An arac / xyls family transcription factor and use thereof

Abstract

The application relates to the field of biomedical technology, and particularly relates to an AraC / XylS family transcription factor and application thereof, which is identified and named as pncR. In a post-transcriptional stage, an A-to-I RNA editing phenomenon exists in a key amino acid site of the pncR gene, thereby generating a new pncR protein variant, and an amino acid sequence of the pncR protein variant is shown as SEQ ID NO:1. By means of gene knockout or inhibition of RNA editing, the generation of the pncR protein variant can be prevented, so that the antioxidant capacity of a carbapenem-resistant Klebsiella pneumoniae is reduced. Therefore, the pncR and related genes can be used as a target of a drug for resisting the carbapenem-resistant Klebsiella pneumoniae.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to an AraC / XylS family transcription factor and application thereof. BACKGROUND

[0002] Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a multi-drug resistant bacterium belonging to Klebsiella pneumoniae. It usually occurs in hospitals and long-term care facilities and is a serious nosocomial pathogen.

[0003] CRKP is resistant to multiple antimicrobial drugs, including carbapenem antibiotics, and has high pathogenicity. Therefore, the treatment options for CRKP infection are limited and the efficacy is poor, leading to high mortality. The incidence of CRKP has been increasing in recent years, causing a huge public health burden worldwide.

[0004] Those skilled in the art hope to develop new antibacterial strategies or drugs to reduce the pathogenicity and / or drug resistance of CRKP, thereby improving the therapeutic effect. SUMMARY

[0005] The present application aims to provide an AraC / XylS family transcription factor and application thereof. The transcription factor is identified and named pncR. In carbapenem-resistant Klebsiella pneumoniae, the active pncR variant is related to the antioxidant capacity of carbapenem-resistant Klebsiella pneumoniae. pncR and its related genes can be used as targets for anti-CRKP drugs.

[0006] To this end, in a first aspect, the present application provides an isolated pncR protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] In a second aspect of the present application, the use of a target in the preparation of an anti-carbapenem-resistant Klebsiella pneumoniae drug is provided, wherein the target is used as an action target of the drug.

[0008] The target includes at least one of the following (A1) to (A4):

[0009] (A1) a pncR protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1;

[0010] (A2) an amino acid site located at position 31 of the pncR protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1;

[0011] (A3) a pncR gene, wherein the pncR gene is KPHS_20140 gene;

[0012] (A4) at least one of the 91st, 92nd, and 93rd nucleotide sites in a pncR gene, wherein the pncR gene is KPHS_20140 gene.

[0013] Further, the pncR gene can express the pncR protein variant in the carbapenem-resistant Klebsiella pneumoniae, wherein the 92nd nucleotide site in the pncR gene is subjected to A-to-I RNA editing during the expression.

[0014] Further, the nucleotide sequence of the KPHS_20140 gene is shown in SEQ ID NO: 2.

[0015] Further, the drug is used for at least one of the following (B1) to (B3):

[0016] (B1) reducing the antioxidant capacity of the carbapenem-resistant Klebsiella pneumoniae;

[0017] (B2) reducing the drug resistance of the carbapenem-resistant Klebsiella pneumoniae;

[0018] (B3) reducing the pathogenicity of the carbapenem-resistant Klebsiella pneumoniae.

[0019] In a third aspect of the present application, a reagent is provided for reducing or completely inhibiting the expression of a pncR protein variant in the carbapenem-resistant Klebsiella pneumoniae, wherein the amino acid sequence of the pncR protein variant is shown in SEQ ID NO: 1.

[0020] Further, the reagent acts on the pncR gene, thereby reducing or completely inhibiting the expression of the pncR protein variant; wherein the pncR gene is KPHS_20140 gene.

[0021] Further, the reagent acts on the pncR gene, and the pncR gene is subjected to mutation, and the protein expressed by the mutated pncR gene has an amino acid sequence different from SEQ ID NO: 1.

[0022] Further, the mutated pncR gene avoids A-to-I RNA editing during the expression, and the protein expressed thereby has an amino acid sequence different from SEQ ID NO: 1.

[0023] Further, the reagent acts on the pncR gene, and the pncR gene is subjected to mutation, and the protein expressed by the mutated pncR gene has an amino acid sequence shown in SEQ ID NO: 3.

[0024] Further, the pncR gene is mutated by CRISPR / Cas9 technology; the reagent comprises a first primer combination; the first primer combination comprises a spacer primer group, a homologous arm primer group:

[0025] The spacer primer group: Spacer-FW, the amino acid sequence of which is shown as SEQ ID NO: 10; Spacer-RV, the amino acid sequence of which is shown as SEQ ID NO: 11;

[0026] The first homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 12; the amino acid sequence of which is shown as SEQ ID NO: 13;

[0027] The second homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 14; the amino acid sequence of which is shown as SEQ ID NO: 15;

[0028] The third homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 16; the amino acid sequence of which is shown as SEQ ID NO: 17.

[0029] Further, the reagent further comprises a pSGKP-spe vector, a pCasKP-hph vector.

[0030] Further, the reagent acts on the pncR gene to knockout or silence the pncR gene.

