or its recombinant expression in other organisms enables the screening and detection of substances which are the specific gene and / or influence the activity of the correlating gene function, preferably inhibit it.

The present invention enables specific and selective primary screening on the basis of a special Helicobacter function (P-ATPase activity) with the aid of recombinant organisms which are classified as non-human pathogens and which are significantly less demanding to handle. Since P-ATPases are of essential importance for the milieu- or germ-specific ion balance of bacterial organisms such as H. pylori, these represent highly selective targets for differential Helicobacter therapy, through which the germ in its natural habitus through appropriate connections in its vital integrity can be decisively inhibited and killed.

All parameters known as a measure of the metabolic activity of bacterial cells are suitable for measuring the metabolic activity of the recombinant organism. In connection with the present invention, the determination of the pH in the immediate vicinity of active cells is particularly suitable. Commercial cytosensor microphysiometers (company. Molecular Devices, Graefelfing) are particularly suitable for the measurement. As a measure of the metabolic activity, the acidification rate, i.e. H. the rate at which the medium is acidified by cellular metabolic activity.

The steps and methods described relate in particular to isolation, analysis, cloning and expression of DNA. These methods and methods have been well understood and described. The experimental work was carried out using these conventional methods of gene or molecular biology (Molecular Cloning, Maniatis et al., Cold Spring Harbor Laboratory Press, 1989). The computer-aided analysis of DNA sequences obtained for the detection of ORFs (open reading frames / open reading frames), homology studies etc. can be carried out with the wide variety of genetic computer programs and databases available (e.g. DNASIS / PROSIS, PC- Gene, UW-GCG).

1. Isolation of DNA fragments from the H. pylori genome which contain P-type ATPase genes and neighboring DNA regions with further Helicobacter genes.

1.1 Description of the gene probe

At the beginning of the work, no P-ATPases were isolated from the HP genome and the corresponding DNA sequence information was therefore not available. A homology comparison of P-ATPase sequences from other pro- and eukaryotic organisms shows that in addition to a remarkable structural homology, this enzyme class also has areas in which the amino acid (AS) primary sequences are highly conserved. A particularly conserved region, the existence of which is the eponym for this class of membrane ATPases, is the phosphorylation site (P site) of the P ATPases. A DNA oligonucleotide was derived from the AS primary sequence of the P site (1282), which represents a selected subpopulation of 16 DNA sequence variations of more than 2000 possible. The 20bp oligonucleotide 1282 represents a mixture of 16 different DNA sequences which code for the DKTGT (I / L) T consensus sequence of P-ATPases. The amino acid sequence is given below in the one-letter and three-letter code above the DNA sequence of the DNA oligonucleotide:

D K T G T I / L (T) Asp Lys Thr Gly Thr Ile (Thr)

Leu

5'- GAT AAA ACC GGC ACC ATC AC - 3 'A G T G

The DNA oligonucleotide 1-282 labeled with digoxigenin was first analyzed in Southern blot analyzes with genomic DNA from HP and E. coli, which had previously been restricted with the restriction clononucleases EcoRI, Hind3 and. Ava2 were digested. The DNA oligonucleotide hybridized efficiently with the HP-DNA and was therefore shown to be suitable for subsequent Gcn screeding. 1-282 also hybridized to E. coli DNA, which was of particular importance for the screening strategy. From the AS primary sequence of the P consensus region, further DNA oligonucleotides were derived which code for the DKTGT (l / L) T consensus sequence of P-ATPascn (P region \ P site). These derived 20 bp long DNA probes represent different subpopulations and thus DNA sequence variations of the P region. Their length corresponds to the DNA oligonucleotide 1-282. 1-405 to 1-409 correspond to the entire DNA sequence pool, which includes all possible sequences of this region. These oligonucleotides can be used according to the procedure at 1-282 for the detection and isolation of potential P type genes by using the usual labeling and hybridization methods.

The DNA sequences of the DNA-oligonucleotide mixtures (DNA probes 1-405, 1406, 1-407, 1-408 and 1-409) are given below in 5'-3 'direction:

GA (TC) AA (AG) AC (AGCT) GG (AGCT) AC (AGTC) AT (TC) AC is 1-405, GA (TC) AA (AG) AC (AGCT) GG (AGTC) AC (AGTC) AT (CA) AC is 1-406, GA (TC) AA (AG) AC (AGCT) GG (AGTC) AC (AGTC) TT (AG) AC is 1-407, GA (TC) AA (AG) AC ( AGCT) GG (AGTC) AC (AGTC) CT (TC) AC is 1-408

and

GA (TC) AA (AG) AC (AGCT) GG (AGTC) AC (AGTC) CT (AG) AC is DNA oligonucleotide 1-409.

