WO2001055397A1

WO2001055397A1 - NOVEL POLYPEPTIDES VraS AND VraR

Info

Publication number: WO2001055397A1
Application number: PCT/JP2001/000510
Authority: WO
Inventors: Keiichi Hiramatsu; Makoto Kuroda; Kyoko Kuwahara
Original assignee: Keiichi Hiramatsu; Makoto Kuroda; Kyoko Kuwahara
Priority date: 2000-01-28
Filing date: 2001-01-26
Publication date: 2001-08-02
Also published as: JPWO2001055397A1

Abstract

Polypeptides represented by SEQ ID NO:3 (VraS) and SEQ ID NO:4 (VraR); polynucleotides (vraS and vraR) respectively encoding these polypeptides; antagonists of these polypeptides; and a detection method with the use of the above polynucleotides. Vancomycin-sensitivity can be restored by using the above antagonists of VraS and VraR. Thus, VraS and VraR are usable as remedies for infection with vancomycin-tolerant MRSA. By using vraR and vraS which are newly found, a vancomycin-tolerant strain can be identified.

Description

Description New polypeptides VraS and VraR Technical field

The present invention relates to novel polynucleotides and polypeptides involved in vancomycin resistance of Staphylococcus aureus, their variants, antagonists thereof, and a method for detecting vancomycin-resistant Staphylococcus aureus using the polynucleotide. Background art

Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to 3-lactam antibiotics such as penicillin, cefm, and canolevanedenem. Treatment of MRSA infection is exclusively for glycopeptides such as vancomycin Rely on antibiotics. It showed low sensitivity to glycopeptides in 1997. ”TMRSA (VRSA; vancomycin-: resistant Staphylococcus aureus or VIbA, vancomycin-intermediate taphylococcus aureus; GISA, glycopeptide- intermediate Staphylococcus aureus A similar strain was also detected in hospitals in Japan according to the present invention, and similar strains were also detected in the United States, South Korea and Europe, and MRSA is no longer a commonly available antibiotic, MRSA infections are a major problem in modern medicine.

If a gene product that causes glycopeptide resistance of Staphylococcus aureus is identified, it is possible to recover the sensitivity of vancomycin by developing an inhibitor for that protein and obtain a therapeutic effect by using it in combination with vancomycin. it can. In addition, the function of the gene product is Inhibitors alone have a therapeutic effect if they have essential functions. Therefore, an object of the present invention is to provide a gene that causes glycopeptide resistance of Staphylococcus aureus, a gene product and an inhibitor thereof, and a method for detecting glycopeptide-resistant Staphylococcus aureus using the gene. That is. Disclosure of the invention

The above object of the present invention is achieved by the following polynucleotides, polypeptides, antagonists and detection methods.

(1) The polynucleotide of SEQ ID NO: 1 or 2.

(2) A polynucleotide comprising a polynucleotide encoding any of the following polypeptides (a) to (c):

(a) A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4.

(b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4 in which one or several amino acids are deleted, substituted or added, and having the polypeptide activity of SEQ ID NO: 3 or 4 .

(c) A polypeptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence of SEQ ID NO: 3 or 4.

(3) A polynucleotide comprising a polynucleotide having 70% or more identity to the polynucleotide of (1) or (2).

(4) A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4.

(5) A polypeptide comprising any one of the following amino acid sequences (d) to (e):

(d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4 in which one or several amino acids have been deleted, substituted or added, and having the polypeptide activity of SEQ ID NO: 3 or 4 Peptide.

(e) at least 50% identical to the amino acid sequence of SEQ ID NO: 3 or 4 A polypeptide of the amino acid sequence

(6) An antagonist that inhibits the activity or expression of the polypeptide of (4) or (5).

(7) A glycopeptide-resistant Staphylococcus aureus characterized in that a reaction operation is performed using a sample and a primer or a complementary polynucleotide that reacts with the polynucleotide of any of the above (1) to (3). Detection method. BEST MODE FOR CARRYING OUT THE INVENTION

As shown below, transcription of the polynucleotide (SEQ ID NO: 2) on the chromosome of S. aureus is increased in vancomycin-resistant S. aureus cells. Furthermore, when this polynucleotide is introduced into vancomycin-sensitive Staphylococcus aureus, vancomycin resistance is developed as shown in the Examples below. Therefore, it is understood that the polypeptide of SEQ ID NO: 4 (VraR) encoded by the polynucleotide vraR is responsible for vancomycin resistance of S. aureus.

