DE102009021681A1 - New Staphylococcus aureus-binding peptide, comprising amino acid sections, useful for the enrichment, immobilization, detection and/or marking of Staphylococcus aureus - Google Patents

Abstract

Staphylococcus aureus-binding peptide (I) comprising at least eight amino acids section including amino acid sequences of SEQ ID NOs. 1 to 13 (comprising a fully defined 12 amino acid sequence as given in the specification), is new. Independent claims are included for: (1) a method for immobilization and/or enrichment of S. aureusfrom a sample comprising binding (I) to a carrier and contacting the carrier-bound (I) with the sample; (2) a method for detecting S. aureusin a sample comprising contacting marked (I) with the sample and detecting the detectable marker of (I); (3) a kit for the enrichment of S. aureuscomprising (I) and a carrier; (4) a kit for detecting S. aureuscomprising (I); and (5) a nucleic acid with a coding section for (I).

Description

The The invention relates to peptides affine to the surface or the extracellular Staphylococcus aureus structures bind to the use of these Peptides for enrichment, immobilization, detection and / or for labeling Staphylococcus aureus as well as methods and kits for the enrichment and detection of Staphylococcus aureus. The The invention further relates to the coupling of therapeutically effective Substances to these peptides.

application areas The invention includes, in particular, medicine, veterinary hospitals, others Health and care facilities as well as other areas with high Hygiene standards, such. B. the food and pharmaceutical industries.

Staphylococcus aureus has a spherical shape Morphology, with a diameter of cells of 0.8-1.2 μm. It belongs to the Gram-positive bacteria and is unable to endospores too form. Staphylococcus aureus is ubiquitous and colonized u. a. Mucous membranes. In certain circumstances Staphylococcus aureus can be pathogenic and induces skin infections, Pneumonia, Endocarditis, toxic shock syndrome and sepsis.

To the Detection of Staphylococcus aureus are currently different, z. T. in the official collection of examination procedures according to §35 LMBG the examination methods used. It acts it is cultural, immunological or molecular biological Detection methods. It is possible, to combine these procedures.

• – Voranreicherung
• – Isolierung auf Selektivnährböden
• – Selektiv-Anreicherung in Flüssigkultur
• – Koagulase-Test
• – Biochemische Identifizierung

The conventional detection of Staphylococcus aureus, even in food, is usually carried out as follows:

• - Pre-enrichment
• - Isolation on selective nutrient media
• - Selective enrichment in liquid culture
• - coagulase test
• - Biochemical identification

Also the MPN is an established method.

To becomes the first result after isolation of the germs on selective nutrient media an examination period of at least 4 to 6 days.

Next the classical tests also include immunological detection methods, such as immunofluorescence tests or ELISA used.

For the molecular biological Detection of Staphylococcus aureus are various methods Available. He can, for example, by means of gene probes in the course of fluorescence in situ hybridization or with the polymerase chain reaction.

The The above-mentioned modern method has the disadvantage that the sensitivity is not is sufficiently high, d. H. the germs still need to be one Cell count greater than the necessary detection limit be enriched.

With Help of superparamagnetic, with appropriate ligands, eg. Antibodies, coated particles is a concentration of the germs possible.

For the proof and the characterization of microorganisms and their permanent forms (Spores) are to be detected as target molecules next to those of the cells excreted metabolites and other cell components or molecules also used the cells themselves. For this, the cells are after the prior art with specific immunoglobulins (antibodies), but also other binding molecules (eg lectins). These molecules What they all have in common is that they recognize and recognize special cell structures tie. Here is the selectivity of the detection method very much of those used for labeling molecules dependent, why z. B. the use of lectins in systems with a high diversity the material composition is unsuitable.

