Application of virK gene in regulation of virulence of klebsiella pneumoniae

Abstract

The application belongs to the field of genetic engineering and medical engineering, and discloses virK Application of gene in regulation of Klebsiella pneumoniae virulence. The application finds that the expression level of the gene is significantly increased in polymyxin-resistant mutant strains through transcriptome analysis, virK Further research shows that the expression of the gene is regulated by the PhoP / PhoQ two-component regulatory system. The application constructs virK Gene deletion mutant strains and complementary strains, and compares the virulence difference of the strains in an animal infection model, finds that virK The virulence of the deletion strain is significantly reduced, and the complementary strain can restore the virulence phenotype, indicating that virK The gene plays an important role in the regulation of Klebsiella pneumoniae virulence. The application first finds that PhoP can regulate virK Gene expression, and affects the virulence phenotype of Klebsiella pneumoniae through the regulation pathway, thereby revealing a new virulence regulation mechanism, and providing a theoretical basis for subsequent targeted intervention.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and medical engineering, specifically, it relates to... virK Application of genes in the regulation of virulence in Klebsiella pneumoniae. Background Technology

[0002] In recent years, the clinical prevalence of multidrug-resistant Gram-negative bacteria has become a significant challenge in global public health. Among them, Klebsiella pneumoniae... Klebsiella pneumoniae As an important opportunistic pathogen, it is one of the main pathogens of hospital-acquired infections, especially in intensive care units where it can cause serious diseases such as pneumonia, sepsis and urinary tract infections.

[0003] Polymyxin antibiotics (such as polymyxin) are considered a treatment for carbapenem resistance. Klebsiella pneumoniae Drugs are the “last line of defense” against infection [1]. However, with the emergence of polymyxin-resistant strains, their therapeutic efficacy is seriously threatened.

[0004] In Gram-negative bacteria, the PhoP / PhoQ two-component regulatory system is an important signaling pathway for regulating lipopolysaccharide modification, antimicrobial peptide tolerance, and virulence expression [2]. PhoQ, as a membrane receptor, undergoes autophosphorylation upon sensing stimuli such as low magnesium or antimicrobial peptides, and transfers its phosphate group to the transcriptional regulator PhoP, thereby activating or inhibiting the expression of downstream genes. Previous studies have shown that PhoP participates in regulating the expression of lipopolysaccharide modification genes, thereby enhancing bacterial tolerance to polymyxins [3,4].

[0005] However, there is currently a lack of systematic research on whether PhoP affects bacterial virulence, membrane stability, and cell morphology by regulating other unknown membrane proteins. Especially in the context of drug resistance, the existence of new molecular linkage mechanisms between drug resistance and virulence remains unclear.

[0006] Currently, research on drug resistance mechanisms in Gram-negative bacteria mainly focuses on the signal transduction mechanisms of lipopolysaccharide (LPS) modification pathways and two-component regulatory systems. For example, the PhoP / PhoQ system has been shown to participate in regulating the expression of LPS modification-related genes, thereby enhancing bacterial resistance to antimicrobial peptides such as polymyxins.

[0007] However, existing research mainly focuses on known lipopolysaccharide (LPS) modification gene clusters and their drug resistance phenotypes, with insufficient attention paid to whether PhoP participates in bacterial virulence regulation by modulating other unknown membrane protein genes. Especially in the context of polymyxin resistance, the molecular mechanisms underlying whether increased resistance is accompanied by altered virulence remain incompletely elucidated. Summary of the Invention

[0008] The purpose of this invention is to providevirK Application of genes in the regulation of virulence in Klebsiella pneumoniae.

[0009] In polymyxin-resistant Klebsiella pneumoniae Klebsiella pneumoniae Transcriptome analysis of the strain revealed significantly high expression of a conserved membrane protein encoding gene (kpn2146_RS17285) belonging to the VirK / YbjX family. This gene encodes a conserved membrane protein, which was named... virK ( vi rulence r equired for K lebsiella pneumoniae VirK protein homologs are conserved in a variety of Gram-negative bacteria, but their specific biological functions remain unclear. Figure 2 Existing research largely focuses on the potential involvement of its homologs in membrane protein homeostasis regulation or envelope stress responses, but direct evidence is lacking. Therefore, it is necessary to clarify the role of the PhoP-VirK regulatory axis in bacterial virulence regulation and explore its potential application value as a target for anti-infection intervention or virulence regulation.

