AraC / XylS family transcription factor and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to an AraC / XylS family transcription factor and application thereof, and the transcription factor is identified and named as pncR. In the post-transcriptional stage, the key amino acid site of the pncR gene has an A-to-I RNA editing phenomenon, so that a new pncR protein variant is generated, and the amino acid sequence of the pncR protein variant is as shown in SEQ ID NO: 1. Through gene knockout or RNA editing inhibition, generation of pncR protein variants can be prevented, so that the oxidation resistance of carbapenem drug-resistant klebsiella pneumoniae is reduced. Therefore, the pncR and related genes thereof can be used as targets of drugs for resisting carbapenem drug-resistant klebsiella pneumoniae.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to an AraC / XylS family transcription factor and its application. Background Art

[0002] Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a multi-drug resistant bacterium and belongs to a type of Klebsiella pneumoniae. It usually appears in hospitals and long-term care facilities and is a serious hospital infection pathogen.

[0003] CRKP is resistant to a variety of antibacterial drugs including carbapenem antibiotics and has high pathogenicity. Therefore, the treatment options for CRKP infection are limited and the curative effect is poor, resulting in a high mortality rate. The incidence of CRKP has been on the rise in recent years, bringing a huge public health burden to the world.

[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 treatment effect. Summary of the Invention

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

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

[0007] In the second aspect of the present invention, there is provided the use of a target in the preparation of a drug against carbapenem-resistant Klebsiella pneumoniae, and the target is used as the action target of the drug;

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

[0009] (A1) A pncR protein variant, whose amino acid sequence is as shown in SEQ ID NO: 1;

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

[0011] (A3) The pncR gene, and the pncR gene is the KPHS_20140 gene;

[0012] (A4) At least one of nucleotide sites 91, 92, and 93 of the pncR gene, and the pncR gene is the KPHS_20140 gene.

[0013] Further, the pncR protein variant can be expressed from the pncR gene in carbapenem-resistant Klebsiella pneumoniae. During the expression process, an A-to-I RNA editing occurs at nucleotide site 92 of the pncR gene.

[0014] Further, the nucleotide sequence of the KPHS_20140 gene is as 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 carbapenem-resistant Klebsiella pneumoniae;

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

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

[0019] In the third aspect of the present invention, a reagent is provided. The reagent is used to reduce or completely inhibit the expression of the pncR protein variant in carbapenem-resistant Klebsiella pneumoniae, and the amino acid sequence of the pncR protein variant is as shown in SEQ ID NO: 1.

[0020] Further, the reagent acts on the pncR gene to reduce or completely inhibit the expression of the pncR protein variant; the pncR gene is the KPHS_20140 gene.

[0021] Further, the reagent acts on the pncR gene to cause the pncR gene to mutate, and the protein expressed from the mutated pncR gene has an amino acid sequence different from that of SEQ ID NO: 1.

[0022] Further, during the expression process of the mutated pncR gene, A-to-I RNA editing is avoided, so that the expressed protein has an amino acid sequence different from that of SEQ ID NO: 1.

[0023] Further, the reagent acts on the pncR gene to cause the pncR gene to mutate, and the amino acid sequence of the protein encoded and expressed by the mutated pncR gene is as shown in SEQ ID NO: 3.

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

[0025] Spacer primer group: Spacer-FW, whose amino acid sequence is as shown in SEQ ID NO: 10; Spacer-RV, whose amino acid sequence is as shown in SEQ ID NO: 11;

[0026] First homologous arm primer group: whose amino acid sequence is as shown in SEQ ID NO: 12; whose amino acid sequence is as shown in SEQ ID NO: 13;

[0027] Second homologous arm primer group: whose amino acid sequence is as shown in SEQ ID NO: 14; whose amino acid sequence is as shown in SEQ ID NO: 15;

[0028] Third homologous arm primer group: whose amino acid sequence is as shown in SEQ ID NO: 16; whose amino acid sequence is as shown in SEQ ID NO: 17.

[0029] Furthermore, the reagent also includes the pSGKP-spe vector and the pCasKP-hph vector.

