EP1259639A2 - Genes et produits geniques essentiels pour l'identification, le developpement et l'optimisation de principes actifs immunologiques et pharmacologiques pour le traitement d'infections microbiennes - Google Patents

Genes et produits geniques essentiels pour l'identification, le developpement et l'optimisation de principes actifs immunologiques et pharmacologiques pour le traitement d'infections microbiennes

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Publication number
EP1259639A2
EP1259639A2 EP00938725A EP00938725A EP1259639A2 EP 1259639 A2 EP1259639 A2 EP 1259639A2 EP 00938725 A EP00938725 A EP 00938725A EP 00938725 A EP00938725 A EP 00938725A EP 1259639 A2 EP1259639 A2 EP 1259639A2
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EP
European Patent Office
Prior art keywords
gene
genes
microorganisms
essential
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00938725A
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German (de)
English (en)
Inventor
Heiko Apfel
Thilo M. Fuchs
Carol P. Gibbs
Christoph J. Hueck
Thomas F. Meyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Creatogen AG
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Creatogen AG
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Publication date
Priority claimed from DE19924965A external-priority patent/DE19924965A1/de
Priority claimed from DE19927740A external-priority patent/DE19927740A1/de
Priority claimed from DE19934029A external-priority patent/DE19934029A1/de
Application filed by Creatogen AG, Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Creatogen AG
Publication of EP1259639A2 publication Critical patent/EP1259639A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a method for identifying and characterizing essential genes of pathogenic microorganisms, their use for finding new immunological and pharmacological active substances for prophylaxis, therapy and diagnosis of bacterial infections, and the further development and optimization of these active substances.
  • Included in the invention are the corresponding nucleic acids, which code for the essential gene products, and the polypeptides coded thereby.
  • the invention relates to vectors which contain the nucleic acids according to the invention, cells transformed with these vectors and antibodies specific for the polypeptides. These nucleic acids and polypeptides can be used for the diagnosis, prevention and treatment of microbial infections, in particular they can be used for the development of antibodies, vaccines and inhibitors.
  • the first step is to identify all genes on the genome. This is usually done with the help of computer-assisted search programs that can predict potential genes with a certain degree of certainty. In this way, gene maps can be created which, however, are still very inaccurate. If the genes identified by the search program cannot be assigned to a known gene, the functionality of these hypothetical genes must be verified by physical detection of the gene products in the original cell.
  • Another strategy which is also based on the use of special search programs, is aimed at the identification of possible gene families which are linked to special biological properties, which in turn are considered as an active substance target based on further assumptions.
  • the search criteria are geared towards characteristic structural features, which are usually derived from known genes.
  • the result of such a search can, depending on the approximation of the specifications to the actual state, provide a high hit rate.
  • the inaccuracy of these methods is relatively high, and the real biological property of the gene or its gene product must in any case be confirmed experimentally.
  • Another strategy captures the expression products of a cell, whereby the genes active for the respective developmental state can be identified. If one compares different developmental states with each other, the interaction of the genes can be deduced and in some cases the biological function of unknown genes can partially be decrypted. If corresponding comparative studies are carried out with cells that have a pathological appearance, it is even possible to identify disease-causing genes and use them as potential drug targets for drug development.
  • Helicobacter pylori is a pathogen of particular medical interest. This germ is a gram-negative, spiral-shaped bacterium with a high pathogenic potential, which has become increasingly resistant to a number of therapeutically relevant antibiotics in recent years and is therefore of great clinical importance. It is characterized by extremely high mobility due to its flagella and the unusual ability to survive in the strongly acidic environment (up to pH 1.5) of the stomach (Goodwin et al., 1,989).
  • H. ylori also has a causal role in the development of gastric and duodenal ulcers (ulcer ventriculi and duodenal ulcer) as well as in some forms of gastric carcinoma (adenocarcinoma) (Lee et al., 1 993; Solnick and Tompkins, 1,993).
  • the bacteria After ingestion, the bacteria first enter the extremely acidic stomach lumen (pH 1 to 2). There is through the production of the enzyme Urease, which leads to the cleavage of the existing urea and thus to the local neutralization of the acidic pH in the stomach, which enables the survival of the bacteria.
  • Urease By means of chemotactic orientation and flagella-dependent motility, the germs then move into the bicarbonate-buffered mucus layer of the antrum region of the stomach, their actual natural habitat. There they are in a unique ecological niche, which due to the acid barrier is only accessible to a few competing types of bacteria.
  • the bacteria are probably based on the pH gradient between the lumen (pH 1 -2) and the epithelial cell surface (pH 6-7) in order to reach the epithelium.
  • pH 1 -2 the pH gradient between the lumen
  • epithelial cell surface pH 6-7
  • these germs are optimally adapted to the living conditions in this habitat. They mostly stay in the deep crypts of the antrum region, where they are protected against external influences such as acid, pepsin, but also against medication to eradicate them, such as antibiotics.
  • Part of the bacterial population (approx. 20%) is closely associated with epithelial cells, especially with mucus-producing cells.
  • Provided gastric metaplasia i.e.
  • duodenal ulcer due to their ability to be adherent, complete elimination of the Helicobacter with the rejected mucus is probably prevented, so that the bacteria can persist for years, decades or even for life (chronic infection).
  • H. ylori Before the existence and importance of H. ylori for ulcer diseases were known, they were treated by so-called antacids, or H 2 -receptor antagonists. These are substances that inhibit acid secretion of the gastric parietal cell. Under the influence of these medicinal products, it usually heals Ulcers, however, since one of the causes of these ulcers, namely the p / or infection, is not eliminated, in most cases the ulceration recurs after a short time (recurrence).
  • Another therapy that is often used for ulceration is bismuth treatment.
  • Different bismuth salts CBS, BSS
  • CBS, BSS bismuth salts
  • a significant disadvantage of this form of therapy is, however, that total eradication of the germ is only achieved in a very small percentage of cases (8 to 32%).
  • Another disadvantage of bismuth treatment is that prolonged therapy with high doses leads to an accumulation of this substance in the liver, kidney and nervous system and has considerable neurological side effects (Malfertheiner, 1 994).
  • the dosage form is also of particular interest since the active ingredient must be effective in the stomach, ie in an extremely acidic environment. Connections with proton blockers, e.g. B. before the administration of the prophylactic or therapeutic agent can be of great benefit.
  • antibiotics bismuth subcitrate, erythromycin, amoxicillin, metronidazole
  • erythromycin bismuth subcitrate, erythromycin, amoxicillin, metronidazole
  • a high O 2 partial pressure or sublethal administration of antibiotics can be induced (Donelli et al., 1 998; Bode et al., 1 993; Sorberg et al., 1 996 ; Berry et al., 1,995).
  • Binding to epithelial cells and the ability to signal transduction seem to be preserved in coccoid forms comparable to the vegetative forms (Khin et al., 1 996; Segal et al., 1 996).
  • H. heilmannii can colonize the stomach of humans, e.g. B. H. heilmannii and H. felis.
  • H. heilmannii can also be associated with pathological ulcer diseases. The causal transmission probably takes place from pets to humans.
  • H. ylori ⁇ n which occurs frequently in humans, has been suspected of playing a role in the development of gastric cancer.
  • H. ylori ⁇ n which occurs frequently in humans, has been suspected of playing a role in the development of gastric cancer.
  • These doubts are particularly supported by more recent data from Helicobacter heilmannii, which attribute greater carcinogenic potential to them and emphasize their importance in the development of gastric MALT lymphoma (Regimbeau et al., 1 988).
  • H. ylori or heilmannii The identification of essential genes of Helicobacter, in particular H. ylori or heilmannii and possible Helicobacter Persistence forms for the development and optimization of new therapeutic, preventive and / or diagnostic agents, such as. B. Vaccines and pharmacological agents is therefore an aim of the invention.
  • In the foreground is the discovery of essential microbial genes, and homologous proteins of various pathogenic germs can be identified. With the help of an active ingredient, several pathogenic germs could then be eliminated at the same time as with classic antibiotics.
  • Helicobacter focuses in particular on genes that fulfill vital functions in the infection process, as well as genes that are involved in the development and reactivation of coccoid forms.
  • Essential genes that code for secreted gene products since these can be reached particularly well for immunological and pharmacological active substances due to their exposed location and are therefore good candidates for active substance development.
  • Essential genes that code for gene products that are involved in the development and maintenance of forms of persistence are also of interest.
  • Another task is to find essential microbial genes, whereby homologous proteins of various pathogenic germs can also be identified. With the help of an active ingredient, several pathogenic germs could then be eliminated at the same time as with classic antibiotics.
  • A Identifying essential genes and the corresponding polypeptides by producing gene-deficient microorganisms by conditional antisense inhibition (CAI) or / and subtractive recombination mutagenesis (SRM) and determining the viability and survivability of the gene-deficient microorganisms in one
  • CAI conditional antisense inhibition
  • SRM subtractive recombination mutagenesis
  • Test system Identification of specific active substances which are directed against the essential polypeptides and which inactivate the microorganisms or the microorganisms used.
  • Another aspect of the present invention is thus also a method for identifying essential microbial genes, which comprises the following steps:
  • CAI is the abbreviation for "conditional antisense inhibition", i.e. conditional antisense inhibition. It is a process which is described in more detail below.
  • SRM stands for "subtractive recombination mutagenesis", which is also described below.
  • gene deficient means that the deficient organism is unable to produce or use one or more of its gene products.
  • the production of the corresponding gene product can be prevented on the one hand by mutagenesis of the corresponding gene, or it can be inhibited during expression, for example by means of antisense nucleic acids.
  • Mutagenesis can be used to mutate a gene in the genome of the microorganism, or to incorporate a mutated gene into the microorganism, and homologous recombination can also be used.
  • a protein-coding section of a nucleic acid sequence is, for example, a gene or part of a gene which allows the expression of a polypeptide.
  • essential gene means a gene which codes for a gene product without which an organism cannot survive or can only survive to a limited extent.
  • Essential genes can be divided into two classes: obligatory essential and facultative essential genes.
  • An obligatory essential gene codes for a protein that is essential for the survival or reproduction of an organism under all circumstances.
  • an optional essential gene codes for a protein that is only necessary under certain conditions for the survival or reproduction of the organism, such as the ability of the organism to survive within cultured mammalian cells or in animals. In both cases, the survival or reproduction of the organism is severely impaired or prevented by the inactivation of a gene essential for it or the inhibition of a gene product essential for it.
  • a bacterium is no longer viable after inactivation of a particular gene or is restricted in its reproduction, this can be taken as a first indication that essential properties are imparted by this gene.
  • the significance of such Findings must, however, be supported by accompanying control experiments, for example, such a lethal mutation should be able to be abolished in a second step by appropriate complementation of the gene or gene product.
  • Obligatory essential genes are therefore those whose non-expression or absence, for example by mutagenesis or deletion, means that the organism is neither viable in a natural environment nor on a full medium that is ideally matched to the needs of the microorganism. If a microorganism is deficient in an optionally essential gene, it is usually still capable of growth on such a full medium, depending on the organism, but can no longer survive in its natural environment, ie in its natural host or cells or tissue cultures of its natural host.
  • the new method enables essential genes of microorganisms to be identified regardless of their special function. This method is preferably used to identify essential genes from Helicobacter and related microorganisms.