[0031] Further, the reagent acts on the pncR gene to knockout the pncR gene by CRISPR / Cas9 technology, and the reagent comprises a second primer combination; the second primer combination comprises a spacer primer group, a homologous arm primer group:

[0032] The spacer primer group: Spacer-FW, the amino acid sequence of which is shown as SEQ ID NO: 10; Spacer-RV, the amino acid sequence of which is shown as SEQ ID NO: 11;

[0033] The fourth homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 12; the amino acid sequence of which is shown as SEQ ID NO: 19;

[0034] The fifth homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 20; the amino acid sequence of which is shown as SEQ ID NO: 17.

[0035] Further, the reagent further comprises a pSGKP-spe vector, a pCasKP-hph vector.

[0036] In a fourth aspect of the present application, the use of the agent of the third aspect of the present application in the preparation of a drug for treating carbapenem-resistant Klebsiella pneumoniae is provided.

[0037] Further, the drug is used for at least one of the following (B1) to (B3):

[0038] (B1) reducing the antioxidant capacity of carbapenem-resistant Klebsiella pneumoniae;

[0039] (B2) reducing the drug resistance of carbapenem-resistant Klebsiella pneumoniae;

[0040] (B3) reducing the pathogenicity of carbapenem-resistant Klebsiella pneumoniae.

[0041] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0042] The present application innovatively reveals the A-to-I RNA editing phenomenon of a key amino acid site of a novel AraC / XylS family transcription factor in CRKP from the perspective of post-transcriptional regulation, and functionally characterizes the transcription factor, which is named pncR, and the corresponding KPHS_20140 gene encoding the transcription factor is named pncR gene. By constructing a specific gene mutant, it is confirmed that the A-to-I RNA editing of pncR has an important influence on the oxidative stress adaptability of bacteria, and the deletion of the edited pncR protein variant significantly reduces the antioxidant capacity of the bacteria and weakens their ability to cope with the host immune system and oxidative antibiotics. This finding provides a new perspective for the prevention and control of CRKP and proposes a therapeutic target, laying a foundation for the development of new antibacterial strategies and drugs. BRIEF DESCRIPTION OF DRAWINGS

[0043] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included only to illustrate preferred embodiments and are not to be considered as limiting of the present application. In the drawings:

[0044] Figure 1 : results of KPHS_20140 gene function analysis by chromatin immunoprecipitation combined with sequencing (ChIP-seq) technology;

[0045] Figure 2 : phnC gene expression of the KPHS_20140 gene overexpression strain is significantly reduced compared with the wild type strain;

[0046] Figure 3 : growth curves of wild type CRKP and 31Tyr strain under H2O2 action conditions;

[0047] Figure 4 Survival rates of wild-type CRKP and 31Tyr strains after 60 min of H2O2 treatment;

[0048] Figure 5 Total antioxidant capacity of wild-type CRKP and 31Tyr strains under H2O2 conditions;

[0049] Figure 6 Survival ability of wild-type CRKP and 31Tyr strains in macrophages;

[0050] Figure 7 Growth curves of wild-type CRKP and ΔpncR strains under H2O2 conditions. Detailed Implementation

[0051] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0052] Terminology

[0053] As used herein, the “pncR protein variant” is a protein that was first discovered, isolated, and identified in this invention, with the amino acid sequence SEQ ID NO: 1. The pncR protein variant is expressed by the KPHS_20140 gene in carbapenem-resistant Klebsiella pneumoniae. However, the amino acid sequence of the pncR protein variant does not perfectly match the amino acid sequence of the KPHS_20140 gene obtained through the codon table. This is because A-to-I RNA editing occurs during the expression of KPHS_20140. The pncR protein has been characterized as an AraC / XylS family transcription factor. Based on the above, the KPHS_20140 gene expressing the pncR protein has been renamed the pncR gene.

[0054] As used in this article, "pncR gene" is the renamed name of the KPHS_20140 gene, and the two are actually the same.

[0055] As used herein, “A-to-I RNA editing” is a genetic information modification mechanism, which refers to the process in which adenosine (A) is deaminated to inosine (I) in an RNA molecule. A-to-I RNA editing occurs after gene transcription, and subsequently, inosine (I) is recognized as guanosine (G) during translation, thus A-to-I editing can cause A→G mutation in genetic information. During the expression of KPHS_20140 gene, A-to-I RNA editing occurs at position 92, which is equivalent to mutating the corresponding codon from UAC to UGC. Therefore, the KPHS_20140 gene expressed in bacteria will generate a pncR protein variant in which the 31st tyrosine is replaced by cysteine.

[0056] As used herein, “antioxidant capacity” refers to the ability of bacteria to resist or slow down the process of oxidation. Reducing the antioxidant capacity of bacteria can make them more susceptible to oxidative stress, thereby destroying their viability. In known antibacterial strategies, reducing the antioxidant capacity of bacteria through drugs is included to achieve the effect of killing bacteria.

[0057] As used herein, “pathogenicity” refers to the ability of bacteria to cause infection, specifically the ability of bacteria to break through the host's defense function after invading the host's body and cause varying degrees of pathological changes in the body. Phagocytes are an important part of the immune system of the body, and they have the ability to recognize and phagocytose foreign pathogens. When bacteria invade the body, phagocytes will quickly recognize and surround the bacteria, and through phagocytosis, they will be taken into the cells, and the bacteria will be killed and digested by the digestive enzymes in the cells. This process is an important defense mechanism of the body against bacterial infection. If the bacteria are easily killed by phagocytes, it indicates that the pathogenicity of the bacteria is relatively weak.