The use of antibodies for detection methods also has some disadvantages. First, have to spe zifische antibodies are produced. This is done first by immunizing animals with the target antigen or a component thereof. However, this does not ensure that the desired immune response is achieved. If an immune response has taken place and the serum has a sufficiently high titre of specific antibodies to the target antigen after some time, B lymphocytes and antibodies are isolated from the blood of the animal and purified by known methods. These antibodies are called polyclonal antibodies regardless of their purity. They respond to all antigens to which the animal had contact until the time of isolation and to which an immune response has occurred. Unwanted cross-reactions with foreign antigens that are not necessarily related to the target antigens are therefore frequently observed and represent a significant disadvantage. Although the antibodies can be enriched in further working steps for a particular antigen, this is extremely time-consuming and involves high costs. Another disadvantage of polyclonal antibodies lies in the limited lifespan of B lymphocytes. These can not be cultured and die off after a period of time. Therefore, there are always undesirable quality fluctuations between different batches of polyclonal antibodies.

Of Furthermore, it is possible using appropriate fusion techniques individual antibody producing B lymphocytes with a tumor cell line to hybridoma cell lines too merge. The resulting hybridoma cells can be transformed into be cultured in vitro. Thus, monoclonal antibodies can be in Win cell cultures. However, the hybridoma techniques are tedious and expensive. The development of new monoclonal antibodies takes place in several steps: immunization, fusion, selection, screening and characterization. There may be further modifications, which improve the properties of the monoclonal antibodies. In addition is not in all cases without problems, antibody to win against the target antigen, especially if it is is about whole, intact cells. Partly it also dies the hybridoma cell line. This represents another disadvantage since corresponding coupling protocols for detection procedures time consuming new must be established.

task The invention is simple, sensitive, specific and possible fast feasible Method for enrichment, immobilization, labeling and / or to indicate the presence of Staphylococcus aureus not on the use of antibodies based. It is another object of the invention to provide the corresponding Specify detection reagents and kits to perform these procedures.

According to the invention Task solved by one or more Staphylococcus aureus binding peptides, the or the one at least twelve amino acids long section of an amino acid sequence according to a the sequences SEQ ID NO. 1 to SEQ ID NO. 13 contains.

Preferably is the amino acid sequence of the peptide one of the sequences SEQ ID NO. 1 to SEQ ID NO. 13th

The peptides according to the invention bind surprisingly affinely and specifically to the surface or on Extracellular structures of Staphylococcus aureus and are therefore suitable to be used in enrichment or detection methods for the enrichment and / or detection of Staphylococcus aureus.

The complex Production and use of antibodies in appropriate detection methods can be avoided in an advantageous manner. The peptides of the invention can also be used with conventional Produce synthesis process easily and in very high purity. Therefore, a consistent, batch-independent product quality can be achieved especially with regard to purity and stability. The handling to be used in immobilization and / or detection methods Reagents are also simplified, allowing total immobilization and / or the detection of Staphylococcus aureus faster, with higher reproducibility and with less total effort. It's also easier Miniaturize detection and / or immobilization and to automate, in which instead of antibodies capture peptides are used.

Prefers are those peptides which are at least 10 amino acids, preferred 12 amino acids, long section of an amino acid sequence according to SEQ ID NO. 2 and / or SEQ ID NO. 4 and / or SEQ ID NO. 8 include. These Peptides have been found to be particularly suitable for binding to Staphylococcus aureus exposed. You can also in complex samples specific and with high yield of Staphylococcus tie aureus.

Especially Peptides of the amino acid sequence SEQ ID NO. 2 and SEQ ID NO. 4 and SEQ ID NO. 8 These peptides are particularly specific for Staphylococcus aureus and have a high affinity for this microorganism. They are therefore particularly suitable for Staphylococcus aureus to bind in samples.

The peptides according to the invention have an affinity for Staphylococcus aureus. They are therefore good for use as probes for Staphylococcus aureus can be used. For this they are advantageously with a provided easily detectable markers. Suitable markers (such as eg Biotin, fluorescent dyes or radionucleotides) are the Skilled in numerous applications, for example from ELISA, known.

component The invention therefore also relates to a peptide according to the invention which is a detectable marker contains.

By the coupling to the detectable marker are the peptides of the invention advantageous for specific labeling and specific detection of Staphylococcus aureus

at the detectable marker is, for example, particulate marker (eg, gold particles), fluorochromes, isotopes, marker proteins, such as z. For example, fluorescent proteins (eg green fluorescent protein (GFP)), Amplifiers, e.g. B. enzymes that catalyze a reaction, which a signal in the form of a visible dye precipitate, a visible one Dye cleavage or chemo- or bioluminescence provides.