  • a first step the entire genome of a bacterial pathogen is searched for essential genes using a molecular genetic approach.
  • This sub-step does not require any knowledge of the primary structure of the genome or individual genes, but takes place solely on the basis of biological criteria. If a gene is identified as an essential determinant, its identity is determined. The raw sequence data of the genomic sequencing can be used. Based on the gene sequence determined z. B. isogenic variants are determined or whether the gene found is located in an operon in which there may be other essential genes.
  • the identified genes are converted into special genetic systems which serve to deliver the genes or their gene products to a direct active substance screening and / or the genes or gene products are used to further optimize already identified active substances.
  • the main advantage of the overall method is based on the rapid successive execution of the gene and active substance screening in meaningful biological systems, so that the potential active substance targets are identified and produced from the complete gene set of a pathogenic microorganism in a relatively short time, and these are used directly for the active substance screening or Optimization can be used.
  • any gene bank can preferably be subjected to a preselection.
  • the preselection for genes which code for polypeptides with a specific function can preferably be carried out, for example with the aid of homology analyzes.
  • the preselection can also be carried out for genes which are only expressed in certain development stages.
  • a marked reduction in the genetic material to be examined can be achieved by selection steps. For example, through an enrichment step for genes that code for exported or secreted gene products.
  • the DNA sections of a gene bank are mutagenized by a pathogen, which can be done, for example, by cloning such a DNA section into a plasmid, transformation into a preferably heterologous host organism and subsequent mutagenesis.
  • the resulting expression product can then be detected.
  • the mutagenesis can be carried out, for example, by inserting a marker sequence which is at Expression of the mutagenized sequence in a host organism leads to a fusion polypeptide that can be selected for.
  • the insertion of the marker sequence is not limited to transposon insertion, but can also be carried out in other ways, for example by homologous recombination or infection and recombination using bacteriophages.
  • the marker sequence used for the purposes of the present invention is generally a gene which codes for a gene product which allows selection for those host organisms which express this sequence.
  • these marker sequences are resistance genes which confer resistance to certain antibiotics or which allow the host organism to survive and multiply in a selection medium.
  • the gene marker preferably does not have its own expression signals, but is directly dependent on an upstream promoter, e.g. the transcription promoter on the promoter segment or a promoter which lies on the cloned heterologous DNA fragment to be identified.
  • enzymes can also be used as gene markers. In these cases, the successful insertion is determined by a certain biochemical reaction, e.g. a color reaction, which allows the manual isolation of the corresponding bacterial clone.
  • DNA material can be isolated from the selected bacterial clones and the DNA sequence which codes for the fusion product can be determined by known methods become. This allows a reading frame to be assigned to the DNA fragment to be identified. It is then possible to conduct comparative studies with commonly available DNA sequence databases to verify the identity of the to clarify the identified gene and, if necessary, to obtain evidence of a biological function.
  • CAI and SRM a targeted reduction of the sample volume can be achieved.
  • upstream selection processes that target specific gene groups, e.g. the use of subtractive gene banks from pathogenic and non-pathogenic representatives.
  • pathogen-mediating gene areas are enriched.
  • subtraction methods can also be used to identify genes specific to certain organisms, for example by comparing and subtracting the genomes of H. ylori and H. heilmannii.
  • Gene groups are identified that are only expressed in a certain development step.
  • the array method for example, in which the individual gene samples of the pathogen are applied in a grid-like manner to a carrier is to be emphasized.
  • the individual application points are known, so that in the case of a positive hybridization reaction with the development-specific transcription products or cDNAs or subtractive cDNAs or fragments thereof, the respective genes can be identified and then cloned.
  • Other methods that capture development-specific gene groups are comparative proteome and differential display analyzes.
  • microorganisms are produced which are deficient in the sequences which correspond to the identified gene sequences.
  • the deficient microorganisms are then tested on various growth media or cell cultures or in an animal model or in the natural host, and the defective genes can then be used as required Growth ability can be assigned to a category of non-essential, obligatory essential or optional essential genes.
  • genes that control the development from the vital to the survival form and vice versa has already been mentioned at the beginning. It is therefore particularly preferred to carry out a preselection for such genes.
  • methods such as CAI or SRM can be used and the gene-deficient microorganisms can then be examined on certain nutrient media, which trigger the transition from one form to the other.
  • the Schivo medium is particularly preferred, which enables the coccoid form to be reactivated into the vital spiral form.
  • Deficient microorganisms can be generated in several ways.
  • a number of molecular genetic methods are available for mutagenizing the genome of a bacterial pathogen in such a way that a mutant is available for each gene.
  • the most common method of mutagenesis is based on the inactivation of genes, e.g. B. by randomly inserting transposons in the genome, which are selected using appropriate markers.
  • This method can be applied to different organisms. (Joyce and Grindley, 1 984; Akerley, et al., 1 998). With the help of the inserted transposons, the mutagenized gene can also be precisely located in the genome.
  • a gene mutant with a detectable biological effect has been generated, for example a reduced growth of the cells in a certain milieu, the clear coupling of the gene or the gene product with this property must be demonstrated in a second step. This is usually done through complementation experiments. In this case the original gene is introduced and expressed in the organism with the specific mutant gene. If the original property of the organism can be regenerated in this way, the necessary evidence has been provided. However, this method cannot be used for the characterization of lethal mutants, ie mutants of essential genes. One way out is to use conditional mutations for complementation.
  • chemical-sensitive mutagenesis of the gene under investigation can produce temperature-sensitive mutants that bring the gene product into an inactive state at the normally optimal growth temperature and produce a biologically active gene product at lower temperatures (Das, et al., 1 976; Harris, et al. , 1,992; Hou, et al, 1,994; Polissi and Georgopoulos, 1,996).
  • the wild-type complementations by exogenous substances so-called. Controlled inductors. These inducers activate the expression of the complementing gene which is introduced into the gene-specific mutant on an episome and which, after induction, replaces the missing gene product (Murphy, et al., 1995-. Chow and Berg, 1 988; Arigoni, et al. , 1 998).
  • CAI Conditional Antisense Inhibition
  • SRM Subtractive Recombination Mutagenesis
  • the CAI method is based on the conditional inhibition of the translation of one or more genes which are located on a cloned genome fragment (which then serves as a template or template) and are propagated via a plasmid in the germ to be examined.
  • asRNA specific antisense RNA
  • the antisense nucleic acid sequences can then be synthesized in large quantities in the microorganism and bind to the original mRNA, whereby this mRNA can no longer be translated and is therefore withdrawn from the expression apparatus.
  • the result is that either no gene product or only small amounts of it are formed.
  • the synthesis of the asRNA is subject to control by a promoter (asPromoter) whose activity is conditionally controlled by defined external signals. This conditional inhibition of the expression of a gene or operon thus takes place via the regulation of the synthesis of the asRNA by the inducible asPromoter.
  • the survival and reproduction rate of a clone in which the synthesis of the asRNA is induced with its survival - ⁇ / rate of increase compared to uninduced asRNA synthesis. If the clone's survival / reproduction rate is reduced when inducing asRNA synthesis, the inhibited gene or operon is an (obligatory or optional) essential gene.
  • These growth analyzes can be carried out automatically, so that a very large number of genes can be examined within a short time can be.
  • the plasmid is isolated from these clones and the DNA sequence of the cloned genome fragment, which serves as a template for the asRNA synthesis, is determined and the structure of the essential gene is subsequently determined.
  • a plasmid vector suitable for the CAI method is shown in Figure 1. It contains a genomic or subgenomic DNA fragment from the microorganism to be examined under the control of an inducible promoter (P,) and other conventional expression signals, and an mRNA-stabilizing element, so that the DNA fragment can be expressed in the form of antisense RNA (asRNA) and has a long biological activity.
  • a suitable promoter is e.g. the Tet promoter.
  • the CAI vector additionally encodes a gene for a regulatory protein which regulates the promoter, in this case, e.g. the Tet repressor, which is triggered by an exogenous or extracellular signal, e.g. Tetracycline, can be controlled.
  • the CAI vector of the particular embodiment of Figure 1 further contains one or more selectable marker genes and two origins of replication (ori), one for the microorganism to be examined (referred to here as a pathogen) and another for a conventional cloning host, e.g. E. coli.
  • ori origins of replication
  • a pathogen a conventional cloning host
  • antisense libraries can be created from entire microbial genomes.
  • FIG. 2 shows a schematic representation of a preferred CAI method.
  • AsRNA is synthesized from a CAI plasmid, which contains small fragments of a genomic library of the microorganism to be investigated, from an inducible promoter (P,) under the control of an extracellular signal (see FIG. 1).
  • the asRNA hybridizes in a sequence-specific manner to the mRNA of the gene which corresponds to the cloned DNA fragment on the CAI plasmid.
  • the translation of this mRNA is reduced or prevented by the formation of the asRNA-mRNA hybrid.
  • the result is a deficient microorganism that is unable to to form the gene product in question.
  • the viability of the corresponding clone is restricted or prevented.
  • the viability of the microorganism is determined below on the basis of its life or survival or reproduction rate in a defined biological system. If there is no induction of asRNA synthesis (B), or if the CAI plasmid contains the fragment of a non-essential gene (C), the clone of the microorganism is normally viable and capable of reproduction.
  • RNA plasmid banks are analyzed from genomic fragments of the microorganism to be examined (see Figure 3).
  • a genomic library with CAI plasmids (see Fig. 1) is transferred into the homologous microorganism to be examined under non-inducing conditions and the plasmid-bearing clones are selected using a plasmid-coded marker.
  • the viability of the individual clones, each of which receives a specific CAI plasmid from the gene bank is then examined in direct comparison on the basis of their multiplication rate under induced or non-induced conditions (+ and - in the figure), based on the asRNA synthesis.
  • CAI plasmids are isolated from these clones.
  • the essential genes are identified by sequencing the genomic fragments in the isolated CAI plasmids.
  • This approach can also preferably be combined with a subtractive method (SCAI), an embodiment of which is illustrated in FIG. 4 for illustration.
  • SCAI subtractive method
  • a genomic library with CAI plasmids (see Fig. 1 and 3) is transferred into the homologous microorganism to be examined and the resulting individual clones are called bacterial CAI-Bank combined in one pool. This pool is split into two identical groups (the driver and the tester pool) for selection.
  • driver is used here for the pool of bacterial clones which is treated in such a way that the inducible promoter is activated and expresses asRNA from the CAI vector.
  • the "tester” pool contains an identical set of clones with CAI plasmids, but which is kept under non-inducing conditions and thus has wild-type properties.
  • the "Driver” pool is used for selection (e.g. in animals), while the “Tester” pool is kept untreated.
  • both groups can also be subjected to a selection, with only the “driver” pool being induced by administration of the signal (e.g. tetracycline).
  • the signal e.g. tetracycline.
  • the cloned genomic fragments are then amplified by PCR, using oligonucleotide primers which hybridize with vector sequences to the side of the cloned genomic fragments. Those amplified DNA fragments that are parts of essential genes are enriched and isolated by subtractive hybridization (see Fig. 8).
  • a promoter suitable according to the invention for a CAI vector is, for example, the Tet promoter, the activity of which can be controlled via a regulatory protein (in this case the Tet repressor) and can be induced by an extracellular signal (tetracycline).