The polynucleotide (vraS) shown in SEQ ID NO: 1 is adjacent to the polynucleotide (vraR) shown in SEQ ID NO: 2 on the chromosome, and a new homology analysis using an existing database It is likely that this is a two-component control system. Therefore, the polypeptide of SEQ ID NO: 3 (VraS), encoded by the polynucleotide of SEQ ID NO: 1 (vraS), is presumed to be involved in vancomycin resistance of S. aureus through regulation of VraR transcriptional expression. Is done. Therefore, by inhibiting the action of VraS, it is possible to suppress the expression of vancomacin resistance.

In the present invention, the polypeptide is not limited to the polypeptide of SEQ ID NO: 3 or 4, and includes a polypeptide having an amino acid sequence in which one or several amino acids having this polypeptide activity are deleted, substituted or added. SEQ ID NO: 3 Or a polypeptide having at least 50%, preferably at least 70%, more preferably at least 90% identical to the amino acid sequence of 4, and having the polypeptide activity of SEQ ID NO: 3 or 4. Including.

The present invention has not only the polynucleotides of SEQ ID NOS: 1 and 2 but also the polypeptides of SEQ ID NOs: 3 and 4 or the polypeptides of SEQ ID NOs: 3 or 4, A polynucleotide encoding a polypeptide that is preferably at least 70%, more preferably at least 90%, or has 70% or more, preferably 90% or more identity thereto. Also includes isolated polynucleotides, including polynucleotides.

The polypeptide of the present invention can be obtained by a known method using the polynucleotide of SEQ ID NO: 1 or 2. For example, the polynucleotide of the present invention can be produced by inserting the present nucleotide downstream of the promoter on the expression vector plasmid of Escherichia coli and introducing it into Escherichia coli by a transformation method.

Another form of the invention is an inhibitor for these polypeptides, the antagonist. Antagonist can be obtained by several known methods. For example, (1) First, a plurality of recombinant plasmids expressing a polypeptide in which a site-specific amino acid mutation has been introduced into these polypeptides are prepared and introduced into a vancomycin-sensitive Staphylococcus aureus strain. By measuring vancomycin resistance of each transformant, sites essential for the activity of these polypeptides can be estimated. Next, a crystal of the purified polypeptide is prepared and its three-dimensional structure is determined by X-ray structure analysis. Then, the structure of the chemical substance that can bind to the three-dimensional structure of the part corresponding to the active site is estimated and synthesized. Alternatively, (2) reacting these purified polypeptides with candidate antagonist chemicals, for example, by measuring the quenching of fluorescence due to binding, Chemicals that bind with high affinity to the polypeptide can be obtained. Whether this substance inhibits the activity of these polypeptides depends on the degree of resistance of the transformed strain into which the vraR gene has been introduced and the degree of resistance to vancomycin has been increased. This can be confirmed by observing the decrease when a sensitivity test (for example, MIC measurement) is performed.

Since the antagonist of the present invention inhibits the expression of VraS or VraR activity, it can restore vancomycin sensitivity. Therefore, the infection with vancomycin-resistant Staphylococcus aureus can be treated by using it in combination with vancomycin.

Still another embodiment of the present invention is a genetic diagnosis of glycopeptide resistance. Using a primer or a complementary polynucleotide that reacts with the polynucleotide provided by the present invention, the presence or absence of expression of these polynucleotides in the strain, or the quantification of the expression, allows the presence or absence of glycopeptide sensitivity of the strain, It is possible to predict the degree of susceptibility and resistance, and it is highly significant to contribute to clinical antibiotic treatment. Examples of such a method include, for example, RT-PCR, which uses a part of the polynucleotide of SEQ ID NO: 1 or 2 to prepare a primer and quantifies the mRNA of these genes, and RNA. Examples include a method based on hybridization, a method based on LCR (ligase chain reaction), and a method based on NASBA ^ nucleic acid sequence-based amplification.

Hereinafter, the present invention will be described with reference to Examples.