[0010] VirK / YbjX family proteins are conserved in a variety of Gram-negative bacteria. Figure 3 Existing technologies have limited understanding of the function of this type of protein, and direct experimental evidence regarding its role in virulence regulation is lacking. Previous reports have largely speculated on its potential involvement in envelope stress or membrane protein homeostasis, but its involvement in virulence expression regulation remains unclear. Therefore, this invention aims to clarify: 1) whether PhoP regulates... virK Genes are involved in the regulation of bacterial virulence; 2) Establish the functional link between the PhoP-VirK regulatory axis and virulence; 3) Provide a way to... virK Technical solutions for potential antiviral intervention targets.

[0011] To achieve the objectives of this invention, in a first aspect, this invention provides a missing... virK Genes in reducing Klebsiella pneumoniae ( Klebsiella pneumoniae Application of toxicity.

[0012] The present invention virK The gene is the gene that encodes either (a) or (b) the following protein: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or, (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0013] Furthermore, the term "deficit" refers to knockout or non-expression.

[0014] Secondly, the present invention provides virK The application of genes as drug targets in the preparation of drugs or compositions that reduce the virulence of Klebsiella pneumoniae; the polypeptide antibiotic is polymyxin.

[0015] Preferably, the Klebsiella pneumoniae is ATCC BAA-2146.

[0016] Thirdly, the present invention provides virK Application of genes or their encoded proteins in the preparation of products for screening antiviral drugs against Klebsiella pneumoniae.

[0017] Fourthly, this invention provides PhoP protein and virK Application of gene promoter binding regulatory axes in the preparation of products for screening antiviral drugs against Klebsiella pneumoniae.

[0018] Furthermore, the aforementioned virK The nucleotide targeting sequence in the gene promoter that specifically binds to the PhoP protein is: 5′-ACACCTCAATCAATTTTAA-3′.

[0019] Fifthly, the present invention provides a method for screening potential antiviral drugs for Klebsiella pneumoniae, comprising the following steps: (1) In the presence of PhoP protein and virK Add candidate substances to the reaction system of gene promoter fragments; (2) Detect the effect of the candidate substance on PhoP protein and virK The influence of gene promoter binding ability; (3) If the candidate substance can inhibit PhoP protein and virK If the gene promoter is bound, it is determined to be a potential antiviral drug for Klebsiella pneumoniae.

[0020] Among them, the virK The gene promoter fragment contains the nucleotide sequence: 5′-ACACCTCAATCAATTTTAA-3′.

[0021] Sixthly, the present invention provides a detection method. virK Application of gene expression level reagents in the preparation of kits for in vitro assessment or prediction of polymyxin-resistant Klebsiella pneumoniae virulence levels.

[0022] Furthermore, the reagents include those for specific amplification. virK Primers for genes.

[0023] Furthermore, the assessment or prediction is based on the detection of polymyxin-resistant Klebsiella pneumoniae. virK Upregulation of gene expression levels indicates enhanced virulence of the polymyxin-resistant Klebsiella pneumoniae.

[0024] Seventhly, the present invention provides a missing... virK Application of gene in reducing the resistance of Klebsiella pneumoniae to polypeptide antibiotics; the polypeptide antibiotic is polymyxin.

[0025] Eighthly, the present invention provides virK The application of genes as drug targets in the preparation of drugs or compositions that reduce the resistance of Klebsiella pneumoniae to polypeptide antibiotics; wherein the polypeptide antibiotic is polymyxin.

[0026] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) First elucidation of direct regulation by PhoP virK molecular mechanism This invention, through genetics, molecular biology, and protein-DNA interaction experiments, is the first to demonstrate that the PhoP protein can directly bind to... virK It promotes specific regions of the promoter and positively regulates their expression. The clarification of this mechanism provides a theoretical basis for subsequent targeted interventions.

[0027] Unlike existing methods that only focus on expression correlation analysis, this invention establishes a clear regulatory relationship at the molecular level based on direct binding evidence (EMSA), in vivo enrichment evidence (ChIP-seq), and precise binding site localization (DNaseI footprinting analysis).

[0028] (ii) First time clearly defined virK Positive regulator of Klebsiella pneumoniae virulence This invention is achieved through virK Gene knockout and animal infection model validation confirmed virK The deletion significantly reduced the virulence of the strain, and the virulence was restored after the deletion was corrected; this demonstrates that... virK It directly participates in the bacterial pathogenesis process. This result is a breakthrough. virK The recognition of proteins that were previously considered to have unknown functions has been clarified in terms of their toxicological properties.