[0058] As used herein, “drug resistance” refers to the relative resistance of bacteria to antibacterial drugs. In reported studies, it has been found that oxidative stress response is related to the bactericidal mechanism of some antibiotics (such as aminoglycosides, quinolones). If the antioxidant capacity of bacteria is reduced, they can be more sensitive to these antibiotics.

[0059] Carbon penem-resistant Klebsiella pneumoniae (CRKP) is resistant to multiple antibacterial drugs including carbon penem antibiotics, and has high pathogenicity, so the treatment options for CRKP infection are limited and the efficacy is poor, leading to high mortality.

[0060] The application discloses a novel A-to-I RNA editing phenomenon in a key amino acid site of a transcription factor of an AraC / XylS family in CRKP from the perspective of post-transcriptional regulation, and the transcription factor is named pncR, and the gene KPHS_20140 encoding the transcription factor is named pncR gene. By constructing a specific gene mutant, it is confirmed that the A-to-I RNA editing of pncR has an important influence on the oxidative stress adaptability of bacteria, and the deletion of the edited pncR protein variant significantly reduces the antioxidant capacity of the bacteria and weakens the ability of the bacteria to cope with the host immune system and oxidative antibiotics. The finding provides a new perspective for the prevention and treatment of CRKP, and proposes a therapeutic target, and lays a foundation for the development of a novel antibacterial strategy and drug.

[0061] In some embodiments of the application, an isolated pncR protein variant is provided, and the amino acid sequence of the pncR protein variant is shown in SEQ ID NO: 1. The pncR protein is characterized as belonging to the AraC / XylS family of transcription factors, and has a significant influence on the oxidative stress capacity of CRKP. The protein or the gene thereof can be used as a target to develop a new drug against CRKP.

[0062] In some embodiments, a target is provided for use in the preparation of a drug against carbapenem-resistant Klebsiella pneumoniae, and the target is used as an action target of the drug.

[0063] The target includes at least one of the following (A1) to (A4):

[0064] (A1) a pncR protein variant, and the amino acid sequence of the pncR protein variant is shown in SEQ ID NO: 1;

[0065] (A2) an amino acid site located at the 31st position of the pncR protein variant, and the amino acid sequence of the pncR protein is shown in SEQ ID NO: 1;

[0066] (A3) a pncR gene;

[0067] (A4) at least one of the 91st, 92nd and 93rd nucleotide sites of the pncR gene.

[0068] In some embodiments, the drug acts on the target, thereby reducing or completely inhibiting the expression of the pncR protein variant in carbapenem-resistant Klebsiella pneumoniae.

[0069] In some embodiments, the drug acts on the target to mutate the pncR gene, and the mutated pncR gene expresses a protein having an amino acid sequence different from SEQ ID NO: 1. In some embodiments, the mutated pncR gene avoids A-to-I RNA editing during expression, so that the expressed protein has an amino acid sequence different from SEQ ID NO: 1. In some embodiments, the amino acid sequence of the protein expressed by the mutated pncR gene is shown in SEQ ID NO: 3.

[0070] In some embodiments, the pncR gene encodes the expression of the pncR protein variant in carbapenem-resistant Klebsiella pneumoniae, and the pncR gene is subjected to A-to-I RNA editing at the 92nd nucleotide site during the encoding expression.

[0071] In some embodiments, the nucleotide sequence of the pncR gene is shown in SEQ ID NO: 2.

[0072] In some embodiments, the drug acts on the target to knockout or silence the pncR gene.

[0073] In some embodiments, the drug is used to reduce the antioxidant capacity of CRKP.

[0074] In some embodiments, the drug is used to reduce the drug resistance of CRKP.

[0075] In some embodiments, the drug is used to reduce the pathogenicity of CRKP.

[0076] In some embodiments, a reagent is provided for reducing or completely inhibiting the expression of a pncR protein variant in carbapenem-resistant Klebsiella pneumoniae, and the amino acid sequence of the pncR protein variant is shown in SEQ ID NO: 1.

[0077] In some embodiments, the reagent acts on the pncR gene to reduce or completely inhibit the expression of the pncR protein. Those skilled in the art know common methods for inhibiting gene expression, and select the reagent according to the actual situation. For example, based on the disclosure of the pncR gene sequence herein, those skilled in the art can reduce or completely inhibit the expression of the pncR protein by methods such as gene knockout, gene silencing (such as using RNA interference technology), gene mutation, etc.

[0078] In some embodiments, the reagent acts on the pncR gene to mutate the pncR gene, and the mutated pncR gene expresses a protein having an amino acid sequence different from SEQ ID NO: 1.

[0079] In some embodiments, the mutated pncR gene avoids A-to-I RNA editing during expression, so that the expressed protein has an amino acid sequence different from SEQ ID NO: 1.

[0080] In some embodiments, the reagent acts on the pncR gene to mutate the pncR gene, and the mutated pncR gene expresses a protein having an amino acid sequence as shown in SEQ ID NO: 3.