Positions in parentheses with multiple bases represent variable positions. The molecules were marked for the hybridization experiments using the digoxigcin 3 'end marking kit (Boehringer Mannheim) in accordance with the manufacturer's instructions. From the direct sequence comparison of these DNA oligonucleotides (1-405 to 1-409) with the base sequence and complexity of the DNA probe 1-282 it can be seen that the probes 1-405 to 1-409 are each significantly more degenerate, i.e. are more variable in their sequence. The greater complexity of these probes, if all of them are used, should make it possible to isolate the entire P type ATPase family. The increased degeneration of the probes compared to 1-282 increases the risk of isolating "false positive" plasmids from the H. pylori library. which, however, can be clearly separated from the P type ATPase genes at the level of DNA sequencing.

Once a specific P type ATPase has been isolated from the H. pylori genome with a DNA oligonucleotide, additional copies can be isolated on the basis of the clear sequence information obtained, again using the known standard methods of molecular biology. This procedure is particularly advantageous if only a partial DNA sequence of a P type ATPase is present on an isolated plasmid. 1.2 Description of the HP bench

The HP bank was developed by Dr. Rainer Haas (Tübingen) designed and made available.

The HP gene bank was created on the basis of HP isolate 69A. For this, chromosomal DNA was isolated from HP69A and partially digested with Sau3A. DΝA fragments> 3 kb were enriched by gel electrophoresis.

PRH160 was used as the cloning vector (FIG. 1), which has a tetracycline resistance marker (Tet ^R ). The 2.44 kb plasmid also has a unique Bgl2 cli interface in the polylinker region. This BgI2 site was used to clone the HP-DΝA fragments. The recombinant plasmids were transformed into E. coli HB101 and cloned. 2 x 10 "independent transformants were obtained. After amplification, 200 μl aliquots with 3.7 x 10 ⁶ cfu (colony forming units) each were filled and stored. The HP-DNA insertions inserted into the plasmid can be double-digested with EcoRI and cut Xhol out of the vector again.

1.3 Plasmid pRH439

1.3.1. Isolation of pRH439

Since the DNA probe 1-282 also reacts with E. coli DNA (1.1), the screening was carried out directly on the plasmid DNA. For this purpose, an aliquot from the gene bank was diluted and distributed as an inoculum to 20 culture tubes in such a way that about 70 independent clones from the gene bank were represented in each tube.

A total of 1400 clones (of the 20,000 available) of digoxigenin-labeled oligonucleotide 1-282 were searched for P-ATPases by this procedure. With an average size of the HP-DNA inserts of approx. 3000 bp, the 1400 plasmids represented approximately 2-3 H. pylori genome equivalents.

One plasmid preparation was made from each of the 20 "mixed plasmid cultures". An aliquot of the bacterial cultures was frozen away as glycerol consens. After restriction with EcoRI / Xhol, the plasmid preparations were subjected to a Southern blot analysis with 1-282 as a probe. The plasmid mixture No. 4 gave a clear signal. An aliquot of the corresponding glycerol preserve was therefore plated on LB agar plates. Plasmid preparations were again made from statistically selected colonies, which were examined by means of Southcm blot analysis as in the primary screen. The plasmid preparation No. 39 proved positive. This procedure led to the isolation of plasmid pRH439. 1.3.2. Sequence analysis of the isolated plasmid pRH439

The plasmid carries an HPRI insert which can be cleaved with EcoRI-Xhol and is approximately 3.4 kb in length. DNA sequencing led to the result that the HP-specific DNA fragment contains an ORF (Open Reading Frame) of 2058 bp. This codes for a protein of 686 amino acids. 2a-e shows the DNA sequence obtained and the amino acid sequence resulting therefrom.

In position D-388 (Asp-388) to T-394 (Thr-394), the polypeptide chain contains the conservative box used for the isolation of the gene, which contains the P-site characteristic of P-ATPases. The hydrophobicity analysis shows a whole range of possible transmembrane Hclices, which are necessary for the special membrane topology of the enzyme and which are characteristic of P-ATPases and other membrane enzymes.