[0029] (III) Revealing the regulatory relationship between drug resistance and enhanced toxicity This invention discovers mgrB Truncation leads to persistent Phop activation; Phop upregulation virK ; virK This study promotes increased virulence, revealing a molecular mechanism that may accompany polymyxin resistance. This finding has significant implications for clinical risk assessment of drug-resistant bacteria.

[0030] (iv) Providing new targets for antiviral intervention The PhoP-VirK regulatory axis established in this invention provides a new molecular target for antiviral therapy, which can be used for: 1) inhibiting PhoP and... virK 1) Promoter binding; 2) Inhibition virK Expression; 3) Development of small molecule inhibitors targeting VirK; 4) Establishment of a system based on virK A model for predicting virulence based on expression levels.

[0031] Compared with the traditional strategy of directly killing bacteria with antibiotics, the antiviral regulation approach proposed in this invention is: (1) not directly applying survival pressure; (2) theoretically reducing drug resistance selection pressure; and (3) beneficial in delaying the development of drug resistance.

[0032] (v) The technical effects are stable and repeatable. The technical effects of this invention have been cross-validated by multiple molecular biology methods and verified by animal models. The regulatory relationship is clear, the logical closed loop is complete, and it is reproducible and scalable. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the PhoP-VirK regulation mechanism of the present invention.

[0034] Figure 2 According to the GenBank database, VirK is homologous to other VirK / YbjX families (the results were visualized using ESPript 3.0).

[0035] Figure 3 The VirK homologs in the VirK / YbjX family of this invention have the same conserved amino acid sequence at the α-helix.

[0036] Figure 4 For the present invention virK Schematic diagram of the upstream region sequence. The predicted promoter is marked in blue, and the PhoP protein binding site is marked in red.

[0037] Figure 5 The secondary structure of the VirK protein in this invention was predicted using PSIPRED. α-helices and β-sheets are represented by pink and yellow cylinders, respectively.

[0038] Figure 6 The present invention uses the VirK protein structure predicted by AlphaFold (AFDB accession number: AF-A6TB10-F1).

[0039] Figure 7 In a preferred embodiment of the present invention, RT-qPCR was used to detect different strains of [the virus / organism]. mgrB , phoP and virK Changes in relative gene expression levels.

[0040] Figure 8 As a preferred embodiment of the present invention, the IGV genome browser demonstrates Phop in... K. pneumoniae Whole genome virK The binding region surrounding the gene. IP: Experimental group, i.e., immunoprecipitation (IP) sample; IN: Control group, i.e., input (IN) sample; the numbers indicate three independent replicate experiments.

[0041] Figure 9 The SDS-PAGE electrophoresis results of purified His-PhoP protein in the preferred embodiment of the present invention are shown. The amount of protein loaded in each lane decreases sequentially from left to right. The His-PhoP protein is represented in the red dashed box.

[0042] Figure 10 In a preferred embodiment of the present invention, EMSA experiments were used to detect Phop and virK The promoter binding capacity; green arrows indicate bound DNA fragments, and red arrows indicate free DNA fragments.

[0043] Figure 11 In a preferred embodiment of the present invention, DNase I footprint analysis was used to detect Phop and virK Promoter binding. The region shown in the black dashed box (ACACCTCAATCAATTTTAA) is a sequence protected by PhoP protein from DNase I cleavage.

[0044] Figure 12 In a preferred embodiment of the present invention, bacterial inner and outer membrane proteins were separated by ultracentrifugation with sucrose concentration gradient. The upper dark region is bacterial inner membrane protein (IM), and the lower dark region is bacterial outer membrane protein (OM).

[0045] Figure 13 In a preferred embodiment of the present invention, Western blotting was used to detect the composition of inner and outer membrane proteins. The upper layer represents the separated inner and outer membrane proteins, and His-VirK was detected using an anti-His antibody. His-VirK protein was detected only in the outer membrane protein sample, which proves that VirK exists in the bacterial outer membrane. The lower layer serves as a control, representing the separated inner and outer membrane proteins, and OmpA protein was detected using an anti-OmpA antibody. Similarly, OmpA protein was detected only in the outer membrane protein sample. This result is consistent with the fact that OmpA exists in the bacterial outer membrane. Detailed Implementation

[0046] This invention aims to provide a PhoP regulation method. virK The technical solution for gene expression is clearly defined. virKThe study aims to establish a functional link between the PhoP-VirK regulatory axis and bacterial virulence, thereby addressing the lack of clarity regarding the mechanisms of drug resistance regulation and virulence changes in existing technologies, and providing new molecular targets for antiviral intervention.