[0081] In some embodiments, the pncR gene is mutated by CRISPR / Cas9 technology; the reagent comprises a first primer combination; the first primer combination comprises a spacer primer group, a homologous arm primer group:

[0082] The spacer primer group comprises Spacer-FW, whose amino acid sequence is shown in SEQ ID NO: 10; and Spacer-RV, whose amino acid sequence is shown in SEQ ID NO: 11;

[0083] The first homologous arm primer group comprises, whose amino acid sequence is shown in SEQ ID NO: 12; and whose amino acid sequence is shown in SEQ ID NO: 13;

[0084] The second homologous arm primer group comprises, whose amino acid sequence is shown in SEQ ID NO: 14; and whose amino acid sequence is shown in SEQ ID NO: 15;

[0085] The third homologous arm primer group comprises, whose amino acid sequence is shown in SEQ ID NO: 16; and whose amino acid sequence is shown in SEQ ID NO: 17.

[0086] In some embodiments, the reagent further comprises a pSGKP-spe vector and a pCasKP-hph vector, which are commercially available vectors; the first primer combination and the vectors can be used to mutate the pncR gene by CRISPR / Cas9 technology, according to the instructions of the vectors, and the mutated pncR gene expresses a protein having an amino acid sequence as shown in SEQ ID NO: 3.

[0087] In some embodiments, the reagent acts on the pncR gene to knockout the pncR gene. In some embodiments, the pncR gene is knocked out by CRISPR / Cas9 technology; the reagent comprises a second primer combination; the second primer combination comprises a spacer primer group, a homologous arm primer group:

[0088] the spacer primer group: Spacer-FW, the amino acid sequence of which is shown as SEQ ID NO: 10; Spacer-RV, the amino acid sequence of which is shown as SEQ ID NO: 11;

[0089] the fourth homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 12; the amino acid sequence of which is shown as SEQ ID NO: 19;

[0090] the fifth homologous arm primer group: the amino acid sequence of which is shown as SEQ ID NO: 20; the amino acid sequence of which is shown as SEQ ID NO: 17.

[0091] In some embodiments, the reagent further comprises a pSGKP-spe vector, a pCasKP-hph vector.

[0092] In some embodiments, the reagent provided in the embodiments of the present application can be used to prepare an anti-carbapenem-resistant Klebsiella pneumoniae drug.

[0093] Hereinafter, examples of the present application will be listed, and the advantages and various effects of the present application will be more clearly presented. Those skilled in the art should understand that these examples are used to illustrate the present application, not to limit the present application.

[0094] Example 1

[0095] In this embodiment, the CRKP strain is sequenced and analyzed, and an A-to-I RNA editing site is found in the KPHS_20140 gene, which causes a missense mutation of the gene and expresses a protein that has not been reported. By analyzing the protein, it is shown that it is a transcriptional repressor of the phnC gene, so it is named pncR, and the KPHS_20140 gene expressing it is renamed as the pncR gene (the above name is continued to use in the subsequent examples). Details are as follows:

[0096] I. A-to-I RNA editing site identification

[0097] The whole genome and transcriptome of the CRKP strain are sequenced to obtain DNA-seq and RNA-seq data of the strain, and the identification of the A-to-I RNA editing site is carried out according to the following process:

[0098] (1) RNA-seq data cleaning by fastp;

[0099] (2) Indexing the reference genome sequence using BWA;

[0100] (3) Aligning RNA-seq data to the reference genome using BWA;

[0101] (4) Aligning WGS sequencing data of the strain to the reference genome using BWA;

[0102] (5) Converting sam files to bam files and sorting using samtools;

[0103] (6) Retrieving SNVs in bam files using bcftools;

[0104] (7) Building a library to annotate editing sites by snpEff.

[0105] The analysis results show that an A-to-I RNA editing site (c.92A>G) is found in the gene KPHS_20140 encoding a hypothetical protein with unknown function. The editing site causes a missense mutation in the gene, replacing the 31st tyrosine (Tyr) in the encoded protein with cysteine (Cys).

[0106] The gene sequence of KPHS_20140 is as follows, where the bold letter "A" shows the corresponding position of the A-to-I RNA editing site.

[0107] SEQ ID NO: 2

[0108] ATGTCTCAGCAGCAGAGCGCGGACTGGGTCAGGCTGGCGCAATCGCCCAGCCGGACGGAACGCATCGAAGCCTTCTTCGGCGGCCACGGC TACGAACCGCACCGGCATGATACCTACGCCATCGGCCAGACGATCGCCGGCGTGCAGAGCTTTCACTATCGCGGCGGACTCCAGCACAGCCTGCCCGGCGGAACGATGGTGCTTCATCCGGACGAAATACACGATGGCGAGGCGGGCACCGAGGCGGGGTTTCACTACCGTATGGTTTATATCGAACCGGCGCTGATCCAGAAGATACTCGGCGGCAGGCCACTACCGTTTATTCCCGGCGGCCTGTCGGCGGATCCGCGCCTGCGGTGCGCCGCGCTGCCGTTATTAAAAGCGGTCACGGACACCTTTGAGCCGCTGGAGGAAGAGGACGCCCTGTACGACCTGGCGCAGACCCTGGCCGTTGTCGGCGGCCAACGTTCCCGTCGCCAGGCGTTTGATTATCAGGCGGCGGAGAGGGCCCGGGAGTATATCCATGCCTGTTTTATGCAGGATATGACCCTCGACACCCTGTCGCAGGTCAGCGGCCGGGACCGCTGGAGCCTGAGCCGGGATTTCAGAACGCTGTACGGCACCAGCCCGTGGCGCTACGTCATGATGCGCCGGCTCGATTTTTGCCGCCAGCGGATGCGCGCCGGGGAACGTCTGGTGGATATTGCCGCCGACGCGGGCTTTGCCGATCAGAGCCACATGACGCGCCAGTTTATCAGCCGCTTTGGTCTCTCTCCGGGCCGCTGGCTGCGGGCGATCCGCGGCTAG

[0109] In the expression of the KPHS_20140 gene, the amino acid sequence of the expressed protein is as follows, in which the 31st position is replaced by cysteine (Cys) from tyrosine (Tyr) due to A-to-I RNA editing.