A special feature of the isolated HP-ATPase. The high concentration of cysteine and histidine residues is different from the P-ATPases isolated so far. Cys and His residues occur massively in the range from AS position 420 to 550. In addition, the enzyme is distinguished by an N-terminal HIHNLDCPDC ion binding site and has an internal CPC motif.

In the helicobacter-specific DNA insertion of the plasmid pRH439, an additional ORF was found before the ATPase gene (ATPase-439), which is terminated by the cloning sequence and is therefore incomplete. However, the Helicobacter-specific DNA insertion of pRH439 overlaps at the 5 'end with the insertion of plasmid pRH514, so that the ATPase-associated ORF could be completed using plasmid pRH514.

1.4. Plasmid pRH514

1.4.1. Isolation of pRH514

This plasmid was isolated from the H. pylori 69A library as described for pRH439. However, the DNA oligonucleotide 1-407 was used as the probe.

Sequencing of the H. pylori-specific DNA insert from pRH514 revealed that it comprises 3097 bp. The DNA sequence as well as the amino acid sequence of the complete ORF is shown in FIG. 3. 1.4.2 Sequence analysis of the isolated plasmid pRH514

The DNA sequence was determined using DNA sequencing. The pRH514 insertion DNA was found to extend the DNA sequence of the plasmid pRH439 after 5 '. The reading frame interrupted in pRH439 by the cloning site and preceding the P-type ATPase is completed. Since this gene upstream of the ATPase codes for a protein product and is apparently located on the chromosome of H.pylori 69A adjacent to the described P-type ATPase, the derived gene product was designated as ATPase-associated protein (AA-protcin, in short: AAP) .

The AAP-encoding ORF of pRH514 predicts an amino acid chain consisting of 506 residues. 24 bp downstream is the ORF for ATPase 439, which is incomplete in the 3 'range on pRH514, however.

The AA protein is characterized by a significant homology to response regulators of bacterial two-component systems. These systems of signal transduction usually consist of a membrane-based sensor kinase and a cytosolic response regulator protein. The AAP from H.pylori shows an identity of approx. 15-20% with E.coli NtrC and K.pneumoniae NifA, two known response regulator proteins, over the entire anioic acid sequence.

H.pylori-AAP can be divided into three domains according to the known response regulators: a regulative N-terminal domain with an aspartate residue in position 56, a central domain with a so-called Walker A ATP binding motif (Gly-Ser-Pro -Gly-Cys-Gly-Lys-Ser) and the C-terminal domain with helix-turn-helix molecules, which indicate the possibility of interaction with DNA. It is striking that the central domain of AAP contains four symmetrically arranged Cys residues (positions 358, 360, 370, 372).

1.5. pRH948

1.5.1. Isolation of pRH948

This DNA clone was isolated from the H.pylori library using DNA probe 1-408. The isolation strategy is under 1.3.1. described.

1.5.2. Sequence analysis of the isolated plasmid pRH948

The plasmid contains an H. pylori-specific DNA insertion of approximately 4.5 kbp. The DNA sequence was determined by means of DNA sequencing. The DNA sequence contains 4 complete ORFs (ORFs 2-5) and 2 incomplete permanent, terminal ORFs (ORFI and 6). 4 shows the complete DNA sequence and the deduced amino acid sequences of the corresponding ORFs. ORF 4 encodes another P type ATPase from H.pylori, here called ATPasc-948.

1.5.3. Detailed description of ATPase-948 (ORF 4)

Starting from DNA base position 1872, the DNA contains an ORF (open reading frame) which codes for a protein consisting of 741 amino acids. The protein has the conserved phosphorylation site required for P-type ATPases. Sequence motifs which are characteristic of P-type ATPases, in particular from the family of metal-ion-transporting p-type ATPases, are contained in the amino acid sequence predicted by pRH948-DNA.

The protein is characterized by: a conserved phosphorylation site, an ATP binding region, an N-terminal Cys-x-x-Cys sequence, a Cys-Pro-Cys sequence, associated with a region of hydrophobic amino acids.

The DNA sequence and the amino acid sequence derived therefrom for the P-type ATPAse encoded by pRH948 is shown in FIG. 5a.

These sequence motifs were also found in the pRH439-encoded, helicobacter-specific P-type ATPase described above.

ATPase948 shows sequences, especially with ATPase439 from H. pylori, the CopA / B-ATPases from Enterococcus hirae, the Cd pump from Staphylococcus aureus (approx. 30% sequence identity).