[0047] The present invention adopts the following technical solution: This invention provides a PhoP regulation virK Regulatory mechanisms of gene expression affecting the virulence of Klebsiella pneumoniae and their application methods.

[0048] In this invention virK The gene originates from Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC BAA-2146 strain genome ( Figure 4 This gene encodes a membrane-associated protein belonging to the VirK / YbjX family. This invention... virK The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. The predicted secondary and tertiary structures of the VirK protein in this invention are as follows: Figure 5 and Figure 6 As shown. The present invention also includes protein sequences having the same or similar biological functions formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:2, and nucleotide sequences encoding these amino acid sequences.

[0049] Transcriptome analysis revealed that in polymyxin-resistant mutant strains, virK Gene expression levels were significantly elevated. Further research indicated that gene expression was regulated by the PhoP / PhoQ two-component regulatory system. PhoP, as an important transcriptional regulator, can regulate the expression of various genes related to bacterial physiological functions, but whether it regulates... virK The genes and their biological significance were previously unclear.

[0050] This invention constructs phoP Gene mutant strains and related complementary strains were detected. virK Changes in gene expression levels indicate that in phoP Missing conditions virK Gene expression decreased significantly, while phoP Its expression level was restored after complementation, indicating that virK Gene expression is positively regulated by PhoP.

[0051] Furthermore, the present invention constructs virK Gene deletion mutants and their complementary strains were studied, and their virulence differences were compared in animal infection models. virKThe virulence of the deletion strains was significantly reduced, while the complementary strains were able to restore their virulence phenotype, indicating that... virK Genes play an important role in the regulation of virulence in Klebsiella pneumoniae.

[0052] Specifically, the technical solution of the present invention includes: First, using Klebsiella pneumoniae ATCC BAA-2146 strain as the research object, its genome was amplified to obtain... virK Genes were identified, and their sequences were determined and analyzed.

[0053] Secondly, constructing through homologous recombination method phoP Gene deletion mutants and virK Gene deletion mutants were developed, and corresponding gene-complementary strains were constructed.

[0054] Subsequently, real-time quantitative PCR was used to detect the presence of different strains. virK The transcriptional expression level of the gene was used to determine the effect of PhoP on... virK The regulatory role of gene expression.

[0055] Furthermore, by establishing a mouse infection model, the wild-type strain, virK The difference in virulence between deletion and complementary strains was used to verify... virK The function of genes in the regulation of Klebsiella pneumoniae virulence.

[0056] This invention is the first to discover that PhoP can regulate virK Gene expression was investigated, and this regulatory pathway influenced the virulence phenotype of Klebsiella pneumoniae, revealing a novel virulence regulation mechanism.

[0057] This invention is the first to clearly demonstrate the direct regulation of PhoP. virK Gene expression; PhoP protein can directly bind virK It promotes the activation of subregions and positively regulates their expression.

[0058] This invention establishes the PhoP-VirK virulence regulation axis; mgrB Inactivation → PhoP activation → virK Upregulation of expression → increased bacterial virulence.

[0059] In this invention, mgrB The protein encoded by the gene has the reference sequence number WP_002911375.1 in NCBI.

[0060] This invention is demonstrated for the first time. virK It is a positive regulator of the virulence of Klebsiella pneumoniae.

[0061] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0062] Unless otherwise specified, the percentage sign "%" used in this invention refers to mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0063] The materials used in the following embodiments are shown in Tables 1 to 5: Table 1. Strains and plasmids used in this invention

[0064] Note: Plasmids pCasKP-apr and pSGKP-spe were purchased from Addgene (https: / / www.addgene.org / ).

[0065] Table 2 Primers used in this invention

[0066] Table 3. Knockout in this invention phoP Primers used in gene generation

[0067] Note: ssDNA- phoP -1, ssDNA- phoP -2 is Kpn2146Δ phoP and Mut-SΔ phoP Nucleotide sequences involved in single-stranded DNA (ssDNA) repair templates.

[0068] Table 4. Knockout in this invention virK Primers used in gene generation

[0069] Note: ssDNA- virk -1, ssDNA- virk -2 is Kpn2146Δ virK and Mut-SΔ virK Nucleotide sequences involved in single-stranded DNA (ssDNA) repair templates.