[0110] SEQ ID NO: 1

[0111] MSQQQSADWVRLAQSPSRTERIEAFFGGHG CEPHRHDTYAIGQTIAGVQSFHYRGGLQHSLPGGTMVLHPDEIHDGEAGTEAGFHYRMVYIEPALIQKILGGRPLPFIPGGLSADPRLRCAALPLLKAVTDTFEPLEEEDALYDLAQTLAVVGGQRSRRQAFDYQAAERAREYIHACFMQDMTLDTLSQVSGRDRWSLSRDFRTLYGTSPWRYVMMRRLDFCRQRMRAGERLVDIAADAGFADQSHMTRQFISRFGLSPGRWLRAIRG

[0112] II. KPHS_20140 gene function analysis

[0113] By Uniprot database comparison, it was found that the putative protein encoded by KPHS_20140 gene was AraC / XylS family transcription factor, but its specific function was not clear. In order to further clarify its function, chromatin immunoprecipitation combined with sequencing (ChIP-seq) technology was used for analysis. The specific process is as follows:

[0114] (1) Construction of overexpression strain

[0115] In order to facilitate the detection of the protein encoded by KPHS_20140 gene, a c-myc tag was introduced at the amino terminal end of the amino acid sequence encoded by the gene, which contains 10 amino acid residues and the sequence is EQKLISEEDL (SEQ ID NO: 4). The protein with c-myc tag is referred to as PD-myc.

[0116] In the experiment, the genome of clinically isolated CRKP strain C789 was used as the template for the first round of PCR amplification, and the product was purified and recovered, and then used as the template for the second round of PCR amplification. The complete c-myc tag was gradually introduced by two rounds of primer design. The strain information is shown in Table 1. The first round of PCR amplification primers are myc-1-F (SEQ ID NO: 5) and myc-1-R (SEQ ID NO: 6), and the second round of PCR amplification primers are myc-2-F (SEQ ID NO: 7) and myc-2-R (SEQ ID NO: 8). The coding gene sequence of PD-myc obtained by PCR is SEQ ID NO: 9.

[0117] Table 1

[0118] Strains ST type Serotype Date of isolation Sample type Carbapenemase pLVPK-like plasmid C789 11 K64 2016-4-14 Blood KPC-2 +

[0119] After synthesizing the coding gene of PD-myc, the coding gene of PD-myc was inserted into the pET-28a(+) vector to construct the overexpression strain. The overexpression strain was constructed as follows: HiFi DNA Assembly Master Mix (#E2621, NEB) was used to insert the gene into the vector pEasy plasmid (#CT101, Beijing Quanshijin) by the method of Assembly. After screening and verification, the synthesized plasmid was electroporated into the C789 strain to construct the overexpression strain of KPHS_20140 gene. The overexpression strain was cultured to obtain bacterial liquid for subsequent experiments.

[0120] (2) Formaldehyde cross-linked protein-DNA complex

[0121] The H2O2-treated bacterial liquid was added with 37% formaldehyde for cross-linking, followed by adding a corresponding volume of 2M glycine to terminate cross-linking. After two washes with cold PBS, the protein-DNA complex was obtained by resuspending in PBS containing protease inhibitors.

[0122] (3) Ultrasonic disruption of protein-DNA complex

[0123] The nucleic acid lysis buffer was added to the protein-DNA complex, and the ultrasonic disrupter was used for ultrasonic disruption. The disrupted sample can be detected by agarose gel electrophoresis.

[0124] (4) Antibody precipitation of protein-DNA complex

[0125] The myc antibody (1:1000 dilution) was used to bind to the target protein, and then the protein-chromatin complex was precipitated by protein G beads magnetic beads.

[0126] (5) De-cross-linking and purification of DNA fragments

[0127] The cross-linking in the antibody-protein-DNA complex was removed, and the DNA was released and purified for recovery.

[0128] (6) Library construction and high-throughput sequencing

[0129] The DNA sample after immunoprecipitation was subjected to end repair and "A" addition according to the kit, and the product was purified by magnetic beads. Subsequently, the library was subjected to high-throughput sequencing using the Illumina platform.

[0130] (7) Data analysis

[0131] The data obtained by sequencing were analyzed, including sequence alignment, peak identification, and enrichment region labeling, to obtain information about protein-chromatin interaction, such as protein binding sites and chromatin enrichment regions.

[0132] The ChIP-seq results are shown in Figure 1, two biological repeat samples both contain Peak within 1 kb upstream of phnC (ANKHBFPF_04185) gene, suggesting that KPHS_20140 gene may be involved in the transcriptional regulation of phnC gene. And, referring to Figure 2 , the phnC gene expression of KPHS_20140 gene overexpression strain is significantly lower than that of wild type strain. The above results show that the protein expressed by KPHS_20140 gene is a transcriptional repressor of phnC gene, so it is named pncR.