The 607 C-terminal amino acids are characterized by a 93.7% sequence identity with that of Tylor et al. published hpCopA-ATPase from H. pylori. However, the hpCopA gene product contains no N-terminal Cys-x-x-Cys motif and the putative membrane-associated amino acid triplet Cys-Pro-Cys is also not present in hpCopA. Instead, the sequence Cys-Pro-Ser can be found in hpCopA. In contrast, the pRH948-encoded ATPase has an N-tcπninalale with the characteristic amino acid sequence Cys-x-x-Cys.

The gene for ATPasc-948, like that of ATPase-439, can be expressed in heterologous form. Suitable for this the methods and strategies described in detail in Chapters 2 and 3. ATPase-948 is also particularly suitable as the basis for the active substance screening model described below using the example of ATPase-439.

1.5.4. Description of other ORFs located on pRH948

The amino acid sequences of the different ORFs can be seen in FIG. 4.

ORF 1 codes for the C-terminal part of a protein and therefore represents a C-terminal partial sequence. This comprises 309 amino acids starting with an aspartate residue and ending with a serine. The sequence starts immediately with the beginning of the insertion DNA. In the homology studies, the derived protein product shows significant homology with so-called AAA-type ATPases, which also include the well-studied FtsH protein from E. coli.

ORF 2 starts at base position 1162 of the pRH948 DNA sequence. Here is the ATG start codon for methionine. This ORF, consisting of a total of 237 triplets, ends at position 1872 with the last base of the terminal alanine triplet. The derived protein is characterized by significant homology to bacterial phosphatidyl serine synthetases (PSS).

ORF 3 is localized within ORF 2. It begins with base A at position 1710 as part of the ATG start codon and codes for a peptide of 48 amino acids.

ORF 4 codes for the P-type ATPase-948 already described above. This ORF begins at position 1872 of the DNA sequence. The last base of the ORF coding for the PSS homologous protein thus simultaneously represents the starting point of the coding sequence for the P-type ATPase. ORF 4 ends at position 4094. The terminating TGA stop codon follows immediately.

ORF 5 immediately follows the TGA stop codon of the P-type ATPase starting with an ATG start codon (base positions 4098-4100). The ORF predicts a protein of 66 amino acids. The protein is characterized by a CxxC motif found in the N-terminal region, CNHC. It is homologous to the CopZ protein, which is part of the Cop operon in Entcrococcus hirae.

Like ORF 1, ORF 6 is incomplete. It begins at position 4401 and terminates after 27 triplets terminates with the end of the hclicobactcr-specific DNA sequence in pRH948. 2. Heterologous expression of Helicobacter gene

The isolated and characterized H.pylori genes can be fully or partially expressed in heterologous systems (bacteria, yeast, eukaryotes). The conventional expression plasmids with bacterial promoters such as e.g. lac, tac and tφ. E.coli kl2 host cells and their derivatives are particularly suitable as host cells. The heterologous expression of H. pylori genes is suitable for the identification and development of helicobactericidal active substances and for the induction of antibodies and antisera.

The functional expression of essential H.pylori-Gcncn in E.coli is suitable for the construction of screening models, with the help of which special H.pylori enzyme activities can be selectively measured. Such models can be used to identify and develop substances that inhibit the function of the cloned and heterologously expressed H.pvlori-Protcinc and are therefore potentially important as helicobactericidal drugs in the therapy of helicobacter-associated diseases.

The experimental procedure for the heterologous expression of H. pylori genes in E. coli for the construction of a screening model is shown below using the example of P-type ATPase-439, encoded by pRH439.

This procedure leads to the functional and measurable expression of Helicobacter genes in a suitable host cell. The measurability of helicobactic-specific activity is based on a change in the overall metabolic performance of the host cells.

It has now been found that expression of an H. pylori P-type ATPase in E. coli significantly changes the metabolic activity. The system described below shows that the metabolic performance of the host cell, which can be measured by the expression of the Rpylori gene product and which can be measured, can be used to identify inhibitors of specific Hclicobacter activity. Active substances that influence the isolated Hclicobacter activity are detected by preventing or canceling the change in the metabolic performance of the host cells.