The Various detectable markers are determined by known methods to the peptide of the invention coupled. Be, for example, GFP or enzymatically active amplifiers used as detectable markers, the coupling of the peptide takes place to the marker, suitably by fusion of the DNA sequence coding for the peptide with the DNA sequence of GFP or the enzymatically active Amplifikators by conventional genetic engineering process.

It is possible, too, the marker only after binding to Staphylococcus aureus on the peptide to install. This can be done, for example, by the intermediary of a linker like biotin-streptavidin or biotin-avidin done by the peptide z. B. biotinylated is. After binding of the peptide to Staphylococcus aureus Streptavidin and the biotinylated catalytic moiety added. Streptavidin then mediates the binding of the catalytic acting entity to the peptide.

In a preferred embodiment According to the invention, at least one peptide according to the invention is bound to a carrier.

at the carrier it is preferably a solid phase, in particular the solid used in known enrichment or detection methods carrier be used, for. B. microtiter plates, biochips or dip sticks.

When solid carriers suitable are metal body, For example, gold foils and gold-coated supports such as z. In surface plasmon resonance spectroscopy (Surface Plasmon Resonance, SPR) systems. Furthermore, plastic body or plastic-coated body as solid carriers used, for example. Polystyrene or polymethyl methacrylate carrier. Besides can also glass carrier, Gels or other carriers suitable for the production of biochips become.

By doing the peptide of the invention to a solid support is bound, it is possible in an advantageously simple manner, Staphylococcus aureus from samples to immobilize, gain and / or z. B. for the to enrich subsequent detection or Staphylococcus aureus too detect.

The peptide of the invention is solidified by various methods known to those skilled in the art carrier bound, for example by adsorption to the solid support. One Coated carrier also causes the bond of the peptide, e.g. For example, a streptavidin-coated carrier binds the peptide after its biotinylation. Other possible coatings for bonding a peptide are z. B. serum albumin or cystamine dihydrochloride optionally in combination with N-succinimidyl-3-maleimidopropionate. The peptide can also work with a nucleic acid linker be connected and bound in this form to the carrier. The peptide may be further connected to an oligonucleotide having a on the solid support bound complementary Oligonucleotide mediates the binding of the peptide to the carrier.

Especially the immobilization of the peptides according to the invention is advantageous to a mobile solid phase as a carrier, such. B. in the biomagnetic Separation. In this case, the peptides by means of covalent bonding for example about Amino or carboxyl groups on superparamagnetic micro- or Coupled nanoparticles (beads). The beads can be applied by applying an external magnetic field easily with the over the peptides of the invention immobilized Staphylococcus aureus cells from solutions and Separate aerosols. In addition, the beads have a high mass-specific surface, so that a high proportion of the sample volume comes into contact with the carrier. The materials used are inexpensive and handling is In a further embodiment, the peptide according to the invention is linked to a pharmaceutically active agent. This one can too be included, for. In microcapsules or hydrogels.

The The invention further relates to the use of these in pharmaceutical effective agents coupled peptides for therapeutic purposes.

The Invention also includes methods for enrichment, immobilization, for the detection and / or labeling of Staphylococcus aureus The execution This process is preferably carried out in solid and liquid material systems for the diagnosis and analysis of Staphylococcus aureus

• a) Bindung mindestens eines erfindungsgemäßen Peptides an einen Träger und
• b) Kontaktieren des trägergebundenen Peptides mit der zu untersuchenden Probe, um ein Binden des Peptides an Staphylococcus aureus zu ermöglichen.

A preferred method for the enrichment or immobilization of Staphylococcus aureus from a sample comprises the following steps:

• a) binding of at least one peptide of the invention to a carrier and
• b) contacting the supported peptide with the sample to be assayed to allow binding of the peptide to Staphylococcus aureus.