[0070] Table 5 DNase I Footprint Measurement Probe

[0071] Example 1 phoP Construction of gene deletion mutants Following the previously described method, the pCasKP-pSGKP genome editing system was successfully used to obtain polymyxin S2 resistant strain Kpn2146 (Mut-S). phoP Gene deletion mutants [5]. In wild-type Kpn2146 phoP Gene knockout was first achieved by transforming wild-type Kpn2146 cells with the pCasKP-apr plasmid via electroporation in 2 mm cuvettes using parameters of 2500 V, 200 Ω, and 25 μF. Subsequently, cells containing the pCasKP-apr plasmid were induced with L-arabinose to prepare competent cells. Annealed cells were then assembled using GoldenGate. phoP The spacer region (designed by CRISPOR[8]) oligonucleotides were inserted into the BsaI site of the pSGKP-spe plasmid. Then, the introduced oligonucleotides were... phoP The pSGKP-spe plasmid of the spacer and the corresponding 90 nucleotide (nt) single-stranded DNA (ssDNA) repair template were co-electroplated into competent cells containing pCasKP-apr. Colonies were selected at 30°C on LB agar plates containing apramycin (50 μg / mL) and spectinomycin (1 mg / mL). Genetic and phenotypic analyses by PCR and sequencing confirmed the presence of pSGKP-spe in Mut-S cells. phoP Successful deletion of a gene is denoted as Mut-S-Δ. phoP .get phoP After the deletion mutant was established, pCasKP-apr and [other mutants] were lost by culturing the cells at 37°C with 5% (wt / vol) sucrose. phoP pSGKP-spe plasmid introduced by spacers.

[0072] Kpn2146Δ was created using the same method. virK and Mut-SΔ [[ID=1l7]]virK strains.

[0073] Example 2 phoP Gene complementation strain construction Use primers phoP -CF and phoP -CR amplifies the full-length polymyxin-sensitive Kpn2146 via PCR. phoP Genes were ligated from pTOPO-mgrB (Apr) using primers pTOPO-PF and pTOPO-PR. rThe amplified fragment was assembled using Gibson Assembly MasterMix (catalog number E2611S, NEB, Gene Company, China) according to the manufacturer's instructions, and transformed into electroporated E. coli top 10 strains (Tsingke Biotechnology, China) via electroporation. Transformants were selected by overnight incubation at 37°C on Mueller-Hinton agar supplemented with apramycin (50 mg / L, Cat. HY-B1329, MedChemExpress, China). Plasmids were isolated using the RapidLyse Plasmid Mini Kit (catalog number DC211-02, Vazyme, China) and confirmed by KBSeq (Sangon, China), and then the correct pTOPO- was transferred via electroporation. phoP (Apr) r The plasmids were introduced into Mut-S. Transformants were selected on plates containing 50 mg / L apramycin. pTOPO-flag- was constructed. phoP (Apr) r The plasmid was verified by ChIP-seq experiments. phoP right virk Regulation of gene expression. Using primers. phoP -FF and phoP -FR was generated by PCR amplification of the full-length polymyxin-sensitive Kpn2146. phoP Genes, and linked from pTOPO- through primers pTOPO-PFF and pTOPO-PFR. mgrB (Apr) r The amplified fragment is then electroporated to obtain the correct pTOPO-flag verified by sequencing. phoP (Apr) r The plasmid Kpn2146 was introduced to construct Kpn2146-Flag- phoP strains.

[0074] Example 3 virK and virK Construction of strains with -his6 gene complementation Use primers virK -CF and virK -CR amplifies the full-length polymyxin-sensitive Kpn2146 via PCR. virK Genes were ligated from pTOPO-mgrB (Apr) using primers pEAST-VF and pEASY-VR. r The amplified fragment was selected on a plate containing 50 mg / L alpramycin.virK (Apr r Transformants of ) were obtained. Using 5'NcoI and 3'XhoI restriction enzymes, the transformants were... virK The gene was cloned into plasmid pET28a, producing a gene carrying a C-terminal 6×His marker. virK (pET28a- virK The pET28a plasmid (-his6) was used. Sequencing from the TSINGKECorporation confirmed the presence of pET28a-... virK -His6 plasmid. Then use primers. virK -CF and virK -CR is derived from pET28a- via PCR amplification. virK -Full length of His6 plasmid virK -His6 region, then connected to pTOPO-mgrB (Apr r The amplified fragment was constructed using primers pEASY-VHF and pEASY-VHR. virK -His6 (Apr) r Its program and construction of pTOPO- phoP (Apr) r Similar to pTOPO-. Correct pTOPO- was verified through sequencing. virK (Apr) r ) and pEASY- virK -his6 (Apr r The plasmid was introduced into Kpn2146-Δ by electroporation. virK .