  • a regulatory protein in this case the Tet repressor
  • tetracycline extracellular signal
  • the high efficiency of the CAI method in the inactivation of single genes in an organism results from the overlapping cloning of small genomic fragments and the associated synthesis of different asRNA derivatives for a specific gene area. In this way, the probability of obtaining an asRNA which efficiently inhibits the translation of the target gene sought is greatly increased.
  • Such investigations can be aimed at the complete genome of a pathogen, which requires the examination of a very large number of individual genomic fragments.
  • equipment aids are advantageous in order to achieve a high sample throughput.
  • only certain conditions can be examined in these cases, e.g. B. the growth of cells in a particular medium.
  • SCAI subtractive conditional antisense inhibition
  • Subtractive recombination mutagenesis is preferred to identify facultative essential genes.
  • SRM Subtractive recombination mutagenesis
  • permanent gene mutations are generated, whereby the enrichment of essential genes is achieved via a subtractive step.
  • the SRM method can be carried out with complete or partial libraries of pathogenic microorganisms.
  • the SRM method is based on the inactivation of individual genes in the genome of a pathogen by complete insertion of a certain suicide plasmid, which is not or only in the organism to be examined can replicate under certain conditions, such as permissive temperature, which is part of a gene bank.
  • the plasmids are inserted into the genome by homologous recombination. Successful insertion is indicated by expression of a plasmid-encoded antibiotic resistance marker.
  • Figure 5 shows a suitable SRM vector which, like the CAI vector, contains a genomic or subgenomic DNA fragment of the microorganism to be examined, and an origin of replication (ori) for a cloning host (eg E. coli), one or more selectable ones Marker genes and another conditionally active origin of replication for the microorganism to be examined, for example a temperature-sensitive origin or an origin, the activity of which depends on a replication factor present in trans and which can also be introduced into the system.
  • an origin of replication eg E. coli
  • the SRM plasmid contains a genomic sequence of the microorganism to be examined, when this vector is transfected into this microorganism, there is a homologous recombination in which the entire SRM plasmid is inserted into the genomic gene of the microorganism and the corresponding gene, if it is one, inactivated. This leads to an insertion mutant.
  • Suitable inducible origins of replication are temperature-sensitive oris or those that can be controlled by a factor, such as e.g. the RGK factor pir or the pWV factor repA, which is fed into the system in trans.
  • An SRM plasmid (see FIG. 5) is inserted into the genome of the microorganism to be investigated via homologous recombination between the genomic fragment of the cloned in the plasmid
  • a bank of insertion plasmids can be transferred from genomic fragments of the microorganism to be examined into this microorganism and genomic insertion mutants can be formed.
  • This preferred embodiment of the SRM method is shown in Figure 7.
  • a bank of SRM plasmids, which contain individual genomic or subgenomic fragments, is transferred into the homologous microorganism to be examined.
  • genomic insertion mutants are selected with the aid of a plasmid-coded marker (see FIG. 5). Only insertion mutants that are mutated in a non- or optional essential gene can survive in this step, since mutants of an essential gene are not viable.
  • the individual insertion mutants are combined in a pool and this pool is then divided into two identical groups, the driver and the tester pool.
  • the driver pool is selected, for example, by infection of an animal.
  • the tester pool remains untreated.
  • those clones are lost from the driver pool that insert in an optionally essential gene (that for survival and reproduction among the Selection conditions is necessary).
  • the plasmids inserted into the genome of the microorganism are recircularized and recovered under permissive conditions.
  • Such plasmids are missing from the driver pool which contain fragments of facultatively essential genes.
  • the fragments cloned in the SRM plasmids are then amplified in both pools by PCR (see FIG. 4). Those amplified DNA fragments, which are parts of optional essential genes, are enriched and isolated by genetic subtraction (see Fig. 8).
  • FIG. 8 A special embodiment, which takes advantage of subtractive hybridization for enrichment in fragments of essential genes, is illustrated by way of example in FIG. 8.
  • A PCR-based genetic subtraction.
  • the tester DNA fragments (see Figs. 4 and 7) are ligated with an adapter oligonucleotide in such a way that the adapter is only covalently linked to one of the two DNA strands of a double-stranded tester DNA fragment, which e.g. by ligation of a double-stranded, non-phosphorylated adapter to the 3'-phosphorylated DNA fragments of the tester DNA.
  • These tester DNA fragments are then mixed with a molar excess of driver DNA fragments. The mixture is denatured and slowly rehybridized. Then overhanging
  • tester tester double strands are separated from the tester homoduplexes by extraction with carrier-coupled streptavidin. The latter are isolated by cloning.
  • the insertion mutants generated, for example, by SRM are examined in animal experiments or cell culture systems with regard to their changed biological properties.
  • special host cells e.g. cultured macrophages or host tissues, e.g. B. spleen
  • gene groups can be selected that determine essential properties of the pathogen, e.g. the colonization of certain host cells. If the surviving mutants are isolated from the cells, the mutants of essential genes are missing. If you subtract the surviving mutants from the complete gene bank, you get the mutants of the essential genes.
  • the CAI or the SRM method is a very efficient method for the unique identification and characterization of essential genes. Since essential genes are a natural target for inhibitory agents, the methods presented offer an ideal basis for the development of new agents.
  • the identified genes are used directly for active substance screening, in comparison to conventional methods, complex purification steps can be dispensed with. These procedures focus on bacterial carrier cells that can be used for screening for prophylactic and therapeutic agents.
  • the gene-deficient microorganisms produced are then tested for their ability to grow or to survive. Suitable test systems are / nv / fro systems, cell culture systems, tissue culture systems and animal models as a natural environment. If the method is applied to H. ylori, the organisms are grown on the one hand on a so-called full medium, the full medium providing the best possible conditions for growth for .py / or /. At the same time, the deficient H.
  • ylori organisms are cultivated in a culture which should correspond as closely as possible to the natural environment of H. pylori.
  • cell cultures based on primary cultures or cell lines from gastrointestinal tissue are used, or differentiated primary tissue (spheroids) in culture medium.
  • Another way to simulate the natural environment of H. pylori is to use stimulated macrophages, because H. pylori has the ability to not be absorbed and metabolized by them.
  • it can also be checked whether the deficient H. ylori organisms are able to establish themselves in immunodeficient mice over a certain period of time.
  • the gene deficient in this organism is referred to as an optional essential gene.
  • essential genes of microorganisms can be assigned to one of these categories. From these results, the sequences in mutated or / suppressed by asRNA can then be identified and each assigned to one of these two categories, or the category of non-essential genes if the gene-deficient organism shows no impairments in its ability to grow.
  • Another object of the present invention is to provide a genetic method for isolating and cloning the identified essential genes from various clinical Helicobacter isolates or from heterologous pathogenic germs of clinical importance.
  • the method according to the invention therefore further comprises the steps (v) producing primers for the amplification and detection of homologous gene sequences in heterologous microorganisms (VI) identifying the homologous gene sequences.
  • a preferred implementation of these further process steps consists in producing so-called megaprimers of the identified essential Helicobacter genes by means of PCR (polymerase chain reaction), the sequence of which can be derived directly from the corresponding plasmids of the mutagenized DNA sections. These primers can then be used to isolate the already identified essential genes from various e // co / -) ac-'er isolates. If these essential genes have counterparts in other microorganisms, the primers may also be used to isolate these genes from microorganisms other than Helicobacter. Furthermore, the exact DNA sequence of the isolated genes and the determination of the gene variance within the different He / icobacter- ⁇ so ⁇ ate or between the different microorganisms can then be determined. DNA fragments with variable 3 'ends are produced during the production of the megaprimer. Because of this property, it is possible to use the DNA fragments for the isolation of variable or related genes by means of the described PCR method. Identify specific active ingredients
  • Bacterial carriers are used very effectively to identify new immunological agents from the pool of identified essential genes of a pathogen or to further develop these agents, since the identified genes can be cloned directly into these carrier systems and expressed there.
  • the active ingredient screening is then carried out directly with the aid of these recombinant, bacterial carriers.
  • Attenuated bacteria such as e.g. Salmonella, used because they have a natural potential for immune stimulation. If these attenuated bacteria are used as carriers or producers for the identified essential genes of the pathogenic germs and if an immunization is carried out on a mammal with these vaccine strains, a sustainable immune response can be triggered.
  • the bacterial carrier systems can be equipped with efficient gene expression systems, which the production also allow problematic antigens (PCT / EP91 / 02478, EP981 1 6827.1). Due to the direct subcloning of the isolated essential genes and the simple handling of the bacterial carriers during the production and vaccination, a large number of antigens can be tested for their immunogenic and protective potential in a relatively short time. In conventional methods, on the other hand, the test antigens have to be subjected to time-consuming purification processes, difficulties often arising during the genetic engineering of the selected antigens in bacteria, which are linked to the toxic effect of these antigens on the producing bacterial strain.
  • the immunogenic polypeptides are used together with suitable additives for immunization in vivo.
  • suitable additives for immunization in vivo.
  • Various adjuvants, bacterial toxins, cytokines or a polypeptide according to the invention are used as the live vaccine.
  • the immune response is then tested to determine whether it has a protective effect against further homologous infections (e.g. infections with different H. pylori strains) after administration of a specific polypeptide of the invention in combination with corresponding additives after infection with the homologous germ.
  • a further possibility for testing the immunogenicity consists in an immune response against in the animal model (eg mouse, rabbit) To trigger Helicobacter or other microorganisms and to obtain antibodies from the immunized animals / which can then be used in a further Western blot analysis. At the same time, patient biopsies must be examined in situ immunologically using the same antibodies, since Helicobacter and other microorganisms in culture can lose or gain certain proteins.
  • the screening for prophylactically or therapeutically effective, immunological substances can proceed according to the following scheme, although compliance with the individual steps is not mandatory:
  • Hyperimmune serum the original gene product in the pathogenic germ should recognize. It may be possible that the original antigen is only produced by the pathogen in a certain development period. 3.
  • the protective effect of the individual antigens in the prophylactic or / and therapeutic use form is in the
  • antigen-presenting cells are isolated from different donors, for example dentritic progenitor cells, which are expanded in vitro and loaded with the antigens to be tested, the antigens preferably being expressed via corresponding vectors.
  • the identified genes can also be used individually or in defined, combinations in dendritic cells (DC) of uninfected Donors are expressed.
  • DC dendritic cells
  • DC is incubated with T cells from autologous donors, it is possible to determine whether this donor would react to the antigen used if it came into contact with it naturally, for example as part of a vaccination. Based on the immune response of the T cells, a possible immunogenicity of the corresponding antigen can be concluded.
  • Such an immune response consists, for example, of a proliferation of the T cells or a cytokine release, in particular of IL-2 and IL-4.
  • the cytokines can be evaluated, for example, using a commercially available assay kit (for example from Genzyme Cambridge MA).
  • antigens or epitopes identified and characterized in the manner described can be used to develop the first vaccine prototypes.
  • antibodies or antibody fragments with a protective or inhibitory effect are supplied to the vaccine from outside.