Example 1

[vraS and vraR]

The vraS and vraR polynucleotides were obtained as follows. 20 μg of the chromosome DΝ of the heterovancomycin-resistant S. aureus Mu3 strain was digested with the restriction enzyme Hindlll, and a DNA fragment of 1.5 to 7.5 kbp was separated and purified by 1% agarose gel electrophoresis. The obtained DNA fragment was ligated to vector plasmid pKF3 and transformed into Escherichia coli TH2 strain. This Escherichia coli was cultured on an LB agar medium containing 50 mg / l streptomycin and 12.5 mg / l cupramphenicol, and a plasmid DNA was prepared by a simple alkaline * SDS method. This was used as the S. aureus Mu3 gene library. After spotting this plasmid DNA (50 ng) on a nylon membrane, the DNA was fixed by ultraviolet irradiation, and two identical ones were produced. Total RNA was prepared from heterovancomycin-resistant S. aureus Mu3 strain, vancomycin-resistant S. aureus Mu50 strain, and vancomycin-sensitive S. aureus Mu50 strain obtained from a patient who obtained Mu50 strain. A reverse transcriptase (GIBCO BR) was applied to 15 μg of the total RNA and reacted at 42 ° C. for 2 hours or more to prepare cDNA, which was used as a probe and subjected to hybridization with the above nylon membrane.

Differential hybridization was performed on 1,273 clones of the S. aureus Mu3 strain gene library. As a result, it was possible to isolate clones with apparent signal enhancement. The obtained plasmid was analyzed as pM386. Determination of the nucleotide sequence of the DNA fragment inserted into plasmid pM386 indicated the presence of six structural genes.

To examine the mRNA expression patterns of these genes, were subjected to Southern hybrida I internalization analysis, transfer enhancement of the response regulator aerator genes are only two component control systems and homology, ₀ the response observed in vancomycin-resistant bacteria The regulator was named vraRi, van corny cin-resi stance associated genes). A gene highly homologous to the sensor gene of the two-component control system exists upstream of the vraR gene. Was.

Example 2

[Creation of recombinant plasmid pVRAR including vraR]

The hidden fragment containing the open reading frame of vraR, the plasmid pM386 was used as type I DNA, and two synthetic oligonucleotides (5, -AAAAGGATCCGTCATTCAAACGGTA CAAAAG-3 ') (SEQ ID NO: 5) and 5' -TTTTTGGATCCTTAAAAAAGACTAAACACCA AC-3 ' (SEQ ID NO: 6)) was used as a primer and amplified by PCR. The amplified DNA fragment was digested with BamHl and inserted into the BamHl site of shuttle vector plasmid pYT3.

Example 3

[Introduction of pVRAR into vancomycin-sensitive Staphylococcus aureus strain N315P and MIC measurement]

Recombinant plasmid pVRAR was introduced into vancomycin-sensitive Staphylococcus aureus strain N315P by electroporation, and selection was performed in the presence of 1 Omg of tetracycline to obtain transformant N315P (pVRAR). The vancomycin and ticobranin MICs of the transformant N315P (pYT3) and parent strains N315P and N315P (pVRAR) using only the vector were measured. Table 1 shows the results. As shown in Table 1, the degree of resistance of vancomycin and ticopranin was increased only when the polynucleotide containing vraR was introduced.

MIC (mg / L)

Nokunkomycin Tikoff. Lanin

(vancomycin) (te i coplanin)

N315P 0.5 1.0

Ν31δΡ (ρΥΤ3) 0.5 1.0

N315P (pVRAR) 2.0 3.0

Industrial applicability

As described above, VraR and VraS of the present invention are products of the resistance genes vr aR and vr aS to glycopeptide antibiotics such as vancomycin. Therefore, the use of the VraS and VraR antagonists of the present invention restores vancomycin sensitivity and can be used as a therapeutic agent for vancomycin-resistant MRSA infection. In addition, vraR and vraS are newly discovered and can be used to identify vancomycin-resistant bacteria.

Claims

Claims 1. The polynucleotide of SEQ ID NO: 1 or 2.

2. A polynucleotide comprising a polynucleotide encoding any of the following polypeptides (a) to (c):

(a) A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4.

(b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4 in which one or several amino acids are deleted, substituted or added, and having the polypeptide activity of SEQ ID NO: 3 or 4 Puchido.

3. A polynucleotide comprising a polynucleotide having 70% or more identity to the polynucleotide of claim 1 or 2.

4. A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 4.

5. A polypeptide comprising any one of the following amino acid sequences (d) to (e):

(e) A polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence of SEQ ID NO: 3 or 4.

6. An antagonist that inhibits the activity or expression of the polypeptide of claim 4 or 5.

7. A method for detecting dali peptide-resistant Staphylococcus aureus, which comprises performing a reaction operation using a sample and a primer or a complementary polynucleotide that reacts with the polynucleotide of any one of claims 1 to 3. .