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

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

[0032] Spacer primer group: Spacer-FW, whose amino acid sequence is as shown in SEQ ID NO: 10; Spacer-RV, whose amino acid sequence is as shown in SEQ ID NO: 11;

[0033] Fourth homologous arm primer group: whose amino acid sequence is as shown in SEQ ID NO: 12; whose amino acid sequence is as shown in SEQ ID NO: 19;

[0034] Fifth homologous arm primer group: whose amino acid sequence is as shown in SEQ ID NO: 20; whose amino acid sequence is as shown in SEQ ID NO: 17.

[0035] Furthermore, the reagent also includes the pSGKP-spe vector and the pCasKP-hph vector.

[0036] In the fourth aspect of the present invention, there is provided the use of the reagent described in the third aspect of the present invention in the preparation of a drug against carbapenem-resistant Klebsiella pneumoniae.

[0037] Furthermore, 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 solution of the present invention has the following beneficial effects:

[0042] The present invention innovatively starts from the perspective of post-transcriptional regulation, reveals the A-to-I RNA editing phenomenon in the key amino acid sites of a novel AraC / XylS family transcription factor in CRKP, and conducts functional characterization of this transcription factor, named pncR. Correspondingly, its encoding gene KPHS_20140 is named the pncR gene. By constructing specific gene mutants, it is confirmed that the A-to-I RNA editing of pncR has an important impact on the bacterial oxidative stress adaptability. The deletion of the editing results in the deletion of the functional pncR protein variant, thus significantly reducing the antioxidant capacity of the bacteria and weakening its ability to cope with the host immune system and oxidative antibiotics. This discovery provides a new perspective for the prevention and treatment research of CRKP and proposes a therapeutic target, laying a foundation for the development of novel antibacterial strategies and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention. In the drawings:

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

[0045] Figure 2 : In the KPHS_20140 gene overexpression strain compared with the wild-type strain, the expression level of the phnC gene decreased significantly;

[0046] Figure 3 : H 2 O 2Growth curves of wild-type CRKP and 31Tyr strains under the action conditions;

[0047] Figure 4 : H 2 O 2 Survival rates of wild-type CRKP and 31Tyr strains after 60 minutes of action;

[0048] Figure 5 : H 2 O 2 Total antioxidant capacity of wild-type CRKP and 31Tyr strains under the action conditions;

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

[0050] Figure 7 : H 2 O 2 Growth curves of wild-type CRKP and ΔpncR strains under the action conditions. Detailed implementation manners

[0051] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be completely conveyed to those skilled in the art.

[0052] Term

[0053] As used herein, the "pncR protein variant" is a protein first discovered, isolated and identified in the present invention, and its amino acid sequence is 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 exactly conform to the amino acid sequence obtained from the KPHS_20140 gene through the codon table, which is due to A-to-I RNA editing during the expression process of KPHS_20140. The pncR protein has been characterized as an AraC / XylS family transcription factor. On this basis, the KPHS_20140 gene expressing the pncR protein is renamed the pncR gene.

[0054] As used herein, the "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 mechanism for modifying genetic information, 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. Subsequently, inosine (I) is recognized as guanosine (G) during translation. Therefore, A-to-I editing can lead to an A→G mutation in genetic information. During the expression of the 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 tyrosine at position 31 is replaced by cysteine.

[0056] As used herein, "antioxidant capacity" refers to the ability of bacteria to resist or slow down the oxidation process. Reducing the antioxidant capacity of bacteria makes them more susceptible to oxidative stress, thereby destroying their viability. Among the known antibacterial strategies, there is an approach to reduce the antioxidant capacity of bacteria through drugs, thereby achieving a bactericidal effect.

[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 and cause varying degrees of pathological changes in the body after invading the host. Phagocytes are an important part of the body's immune system and have the ability to recognize and phagocytose foreign pathogens. When bacteria invade the body, phagocytes quickly recognize and surround the bacteria, ingest them into the cells through phagocytosis, and use the digestive enzymes in the cells to kill and digest the bacteria. This process is an important defense mechanism of the body against bacterial infection. If 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 the reported studies, it has been found that the oxidative stress response is related to the bactericidal mechanisms of certain antibiotics (such as aminoglycosides, quinolones). If the antioxidant capacity of bacteria is reduced, they may be more sensitive to these antibiotics.