[0133] Example 2

[0134] This example carries out gene editing by CRISPR / Cas9 technology, and the codon at the 31st position of pncR is mutated from TAC to TAT. Through this synonymous mutation, a pncR gene mutant strain that does not undergo A-to-I RNA editing is constructed, i.e. the amino acid sequence of the protein expressed by the pncR gene in the mutant strain is SEQ ID NO: 3. The specific process is as follows:

[0135] (1) Use sgRNAcas9 software to find a suitable spacer sequence, and synthesize the forward and reverse oligos of the designed spacer. As shown below:

[0136] Spacer-FW: TAGTCCAGGCGTTTGATTATCAGG (SEQ ID NO: 10);

[0137] Spacer-RV: AAACCCTGATAATCAAACGCCTGG (SEQ ID NO: 11).

[0138] (2) Synthesis of homologous arm: introduce mutation and enzyme digestion site by PCR primer, take C789 strain genome as template, synthesize three sub-homologous arms respectively, and then synthesize homologous arm containing mutation site by fusion PCR with sub-homologous arm as template and sub-homologous arm upstream and downstream primers as amplification primers. The homologous arm synthesis primers are as follows:

[0139] FHR-F: CGGAATTCCGCGGTTACGGATCAGGGTTTCCAT (SEQ ID NO: 12);

[0140] FHR-Tyr-R: GGTCGCGTTCATAGCCGTGGCC (SEQ ID NO: 13);

[0141] SHR-Tyr-F: GGCCACGGCTATGAACCGCACC (SEQ ID NO: 14);

[0142] SHR-R:

[0143] GCCCTCTCCGCAGCTTGGTAGTCGAAAGCCTGGCGACGGGAACGTT (SEQ ID NO: 15);

[0144] THR-F:

[0145] CGTCGCCAGGCTTTCGACTACCAAGCTGCGGAGAGGGCCCGGGAGT (SEQ ID NO: 16);

[0146] THR-R: GCTCTAGAAGGGATGAAGCAGTTTATTGACAGCC (SEQ ID NO: 17).

[0147] After fusion PCR, the full-length homology arm was obtained, denoted as HR-Tyr, and its nucleotide sequence is shown in SEQ ID NO: 18.

[0148] (3) Recombination vector construction: HR-Tyr was connected into the vector pSGKP-spe (#117234, Addgene) by double digestion with EcoR I and Xba I, and the ligation product was transformed into E. coli Transl-Tl using Transl-Tl Phage Resistant Chemically Competent Cell (#CD501-03, Vazyme) for chemical transformation. After resistance screening, the plasmid pSGKP-HR was obtained by using FastPure Plasmid Mini Kit (#DC201-01, Vazyme) to extract the plasmid. Then, the phosphorylated spacer was connected by Bsa I single digestion, and finally the vector pSGKP-spacer-HR for site-directed mutagenesis was obtained.

[0149] (4) Preparation of bacterial competence and electroporation: 10% glycerol solution was prepared and autoclaved. The C789 bacterial solution in the logarithmic phase was repeatedly centrifuged at 4°C. Then, 1 mL of pre-cooled 10% glycerol was added, mixed well by blowing and sucking, centrifuged and discarded the supernatant, repeated three times. The supernatant was discarded for the last time, and the precipitate was left, which was the prepared C789 competence. pCasKP-hph (#117232, Addgene) was electroporated into the competent bacteria, and the strain carrying pCasKP was obtained by resistance screening.

[0150] (5) Preparation of competent cells and electroporation of pCasKP-carrying strains: The preparation of competent cells carrying pCasKP strains is basically the same as the conventional preparation of competent cells, but there are some differences. The pCasKP plasmid is a temperature-sensitive plasmid, which needs to be cultured at 30°C. The expression of the lambda-Red system needs to be induced during the enrichment to improve the recombination efficiency. The competent cells of C789 strain carrying pCasKP are obtained, and the previously successfully recombined pSGKP-spacer-HR plasmid is subjected to electroporation.

[0151] (6) Verification and screening: single colony bacteria growing on the screening plate are picked and subjected to PCR verification. The Cas9, SacB and site-directed mutation region are all consistent with the expectation, i.e., the colony of pncR gene site-directed mutation is successfully prepared, which is named 31Tyr.

[0152] Example 3

[0153] This example analyzes the oxidative stress adaptability of wild-type CRKP (i.e., C789 strain, denoted as WT) and A-to-I RNA editing-deficient mutant CRKP (i.e., 31Tyr strain, denoted as 31Tyr) of KPHS_20140. Specifically as follows:

[0154] (1) Hydrogen peroxide growth curve

[0155] The logarithmic growth phase bacterial liquid of WT and 31Tyr strains is obtained respectively, which is diluted 100 times. 100 μL of each sample is added to a 96-well plate, and 3 repeats are performed for each strain. 4.6 mM of H2O2 is added to each well to make the final concentration reach 2.3 mM of H2O2. Incubation is performed at 37°C in a microplate reader, and the absorbance at 600 nm is detected every 30 min, with a total time of 24 h. The results are shown in Figure 3 , which shows that the growth rate of 31Tyr strain is significantly decreased.