The peptide of the invention binds both to the wearer as well as to be immobilized or detected Staphylococcus aureus cells. As a result, it provides an advantageously simple way a stable and specific connection between the carrier and the Staphylococcus aureus cells to be immobilized or detected ago. The immobilized Staphylococcus aureus cells can then easily be separated from the sample, for example by the carrier sedimented, centrifuged or separated by applying an external magnetic field becomes.

If the carrier a receptacle the sample itself is, for example in the form of a microtiter plate well, so can the bound Staphylococcus aureus cells by washing the vessel with a corresponding washing solution from the rest Separate sample and thus purified and enriched.

The order in which steps a) and b) are performed is not fixed. It has been found that the binding of peptides to a microorganism to be immobilized may be hindered if the peptide is already bound to the solid support before binding to the microorganism. In such a case, it is advantageous to carry out step b) before or simultaneously with step a), for example by contacting the peptide and the sample to be examined by mixing with one another be brought and at the same time or subsequently the carrier is brought into contact with the mixture. However, if step a) is performed prior to step b), for example by using a supported peptide of the invention as described above, the same support can be used multiple times to immobilize microorganisms from multiple samples or to immobilize microorganisms from a sample stream become. Such a procedure may be advantageous in particular if the samples to be examined have only low concentrations of the microorganism to be detected, or if the samples contain numerous accompanying substances which non-specifically bind to the solid support or otherwise interfere with the binding of the peptides to the microorganism to be detected ,

The enrichment process according to the invention allows it, even from heavily diluted Samples enough to isolate high cell numbers of Staphylococcus aureus to one safe and specific detection of Staphylococcus aureus enable.

The Peptides are preferred by methods known to those skilled in the art to a solid phase as a carrier bound. As a solid phase are preferably superparamagnetic Micro or nanoparticles (beads) used. After contact with a Probe immobilize the peptides by their specific binding ability the Staphylococcus aureus cells contained in the sample to the Carrier phase.

• a) Kontaktierung der Probe mit mindestens einem erfindungsgemäßen Peptid, das einen detektierbaren Marker enthält, um ein Binden des Peptids an die nachzuweisenden Staphylococcus aureus-Zellen zu ermöglichen
• b) Nachweis des detektierbaren Markers des Peptides.

Also part of the invention is a method for detecting Staphylococcus aureus in a sample comprising the steps

• a) contacting the sample with at least one peptide of the invention containing a detectable marker to allow binding of the peptide to the Staphylococcus aureus cells to be detected
• b) detection of the detectable marker of the peptide.

The Detection method allows a specific and sensitive detection of Staphylococcus aureus even in complex samples.

The Method for detecting the detectable marker depends the marker used.

As already in the immobilization and enrichment processes described above is the order in which steps a) and b) are performed, at the discretion of the skilled person. This can be based on the circumstances of the Individual cases can easily decide for yourself which order the best Provides detection results.

In a preferred embodiment of the detection method the Staphylococcus aureus cells present in the sample before the Detection of the detectable marker by means of the immobilization or enrichment process enriched from the sample or on a Carrier immobilized.

To takes place first the immobilization of Staphylococcus aureus cells on carrier-bound and unlabeled peptides. After a washing step for removal unbound sample components (antigens, other cells) another invention not bound to a carrier Peptide with a detectable marker added to the targets on the now also immobilized on the solid phase target cells binds and proves them in the manner described above.

On This way, the microorganism to be detected without much effort also in heavily diluted Samples or in samples with the detection strongly interfering accompanying substances reliably and with high sensitivity be detected.

Conveniently, is the detection method and / or the immobilization or enrichment process in the form of a miniaturized assay. For example, different ones each sample to be examined in a well of a microtiter plate given and there one for the respective well specifically designed detection method be subjected. In this way, large quantities of samples can be processed in parallel become.

component The invention likewise relates to the use of at least one peptide according to the invention Enrichment, immobilization, detection and / or labeling of Staphylococcus aureus.

object Finally, the invention is also test kits for specific labeling, detection, immobilization and for the enrichment of cells, in addition to the usual ingredients at least contain one or more of the peptides of the invention.