[0059] Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to multiple antibacterial drugs including carbapenem antibiotics and has high pathogenicity. Therefore, the treatment options for CRKP infections are limited and the efficacy is poor, resulting in a high mortality rate.

[0060] Innovatively starting from the perspective of post-transcriptional regulation, the present invention reveals the phenomenon of A-to-I RNA editing at key amino acid sites of a novel AraC / XylS family transcription factor in CRKP, and functionally characterizes this transcription factor, naming it pncR. Correspondingly, its encoding gene KPHS_20140 is named the pncR gene. By constructing specific gene mutants, it is confirmed that the A-to-I RNA editing of pncR has an important impact on the oxidative stress adaptability of bacteria. The deletion of editing results in the absence of functional pncR protein variants, thereby significantly reducing the antioxidant capacity of bacteria and weakening their ability to cope with the host immune system and oxidative antibiotics. This discovery provides a new perspective for the prevention and treatment research of CRKP and proposes a therapeutic target, laying a foundation for the development of new antibacterial strategies and drugs.

[0061] In some embodiments of the present invention, an isolated pncR protein variant is provided, and its amino acid sequence is as shown in SEQ ID NO: 1. The pncR protein is characterized as belonging to the AraC / XylS family transcription factor and has a significant impact on the oxidative stress ability of CRKP. New anti-CRKP drugs can be developed by targeting this protein or its gene.

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

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

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

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

[0066] (A3) The pncR gene;

[0067] (A4) At least one of the nucleotide sites at the 91st, 92nd, and 93rd positions 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, causing the pncR gene to mutate, and the protein expressed by the mutated pncR gene has an amino acid sequence different from SEQ ID NO: 1. In some embodiments, during the expression of the mutated pncR gene, A-to-I RNA editing is avoided, 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 as shown in SEQ ID NO: 3.

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

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

[0072] In some embodiments, the drug acts on the target, causing the pncR gene to be knocked out or silenced.

[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, and the reagent is used to reduce or completely inhibit the expression of the pncR protein variant in carbapenem-resistant Klebsiella pneumoniae, and the amino acid sequence of the pncR protein variant is as shown in SEQ ID NO: 1.

[0077] In some embodiments, the reagent acts on the pncR gene, thereby reducing or completely inhibiting the expression of the pncR protein. Those skilled in the art are aware of common methods for inhibiting gene expression and select reagents according to the actual situation. For example, based on the pncR gene sequence disclosed herein, those skilled in the art can reduce or completely inhibit the expression of the pncR protein by 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 protein expressed by the mutated pncR gene has an amino acid sequence different from SEQ ID NO: 1.

[0079] In some embodiments, during the expression of the mutated pncR gene, A-to-I RNA editing is avoided, so that the protein expressed 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 amino acid sequence of the protein expressed by the mutated pncR gene is as shown in SEQ ID NO: 3.

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

[0082] Spacer primer set: Spacer-FW, whose amino acid sequence is as shown in SEQ ID NO: 10; Spacer-RV, whose amino acid sequence is as shown in SEQ ID NO: 11;

[0083] First homologous arm primer set: whose amino acid sequence is as shown in SEQ ID NO: 12; whose amino acid sequence is as shown in SEQ ID NO: 13;

[0084] Second homologous arm primer set: whose amino acid sequence is as shown in SEQ ID NO: 14; whose amino acid sequence is as shown in SEQ ID NO: 15;

[0085] Third homologous arm primer set: whose amino acid sequence is as shown in SEQ ID NO: 16; whose amino acid sequence is as shown in SEQ ID NO: 17.

[0086] In some embodiments, the reagent further includes a pSGKP-spe vector and a pCasKP-hph vector, and the above vectors are vectors that can be purchased commercially; the pncR gene can be mutated by CRISPR / Cas9 technology using the aforementioned first primer combination and the vector, and the amino acid sequence of the protein encoded and expressed by the mutated pncR gene is 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 includes a second primer combination; the second primer combination includes a spacer primer set and a homologous arm primer set:

[0088] Spacer primer set: Spacer-FW, whose amino acid sequence is as shown in SEQ ID NO: 10; Spacer-RV, whose amino acid sequence is as shown in SEQ ID NO: 11;

[0089] Fourth homologous arm primer set: whose amino acid sequence is as shown in SEQ ID NO: 12; whose amino acid sequence is as shown in SEQ ID NO: 19;

[0090] Fifth homologous arm primer set: whose amino acid sequence is as shown in SEQ ID NO: 20; whose amino acid sequence is as shown in SEQ ID NO: 17.