[0156] (2) Hydrogen peroxide sterilization experiment

[0157] The logarithmic growth phase bacterial liquid of WT and 31Tyr strains is obtained respectively, which is diluted 100 times. 100 μL of each sample is added to a 96-well plate, and 3 repeats are performed for each strain. 4.6 mM of H2O2 is added to each well to make the final concentration reach 2.3 mM of H2O2. Incubation is performed at 37°C in a microplate reader, and the absorbance at 600 nm is detected every 30 min, with a total time of 24 h. The results are shown in Figure 4 , which shows that the survival rate of 31Tyr strain is significantly lower than that of WT strain at 60 min (P = 0.0075).

[0158] (3) Total antioxidant capacity detection

[0159] The total antioxidant capacity detection kit (#S0121, Biyun Tian) is used to detect the total antioxidant capacity of WT and 31Tyr strains under 2.3 mM H2O2 stress. The results are shown in Figure 5As shown, the total antioxidant capacity of strain 31Tyr was significantly lower than that of strain WT under 2.3 mM H2O2 pressure (P = 0.0180).

[0160] In summary, in CRKP, eliminating the mutation at position 31 of pncR caused by A-to-I RNA editing significantly reduced the bacteria's ability to adapt to oxidative stress.

[0161] Example 4

[0162] Building upon Example 3, this example further analyzes the pathogenicity of WT and 31Tyr strains using an intracellular survival assay of immortalized mouse bone marrow-derived macrophages (iBMDM). Details are as follows:

[0163] iBMDM cells were loaded at 1×10 6 Cells were seeded at a density of 10 cells / well in 24-well cell culture plates (#3524, Corning) to form a monolayer. Simultaneously, LPS (100 ng / mL) and IFN-γ (20 ng / mL) were added to stimulate the cells. WT and 31Tyr strains were cultured to the logarithmic growth phase and the bacterial culture was adjusted to an OD600 of 0.6. Subsequently, 5 × 10⁶ cells / well were seeded. 6 CFU of bacteria (MOI=5) and 10% mouse serum (#ZI807-1, Zhuang Meng) were co-cultured with iBMDM cells at 37°C and 5% CO2 for 30 min. Extracellular bacteria were then killed by treatment with 500 μg / mL hygromycin for 30 min, followed by cell lysis with 1% Triton X-100 (#SLCF5969, Sigma-Aldrich). The cells were then diluted and cultured on LB agar plates to count intracellular bacteria, recorded as the intracellular bacterial load at time 0. Based on this, bacterial viability within macrophages was assessed by extending the incubation time before lysis (1 and 2 hours). Results are as follows: Figure 6 As shown, after 2 hours, the number of intracellular bacteria in iBMDM cells of strain 31Tyr was significantly lower than that of strain WT (P = 0.0224), indicating that strain 31Tyr had a decreased ability to kill iBMDM cells, i.e., its pathogenicity was weakened.

[0164] Example 5

[0165] This embodiment uses CRISPR / Cas9 technology to knock out the pncR gene, constructing a mutant strain without the pncR gene. Details are as follows:

[0166] (1) The same spacer sequence as the pncR gene site-directed mutation in Example 2 was used, namely SEQ ID NO: 10 and SEQ ID NO: 11.

[0167] (2) Synthesis of homologous arms: enzyme cutting sites were introduced by PCR primers and the upstream homologous arm (UHR) and the downstream homologous arm (DHR) were synthesized, then the upstream and downstream homologous arms were used as templates and the upstream and downstream homologous arm primers were used as amplification primers to synthesize the homologous arm containing no target gene pncR by fusion PCR, which was used as a subsequent homologous recombination repair template. The upstream and downstream homologous arm synthesis primers are as follows:

[0168] UHR-F: CGGAATTCCGCGGTTACGGATCAGGGTTTCCAT (SEQ ID NO: 12);

[0169] UHR-R: AATGGATAACCTGCGACGTTCTT (SEQ ID NO: 19);

[0170] DHR-F: CGCGGTTCAGGTTGCACAATCGTTCAAG (SEQ ID NO: 20);

[0171] DHR-R: GCTCTAGAAGGGATGAAGCAGTTTATTGACAGCC (SEQ ID NO: 17).

[0172] After fusion PCR, the full-length homologous arm was obtained, denoted as HR-△pncR, and its nucleotide sequence is shown in SEQ ID NO: 21.

[0173] (3) Recombinant vector construction: HR-△pncR was connected into the vector pSGKP-spe (#117234, Addgene) by double digestion with EcoR I and Xba I, and the ligation product was transformed into E. coli Trans1-T1 using Trans1-T1 Phage Resistant Chemically Competent Cell (#CD501-03, Vazyme) for chemical transformation. After resistance screening, the plasmid pSGKP-HR2 was obtained by using FastPure Plasmid Mini Kit (#DC201-01, Vazyme). Then, the phosphorylated spacer was connected by Bsa I single enzyme digestion, and finally the vector pSGKP-spacer-HR2 for site-directed mutagenesis was obtained.

[0174] (4) The preparation of bacterial competent cells and the process of electroporation were consistent with the description in Example 2.

[0175] (5) The preparation of competent cells of the pCasKP-carrying strain was consistent with the description in Example 2. The competent cells of the C789 strain carrying pCasKP were obtained, and the pSGKP-spacer-HR2 plasmid obtained by recombination in step (3) was electroporated.