[0075] Example 4 Real-time quantitative PCR The HiScrip tII one-step qRT-PCR SYBR Green kit (catalog number Q221, Vazyme Biotech, China) was used in qTOWER. 3 Quantitative real-time PCR (qPCR) instrument mgrB , phoP , virK RNA levels of 23S rRNA (used as an internal control) were measured. All specific primers were designed based on the genome of Kpn2146 (NCBI reference sequence: NZ_CP006659.2). Relative RNA expression levels were measured according to a 2... ^-ΔΔCt The method was used for calculation and normalized using 23S rRNA levels as a standard. RT-qPCR was performed using three biological replicates and three technical replicates.

[0076] from Figure 7The experimental results show that, compared with the wild-type Kpn2146 strain, the Mut-S strain... phoP and virK The expression of these substances increased approximately 4-fold and 22-fold, respectively. mgrB The expression is down-modified ( Figure 7 A). Compared to Mut-S, Mut-S supplements... mgrB back virK The expression decreased ( Figure 7 B). Knockout in Mut-S phoP This will lead to virK Decreased expression ( Figure 7 C), and supplement mgrB It will not change virK The expression ( Figure 7 D); Conversely, supplement phoP This will lead to virK Elevated expression, at the same time phoP The expression also increased ( Figure 7 E).

[0077] Example 5 ChIP-seq Analysis Kpn2146-Flag- phoPThe strain was washed twice with pre-cooled PBS buffer, cross-linked with 1% formaldehyde for 10 minutes at room temperature, and then the cross-linking reaction was terminated by adding glycine (final concentration 125 mmol / L). Subsequently, the sample was lysed using a lysis buffer containing a mixture of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS, and 1× protease inhibitor, and chromatin was obtained on ice. The chromatin was sonicated to obtain soluble, cleaved chromatin (average DNA fragment length 200–500 bp). 20 μL of chromatin was stored at -20°C as input DNA, and 100 μL of chromatin was immunoprecipitated using an HRP-conjugated anti-Flag antibody. 10 μg of antibody was added to each immunoprecipitation reaction, and the mixture was incubated overnight at 4°C. The next day, 30 μL of protein magnetic beads were added, and incubation continued for 3 hours. The following washing steps were then performed sequentially: First, wash with a buffer containing 20 mM Tris / HCl (pH=8.1), 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS; second, wash with a buffer containing 10 mM Tris / HCl (pH=8.1), 250 mM LiCl, 1 mM EDTA, 1% NP-40, and 1% sodium deoxycholate; followed by two washes with TE buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA). The material bound to the magnetic beads was eluted with 300 μL of elution buffer (100 mM NaHCO3, 1% SDS). The mixture was then treated with RNase A (final concentration 8 μg / mL) at 65°C for 6 hours, followed by incubation overnight at 45°C with proteinase K (final concentration 345 μg / mL). DNA obtained from immunoprecipitation was used to construct sequencing libraries according to the INEXTFLEX® ChIP-Seq LibraryPrep Kit for Illumina® Sequencing (Cat. NOVA-5143-02, BiooScientific) manual, and sequencing was performed on the Illumina NovaSeq 6000 platform in PE150 mode. Low-quality reads were filtered using Trimmomatic (version 0.36). Clean reads were aligned to the correct order using Bwa (version 0.7.15). K. pneumoniaeThe (ATCC®BAA-2146™) genome was analyzed. Potential PCR repetitive sequences were removed using Samtools (version 1.3.1). Peaks were identified using MACS2 software (version 2.1.1.20160309) with default parameters (bandwidth set to 300 bp; modelfold set to 5-50; q-value threshold set to 0.05). Peaks were assigned to genes whose midpoints were closest to the transcription start site (TSS). Motif prediction of peak regions was performed using HOMER (version 3) with default settings and a maximum motif length of 12 bp. GO (GeneOntology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were performed using ClusterProfiler in R packages. GO and KEGG enrichment analyses were calculated based on hypergeometric distributions with a q-value threshold set to 0.05.

[0078] from Figure 8 The experimental results show that in all three independent experiments, virK The detection of partially overlapping peaks (P1, P2, and P3) in the gene promoter region indicates that the PhoP protein can bind to... virK Gene promoter region.