The region coding for the Helicobactcr-ATPase was amplified by means of Pohπierase chain reaction (PCR) from plasmid pRH439 and inserted and cloned into various bacterial expression vectors with E.coli Tac, Trc or Tφ promoters. These include the plasmids pTI2-l, pTrcHisA and pTφ233. In these vectors, the H. pylori PT \ p ATPase gene is under the control of the promoters indicated. The constructed plasmids were transformed into E. coli K12 strains. For expression, the recombinant E. coli strains were grown and the inducer was added before reaching the maximum cell density (IPTG, isopropyl-thiogalactoside. For tac and Trc promoters or .beta.-IAA. Indolacetic acid, for Tφ promoters). The Protein profile of the E. coli strains was analyzed before and after induction. After gene induction, a new protein band appears in the recombinant E. coli strains, which runs in the gel at approx. 70 kDa. The procedure described below for the design of functional expression vectors which allow the synthesis of recombinant Hclicobactcr proteins in E. coli represents a basis for the construction of a screening module. The structure and function of the screening module is described in the next chapter described in detail.

3. Description of the screening module

The model consists of a recombinant E. coli strain which carries the gene sequence of an H. pylori P-type ATPase, which is controllable in its activity, on an expression plasmid. The medium in which the measurement is carried out must be composed such that the activity of HP-ATPases can be induced and measured therein. The activity of ATPases infiltrated into the host cell influences the general metabolic activity of the host cell and is therefore amenable to measurement. The change in the metabolic activity of the recombinant host cell induced by the P-type ATPase activity can be prevented by inhibiting the P-type ATPase activity. The latter is the basis for using the model for screening and optimizing substances.

The function of the model is described in the following using the example of the E. coli slamm PY25.

3.1. Detailed description of the model components

3.1.1. Host cell

The host cell is E. coli MM294. This strain is an E. coli 12 derivative and is therefore classified as non-human pathogenic.

3.1.2. ATPase expression plasmid

Expression vector is plasmid PY25. This is composed of the plasmid pTI2-1 and the HP-P type ATPase gene 439.

pTI2-1 was constructed from the plasmids pKK223-3 and pGc. \ 2T, both obtained from Pharmacia. First, the Pstl interface was deleted in the polylinker from pKK. For this, pKK with Pstl cut and the ends treated with T4 DNA polymer crasc. The vector was then digested with Smal, religated and cloned after transformation into E. coli HB101. The cloning product is pKKδBamHl. The Amp ^R in pKKδBamHl was exchanged for the Amp ^R gene from pBR322, since the latter has an internal Pst 1 interface. For this purpose pKKδBamHl and also pBR322 were double digested with Pvul and Ndel. The smaller band (approx. 1.45 kb) was isolated from the pBR322 digest and the large band (approx. 3 kb) from pKKδBam. The two DNA fragments were ligated and cloned in E. coli HBIOI. The cloning product is pKK (Pst) δBam.

pKK (Pst) δBamH 1 was digested with Pstl and Sspl. The two small DNA bands of approximately 620 and 560 bp were isolated from an agarose gel. pGcx2T was also digested with the enzyme combination Sspl x Pstl. After electrophoretic separation in agaroscgcl, the large 3.2 kb DNA fragment was isolated. The three isolated DNA fragments from pKK (Pst) δBam and pGe. \ 2T were ligated and cloned in E. coli HB101. Cloning product is pTI2. This vector contains a Tac promoter, an Amp ^R gene and the lac repressor gene lacl ^q . Plasmids with a comparable structure can e.g. B. from Pharmacia (pTrc 99A).

pTI2 was linearized with the enzyme combination EcoRI Hind3 and ligated with the double-stranded DNA oligonucleotide MCS1 (Multi Cloning Site 1), which has the corresponding compatible EcoRI or Hind3. After ligation and transformation, plasmid pTI2-1 was obtained. Here MCS1 is cloned behind P _Tlc (Tac promoter) into the EcoRI site from pKK. After cloning in pTI2-1, MCS 1 has the sequence 5 '- GAATTCGTAG GAAGCTCATAT GGTCGACTC TAGACCCGGG CTGCAGAAGCTT-3', which offers the restriction sites EcoRI, Ndcl, Sal1, Xbal, Smal, Pstl and Hind3 for the cloning. 5 shows a plasmid map of the constructed expression vector pT12-1.