One preferred kit for accumulating Staphylococcus aureus contains at least a peptide of the invention and a carrier, wherein the peptide is preferably bound to the carrier.

With Such a kit will be readily available to a person skilled in the art for a reagent composition given to the binding of a peptide according to the invention to a solid support without much effort to enable. Thus, such a kit simplifies the implementation of an immobilization or enrichment process. Particulate solid carriers like z. As magnetic beads (preferably superparamagnetic micro or Nanoparticles) are particularly preferred because of the advantages mentioned.

One preferred kit for the detection of Staphylococcus aureus contains at least a peptide coupled to a detectable marker according to the invention.

component The invention also includes nucleic acids, the at least one for one of the peptides of the invention coding section included. Advantageously, the nucleic acids according to the invention, which enable peptides according to the invention in a simple way, for example by adding the peptides encoding nucleic acids introduced into suitable organisms (eg Escherichia coli) and the peptides are recovered recombinantly.

In particularly preferred nucleic acids is the for the peptide of the invention coding section with a for linked to a marker protein nucleic acid, so that a Fusion protein from the peptide according to the invention and the marker protein, z. As GFP and / or an enzymatically active amplifier becomes.

Preferably, the nucleic acids contain at least one of the sequences SEQ ID NO. 14 to SEQ ID NO. 26:

The nucleic acid sequence according to SEQ ID NO. 14 encodes for a peptide with the amino acid sequence SEQ ID NO. 1, the nucleic acid sequence according to SEQ ID NO. 15 for a peptide with the amino acid sequence SEQ ID NO. 2 etc.

embodiments

Reference to the following figures and embodiments, the invention will be explained in more detail, without this limit.

CFU

Kolonie bildende Einheiten (clony forming units)

M

mol/l

RT

Raumtemperatur

It uses the following abbreviations:

CFU

Colony forming units

M

minor

RT

room temperature

embodiment 1:

According to the invention, a pool of peptides for bioaffinity specific binding to Staphylococcus aureus could be determined by means of the phage display technique. For this purpose, a commercially available M13 phage library (Ph.D.-12, New England Biolabs) was used which presents random peptides with a length of 12 amino acids on the surface of the phages. The peptides are expressed together with a short spacer (Gly-Gly-Gly-Ser) at the N-terminal end of the envelope protein gpIII (type 3 library). According to the manufacturer, the library has a diversity of 2.7 × 10 ⁹ different peptide sequences. In several rounds of panning, the phages binding to Staphylococcus aureus were isolated. In order to increase the specificity also appropriate subtraction rounds were carried out.

The DNA of the specific binding phage was isolated by PCR reproduced, cleaned up and then sequenced by the company Seglab GmbH.

embodiment 2:

The chosen Peptides were synthesized (Seglab GmbH) and paramagnetic Aminobeads (Dynal) immobilized.

embodiment 3:

For the biomagnetic Separation of Staphylococcus aureus cells was initially an overnight culture of Staphylococcus aureus DSM 1104.

The cell concentration of the culture was determined by means of a counting chamber, the cell concentration in the test was adjusted to a value of 10 ³ CFU ml ^-1 by dilution with phosphate-buffered saline (0.01 M, pH 7.4).

Aliquots of 100 μl of the diluted cell suspension were incubated in a 2 ml reaction vessel with 100 μl each of the magnetic beads loaded with peptide SA02 (SEQ ID NO: 2) for 60 min at room temperature. Subsequently, the bead cell assemblies in the magnetic field (MPC ^® -S, Dynal) were separated from the liquid phase. The supernatant was removed. The bead cell pellet was washed 4 times with 500 μl PBS-Tween (0.01 M, pH 7.4, 0.05% Tween 20) and resuspended in 100 μl PBS.

to Determination of the proportion of separated cells were resuspended for each 100 μl of the Bead cell pellets streaked on PCA (Merck) plates. simultaneously 100 μl the diluted overnight culture as a reference for determining the proportion of separated cells struck.

The results of the biomagnetic separation for a series of experiments with the peptide SA02 (SEQ ID NO. 2) are in 1 shown.