[0079] Example 6 phoP Cloning and recombinant protein purification Will phoP The gene was cloned into plasmid pET28a using 5' NdeI and 3' XhoI restriction endonucleases, resulting in a gene with an N-terminal 6×Hi tag. phoP Expression plasmid (pET28a-) phoP -his6). This plasmid was sequenced and verified by GENEWIZ (Suzhou, China) and then transformed into... E. coli BL21(DE3) strain. Carrying pET28a- phoP -his6 E. coli BL21(DE3) was cultured in LB medium until OD 600The concentration was initially set to 0.6, then 0.2 mM isopropyl β-D-thiogalactoside (IPTG) was added for induction, and the cells were cultured at 18°C ​​and 160 rpm with shaking for 5 hours. The cells were collected by centrifugation at 3500 × g (4°C, 20 min) and resuspended in pre-chilled lysis buffer (50 mM Na₂HPO₄, 50 mM NaH₂PO₄, 300 mM NaCl). The cells were then sonicated on ice (3 seconds on, 3 seconds off, 5 min total) and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was collected, mixed with PureCubeNi-NTA agarose (CubeBiotech, Germany), and incubated with gentle shaking at 4°C for 2 hours. The cells were then washed three times with lysis buffer containing 20 mM imidazole, followed by elution of the binding protein with lysis buffer containing 300 mM imidazole. The purified recombinant protein was desalted using Amicon® Ultra centrifugal ultrafiltration tubes (Cat. UFC9010, Sigma, USA), and the quality of the purified Phop protein was assessed by SDS-PAGE and Coomassie Brilliant Blue R-250 staining (Cat. ZD305A, ZOMANBIO, China).

[0080] from Figure 9 The experimental results show that the above method can yield His-PhoP protein with high purity, which meets the experimental requirements.

[0081] Example 7: Electrophoretic Mobility Variation Analysis (EMSA) Amplification using specific primers virK The upstream region of the gene (-464 to 0 bp) and a 16S rDNA fragment (251 bp, as a negative control) were included. In the binding reaction system, the DNA fragment (0.3 pM) was incubated with 6×His-labeled PhoP protein in a buffer containing 10 mM Tris-HCl (pH 8.0), 25 mM MgCl2, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.01% NonidetP40, and 10% glycerol at room temperature for 20 minutes. The reaction mixture was then loaded onto an 8% non-denaturing PAGE gel and electrophoresed at 100 V in native PAGE buffer. Bands were detected using the Bio-Rad ChemiDoc™ XRS+ imaging system.

[0082] from Figure 10 The experimental results show that PhoP protein can effectively bind in vitro in a concentration-dependent manner (0-18 μg). virK Promoter region. In contrast, the negative control 16S rRNA sequence showed no significant binding even at the highest concentration tested.

[0083] Example 8: DNase I Footprint Analysis Experiment Using primers virK -chip-F / virK -chip-R amplification virK The target DNA fragment in the promoter region was identified, and a 5-carboxyfluorescein (FAM)-labeled fluorescent probe was prepared using the amplified product as a template. The probe was incubated with 0, 2, 5, and 10 μg of Flag-PhoP protein at 25°C for 30 min, respectively. Subsequently, 5 μL of a solution containing 10U DNase I (Cat.EN0523, ThermoFisher) and 1 mol of freshly prepared CaCl2 was added, and incubation continued at 37°C for 55 sec. Immediately after the reaction, 10 μL of 0.5M EDTA was added to terminate the reaction (mix thoroughly within 65 sec), and the reaction was inactivated at 65°C for 10 min. The reaction product was extracted with phenol / chloroform and recovered by ethanol precipitation. The precipitate was dissolved in 30 μL of MiniQ water, and then sequenced using a 3130XL DNA analyzer by Sangon Biotech (Shanghai) Co., Ltd.

[0084] from Figure 11 The experimental results show that, virK A DNase I hypersensitive site was detected in the region from -96 bp to -77 bp upstream of the ATG start codon (sequence: ACACCTCAATCAATTTTAA), suggesting that this region is a regulatory region protected by the PhoP protein. This site is consistent with the consensus sequence identified by ChIP-seq data.