[0176] (6) Verification and screening: single colony growing on the screening plate was picked up, and PCR was used to verify that Cas9, SacB and the knockout region were all as expected, i.e. the colony of successful pncR gene knockout was prepared and named as △pncR strain.

[0177] Example 6

[0178] This example analyzes the hydrogen peroxide growth curve of wild type CRKP (i.e. C789 strain, denoted as WT) and pncR gene knockout strain (i.e. △pncR strain, denoted as △pncR) to characterize the oxidative stress adaptability. Specifically as follows:

[0179] WT and △pncR strain liquid in logarithmic growth phase was obtained by culture, and was diluted by 100 times, 100 μL of each sample was added to a 96-well plate, and 3 repeats were made for each strain. 5 mM H2O2 was added to each well to make the final concentration 2.5 mM H2O2. Incubation was carried out in a microplate reader at 37℃, and 600 nm absorbance detection was carried out every 30 min, and the total time was 24 h. As shown in Figure 7 , it is shown that the growth rate of △pncR strain is significantly decreased.

[0180] The above results show that in CRKP, by knocking out the pncR gene, the adaptability of bacteria to oxidative stress is significantly reduced.

[0181] The above describes only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. An isolated pncR protein variant, characterized in that, The amino acid sequence of the pncR protein variant is shown in SEQ ID NO:

1.

2. Use of a target in the preparation of a medicament for treating carbapenem-resistant Klebsiella pneumoniae, characterized in that, The target is the target of the drug; The target is selected from at least one of the following (A1)-(A4): (A1) pncR protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1; (A2) the amino acid site at position 31 of the pncR protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1; (A3) pncR Genes, the ones mentioned pncR The gene is the KPHS_20140 gene; the nucleotide sequence of the KPHS_20140 gene is shown in SEQ ID NO: 2; (A4) pncR at least one of the 91st, 92nd, and 93rd nucleotide sites in the gene, wherein pncR The gene is KPHS_20140 gene; the nucleotide sequence of the KPHS_20140 gene is shown as SEQ ID NO:

2.

3. Use according to claim 2, characterized in that, The drug is used for at least one of the following (B1)-(B3): (B1) reducing the antioxidant capacity of carbapenem-resistant Klebsiella pneumoniae; (B2) reducing the drug resistance of carbapenem-resistant Klebsiella pneumoniae; (B3) reducing the pathogenicity of carbapenem-resistant Klebsiella pneumoniae.

4. An agent, characterized in that, The reagent is used to reduce or completely inhibit the expression of pncR protein variant in carbapenem-resistant Klebsiella pneumoniae, the amino acid sequence of which is shown in SEQ ID NO: 1; The reagent acts on pncR Genes, making the pncR Gene mutation, the pncR The gene is the KPHS_20140 gene; the nucleotide sequence of the KPHS_20140 gene is shown in SEQ ID NO: 2; the mutated... pncR The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO:

3.

5. The agent of claim 4, wherein The mutated pncR gene avoids A-to-I RNA editing during expression, and thus the protein expressed by the mutated pncR gene has an amino acid sequence as set forth in SEQ ID NO:

3.

6. The agent of claim 4, wherein The reagent comprises a first primer combination; the first primer combination comprises a spacer sequence primer combination, a homologous arm primer combination: Spacer sequence primer combination: Spacer-FW, the nucleotide sequence of which is shown in SEQ ID NO: 10; Spacer-RV, the nucleotide sequence of which is shown in SEQ ID NO: 11; The nucleotide sequence of the first homologous arm primer combination is shown in SEQ ID NO: 12; The nucleotide sequence of the second homologous arm primer combination is shown in SEQ ID NO: 13; The nucleotide sequence of the second homologous arm primer combination is shown in SEQ ID NO: 14; the nucleotide sequence of which is shown in SEQ ID NO: 15; The nucleotide sequence of the third homologous arm primer combination is shown in SEQ ID NO: 16; the nucleotide sequence of which is shown in SEQ ID NO:

17.

7. The agent of claim 6, wherein The reagent further comprises a pSGKP-spe vector, a pCasKP-hph vector.

8. The agent of claim 6, wherein The reagent comprises a second primer combination; the second primer combination comprises a spacer sequence primer combination, a homologous arm primer combination: Spacer sequence primer combination: Spacer-FW, the nucleotide sequence of which is shown in SEQ ID NO: 10; Spacer-RV, the nucleotide sequence of which is shown in SEQ ID NO: 11; The nucleotide sequence of the fourth homologous arm primer combination is shown in SEQ ID NO: 12; The nucleotide sequence of the fourth homologous arm primer combination is shown in SEQ ID NO: 19; The nucleotide sequence of the fifth homologous arm primer combination is shown in SEQ ID NO: 20; the nucleotide sequence of which is shown in SEQ ID NO:

17.

9. The agent of claim 8, wherein The reagent further comprises a pSGKP-spe vector, a pCasKP-hph vector.

10. Use of the reagent of any one of claims 4-9 in the preparation of an anti-carbapenem-resistant Klebsiella pneumoniae drug.

11. Use according to claim 10, characterized in that, The drug is used for at least one of the following (B1)-(B3): (B1) reducing the antioxidant capacity of carbapenem-resistant Klebsiella pneumoniae; (B2) reducing the drug resistance of carbapenem-resistant Klebsiella pneumoniae; (B3) reducing the pathogenicity of carbapenem-resistant Klebsiella pneumoniae.

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