  • Antibodies are provided in the form of polyclonal, preferably monoclonal antibodies (MAKs) or recombinant antibodies. These include antibodies which react specifically with polypeptides of the invention or their subunits and fragments and can be used for prophylactic and / or therapeutic use, for example passive immunization. These anti-protein or anti-peptide antisera or monoclonal antibodies can be obtained using standard protocols, for example by immunizing animals such as mice, rats or goats with a purified polypeptide of the invention, a fusion protein or a subfragment of it. In addition, the animals can also be immunized with bacterial vaccine carriers which are equipped with corresponding genes of the invention and which express the encoded polypeptides.
  • MAKs monoclonal antibodies
  • the antibodies are preferably immunospecifically directed against antigenic determinants or epitopes of the He / Zco / jacter polypeptides described or a closely related polypeptide which has a homology of at least 90%. They are not cross-reactive with polypeptides which, for example, have a homology of less than 80%.
  • the coding gene of such an antibody can be used to create chimeric genes that determine antibodies, consisting of an antigen-binding domain from the mouse and the Fc part of a human antibody . These antibodies can be produced in cell lines or transgenic animals.
  • mini-antibodies can also be used, e.g. in a heterologous system like bacteria.
  • miniantibodies can be either monovalent or bivalent and consist of dimerized single chain molecules (Kujau et al., 1 998; Kalinke et al., 1 996; Pack et al., 1 993).
  • Antibodies to the immunogenic polypeptides of the invention can also be generated in plants. Examples of this are e.g. by Hiatt and Ma (1 993), van Engelen et al. (1,994) and Ma et al. (1,994). Depending on the plant used, they can e.g. can be used directly for consumption and thus as an oral vaccine.
  • Another very widely used way of producing antibodies is in animals immunized in milk and eggs. If you give pregnant cows, for example, Suitable antigens for sheep or horses, there are immunoglobulins in milk that can be used to develop a vaccine. The milk can then either be administered directly as a vaccine or a concentrated immunoglobulin extract can be prepared. Antibodies (hyperimmune antibodies) can also be produced in chicken eggs in the same way (Ling et al., 1 998; Sasse et al., 1 998). The described immunogenic polypeptides of the invention can therefore also be used to develop a milk product or chicken eggs which can be used as an oral vaccine.
  • the generated antibodies or their fragments are tested for their applicability.
  • they can be purified by known methods (precipitation, chromatographic methods) and, for example, examined to determine whether they can inhibit the infection process of H. pylori (adhesion assays) or activating for complement or ADCC ("antibody-dependent cell-mediated cytotoxicity", antibody-dependent cell-mediated cytotoxicity).
  • the antibodies generated using the polypeptides of the invention are administered either orally or intragastric.
  • the antibodies are mixed with a bicarbonate buffer.
  • they can also be administered systemically, without having to be buffered.
  • Antibodies are preferably used alone or in combination with other non-immunological agents, e.g. with antibiotics or proton blockers.
  • Active vaccination is based on an immune response that is triggered by the vaccinated organism itself. Dosage forms of vaccines are preferred as antigens, antigen fragments, subunit vaccine, as DNA vaccine, as live vaccine or as food vaccine. Antigens are those polypeptides or their fragments that can elicit an immune response in vivo.
  • a polypeptide is to be provided as a subunit vaccine, it is first broken down into its subunits or structural domains according to its antigenicity pattern (e.g. T and B cell epitopes).
  • This antigenicity pattern can be created with the aid of a computer program, whereby immunogenic regions consisting of a short polypeptide sequence of approximately 8 to 10 amino acids can be recognized (Hughes et al., 1 992).
  • the individual polypeptide pieces can then subsequently be tested for their immunogenicity in the mouse or in primates or in humans.
  • polypeptides can either be administered as purified polypeptides, which have been produced synthetically, in combination with appropriate additives such as an adjuvant, toxin or cytokine, or as a fusion protein to a known immunogenic protein or protein subunit, e.g. the cholera toxin B subunit (Liljeqvist et al., 1 997).
  • immunogenic peptides can be incorporated into outer membrane proteins such as e.g. the OmpS maltoporin from E. coli and heterologously expressed in a vaccine carrier strain. (Lang and Korhonen, 1 997).
  • the polynucleic acid molecules characterized in the invention can be administered “naked” in fusion with a eukaryotic tissue-specific promoter or in the form of a plasmid.
  • the "naked” DNA or the corresponding plasmid is combined with an additive such as a reagent that changes the cellular permeability such as, for example, bupivacaine (WO94 / 1 6737), cationic lipids such as, for example, DOTMA (N- [1 - (2,3- dioleyloxy) propyl] -N, N, N-trimethyl-ammonium chloride, DOTAP (1, 2-bis (oleyloxy) -3-trimethyl-ammonio) propane), DDAB (dimethyl-dioctadecyl-ammonium bromide), DOGS (dioctadecyl-amidolglycyl- spermidine) or cholesterol derivatives, silica, gold or
  • Attenuated Salmonella can also be used to apply the polynucleotide molecules.
  • the bacteria are transformed with eukaryotic expression vectors which contain a polynucleotide molecule of the invention and then administered orally.
  • the plasmid DNA is then transferred from the bacterium to the host (Darji et al., 1 997).
  • filamentous phages can also be used to transform attenuated carrier bacteria. The advantage of these is that they can transmit an extremely high number of plasmids.
  • a live vaccine includes, among others, viral ones, e.g. adenoviral or chickenpox virus vectors, or bacterial vectors such as Salmonella, Shigella or Lactobaci / Ius are available.
  • viral ones e.g. adenoviral or chickenpox virus vectors
  • bacterial vectors such as Salmonella, Shigella or Lactobaci / Ius are available.
  • Attenuated, non-virulent Salmonella typhimurium strains which can be used for the recombinant expression of heterologous antigens and are administered orally, have been characterized many times (Mekalanos, 1 994, WO92 / 1 1361, Cirillo et al., 1 995 and Dorner (1 995).
  • bacterial vectors that can be used as vaccine vectors have been described by Cirillo et al., (1 995) and Dorner (1 995), using a polynucleotide molecule of the invention encoding a therapeutically or prophylactically active polypeptide either stably integrated into the bacterial genome and subjected to a transport system which enables presentation on the bacterial surface (PCT / EP94 / 04286; WO97 / 35022) .
  • the corresponding polynucleotide molecule can also be present in the bacterium in the free state as a plasmid.
  • Vaccines are usually administered with suitable additives such as adjuvants, bacterial toxins, cytokines, etc., which support the protective or therapeutic effect of the immunogenic polypeptide.
  • Adjuvants with minor side effects for use In humans which are suitable for subunit vaccines and live vaccines, but also in part for DNA vaccines, are, for example, aluminum hydroxide, aluminum phosphate, calcium phosphate, N-acetyl-muramyl-L-threonyl-D-isoglutamine, N-acetyl -normuramyl-L-alanyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine , Liposomes, monophosphoryl lipid A, trehalose dimicoloate, fungal polysacc
  • Cholera toxin or its subunits or the heat-labile toxin from E. coli can be used as the bacterial toxin. Although they are highly potent as adjuvants, their toxicity means that they can only be used to a limited extent on humans. However, certain mutagenesis techniques can be used to develop molecules that are active but non-toxic (O'Hagan, 1 998).
  • Another way of optimizing the immune response to the prophylactically and / or therapeutically active substances of the invention is to express the corresponding polypeptide as a fusion protein with an immunogenic protein domain.
  • One possibility is e.g. in using the Pilin DSL domain from Pseudomonas aeruginosa as a fusion partner.
  • fusion proteins which have been fused to glutathione S-transferase or thioredoxin (Hill et al., 1 997; Gabelsberger et al., 1 997).
  • the corresponding fusion protein can be produced and administered as a subunit or live vaccine.
  • the immune response of the identified immunologically active substances can also be modulated in that they are administered in combination with certain cytokines.
  • Co-expression of the polynucleotide sequences of the invention with a specific cytokine in Salmonella or another host bacterium is suitable for simultaneous administration.
  • the corresponding cytokine can either do this a separate plasmid, in series, or as a fusion protein encoded with the desired polynucleotide sequence of the invention and then transformed into the host bacterium.
  • Cytokines which stimulate the immune system such as interleukin-6 (IL-6), interleukin-1 0 (IL-10) or interleukin-1 2 (IL-1 2), are suitable.
  • individual immunogenically active substances of the invention can be used together or in combination with known immunogenic substances such as e.g. VacA or its individual subunits can be combined.
  • the corresponding polypeptide can therefore be used together with at least one further Helicobacter antigen, such as e.g. the native urease or its subunits, fragments, homologs, mutants or derivatives thereof are expressed.
  • Helicobacter antigen such as e.g. the native urease or its subunits, fragments, homologs, mutants or derivatives thereof are expressed.
  • different subunit vaccines can be administered individually or as a fusion protein as described above together.
  • purified polypeptide molecules can be used in combination with a suitable adjuvant, bacterial toxin or cytokine.
  • a vaccine vector of the invention can thus contain one or more polypeptides of the invention, derivatives or fragments of the like.
  • a DNA vaccine with one or more purified subunit vaccines in a suitable carrier, as has already been described above.
  • Bacterial carriers can also be used very effectively to identify new pharmacological agents from the pool of the identified essential genes of a pathogen or to further develop these agents, since the identified genes can be cloned directly into these carrier systems and expressed there. The active ingredient Screening is then carried out directly with the aid of these recombinant, bacterial carriers.
  • Display systems are used to present an expressed polypeptide of the invention on the cell surface of bacteria.
  • a signal peptide at the amino end enables the transport of polypeptides through the inner membrane, while other parts take on the storage and anchoring in the outer membrane.
  • Various outer membrane proteins from E. coli have been described as carrier components, such as PhoE (Agterberg et al., (1,990) or OmpA (Francisco et al., 1,999).
  • fusions with the transport domain of the IgA protease precursor IgAß can also be carried out (Klauser et al., 1 990)
  • Examples of display systems are, for example, the DsbA system (PCT / EP 94/02486) or the car transporter system (AIDA; WO97 / 35022).
  • the polypeptides presented on the surface can then be used for binding studies with peptide or combinatorial chemical substance libraries.
  • the binding studies can be carried out with the aid of a "high through put" system, which enables high test rates, in liquid or bound form.
  • the presented polypeptides are applied, for example, to a chromatophore coupled, which in combination with another chromatophore, which is coupled to the active ingredient peptide or the chemical substance, enables a color reaction.
  • the corresponding active ingredient component can also be labeled, for example, with a fluorescent dye or coupled to a solid carrier matrix. If a “solid phase system” is used, either the polypeptide of the invention used or, conversely, the peptide or combinatorial active substance bank is coupled to the carrier matrix. The respective dye-labeled component then binds to the immobilized component, which in turn enables a color reaction. After the binding reaction, several washing processes must then be carried out before the corresponding substance is isolated.
  • Advantage of the "Solid Phase System" over the binding in Liquid is that the active substance can be isolated more quickly since the unbound substances are washed away.
  • An alternative embodiment of the method according to the invention is to find specific binding partners of the polypeptides encoded by the identified genes.
  • Homology studies can preferably be carried out with Helicobacter and other organisms, e.g. with the aid of computer alignments, Southern blots, PCR and the like and subsequent assignment of the sequences. Based on homologies, it can then be concluded that the proteins are binding partners.