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

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

[0093] Examples of the present invention will be listed below, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these examples are for illustrating the present invention rather than limiting the present invention.

[0094] Example 1

[0095] In this example, the CRKP strain was subjected to sequencing analysis, and an A-to-I RNA editing site was found in the KPHS_20140 gene. This editing site caused a missense mutation in the gene, expressing an unreported protein. Through the analysis of this protein, it was shown that it is a transcriptional repressor of the phnC gene, so it was named pncR. Correspondingly, the KPHS_20140 gene expressing it was renamed the pncR gene (the above names will continue to be used in subsequent examples). Specifically as follows:

[0096] I. Identification of A-to-I RNA editing site

[0097] The CRKP strain was subjected to whole-genome and transcriptome sequencing to obtain the DNA-seq and RNA-seq data of the strain, and the A-to-I RNA editing site was identified according to the following process:

[0098] (1) Clean RNA-seq data using fastp;

[0099] (2) Build an index of the reference genome sequence using BWA;

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

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

[0102] (5) Use samtools to convert the sam file to a bam file and sort it;

[0103] (6) Use bcftools to retrieve SNVs from the bam file;

[0104] (7) Build a library for annotating editing sites using snpEff.

[0105] The analysis results showed that an A-to-I RNA editing site (c.92A>G) was found in the gene KPHS_20140 encoding a protein with a putative unknown function. This editing site caused a missense mutation in the gene, resulting in the replacement of tyrosine (Tyr) at position 31 of the encoded protein with cysteine (Cys).

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

[0107] SEQ ID NO: 2

[0108] ATGTCTCAGCAGCAGAGCGCGGACTGGGTCAGGCTGGCGCAATCGCCCAGCCGGACGGAACGCATCGAAGCCTTCTTCGGCGGCCACGGC TACGAACCGCACCGGCATGATACCTACGCCATCGGCCAGACGATCGCCGGCGTGCAGAGCTTTCACTATCGCGGCGGACTCCAGCACAGCCTGCCCGGCGGAACGATGGTGCTTCATCCGGACGAAATACACGATGGCGAGGCGGGCACCGAGGCGGGGTTTCACTACCGTATGGTTTATATCGAACCGGCGCTGATCCAGAAGATACTCGGCGGCAGGCCACTACCGTTTATTCCCGGCGGCCTGTCGGCGGATCCGCGCCTGCGGTGCGCCGCGCTGCCGTTATTAAAAGCGGTCACGGACACCTTTGAGCCGCTGGAGGAAGAGGACGCCCTGTACGACCTGGCGCAGACCCTGGCCGTTGTCGGCGGCCAACGTTCCCGTCGCCAGGCGTTTGATTATCAGGCGGCGGAGAGGGCCCGGGAGTATATCCATGCCTGTTTTATGCAGGATATGACCCTCGACACCCTGTCGCAGGTCAGCGGCCGGGACCGCTGGAGCCTGAGCCGGGATTTCAGAACGCTGTACGGCACCAGCCCGTGGCGCTACGTCATGATGCGCCGGCTCGATTTTTGCCGCCAGCGGATGCGCGCCGGGGAACGTCTGGTGGATATTGCCGCCGACGCGGGCTTTGCCGATCAGAGCCACATGACGCGCCAGTTTATCAGCCGCTTTGGTCTCTCTCCGGGCCGCTGGCTGCGGGCGATCCGCGGCTAG

[0109] During the expression process of the KPHS_20140 gene, after A-to-I RNA editing, the amino acid sequence of the expressed protein is as follows, in which the 31st amino acid 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. Functional analysis of the KPHS_20140 gene

[0113] By alignment with the Uniprot database, the putative protein encoded by the KPHS_20140 gene is a transcription factor of the AraC / XylS family, but its specific function is not yet clear. To further clarify its function, chromatin immunoprecipitation combined with sequencing (ChIP-seq) technology was used for analysis. The specific steps are as follows:

[0114] (1) Construction of overexpression strains

[0115] To facilitate the detection of the protein encoded by the KPHS_20140 gene, a c-myc tag containing 10 amino acid residues with the sequence EQKLISEEDL (SEQ ID NO: 4) was introduced at the amino terminus of its encoded amino acid sequence. The protein with the c-myc tag was denoted as PD-myc.