For the insertion of the HP-ATPase gene 439 into pTI2-1, DNA primers 1-404 and 1-402 with the sequences 5'-ACCGA CTTGA ATTCA TGCAA GAATA CCACA TT-3 '(1-404) and 5'- CTGCA ACTCA AGCTT AAGCT CTCAT TGCGC GCAT-3 '(1-402) synthesized. 1-404 corresponds to the first 6 amino acid triplets of the ATPase gene and contains an EcoRI site ("sense" DNA oligonucleotide) upstream. 1-402 corresponds to the last 6 amino acid triplts and contains a TAA stop codon and a Hind3 site ("Antisensc" DNA oligonucleotide). The region from pRH439 (1.3) coding for the HP-ATPase was amplified with these DNA oligonucleotides using polymcrase chain reaction (PCR). The PCR product was cut with EcoRI and Hind3 in order to release the ends of the clone, and purified on an agarose sail. pTI2-1 was digested with the same enzyme combination (EcoRI and Hind3). Plasmid and PCR product were ligated and cloned in E. coli MM294 after transformation. The cloning product is plasmid PY25. The DNA sequence of the inserted ATPase gene was verified by means of DNA sequencing. The recombinant E. coli strain is called E. coli PY25. The HP-ATPase gene is located in E. coli P Y25 Control of the Tac promoter. The structure of the vector PY25 is given in FIG. 5.

3.1.3 Measuring device for determining the metabolic activity of E. coli PY25

With the help of a cytosensor microphysiometer (Molecular Devices, Graefcling), it is possible to measure and record the metabolic activity of cells via the acidification rate. The acidification rate is defined as the rate at which the medium is acidified by cellular metabolic activity. The biological principle of operation of the cytosensor is based on the fact that the H ^ concentration in the immediate vicinity of living cells depends on the metabolic activity. The cytosensor is based on a light-controlled sensor (silicon chip), which acts as a highly sensitive and fast pH detector. The sensor is in direct contact with the measuring cell in which the cells are incubated under a manipulable flow of medium. A voltage is applied across the sensor, the current flow being dependent on the proton occupancy of the oxynitrile layer of the silicon chip (McConnel et al., Science 257, 1906-1912, 1992). The sensor detects the smallest metabolic changes in cells that are located in the measuring or sensor chamber. The addition of media, nutrients and test substances is controlled via a computer-controlled system. 8 samples can be measured in parallel.

3.2. Measurement of metabolic activity

3.2.1. Cytosensor test medium

The experiments in the cytosensor were carried out in BSSGG-Mcdium. BSS stands for "Balanced Salt Solution", a weakly phosphate-buffered ion solution, and GG for the addition of glucose and glutamine as a source of nutrients and energy.

BSSGG composition (pH 7.4)

138 mM NaCl

5 mM KC1

0.81 mM Na, HP0

0.11 M NaH, PO _<

1.3 mM CaCU

0.5 mM MgCL

10 mM glucose

1mM glutamine As a source of ammonium ions and sulfur, the medium is also (NH ₄ ), SO _{i |} added (final concentration 10 mM).

3.2.2. Preparation of the cells

M9 minimal medium (Molecular Cloning, Maniatis et al., Cold Spring Harbor Laboratory Press, 1989) is inoculated with recombinant E. coli and incubated overnight in a Schüttciinkubator at 37 ° C. The recombinant strain is E. coli PY25. E. coli PTI2-1, which carries the plasmid without the insertion of the ATPase ("empty vector"), is used as the control strain.

The next day, 70 μl of Zcllsuspcnsion were mixed with 30 μl of melted agarose (Molecular Devices) and 10 μl in each case spotted on the membrane of an insert for the cytosensor measuring chamber. The inserts were cured for 20-40 min to harden the agarose. incubated at + 4 ° C and then transferred into the scanner chambers of the cytoscanner according to the manufacturer's instructions. BSSGG served as the medium.

3.2.3. Preparation of the cytoscanner

The measuring chambers of the cytosensor were flooded with BSSGG with 50% pumping power before inserting the sensor chamber. After 10, the prepared sensor chambers with the agarose-fixed cells were inserted. The following parameters were defined:

Temperature: - Measuring chamber: 37 ° - Dcbubblers Δ: 6 ° C

Pumping and measuring cycle

- Pump phase

Start Stop Speed is Interval 00:00:00 00:00:40 30%

(h: min: s)

- Rate Measurement (measurement of the acidification or alkalisification rate)

Start stop

00:00:43 00:00:58 The pump stops during the measuring phase (speed = 0%). - total cycle: 00:01:00

This is followed by the calibration of the device, that is to say the calibration of the sensor chambers with regard to the activity of the cells used, based on the set parameters. The rest of the test is carried out exactly according to the manufacturer's instructions. Raw and rate data are recorded and saved. The rate data reflect the trend in cellular activity during the measurement phases (rate measurements). It can be seen here to what extent there are induced changes in the metabolism of the cells, which lead to changes in the surface charge on the oxynitrite layer of the sensor. The rate data are given in -μVolt / s.