  • Target-directed screening methods with the aid of substance libraries can also be carried out with preference.
  • the potential binding substances are presented bound in a special arrangement, for example in microtiter plates or other carrier materials.
  • the "target” usually purified, optionally recombinant added in soluble form, which enables the detection of an interaction between the target and certain substances.
  • Indirect detection can be provided by labeled antibodies directed against the substance or by introducing additional elements ("tags") into the target.
  • Another variant of the binding studies is the expression of in recombinant bacteria (e.g. those that do produce fluorescent protein GFP or certain enzymes), which present the proteins on the surface, and then testing a substance library.
  • recombinant bacteria e.g. those that do produce fluorescent protein GFP or certain enzymes
  • the three-dimensional structure of the polypeptides encoded by the genes identified according to the invention or their fragments can also be determined by crystallographic analysis. If the resolution is sufficient, any "pockets" or other binding points can be exactly characterized in their three-dimensional structure. Based on this data, the structure of potential binding partners can be calculated.
  • polypeptides can be identified which bind to the Helicobacter polypeptides of the invention or their fragments, or to other polypeptides identified according to the invention. If a corresponding peptide is found in this way, it can be chemically modified further until the optimal possible binding is achieved.
  • the identified polypeptide can be used, for example, as an inhibitor, for example by coupling it to a toxin and an internalization signal which destroys the pathogenic germ or else as a peptide mimetic to prevent the germ from binding to the cellular surface (EP-41 2,762A and EP-B31, 080A).
  • the identified peptides that bind to the Helicobacter polypeptides of the invention can be coupled to certain ligands, for example for the T cell receptor. If an animal or human infected with pathogens is given these peptides equipped in this way, the body's immune system is specifically attracted and activated. Compliance with the individual steps is not mandatory here, but can be supplemented or replaced by further steps.
  • a vaccine can be further developed by combining several antigenic gene products or parts thereof in one active ingredient and / or by administering them with different carriers or additives.
  • Different attenuated bacterial or viral organisms act as carriers and adjuvants and / or cytokines as additives.
  • a lead structure characterized as being effective is chemically modified further so that the identified gene product is optimally bound and inhibited. Furthermore, the drug should be well tolerated by the patient and have few side effects.
  • a polynucleotide of the invention can e.g. are pre-coupled to a carrier matrix or vice versa, the active substances of the polypeptide or combinatorial substance bank.
  • the following scheme used is the same as that already described for the polypeptides.
  • inhibitory substances can be polypeptides, peptides, but also chemical substances, such as antibiotics.
  • the inhibitory effect can intervene in different stages of replication of the microorganisms to be controlled. Examples are expression inhibitors or enzyme inhibitors or other inhibitors which can influence the natural function of the polypeptides of Helicobacter and related microorganisms.
  • Such inhibitory substances are also the subject of the invention.
  • Another way to find an optimal active ingredient against Helicobacter and other bacterial infections is with the help of special computer programs. Based on crystallographic data obtained from the polypeptides described in the invention, a model can be created that combines steric, electronic, hydrophobic and so-called "resulting binding moments" (RBMs) (Ray et al., 1 998). Using this model, substances can be modeled on the computer that may contain the lead structures that have already been identified, but that have better binding properties. However, completely new active ingredients can also be designed.
  • RBMs resulting binding moments
  • Another aspect of the present invention is a nucleic acid which codes for an essential secretory gene from Helicobacter pylori, which was identified by the inventive method described above.
  • Essential He / icobacter genes were identified with the present method, whose nucleic acid sequences are shown in SEQ ID NO. 1 to 245 (odd numbers) are given.
  • a nucleic acid according to the invention is, for example, characterized in that it (a) one of the nucleic acid sequences shown in SEQ ID NO: n, where n is an odd integer from 1 to 245 inclusive, or a protein-coding section thereof, (b) one of the sequences from (a) nucleotide sequence corresponding to the degeneration of the genetic code or (c) a nucleotide sequence hybridizing with one of the sequences from (a) and / or (b) under stringent conditions.
  • the present invention also includes nucleotide sequences which hybridize with one of the aforementioned sequences.
  • hybridization according to The present invention is in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1 989), 1 .1 01 - 1 .1 04).
  • nucleotide sequence hybridizing under such washing conditions with one or more of the nucleotide sequences according to the invention or with a nucleotide sequence corresponding to these sequences in the context of the degeneration of the genetic code is a nucleotide sequence according to the invention.
  • the nucleotide sequence according to the invention is preferably a DNA. However, it can also comprise an RNA or a nucleic acid analog, such as a peptide nucleic acid.
  • the nucleic acid according to the invention particularly preferably comprises a protein-coding section of the nucleotide sequences shown in the sequence listing or a sequence which has a homology of more than 80%, preferably more than 90% and particularly preferably more than 95% to the nucleotide sequences shown or preferably at least 20 Has nucleotides (nt) and particularly preferably at least 50 nt long section thereof.
  • a nucleic acid according to the invention can code for a secreted polypeptide with signal peptide or for a secreted polypeptide without signal peptide.
  • a nucleic acid according to the invention comprises both the sequence of the coding strand and the sequence complementary thereto.
  • the latter can be used, for example, in the production of antisense nucleic acids.
  • the invention also of course also relates to a gene bank which contains at least 2, preferably at least 20, more preferably at least 100 of the nucleic acids mentioned in clones in vectors.
  • genes that are specified in the sequence listing in their nucleic acid and amino acid sequence are bacterial genes.
  • prokaryotes also use other (alternative) start codons. These are AUU (usually codes for isoleucine, Ile), UUG (usually codes for leucine, Leu) and GUG (usually codes for valine, Val).
  • AUU usually codes for isoleucine, Ile
  • UUG usually codes for leucine, Leu
  • GUG usually codes for valine, Val
  • the amino acid sequences were translated according to the translation code normally used. It is pointed out that the prokaryotic use of alternative start codons must be taken into account when reading the sequence listing, and that the nucleic acid sequences according to SEQ ID NO.
  • SEQ ID NO 247 shows the sequence of the vector pSRM4 ( Figure 13).
  • sequences SEQ ID NO. 1 to 114 are shown in the enclosed sequence listing.
  • the sequences SEQ ID NO. 115 to 246 are shown in Figures 14 and 15.
  • Tables A and B below list the nucleic acid sequences identified as Helicobacter genes (Ge ⁇ -ID), the nucleic acid sequences of which could not be determined in full.
  • the shown e ⁇ era ⁇ t ⁇ r of the determined Helicobacter Ge ⁇ s ⁇ quenze ⁇ is ensured due to the selection for functional fusions with the used indicator, the ⁇ -lactamase (see examples 3 and 4).
  • Table A lists the Helicobacter genes that contain an incomplete S 'end and or 3' end.
  • Table B lists such Helicobacter genes that one or more amino acids cannot be assigned due to the lack of sequence data. In all cases, St ⁇ p codons can be excluded.
  • Another aspect of the present invention is a vector which contains a nucleic acid according to the invention or a section thereof.
  • the nucleic acid or the nucleic acid section can be cloned into the vector in such a way that it can be expressed in either sense or antisense direction.
  • the nucleic acid section preferably has a minimum length of 15 nucleotides, more preferably 20 nucleotides, more preferably 50 nucleotides.
  • This vector can be any prokaryotic or eukaryotic vector on which the DNA sequence according to the invention is preferably located in connection with expression signals, such as e.g. Promoters, operators, enhancers etc. Examples of prokaryotic vectors are chromosomal vectors such as bacteriophages (e.g.
  • the vector according to the invention can also be a eukaryotic vector, e.g. a yeast vector or a vector suitable for higher cells (e.g. a plasmid vector, viral vector, plant vector).
  • a eukaryotic vector e.g. a yeast vector
  • a vector suitable for higher cells e.g. a plasmid vector, viral vector, plant vector.
  • CAI and SRM vectors as described above are also the subject of the invention.
  • Another object of the present invention is a cell which is transformed with a vector or a nucleic acid according to the invention.
  • this cell is a prokaryotic cell, preferably a gram-negative bacterium, for example E. coli.
  • the cell according to the invention can also be a eukaryotic cell, such as a fungal cell, a yeast cell, an animal or a plant cell.
  • the cell is particularly preferably a microorganism, for example Helicobacter or Salmonella.
  • Microorganisms transformed with CAI or SRM vectors have already been described above. These are, as with the above Mutant banks which can be prepared, also a subject of the invention.
  • Another aspect of the invention relates to an essential and preferably secreted polypeptide of H. ylori.
  • this is a polypeptide that
  • (b) comprises a sequence which is immunologically cross-reactive with one of the sequences according to (a).
  • Immunological cross-reacting sequences thus also include muteins, variants and fragments of those shown in SEQ ID NO. 2 to 246 sequences shown. These are to be understood as sequences which differ from the above sequences by substitution, deletion and / or insertion of individual amino acids or short amino acid segments.
  • the identified polypeptides Based on homology analyzes with the help of the FASTA protein program, the identified polypeptides, the sequence of which could be found with the nucleic acid sequence, could be assigned certain features or a putative localization in the bacterium.
  • Some of the polypeptides encoded by the nucleic acids of the invention have a signal peptide and are exported to their destination by the sea-dependent transport mechanism, while others have no signal peptide and are therefore likely to be via a sea-independent transport mechanism, e.g. can be secreted by the ABC transporter system (see Tables I and II).
  • polypeptides according to the invention are preferably prepared by: transforms a cell with a DNA molecule or vector according to the invention, cultivates the transformed cell under conditions in which expression of the polypeptide takes place, and isolates the polypeptide from the cell and / or from the culture supernatant.
  • the polypeptide according to the invention can be obtained both as a fusion polypeptide and as a non-fusion polypeptide.
  • the polypeptide according to the invention can be used as an immunogen for the production of antibodies.
  • the present invention thus also relates to an antibody which is directed against a polypeptide according to the invention.
  • the invention also relates to fragments of such antibodies, e.g. Fab fragments or Fc fragments.
  • Yet another aspect of the invention is an inhibitor of the polypeptides according to the invention, their fragments, or their expression, presentation or / and natural function.
  • This is preferably a molecule which is able to bind specifically to a polypeptide or fragment thereof and / or to influence its expression, presentation or / and natural function.
  • Particularly suitable as inhibitors are proteins or peptides which inhibit a polypeptide according to the invention in its natural function, e.g. enzymes can be inhibited by blocking the active site.
  • Yet another aspect of the present invention relates to a pharmaceutical composition which, as an active ingredient, a DNA molecule according to the invention, a vector according to the invention, a cell according to the invention, a polypeptide according to the invention, an antibody or fragment thereof or / and an inhibitory molecule capable of is specific to one bind inventive polypeptide, optionally together with conventional pharmaceutical auxiliaries, diluents, additives and carriers.
  • a pharmaceutical composition according to the invention can be used in various ways and using individual components thereof as effective substances for inhibiting the reproduction of Helicobacter organisms in a host, especially in humans.
  • the pharmaceutical composition according to the invention can be used on the one hand for the diagnosis of a Helicobacter infection.
  • the diagnosis at the nucleic acid level is preferably carried out by using hybridization probes or primers which have a specific DNA sequence which relates to at least a section of one of the sequences shown in SEQ ID NO. 1 to 245 (odd numbers) sequences is complementary, so that they allow an amplification of the sequences according to the invention.