[0116] In the experiment, the genome of the clinically isolated CRKP strain C789 was used as a template for the first-round PCR amplification. After purification and recovery of the product, the product was used as a template for the second-round PCR amplification. The complete c-myc tag was gradually introduced through two rounds of primer design. The strain information is shown in Table 1. The primers for the first-round PCR amplification were myc-1-F (SEQ ID NO: 5) and myc-1-R (SEQ ID NO: 6), and the primers for the second-round PCR amplification were 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 was SEQ ID NO: 9.

[0117] Table 1

[0118] Strain ST type Serotype Isolation date Sample type Carbapenemase pLVPK-like plasmid C789 11 K64 2016-4-14 Blood KPC-2 +

[0119] After synthesizing the coding gene of PD-myc, use The HiFi DNA Assembly Master Mix (#E2621, NEB) was inserted into the vector pEasy plasmid (#CT101, TransGen Biotech) by the Assembly method. After screening and verification, the synthesized plasmid was electrotransformed into 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] Take the bacterial liquid treated with H 2 O 2 Add 37% formaldehyde to the treated bacterial liquid for cross-linking, then add the corresponding volume of 2M glycine to terminate the cross-linking. After washing twice with cold PBS, resuspend with PBS solution containing protease inhibitor to obtain protein-DNA complex.

[0122] (3) Sonication of protein-DNA complex

[0123] Add nucleic acid lysis buffer to the protein-DNA complex and use an ultrasonic disruptor for sonication. The sonicated sample can be detected by agarose gel electrophoresis.

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

[0125] Use myc antibody (diluted 1:1000) to bind to the target protein, and then precipitate the protein-chromatin complex through protein G beads magnetic beads.

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

[0127] Remove the cross-linking in the antibody-protein-DNA complex, release DNA and perform purification and recovery.

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

[0129] For the immunoprecipitated DNA sample, perform end repair and add "A" according to the kit, and then purify the product with magnetic beads. Subsequently, the library is subjected to high-throughput sequencing using the Illumina platform.

[0130] (7) Data analysis

[0131] Analyze the data obtained by sequencing, including steps such as sequence alignment, peak identification, and calibration of enriched regions, to obtain information on protein-chromatin interactions, such as protein binding sites, chromatin enriched regions, etc.

[0132] The ChIP-seq results show, see Figure 1, both biological replicate samples contained Peaks within 1 kb upstream of the phnC (ANKHBFPF_04185) gene, suggesting that the KPHS_20140 gene might be involved in the transcriptional regulation of the phnC gene. And, see Figure 2 , in the KPHS_20140 gene overexpression strain compared with the wild-type strain, the expression level of the phnC gene decreased significantly. The above results indicate that the protein expressed by the KPHS_20140 gene is a transcriptional repressor of the phnC gene, so it is named pncR.

[0133] Example 2

[0134] In this example, gene editing was performed by the CRISPR / Cas9 technology to mutate the codon at position 31 of pncR from TAC to TAT. Through this synonymous mutation, a pncR gene mutant strain that does not undergo A-to-I RNA editing was constructed, that is, the amino acid sequence of the protein expressed by the pncR gene in this mutant strain is SEQ ID NO: 3. Specifically as follows:

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

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

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

[0138] (2) Synthesize homologous arms: Introduce mutations and restriction enzyme sites through PCR primers. Using the genome of strain C789 as a template, first synthesize three sub-homologous arms respectively, and then use the sub-homologous arms as templates and the upstream and downstream primers of the three sub-homologous arms as amplification primers to perform fusion PCR to synthesize homologous arms containing mutation sites. The homologous arm synthesis primers are as follows:

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

[0140] FHR-Tyr-R: GGTGCGGTTCATAGCCGTGGCC (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 homologous arm was obtained, denoted as HR-Tyr, and its nucleotide sequence is shown in SEQ ID NO: 18.