3.2.4. Conducting experiments

3.2.4.1. Measurement of the activity of E. coli PY25 and E. coli PTI2-1: influence of the HP-ATPase activity

4 the rate data of the two recombinant E. coli strains E. coli PY25 (with HP-ATPase) and PTI2-1 (without HP-ATPase) in BSSGG are supplemented with 10 mM (NH- ^ SO., Both strains initially show a pronounced acidification behavior E. coli PY25 is shown in measuring channel F, strain PTI2-1 in measuring channel B. When adding the inductor isopropyl-thiogalactoside (IPTG, final concentration 1 μM) for the activation of the Tac- In this example, promoters have both strains at a rate of approx. -800 μVolt / s.Around 5 min after the addition of IPTG, only E. coli PY25 shows a drastically decreasing acidification rate, which is at least around 100 μVolt / s and The delayed effect of IPTG induction can be explained by the biosynthesis of HP-ATPase, which takes place initially.

The drop in the acidification rate of E. coli PY25 can be observed under these test conditions only in the presence of NH. If tetracycline, an inhibitor of bacterial protein biosynthesis, is added to your medium at the same time as IPTG, there is no change in the metabolic activity of strain PY25.

3.2.4.2. Inhibition of the IPTG effect by ortho-vanadate

It is known that ortho-vanadate inhibits P-type ATPases in the lower μ-molar concentration range. Fig. 5 shows this restrictive effect. The acidification rate of E. coli PY25 was first recorded in the cytosensor in BSSGG supplemented with 10 mM (NH.) SO. (Mcßkammcrn F, G, H). In F and H, the cells were exposed to vanadate at a final concentration of 10 μM. G contained no inhibitor. After 3.5 Hours of preincubation is switched to BSSGG, 10 mM (NH) _: S0 ₄ with 1 μM IPTG. An additional 10 μM vanadate is present in G (previously without vanadate), as is H (present before induction). No vanadate is present in F with induction, but was in the medium of the pre-incubation. As seen in Fig. 5 (G), the induced inhibition of acidification occurs only in sample G. Samples H and F, which contained 10 μM vanadate, were not inhibited, ie, in contrast to sample G, the acidification rate did not decrease significantly.

This experimental setup shows the effect of the vanadate as an inhibitor of the induced P-type ATPase and shows that the recombinant E. coli on this basis is screened for substances which interact with the expressed ATPase activity. enables.

Figure description

1 shows the plasmid map of pRH160. The plasmid contains a tetracycline resistance marker. The location of the restriction classes for EcoRl, Bglll, Xhol, Kpnl, Smal, Xbal, Clal, Sal l, EcoRV, BamH l. SpH l. Nru l and Pstl is specified. The DNA fragments obtained by partial digestion of genomic H. pylori DNA with Sau3A were cloned into the BglII restriction position of the plasmid.

2a-e show the DNA sequence of the complete pRH439 Xho-EcoRI insert and the derived one

ORF (open reading framc) from HP-ATPase. The DNA sequence starts with the C of the Xhol (CTCGAG) recognition site in position 1 and ends with the C of the EcoRI (GAATTC) restriction site in position 3410. The ORF, which is responsible for an H. pylori P-type ATPase with 686 amino acids encoded, lies between position 1219 with the A of the ATG start codon and position 3275 with the T of the GCT-alanine codon. The ORF is followed by a TAA as a stop codon for translation. The predicted amino acid sequence is shown below the DNA sequence. The consensus sequence containing the phosphorylation site (Asp-388) is underlined.

3a-f show the DNA sequence of the complete pRH514 EcoRI-Xhol insert and the derived ones

Amino acid sequence of the complete ORF. A oleic acid sequences are given in the one-letter code below the coding region. The complete, 5'-located ORF encodes a protein of 506 amino acids with significant homology to bacterial response regulators. pRH 14 overlaps with DNA clone pRH439. that carries the gene for a P-type ATPase. pRH514 has the 5'-tcil of the ATPase gene 439. Since the complete ORF of pRH514 preceded by ATPase, the response regulator protein encoded on pRH514 was designated as ATPasc-Associates-Protcin (AAP).