1 shows the deposition rates as a function of the bead concentration (5 × 10 ⁵ to 1 × 10 ⁷ ) in the test. (n = 3)

In The experiments have shown that it is isolated Ligand peptides possible is to successfully isolate Staphylococcus aureus from the liquid phase.

embodiment 4:

to Application was the SPR analysis system of the Fraunhofer Institute for Applied Optics and Precision Engineering, Jena (IOF-SPR).

• • Reinigung der Goldoberfläche mit 65% HNO₃,
• • Funktionalisierung mit DTSSP bei 4°C über Nacht,
• • Immobilisierung von je 50 μl Protein A (2 μg/μl) bzw. Casein (0,5 μg/μl) für 1 h bei RT,
• • Blocken mit 50 μl Casein (0,5 μg/μl) für 1 h bei RT,
• • Blocken mit 50 μl 0,1 M Tris-HCl für 1 h bei RT.

Messung Parameter Gewählte Versuchsbedingungen Vorwäsche mit PBS 1 ml Probenvolumen (Peptid SA01 (SEQ ID NO. 1) 4 mg/ml) 90 μl Zwischenwäsche mit PBS 105 μl Anzahl der Durchläufe 50 Volumen eines Durchlaufs 65 μl Flussgeschwindigkeit 300 μl/min Temperatur 30°C The chips were prepared as follows:

• • cleaning the gold surface with 65% HNO ₃ ,
• Functionalization with DTSSP at 4 ° C overnight,
• Immobilization of 50 μl protein A (2 μg / μl) or casein (0.5 μg / μl) for 1 h at RT,
• Block with 50 μl of casein (0.5 μg / μl) for 1 h at RT,
• Block with 50 μl of 0.1 M Tris-HCl for 1 h at RT.

Measurement parameter Selected experimental conditions Prewash with PBS 1 ml Sample volume (peptide SA01 (SEQ ID NO. 1) 4 mg / ml) 90 μl Intermediate wash with PBS 105 μl Number of passes 50 Volume of a pass 65 μl flow rate 300 μl / min temperature 30 ° C

2 shows SPR signals depending on the ligand selected (protein A and casein) and peptide SA01 (SEQ ID NO. 1) (4 mg / ml) as analyte.

It has been demonstrated that peptide SA01 (SEQ ID NO: 1) is able to bind to Staphylococcus aureus protein A in contrast to casein ( 2 ).

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform downloaded from the DPMA.

Claims (13)

Staphylococcus aureus binding peptide containing a at least eight amino acids long section of an amino acid sequence according to a the sequences SEQ ID NO. 1 to SEQ ID NO. 13 contains. Peptide according to claim 1, characterized in that the amino acid sequence of the peptide one of the sequences SEQ ID NO. 1 to SEQ ID NO. 13 is. Peptide according to one of claims 1 or 2, characterized that it contains a detectable marker. Peptide according to one of claims 1 or 2, characterized that it is to a carrier is bound. Peptide according to claim 4, characterized in that superparamagnetic micro- or nanoparticles (beads) used as a carrier become. Peptide according to one of claims 1 or 2, characterized that it is bound to a pharmaceutically active agent. Process for immobilization and / or enrichment of Staphylococcus aureus from a sample with the following steps: a. Binding of at least one peptide according to one of claims 1 or 2 to a carrier b. Contacting the carrier-bound Peptides with the sample. Method for the detection of Staphylococcus aureus in a sample with the following steps: a. To contact at least one labeled peptide according to claim 3 with the sample b. Detecting the detectable marker of the peptide. Use of at least one peptide according to one of claims 1 to 5 for enrichment, immobilization, detection and / or for labeling Staphylococcus aureus. Kit for accumulating Staphylococcus aureus, at least including: a. A peptide according to any one of claims 1 to 5 and b. one Carrier. Kit for the detection of Staphylococcus aureus, at least containing a peptide according to claim 3. nucleic acid with a for a peptide according to any one of claims 1 to 2 coding section. nucleic acid according to claim 12, characterized in that the nucleic acid a Section with a sequence according to a the sequences SEQ ID NO. 14 to SEQ ID NO. 26 includes.