[0148] (3) Recombinant vector construction: HR-Tyr was ligated into the vector pSGKP-spe (#117234, Addgene) by double digestion with EcoR I and Xba I. Chemical transformation was performed using Trans1-T1 Phage Resistant Chemically Competent Cell (#CD501-03, TransGen Biotech), and the ligation product was transferred into Escherichia coli Trans1-T1. After resistance screening, the plasmid was extracted using FastPure Plasmid Mini Kit (#DC201-01, Vazyme) to obtain pSGKP-HR. Then, the phosphorylated spacer was ligated by single digestion with Bsa I, and finally, the vector pSGKP-spacer-HR for site-directed mutagenesis was obtained.

[0149] (4) Preparation and electrotransformation of bacterial competent cells: Prepare a 10% glycerol solution and autoclave it. The logarithmic-phase C789 bacterial solution was repeatedly centrifuged at 4°C for enrichment. Subsequently, 1 mL of pre-cooled 10% glycerol was added, and the mixture was thoroughly pipetted and mixed, then centrifuged to discard the supernatant, and this was repeated three times. Finally, the supernatant was discarded for the last time, leaving the precipitate, which was the prepared C789 competent cells. pCasKP-hph (#117232, Addgene) was electrotransformed into the competent bacteria, and after resistance screening, the strain carrying pCasKP was obtained.

[0150] (5) Preparation and electrotransformation of competent cells carrying the pCasKP strain: The preparation of competent cells carrying the pCasKP strain is basically the same as the conventional preparation of competent cells, but there are some differences. The main difference is that the pCasKP plasmid is a temperature-sensitive plasmid and needs to be cultured at 30 °C; when increasing the bacteria, the expression of the λ-Red system needs to be induced to improve the recombination efficiency. The competent cells of the C789 strain carrying pCasKP were obtained, and the previously successfully recombined pSGKP-spacer-HR plasmid was electrotransformed.

[0151] (6) Verification and screening: Single colonies growing on the screening petri dish were picked, and Cas9, SacB, and the site-directed mutation region were verified by PCR to meet the expectations, that is, colonies with successful site-directed mutation of the pncR gene were prepared, and they were named 31Tyr.

[0152] Example 3

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

[0154] (1) Hydrogen peroxide growth curve

[0155] The logarithmic growth phase bacterial solutions of the WT and 31Tyr strains were obtained by culturing respectively, diluted 100 times, and 100 μL of each sample was added to a 96-well plate, and 3 replicates were made for each strain. 4.6 mM H 2 O 2 was added to each well to make the final concentration reach 2.3 mM H 2 O 2 . Incubation was carried out at 37 °C in an enzyme-labeling instrument, and the absorbance at 600 nm was detected every 30 min for a total duration of 24 h. The results are as Figure 3 shown, showing that the growth rate of the 31Tyr strain decreased significantly.

[0156] (2) Hydrogen peroxide bactericidal experiment

[0157] The logarithmic growth phase bacterial solutions of the WT and 31Tyr strains were obtained by culturing respectively, and the bacteria were treated with 5 mM H 2 O 2 , incubated at 37 °C for 15, 30, and 60 min respectively, and then plated for counting to statistically analyze the bacterial survival rate. The results are as Figure 4 shown, showing that the survival rate of the 31Tyr strain was significantly lower than that of the WT strain at 60 min. (P = 0.0075)

[0158] (3) Total antioxidant capacity detection

[0159] Using the Total Antioxidant Capacity Detection Kit (#S0121, Beyotime), the total antioxidant capacity of WT and 31Tyr strains under 2.3 mM H 2 O 2 pressure was detected. The results are as Figure 5 shown, indicating that the total antioxidant capacity of the 31Tyr strain was significantly lower than that of the WT strain under 2.3 mM H 2 O 2 pressure. (P = 0.0180)