  • these amplification primers or probes can also be used for the amplification and thus for the detection of related microorganisms if they have gene sequences which code for the same essential gene. Diagnostics at the protein level are preferably carried out with the aid of the antibodies according to the invention.
  • the pharmaceutical composition is suitable for the prophylaxis and control of / e // co-_> acter infections and infections with related microorganisms.
  • Another important aspect of the present invention is the use of the identified essential genes of Helicobacter pylori ' for the prevention or control of an infection with Helicobacter or related microorganisms.
  • these identified essential genes can be used for the production of vaccines (vaccines) (see above).
  • Another use of the polypeptides of the invention is to purify the antibodies against H. ylori polypeptides and against corresponding polypeptides from related and other microorganisms.
  • Figure 1 shows a schematic representation of a CAI vector.
  • FIG. 2 shows a schematic representation of a conditional antisense inhibition (CAI) method.
  • Figure 3 shows a schematic representation of the investigation of the viability of deficient microorganisms based on their survival rates using the CIA method.
  • Figure 4 shows a schematic representation of the subtractive
  • CAI procedure CAI
  • Figure 5 shows a schematic representation of an SRM vector.
  • Figure 6 shows a schematic representation of the reversible
  • Figure 7 shows a schematic representation of an SRM process.
  • Figure 8 shows a schematic representation of the enrichment of fragments of essential genes by subtractive hybridization.
  • Figure 9 shows an overview map of pSRM4.
  • the "origin of replication" (ori) is shown as a bar, coding regions (for further explanations see below) are shown as arrows. Singular restriction sites are given.
  • Figure 10 shows a schematic representation of the integration
  • Dashed lines indicate the DNA area in which homologous recombination takes place.
  • Figure 11 shows a schematic overview of the steps for constructing a random fragment bank.
  • Figure 12 shows the complete nucleotide
  • Figure 13 shows the nucleotide sequence of the SRM vector pRSM4.
  • Figures 14 and 15 show the nucleotide and amino acid sequences of other essential Helicobacter genes. Examples
  • the SRM method is carried out with the vector pSRM4, which was specially developed for this method (see Figure 9, sequence see SEQ ID No .: 247
  • the plasmid has the following properties:
  • pSRM4 carries a temperature-sensitive replication function (repA Xs , ori +) from the Streptococcus plasmid pWV01 (Kok, et al., 1984; Maguin, et al., 1 992) and replicates at a permissive temperature (30 ° C) in Gram-negative bacteria (E. co / i, Salmonella, etc.) and in Gram-positive bacteria (Bacillus, Lactococcus, Streptococcus, etc.).
  • repA Xs , ori + replicates at a permissive temperature (30 ° C) in Gram-negative bacteria (E. co / i, Salmonella, etc.) and in Gram-positive bacteria (Bacillus, Lactococcus, Streptococcus, etc.).
  • pSRM4 does not replicate either in Salmonella or in E. coli.
  • the experiment is carried out as under (1), with the difference that the incubation takes place at 37 ° C. (without Tet). After 80 to 180 minutes after tO, the number of Tet sensitive clones begins to increase logarithmically, while the number of Tet resistant (plasmid-containing) clones remains the same.
  • pSRM4 codes for a tetracycline resistance that mediates resistance to 1 7.5 pg tetracycline / ml.
  • the tetracycline resistance gene can be exchanged for other resistance genes.
  • several restriction interfaces are available within the vector (see Fig. 9).
  • pSRM4 contains a so-called “multiple cloning site” (mcs) with different, unique recognition sequences for restriction enzymes.
  • mcs multiple cloning site
  • the mcs is located in a part of the lacZ gene which can be used for alpha complementation in E. coli strains such as DH5 ⁇ , XL1-Blue, or EC101 (Law, et al., 1 995).
  • E. coli strains such as DH5 ⁇ , XL1-Blue, or EC101 (Law, et al., 1 995).
  • plasmids which carry cloned DNA fragments in the mcs can be identified by blue / white differentiation on solid medium with X-Gal (80 pg / ml) and IPTG (420 pM IPTG).
  • Clones of EC101 that carry plasmids with inserts form white colonies, while colonies of clones that contain plasmids without an insert are blue.
  • pSRM4 can be transformed with an efficiency of 2x10 7 to 2x10 8 per pg DNA in E. coli DH5 ⁇ or EC101, and with an efficiency of 5x10 6 / pg in S. typhimurium ATCC 14028.
  • the transformation takes place via electroporation according to standard protocols.
  • pSRM4 does not recombine with the S. typhimurium genome.
  • pSRM4 was transformed into S. typhimurium ATCC 14028; the transformants were cultivated for 48 h at 30 ° C. on FeMed with Tet and separated. A single colony (approx.
  • pSRM4 with short genomic inserts is suitable for inactivating genomic genes via homologous recombination (examples: phoP gene and phoN gene from S. typhimurium).
  • phoP-specific primer that leads to a chromosomal p ⁇ o / V or p ⁇ E sequence, but not to the cloned p /? oN or p ⁇ oE fragment is homologous, carried out (primer pairs phoNfor [GCTGTCGACTTTCTACCACTGATCGTAGC] / lacZ2 [CATGCCATGGCTGCGCGTAACCACC] or phoP3 [CCCCAAAGCACCATAATCAACGC] / lacZl [CATGCCCCGGATAG]].
  • DNA is amplified from the plasmid along with a piece of chromosomal DNA that is adjacent to the insertion site.
  • genomically integrated large amounts of PCR fragment are obtained.
  • a PCR with the primers lacZl / SRMgb2 [ATACCGTCGACCTCGAG] is used to check the loss of free plasmid DNA. Both primers hybridize only against sequences of the plasmid that are located laterally to the cloned insert; in the case of a genomically integrated plasmid, this amplification results in only a very small amount of product.
  • Genomically integrated pSRM4 excises at permissive temperature. Cloned genomic inserts can be easily prepared via PCR with different primer pairs (SRMgb2 / SRMgb4 [AACAAAAGCTGGGTACC]; lacZl / SRMgb2).
  • Integrant strains (S. typhimurium ATCC14028 derivatives with integration of pSRM-phoN) are incubated on FeMed with Tet at 30 ° C and at 37 ° C and then plated on FeMed with or without Tet, using different dilutions at both temperatures, in order to Quantify frequency of excision.
  • the quotient of the number of colonies on FeMed without Tet and that on FeMed with Tet indicates in how many cells in a colony the plasmid is free compared to cells with genomically integrated plasmid. The quotient is a measure of the excision rate. Depending on the starting colony, excision rates of 3.2 to 1 7, 1 result.
  • Excised plasmid is again detected in a PCR using the primers SRMgb2 and SRMgb4 or the primers lacZl and SRMgb2. There are large amounts of PCR product.
  • the insertion and excision of pSRM4- / ira ⁇ 7 / 77et7f via homologous recombination is an equilibrium reaction (see Fig. 10).
  • the equilibrium is shifted to the side of the freely replicating plasmid in the cell; Both at 30 ° C and at 37 ° C free and integrated plasmid can be detected via PCR.
  • the respective proportions are shifted according to the temperature sensitivity of the plasmid.
  • a mutant that contains pSRM4-phoP in the phoP gene is stable and attenuated in a cell culture system.
  • the intracellular multiplication of this mutant in murine J774A.1 macrophages is compared with the multiplication of pSRM4-p /? ON, with the wild type S. typhimurium ATCC14028, and with stable phoP and phoN mutants.
  • 4x10 5 macrophages of the cell line J774A.1 are infected with 8x1 0 6 bacteria from a clone at 37 ° C., 5% CO 2 . After 30 min, the extracellular bacteria are removed by washing with PBS and by adding 10 pg / ml gentamicin to the medium.
  • the macrophages are lysed with 0.5% sodium deoxycholate and suitable dilution levels of the cell lysate are plated on FeMed. After incubation, the colony numbers are determined and the number of bacteria per ml culture is calculated. While the wild-type strain lasts for a period of 7 to 24 hours significantly increased in the macrophages, a stable phoP mutant is strongly attenuated. A stable phoN mutant and a pSRM4-p / 7 ⁇ / V insertion mutant behave like the wild type. A pSRM4-p /? ⁇ P insertion mutant behaves like the stable phoP mutant. The propagation rates are summarized in the table below
  • pSRM4 is suitable for the construction of SRM rPCR fragment banks.
  • the rPCR (random PCR) fragment banks are produced in the following way: 1.) Amplification of randomly generated - 500 bp fragments
  • SRM fragment banks are used to construct SRM mutant banks.
  • SRM fragment banks that were cloned in E.coli EC101 are transformed into S. typhimurium ATCC 14028.
  • the transformants are selected at 30 ° C. on FeMed with Tet.
  • the colonies obtained are washed away from the plates in FIMed with Tet.
  • the bacterial suspension is incubated for 3 hours at 37 ° C., and then plated on FeMed with Tet.
  • thousands of genomic insertion mutants are obtained, which in their entirety represent the SRM mutant bank.
  • To reduce the redundancy of the SRM mutant bank several independent designs of the SRM fragment bank are assumed, from which independent mutant banks are generated.
  • 96 SRM mutants are pooled in a pool. These 96 mutants of an SRM pool can also be preserved as individual clones by freezing.
  • SRM mutant banks are selected negatively.
  • the S. typhimurium SRM mutant library is selected in J774A.1 macrophages or in BALB / c mice (other cell lines and other mouse strains can also be used). 1 pool (96 mutants) is selected. After the selection, the bacteria are first cultivated on FeMed with Tet at 37 ° C. This step only multiplies those clones that contain genomically integrated pSRM plasmid.
  • the integrated SRM plasmids are excised from the genome by incubation in FIMed with Tet at 30 ° C. Plasmids obtained from a selected pool of SRM mutants are called "drivers”. Plasmids that come from the identical but not selected pool result in the "tester”. The plasmid DNA is for testers and drivers obtained from the bacteria by plasmid preparation.
  • DNA fragments cloned in pSRM4 can be identified by genetic subtraction after amplification of the tester and driver fragment banks via PCR.
  • the SRM fragments of the selected clones from a pool of 96 are amplified for the tester group by PCR with the primer pair SRMgb2 / SRMgb4.
  • these fragments are amplified with the same primers, the primers for amplifying the driver DNA on
  • Edge sequences of the tester DNA fragments can be eliminated by a subsequent restriction with Kpn ⁇ , so that
  • biotinylated driver DNA and tester DNA are mixed in a ratio of 10: 1 to 1000: 1.
  • the DNA of these batches is precipitated, dissolved in 1 0 FI hybridization buffer (0.05 M HEPES pH7.5, 1 mM EDTA, different NaCl concentration), first incubated for 3 minutes at 95 ° C, followed by an 18-hour incubation at 65 ° C.
  • the biotinylated homo- and heterodimers formed during this hybridization are extracted from the mixture via streptavidin-coupled magnetic particles.
  • the subtraction is repeated two to three times.