4a-i show the complete DNA insertion sequence of the DNA clone pRH948 from the H. pylori

Gene bank. The terminal nuclides, which are highlighted by underlining, still correlate with vector DNA. The non-underlined DNA sequence represents the cloned Helicobacter DNA from plasmid pRH948. The H. pylori-specific DNA sequence comprises a total of 6 ORFs. The two outer ones are incomplete, as they are interrupted by the cloning sites and thus merge into the vector DNA sequence. The derived amino acid sequences are given in the one-letter code under the DNA sequence, taking into account all 6 ORFs. The location, type and nature of the ORFs are described in the text. ORF 4 encodes the P-type ATPase-948

Fig. 5 plasmid map of PY-25. The basic gate is pTI2-l. This contains a tac promoter

(P <TAC>), an ampicillin-Rcsistcnz gene (AP <R>) and a lacI ^Q gene (LACIQ) which expresses the lac repressor. The P-type ATPase gene (ATPASE) from pRH439 is cloned behind the tac promoter via EcoRI / HindIII restriction enzyme. The vector is suitable for the functional expression of Hclicobaktcr genes in E. coli. The location of the existing unique interfaces of EcoRI, Hind3 and Pstl are given.

6 shows the acidification rates obtained for the expression of HP-ATPase in recombinant E. coli. The sensor chamber B contains recombinant E. coli with plasmid pTI2-1 as a control vector. The sensor chamber F contains recombinant E. coli with the plasmid PY25 with inserted HP-ATPasc-Gcn. The cells were incubated as described in 5.2.4.1. After two hours, IPTG was added to the medium. In the presence of plasmid PY25, the acidification rate of E. coli is greatly reduced.

Fig. 7 shows the influence of ortho-vanadate on the acidification rate. The sensor chambers F, G and H contain E. coli with plasmid PY25. In chambers F and H, ortho-vanadate was added to the medium before induction with IPTG. After 3.5 hours, IPTG (F) or IPTG was added to the medium in the presence of 10 μM ortho-vanadate. The reduction in the rate of acidification induced by IPTG was not observed in cells pretreated with ortho-vanadate (F, H).

Claims

1. Screening model for determining the P-type ATPase activity of Helicobacter-inhibiting activity of substances comprising a) a recombinant organism consisting of at least one host cell transformed via a promoter controllable P-type ATPase gene, b) one Inducer for gene activation of the P-type ATPase, c) cations which only impair the metabolic activity of the recombinant organism in the presence of Helicobacter P-type ATPase, and d) a measuring device for determining the metabolic activity of the recombinant organism.

2. Screening model according to claim I, characterized in that E. coli cells are used as host cells.

3. Screening model according to claim 2, characterized in that the host cells are E. coli K12 derivatives, preferably E. coli MM294. be used.

4. Screening model according to claim 1, characterized in that Tac, Trc or Tφ promoters are used as promoters.

5. Screening model according to claim 4, characterized in that IPTG are used as inducers in the case of Tac and Trc promoters and ß-IAA in the case of Tφ promoters.

6. Screening model according to claim 1, characterized in that ammonium ions are supplied as cations.

7. Screening model according to claim 1, characterized in that additional energy and / or nutrient sources are present.

8. screening model according to claim 7. thereby gckcnnzeiclinet. that amino acids, preferably glutamine, and / or glucose are present.

9. screening model according to claim 1, characterized gckci zcichnet that a C loscnsor-Microphysiomclcr is used as Maßiiirrichtung for determining the metabolic activity.

10. Vffiriren to screen the ATPase activity of Helicobacter inhibiting substances, characterized in that the metabolic activity of a recombinant organism, consisting of a host cell transformed with at least one P-type ATPase gene controllable via a promoter from Helicobacter, with the P-type ATPase gene switched on, before and after addition of cations which have the metabolic activity of the recombinant organism only in the presence of Helicobacter P-type impair ATPase, determined.

11. Purified and isolated DNA sequence consisting essentially of the helicobacter-specific ATPase gene 439 according to FIG. 2a-e.

12. Purified and isolated DNA sequence consisting essentially of the helicobacter-specific ATPase gene 514 according to FIGS. 3a-f.

13. Purified and isolated DNA sequence consisting essentially of the helicobacter-specific ATPase gene 948 according to FIG. 4a-i.

14. Vector containing the DNA sequence according to Fig. 2a-e.

15. Vector containing the DNA sequence according to FIGS. 3a-f.

16. Vector containing the DNA sequence according to Fig. 4a-i.