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

[0161] Example 4

[0162] On the basis of Example 3, in this example, the pathogenicity of WT and 31Tyr strains was further analyzed by the intracellular survival experiment of immortalized mouse bone marrow-derived macrophages (iBMDM). The specific steps are as follows:

[0163] The iBMDM cells were seeded in 24-well cell culture plates (#3524, Corning) at a density of 1×10 6 cells / well to form a monolayer of cells. At the same time, LPS (100 ng / mL) and IFN-γ (20 ng / mL) were added to stimulate the cells. The WT and 31Tyr strains were cultured to the logarithmic growth phase and the bacterial suspension was adjusted to an OD600 of 0.6. Subsequently, 5×10 6 CFU of bacteria (MOI = 5) and 10% mouse serum (#ZI807-1, Zhuangmeng) were co-cultured with the iBMDM cells in an environment of 37°C and 5% CO 2 for 30 min. Then, 500 μg / mL of hygromycin was used for 30 min to kill extracellular bacteria, and the cells were lysed with 1% Triton X-100 (#SLCF5969, Sigma-Aldrich), and then diluted and cultured on LB agar plates to count the intracellular bacteria, which was recorded as the intracellular bacterial load at 0 h. On this basis, the survival ability of bacteria in macrophages was evaluated by extending the incubation time before lysis (1, 2 h). The results are as Figure 6 shown, indicating that the number of intracellular bacteria in iBMDM cells of the 31Tyr strain was significantly less than that of the WT strain at 2 h (P = 0.0224), suggesting that the ability of the 31Tyr strain to resist killing by iBMDM cells decreased, that is, its pathogenicity weakened.

[0164] Example 5

[0165] In this example, the pncR gene was knocked out by CRISPR / Cas9 technology to construct a mutant strain without the pncR gene. The specific steps are as follows:

[0166] (1) The same spacer sequences as those used for site-directed mutagenesis of the pncR gene in Example 2 were adopted, namely SEQ ID NO: 10 and SEQ ID NO: 11.

[0167] (2) Synthesis of homologous arms: Restriction enzyme sites were introduced through PCR primers to synthesize the upstream homologous arm (UHR) and the downstream homologous arm (DHR). Then, using the upstream and downstream homologous arms as templates and the primers at both ends of the upstream and downstream homologous arms as amplification primers, fusion PCR was performed to synthesize the homologous arm without the target gene pncR, which was used as the template for subsequent homologous recombination repair. The primers for synthesizing the upstream and downstream homologous arms are shown 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, denoted as HR-△pncR, was obtained, and its nucleotide sequence is as shown in SEQ ID NO: 21.

[0173] (3) Construction of the recombinant vector: HR-△pncR was ligated into the vector pSGKP-spe (#117234, Addgene) by double digestion with EcoR I and Xba I. Chemical transformation was performed using Trans1-T1 Phage Resistant Chemically Competent Cell (#CD501-03, TransGen Biotech), and the ligation product was transferred into Escherichia coli Trans1-T1. After resistance screening, the plasmid was extracted using FastPure Plasmid Mini Kit (#DC201-01, Vazyme) to obtain pSGKP-HR2. Then, the phosphorylated spacer was ligated by single digestion with Bsa I, and finally, the vector pSGKP-spacer-HR2 for site-directed mutagenesis was obtained.

[0174] (4) The preparation of competent cells of the strain and the electrotransformation process are the same as those described in Example 2.

[0175] (5) The preparation of competent cells of the strain carrying pCasKP is the same as that described in Example 2. The competent cells of C789 strain carrying pCasKP are obtained, and the pSGKP-spacer-HR2 plasmid recombined in step (3) is electrotransformed.

[0176] (6) Verification and screening: Pick the monoclonal colonies growing on the screening plate, and verify by PCR that Cas9, SacB and the knockout region all meet the expectations, that is, the colonies with successful pncR gene knockout are prepared, and they are named △pncR strains.