  • SRM fragments For the isolation and subsequent identification of the enriched tester DNA fragments, these are cloned into the pSRM4 vector via the p / 71 interface. Identification of SRM fragments by Southern (Dot) blot. As an alternative to genetic subtraction, a blotting procedure can be used to identify SRM fragments of essential genes. For this purpose, the SRM fragments from 96 SRM mutants each with free pSRM plasmids are amplified via PCR (primer SRMgb2 / SRMgb4) and fixed individually on a membrane filter. (1) PCR-amplified SRM fragments from the driver and (2) PCR-amplified SRM fragments from the tester are used as the probe for hybridization. DNA fragments which give a signal with the probe from the driver but not with the probe from the tester are the fragments of essential genes sought.
  • the SRM method can be used in all organisms in which pSRM4 shows temperature-sensitive replication.
  • the SRM method can be adapted for other organisms such as, for example, Helicobacter spp., Campylobacter spp., Or others, by constructing SRM vectors for these organisms.
  • SRM vectors for Helicobacter spp.
  • a plasmid is found in Helicobacter spp. replicated, e.g. pHel (Heuermann and Haas, 1 995; Heuermann and Haas, 1 998).
  • the plasmid replicates according to the "rolling circle” mechanism, and the RepA proteins of both proteins are homologous (del Solar, et al., 1 993; Kleanthous, et al., 1 991).
  • the plasmid is used according to the method described by Maguin et al. described method (Maguin, et al., 1 992) produced a temperature-sensitive variant.
  • the plasmid is mutagenized in vitro with hydroxylamine according to standard methods and transformed into H. pylori at a permissive temperature.
  • only the repA gene is mutagenized and the mutagenized DNA is then cloned into a rep-free plasmid.
  • Replica plating of H. pylori clones which contain mutagenized plasmid or repA and selection of the clones by means of a plasmid-encoded antibiotic resistance identify variants of the plasmid or repA gene which have a Allow replication at 30 ° C, but not at 37 ° C.
  • the mutation (s) that mediated temperature sensitivity are identified by sequencing the plasmid (or the repA gene).
  • the temperature sensitivity of the plasmid is further characterized as described above for pSRM4.
  • the plasmid is subsequently used as pSRM4 as a vector for the subtractive recombination mutagenesis in Helicobacter spp. used.
  • the CAI method can be carried out with different vectors which can replicate and be selected in the organism to be examined.
  • PCAl is used as the vector.
  • pCAl contains the replication and resistance functions of pWSK29 (Wang and Kushner, 1 991), or of pBluescript, or of pUC, or of other plasmids.
  • the respective vector contains an adjustable CAI promoter and its ice and -1 ra / 7S regulatory components. The transcription from this promoter is controlled by exogenous factors (inducers).
  • the regulated CAI promoter is the tet promoter, which is activated by the tef operator and the Tet repressor only in the presence of tetracycline in the growth medium (Hillen and Berens, 1 994).
  • the ara promoter is used, which is controlled via the ara operator and AraC, an activator protein, and is only activated in the presence of arabinose in the growth medium (Guzman, et al., 1 995).
  • any other promoter that can be regulated by exogenous signals can also be used.
  • RNA stabilizing elements (RSE) can be inserted (Carrier and Keasling, 1 997a; Carrier and Keasling, 1 997b; Carrier and Keasling, 1 999).
  • RSE RNA stabilizing elements
  • the RSE are cloned in such a way that after the insertion of CAI fragments at the 5 'and / or at the 3' end of the transcribed RNA fusions with the RSE result.
  • a Kpn ⁇ and other restriction sites (mcs) are inserted between the promoter and the 5'-RSE sequence on the one hand and the 3'-RSE sequence on the other hand, which are later used for the insertion of CAI fragments.
  • a bank with genomic fragments of the organism to be examined is constructed in the CAI vector. For this purpose, cloned into the mcs genomic DNA fragments, which were prepared according to the rPCR method described in Example 1.
  • the CAI gene bank obtained in this way is transformed into the organism to be examined and individual clones are selected using the plasmid-coded resistance marker,
  • the CAI method can be used with various selection and / or screening methods, which are described below.
  • Fragment banks of (1) the aroA gene, (2) the phoN gene, and (3) the phoP gene were constructed in pCAl. To construct these fragment banks, the genomic regions of these genes, including neighboring ones, are made
  • the PCR products thus obtained are used as templates for the generation of rPCR fragments according to the method described above.
  • the rPCR fragments are made without fractionation of different lengths of DNA
  • the clones of the aroA fragment bank are plated on plates with solid medium (M9 minimal medium with casaminoacids [CAA, amino acid mixture] without inductor).
  • the function of the aroA gene is necessary so that the bacteria can multiply on M9 without CAA.
  • the colonies obtained are transferred by replica plating to M9 with CAA and to M9 without CAA, each with an inductor.
  • the clones of the phoN fragment bank are transferred by replica plating to M9 medium with BCiP and with inductor, and to M9 with BCiP without inductor.
  • the activity of PhoN converts BCiP to a blue dye.
  • Clones in which the translation of the phoN gene is inhibited by asRNA remain whitish on M9 medium with BCiP with inducer, while they turn blue on M9 medium with BCiP without inducer.
  • the function of the phoP gene is necessary so that the bacteria can multiply in macrophages.
  • the clones of the p / .oP fragment bank are combined in a pool.
  • the pool is divided into two identical groups, which are passaged in J774A.1 macrophages.
  • the inducer becomes the culture medium during the infection Macrophages added.
  • the second group is passaged in J774A.1 macrophages without added inductor. (For a description of the infection experiment, see Example 1.)
  • the pCAl plasmids are prepared from both batches.
  • the CAI procedure is then used with a complete genomic fragment library in S. typhimurium.
  • the clones with the CAI fragment bank are tested according to the three methods (1-3) described above.
  • the H. pylori wild-type strain 69A serves as the starting strain.
  • the bacteria are grown on serum plates (see Westblom et al., 1 991) at a temperature of 37 ° C. in an atmosphere of 5% O 2 , 10% CO 2 , 85% N 2 .
  • the chromosomal DNA is isolated by the method of Leying et al. (1 992), the DNA finally being purified using a cesium chloride gradient.
  • chromosomal DNA 50 pg of the purified, chromosomal DNA is partially cleaved with the restriction endonucleases Sau3A and Hpall, the DNA fragments separated in an agarose gel and the fragments in a size of 3 to 6 kbp eluted from the gel using the Geneclean II kit (Bio1 01) .
  • the isolated DNA fragments are cloned into the BglII and Clal-cut pMin2 vector (Kahrs et al., 1 995) and transformed by electroporation into the E. coli strain E1 81, previously the Plasmid pTnMax9 was transferred. In total, approximately 4000 clones are generated using such an approach.
  • coli strain E1 81 is a derivative of the HB101 strain (Boyer and Roulland-Dussoix, 1 969) and contains the lysogenic ⁇ phage ⁇ CH61 6 for replication of the pTnMax9 plasmid.
  • Cysteine HC1 (C 3 H, NO.S x HCl x HO) 26 g Cocarboxylase 100 mg in 50 ml
  • L-Arginine HC1 150 mg (sterile filter, fill 13 mg portions in 10 ml p-aminobenzoic acid + freeze)
  • the transposon TnMaxS located on pTn axS is equipped with the genetic marker? -Lactamase. This marker is placed on the transposon in such a way that it enables the selection of successful transposon insertions if this is in the correct reading frame of genes whose products are secreted or exported by E. co // strain E145 nf .
  • the insertion of the transposon into such a gene leads to a gene fusion between the target gene and the marker, whereby an im
  • the fusion protein is ejected from the cell and the activity of the integral reporter gene is unfolded.
  • the clones can be directly via the
  • the gene bank transposon mutagenesis is carried out in pools of up to 20 individual clones.
  • the respective pools are plated on LB plates which are mixed with 100 pM IPTG, 15 pg / ml chloramphenicol and 15 pg / ml tetracycline.
  • the mutagenized pMin2 plasmids transferred via conjugation into the E. coli strain E1 45 r ⁇ f , since these are equipped with a corresponding mob signal (oril).
  • the pTn / WaxS plasmids are not transferred.
  • a specific duplication of the mutagenized pMin2 gene bank occurs in E. coli E 1 45 nf .
  • transconjugants are on LB medium with 1 5pg / ml chloramphenicol, 1 5pg / ml tetracycline and 100 pg / ml rifampicin selected.
  • a total of 500-1000 transconjugants are combined in 2 ml LB medium and grown in appropriate dilutions (10 ' 1-10 ⁇ 2 ) on LB plates which are mixed with 50 pg / ml ampicillin. After culturing the plates for 36 hours at 37 ° C., 200-300 ampicillin-resistant clones with transposon-inserted plasmids are obtained in the entire batch.
  • the TnMax3 mutated H. pylori genes coding for secreted / excreted polypeptides into an H. pylori wild-type strain
  • gene-specific mutants can be generated. Due to the cloned H. pylori gene sequences on the plasmids, in the event of a double homologous recombination event, the TnMaxS transposon is genomically inserted into the chromosomal target gene and thus inactivated. This process can be selected using the genetic marker on the transposon, since the pMin2 plasmid is not replicated in H. pylori.
  • the H. pylori wild-type strain 69A is transformed in individual batches with the individual plasmids obtained, taking advantage of the bacteria's natural competence to take up DNA (Haas et al., 1 993).
  • the bacteria are taken up in BHI medium and grown to an optical density at 550 nm of 0.1 at 37 ° C. under microaerophilic conditions.
  • the individual culture batches are each mixed with 100 to 500 ng of purified plasmid DNA and the culture is continued overnight. Characterization of the biological function of the gene-deficient H. pylori mutants in the growth test:
  • the individual batches are plated on serum plates which are mixed with 4pg / ml chloramphenicol.
  • three categories can be distinguished: (1) mutants that do not grow; (2) mutants that form smaller colonies; (3) mutants that develop normal colony size. These results can be reproducibly achieved for each starting plasmid.
  • the CAI method is used to clearly assess the biological importance of the genetically deficient mutants of category 1.
  • the gene-deficient mutants of category 2 and 3 are analyzed in the other biological test systems.
  • the respective starting plasmids from the E. coli strain are used to determine the primary structure of the identified H. pylori genes.
  • Plasmids are isolated from these strains and the nucleotide sequence of the
  • Target genes by sequencing the areas above and below the
  • Target gene can be determined directly since the transposon-encoded SS lactamase gene actively fuses with the gene product of the target gene
  • the Genebank database of the GCG program is used, for example for the identification of known, homologous genes of other microorganisms (FASTA), for identification potential signal peptide regions (SPSCAN) or for the identification of lipoproteins (MOTIFS).
  • FASTA homologous genes of other microorganisms
  • SPSCAN identification potential signal peptide regions
  • MOTIFS lipoproteins
  • the full gene sequence could not be determined for some of the identified clones, since the cloned DNA fragment did not contain the entire gene.
  • the available DNA fragment is used to isolate a DNA fragment with a complete gene from the original gene bank.
  • the missing gene sequences can then be amplified from these clones using a gene-specific primer and a vector-specific primer and then sequenced directly.
  • chromosomal DNA is isolated from the H. pylori strain 69A, 25-35 pg of which is partially digested with Bsp143l (Sau3AI-isoschizomer) and fragments of a size of 2 to 8 kbp are isolated.