[0177] Example 6

[0178] In this example, the growth curves of wild-type CRKP (i.e., C789 strain, denoted as WT) and pncR gene knockout strain (i.e., △pncR strain, denoted as △pncR) in hydrogen peroxide were analyzed to characterize their oxidative stress adaptability. Specifically as follows:

[0179] The logarithmic growth phase bacterial solutions of WT and △pncR strains are cultured separately, diluted 100 times, and 100 μL of each sample is added to a 96-well plate, with 3 replicates for each strain. 5 mM H 2 O 2 is added to make the final concentration reach 2.5 mM H 2 O 2 . Incubate at 37 °C in an enzyme-labeling instrument, and measure the absorbance at 600 nm every 30 min for a total duration of 24 h. The results are as Figure 7 shown, showing that the growth rate of the △pncR strain has decreased significantly.

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

[0181] The above is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention 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 drug for treating carbapenem-resistant Klebsiella pneumoniae, characterized in that: The target serves as the target of action of the drug; The target includes at least one of the following (A1) to (A4): (A1) pncR protein variant, whose amino acid sequence is shown in SEQ ID NO: 1; (A2) an amino acid site located at position 31 of a pncR protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1; (A3) pncR gene, wherein the pncR gene is KPHS_20140 gene; (A4) at least one of the 91st, 92nd and 93rd nucleotide positions of the pncR gene, wherein the pncR gene is the KPHS_20140 gene.

3. The use according to claim 2, characterized in that The nucleotide sequence of the KPHS_20140 gene is shown in SEQ ID NO:

2.

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

5. A reagent, characterized in that The reagent is used 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.

6. The reagent according to claim 5, characterized in that The reagent acts on the pncR gene, thereby reducing or completely inhibiting the expression of the pncR protein variant; the pncR gene is the KPHS_20140 gene.

7. The reagent according to claim 5 or 6, characterized in that The reagent acts on the pncR gene to cause the pncR gene to mutate, and the protein expressed by the mutated pncR gene has an amino acid sequence different from that of SEQ ID NO: 1; Preferably, 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; Preferably, the amino acid sequence of the protein expressed by the mutated pncR gene is as shown in SEQ ID NO: 3; Preferably, the reagent comprises a first primer combination; the first primer combination comprises a spacer sequence primer set and a homology arm primer set: Spacer sequence primer set: Spacer-FW, whose amino acid sequence is shown in SEQ ID NO: 10; Spacer-RV, whose amino acid sequence is shown in SEQ ID NO: 11; The first homology arm primer set: its amino acid sequence is shown in SEQ ID NO: 12; Its amino acid sequence is shown in SEQ ID NO: 13; The second homology arm primer set: its amino acid sequence is shown in SEQ ID NO: 14; its amino acid sequence is shown in SEQ ID NO: 15; The third homology arm primer set: its amino acid sequence is shown in SEQ ID NO: 16; Its amino acid sequence is shown in SEQ ID NO: 17; Preferably, the reagent also includes a pSGKP-spe vector and a pCasKP-hph vector.

8. The reagent according to claim 5 or 6, characterized in that The reagent acts on the pncR gene to knock out the pncR gene; Preferably, the reagent comprises a second primer combination; the second primer combination comprises a spacer sequence primer set and a homology arm primer set: Spacer sequence primer set: Spacer-FW, whose amino acid sequence is shown in SEQ ID NO: 10; Spacer-RV, whose amino acid sequence is shown in SEQ ID NO: 11; The fourth homology arm primer set: the amino acid sequence of which is shown in SEQ ID NO: 12; Its amino acid sequence is shown in SEQ ID NO: 19; The fifth homology arm primer set: the amino acid sequence of which is shown in SEQ ID NO: 20; Its amino acid sequence is shown in SEQ ID NO: 17; Preferably, the reagent also includes a pSGKP-spe vector and a pCasKP-hph vector.

9. Use of the reagent according to any one of claims 5 to 8 in the preparation of a drug for treating carbapenem-resistant Klebsiella pneumoniae.

10. The reagent according to claim 9, characterized in that The drug is used for at least one of the following (B1) to (B3): (B1) Reduce the antioxidant capacity of carbapenem-resistant Klebsiella pneumoniae; (B2) Reduce the drug resistance of carbapenem-resistant Klebsiella pneumoniae; (B3) Reduce the pathogenicity of carbapenem-resistant Klebsiella pneumoniae.

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