  • the vector pACYCI 84 is cut with BamHI, dephosphorylated with shrimp alkaline phosphatase and then ligated with the isolated fragments. After transformation into E. coli strain 0466 and selection for 30 pg / ml chloramphenicol, 5560 individual colonies are isolated and analyzed using the polymerase chain reaction.
  • Primers for a product size of less than or equal to 700 bp are derived from the known gene fragments as closely as possible adjacent to the missing gene piece. These primers are used to produce amplification products which contain a clone which comprises both primer binding sites. The plasmids are then isolated from the positive clones and are verified either by sequencing or by dot blot analysis. Sequencing
  • the technique of primer walking is used to complete the DNA sequence of genes, which cannot be determined completely after the sequence reactions with the universal primers M 1 3F and M 1 3RP1.
  • an oligonucleotide binding approximately 50 to 200 bp upstream of the 3 'end of the known sequence section is derived and used as a primer for a further sequence reaction according to the dye terminator principle (Amersham Pharmacia Biotech).
  • the dye terminator principle Amersham Pharmacia Biotech.
  • Mu052a 5'-AGGCTAAAGACGTGTTAG-3 '
  • Mu052b 5'-CTAGCGTGGAATTAGCC-3'
  • the gene sequences are heterologously expressed in attenuated Salmonella vaccine strains.
  • the polymerase promoter system of the bacteriophage T7 (Tabor & Richardson 1 985) is used for this.
  • the gene to be expressed is amplified by polymerase chain reaction (PCR) with specific oligonucleotide primers from chromosomal DNA.
  • PCR polymerase chain reaction
  • the selection of the gene-specific oligonucleotide primers depends on the presence of a signal peptide for the sea-dependent translocation of the gene product into the periplasm. If there is one, the oligonucleotide primer pair is chosen to amplify a fragment of the gene to be expressed which encodes the mature protein without signal peptide. If there is no signal sequence, the entire open reading frame is amplified.
  • the amplified gene fragments are inserted into a plasmid vector derived from pBR322.
  • the insertion takes place in such a way that the cloned
  • RGSHis 4 coding sequence section under the transcriptional control of the T7 bacteriophage promoter The expression of the gene products is investigated in the Salmonella typhimurium vaccine strain SL3261 :: pYZ84 (Yan & Meyer 1 996), which is transformed with the expression vectors prepared in the manner described above.
  • the RGSHis 4 fusion proteins are detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then Western blot analysis with a monoclonal antibody (RGS.His) specific for the RGSHis 4 epitope. Expression cloning is used as an example using the gene sequence H PC001 with subsequent Western blot analysis.
  • the gene fragment of the hpcOOl gene which codes for the mature HPC001 protein (without signal peptide), using the oligonucleotide primers LAT50 (5'-GATCAGATCTACATATGTTTATCATTCCCTCTCGC-3 ') and LAT51 (5'- GATCGGTACCAAAACTCT) amplified by means of PCR.
  • LAT50 5'-GATCAGATCTACATATGTTTATCATTCCCTCTCTCGC-3 '
  • LAT51 5'- GATCGGTACCAAAACTCT
  • the 0.9 kb amplicon is hydrolyzed using Bgl ⁇ and Acc65 ⁇ and inserted into the vector pLAT289 hydrolyzed with Bam ⁇ ⁇ ⁇ and Acc65 ⁇ .
  • the resulting expression vector pMSC34 codes for a fusion protein which consists of the hpcOOl gene which lacks the signal sequence of HPC001 and the RGSHis epitope.
  • the expression product has a molecular mass of 34 kDa.
  • the experimental conversion of the spiral Helicobacter form into a coccoid form can be done in different ways.
  • the conversion can be observed under stress conditions, for example in the presence of sublethal doses of an effective antibiotic, in the absence of a substrate or in a normal air atmosphere.
  • the conversion process can thus be understood as a protective reaction of the bacteria.
  • these shapes can also be found in tissue watch infected people. It may be a form of persistence. Loss or the delayed or incomplete expression of this property, for example by dropping or inactivating a gene or gene product, can have fatal disadvantages for the bacterium in the struggle for survival.
  • the experimental conversion of the spiral Helicobacter into a coccoid form is usually started from a fresh plate culture that was grown under ideal conditions (serum plates, 37 ° C, microaerophilic atmosphere (5% O 2 , 10% CO 2 , 85% N 2 )) and therefore mainly contains spiral Helicobacter.
  • the harvested bacteria are transferred to a minimal medium that consists of water, eg sterilized tap water, and x% (v / v) of a complex protein solution, eg fetal calf serum (FCS).
  • FCS fetal calf serum
  • the bacteria are cultivated in a defined number, for example with an OD 550 of 0.2, in 5 ml of minimal medium at 37 ° C. in a cell culture cabinet, the CO 2 content in the air being adjusted to 10%.
  • the progressive development process is documented microscopically and through growth determinations.
  • Microscopically, in phase contrast, the morphology (coccoid _ spiral) and the mobility (coccoid + immobile; spiral + movable; spiral + immobile) of the bacteria in the culture batch is determined in part, e.g. B. in a counting chamber.
  • specific staining is carried out in order to indirectly detect structural changes, for example in the bacterial shell, or to determine the proportion of dead germs. Since cocoidal forms do not grow on conventional media, for example serum plates, the proportion of vital or still reactivable state forms (colony forming units; CFU) can be determined at different times. Reactivation of coconut forms.
  • the coccoid bacteria are activated in a medium which is described in EP 0900839.
  • the bacteria are in a defined number, e.g. 500 - 1,000 bacteria, plated on this medium and incubated under microaerophilic conditions at 37 ° C for several days. In this approach too, the development process is documented microscopically (see above) and by determining the CFU.
  • the coccoid forms produced are administered in a defined number, for example 10 8 bacteria, to a mouse, for example Balb / c, orally in an aqueous solution, for example using a gastric tube.
  • a certain time for example after 2-4 weeks, the infected animals are sacrificed, the stomach is removed, the contents are removed and histologically and by growth tests (see above) are examined for colonization.
  • the proportion of coccoid and spiral bacteria can be determined in the histological examination.
  • the experiment should preferably be carried out with Type I Helicobacter strains because the Type II strains are less virulent.
  • the described biological systems can be used, for example, for the analysis of defined Helicobacter mutants which were produced using the described methods. The investigation is carried out in each case in comparison to the respective wild-type strain. If, for example, a mutant is not able to convert to the coccoid form, or if the coccoid form does not reactivate or if the conversions occur with great delays, one can do so for the mutated gene optional essential function can be derived. When reactivating coccoid forms in animal models, several mutants can be used at the same time per test batch, for example 10 strains. The prerequisite is that the recognition of these strains in the tissue is ensured by suitable methods, for example by appropriate PCR methods or by in situ hybridization.
  • Tn ⁇ tad a derivative of transposon Tn5 that generates conditional mutations.
  • mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class ll-restricted responses. J. Exp. Med. 1 88 (2): 2277-2288.
  • McPC603scFvDhlx in cell-wall-less L-form strains-of Proteus mirabilis and Escherichia coli a comparison of the synthesis capacitites of L-form strains with an E. coli producer strain. Appl. Microbiol. Biotechnol. 49: 51-58.
  • Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 65: 3672-3679.

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  • Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne un procédé servant à préparer des agents thérapeutiques, préventifs ou diagnostiques contre des infections microbiennes et à identifier et caractériser des gènes essentiels à partir de Helicobacter pylori. L'invention concerne également les acides nucléiques identifiés codant pour les produits géniques essentiels, ainsi que les polypeptides codés à partir d'eux.
EP00938725A 1999-05-31 2000-05-31 Genes et produits geniques essentiels pour l'identification, le developpement et l'optimisation de principes actifs immunologiques et pharmacologiques pour le traitement d'infections microbiennes Withdrawn EP1259639A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE19924965A DE19924965A1 (de) 1999-05-31 1999-05-31 Essentielle Gene und Genprodukte zur Identifizierung, Entwicklung und Optimierung von immunologischen und pharmakologischen Wirkstoffen zur Behandlung mikrobieller Infektionen
DE19924965 1999-05-31
DE19927740A DE19927740A1 (de) 1999-06-17 1999-06-17 Essentielle Gene und Genprodukte zur Identifizierung, Entwicklung und Optimierung von immunologischen und pharmakologischen Wirkstoffen zur Behandlung mikrobieller Infektionen
DE19927740 1999-06-17
DE19934029A DE19934029A1 (de) 1999-07-21 1999-07-21 Essentielle Gene und Genprodukte zur Identifizierung, Entwicklung und Optimierung von immunologischen und pharmakologischen Wirkstoffen zur Behandlung mikrobieller Infektionen
DE19934029 1999-07-21
PCT/EP2000/005024 WO2000073502A2 (fr) 1999-05-31 2000-05-31 Genes et produits geniques essentiels pour l'identification, le developpement et l'optimisation de principes actifs immunologiques et pharmacologiques pour le traitement d'infections microbiennes

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WO2002083940A2 (fr) * 2001-04-06 2002-10-24 Creatogen Aktiengesellschaft Methode de criblage pour cibles medicamenteuses antimicrobiennes par mutagenese a saturation du genome
ATE345386T1 (de) * 2001-05-17 2006-12-15 Creatogen Ag Methode zum auffinden von abgeschwächten oder virulent-defekten mikroben

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EP0174096A3 (fr) * 1984-08-02 1987-11-04 Biotechnica International, Inc. Vecteurs pour l'introduction d'ADN dans les chromosomes d'une bactérie ou pour la suppression d'ADN de celle-ci et pour la production de protéine
US5639595A (en) * 1990-05-01 1997-06-17 Isis Phamaceuticals, Inc. Identification of novel drugs and reagents
US5217889A (en) * 1990-10-19 1993-06-08 Roninson Igor B Methods and applications for efficient genetic suppressor elements
US5434065A (en) * 1993-05-06 1995-07-18 President And Fellows Of Harvard College In vivo selection of microbial virulence genes
US5527678A (en) * 1994-10-21 1996-06-18 Vanderbilt University CagB and CagC genes of helicobacter pylori and related compositions
AU1055497A (en) * 1995-11-17 1997-06-11 Astra Aktiebolag Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics
US5756305A (en) * 1996-05-06 1998-05-26 Millennium Pharmaceuticals, Inc. Identification of essential survival genes
EP0837142A1 (fr) * 1996-10-15 1998-04-22 Smithkline Beecham Corporation Procédé de détermination de gènes essentiels dans des agents pathogènes
ES2281917T3 (es) * 1996-11-06 2007-10-01 Smithkline Beecham Corporation Procedimientos para identificar genes para el crecimiento de un organismo.
EP1021458A4 (fr) * 1996-11-14 2001-12-12 Merieux Oravax Polypeptides helicobacter et molecules de polynucleotides correspondantes
AU756010B2 (en) * 1997-04-01 2003-01-02 Human Genome Sciences, Inc. Identification of polynucleotides encoding novel helicobacter polypeptides in the helicobacter genome

Non-Patent Citations (1)

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Title
See references of WO0073502A2 *

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WO2000073502A2 (fr) 2000-12-07
AU5399800A (en) 2000-12-18
CA2385822A1 (fr) 2000-12-07
WO2000073502A3 (fr) 2002-10-03

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