[0085] Example 9: EDTA-free sucrose density gradient centrifugation to separate the inner and outer membranes Bacterial cells were collected by centrifugation at 5,000 × g for 10 minutes at 4 °C. The pellet was resuspended in 40 mL of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol (v / v), pH 7.8) with the addition of UltraNuclease (25 U / mL, Cat. 20156ES50, YEASEN). Cells were lysed twice using a French Press (GLENMILLS) at 15,000 psi. Cell debris was then removed by centrifugation at 5,000 × g for 10 minutes at 4 °C. Total membrane fractions were separated by centrifugation at 185,000 × g for 2 hours using an Optima™ MAX-XP ultracentrifuge (Beckman) with an MLA-55 rotor, and the membrane pellet was resuspended in 20% (w / v) sucrose solution (20 mM Tris-HCl, pH 7.8). Subsequently, a 73%-53%-20% sucrose gradient was used, and the inner membrane (IM) and outer membrane (OM) were separated by ultracentrifugation at 250,000 × g for 18 hours at 4°C using an MLS-50 rotor (Beckman). The upper IM and OM bands were collected separately using pipettes and transferred to ultracentrifuge tubes. Dilution buffer (50 mM Tris-HCl, pH 7.8, 300 mM NaCl) was added to bring the final sucrose concentration below 10%. The IM and OM were then collected by ultracentrifugation at 185,000 × g for 2 hours at 4°C using an MLA-55 rotor. The purity of the separated membrane components was assessed, and the subcellular localization of VirK protein was detected using Western blot with anti-outer membrane protein OmpA antibody and anti-His antibody.

[0086] from Figure 12 The experimental results show that the upper bands correspond to inner membrane (IM) proteins, while the lower bands correspond to outer membrane (OM) proteins.

[0087] Example 10 SDS-PAGE and Western Blot Analysis Proteins were separated using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined using the Thermo Scientific Pierce BCA Protein Quantification Kit (Cat. 23227, ThermoFisher), a high-precision and detergent-compatible method for total protein quantification. Protein samples were diluted with 5×SDS-PAGE loading buffer and then denatured by boiling at 95°C for 10 minutes. Proteins were separated in a 15% polyacrylamide gel (Cat. E305, Vazyme Biotech) and subsequently electrotransferred to a PVDF membrane using Western Rapid transfer buffer (Cat. P0572, Beyotime). The membrane was blocked in QuickBlock™ blocking buffer (Cat. P0252, Beyotime) at room temperature for 15 minutes, then primary antibody diluted 1:3000 in primary antibody dilution buffer (Cat. P0256, Beyotime) was added, and the membrane was incubated overnight at 4°C. Subsequently, HRP-labeled secondary antibody diluted 1:5000 in secondary antibody dilution buffer (Cat. P0258, Beyotime) was added, and the membrane was incubated at room temperature for 1 hour. After secondary antibody incubation, the membrane was reacted with the SuperFemtoECL chemiluminescence detection kit (Cat. E423, Vazyme Biotech) for color development. Images were acquired using the Bio-Rad™ ChemiDoc imaging system.

[0088] The antibodies used include: Outer membrane protein A (OmpA) antibody (1:3000 dilution, Catalog No. abx345696, abbexa); DYKDDDDKTag (D6W5B) rabbit monoclonal antibody (HRP conjugated) (1:3000, Catalog No. 86861, CellSignaling Technology). His-Tag (D3I1O) XP® rabbit monoclonal antibody (HRP conjugated) (1:3000, Catalog No. 12688, CellSignaling Technology). Anti-rabbit IgG, HRP-labeled secondary antibody (1:3000, Cat.7074, Cell Signaling Technology).

[0089] from Figure 13The experimental results show that, after SDS-PAGE electrophoresis, OmpA, a protein known to be located in the outer membrane, was used as a positive control. The results indicate that, similar to OmpA, VirK-His is present in the outer membrane (OM), while neither VirK-His nor OmpA was detected in the inner membrane (IM).

[0090] Example 11 Virulence determination of strains in a mouse systemic infection model In vivo virulence comparisons of the strains were conducted using a mouse systemic infection model, via intraperitoneal injection in the presence of 5% mucin. CD-1 (ICR) mice (weighing 19-21 g, half male and half female) were purchased from Vital River Laboratories. All animals were housed in a controlled environment: humidity 30%-70%, temperature 22±3℃, and a 12-hour light-dark cycle. Cell pellets obtained from fresh overnight cultures were appropriately diluted with physiological saline, followed by 10-fold serial dilutions in 5% mucin. Five infection doses were set for each strain. 0.5 mL of bacterial suspension containing 5% mucin was injected intraperitoneally into mice randomly assigned to different experimental groups. During the virulence study, mice were monitored twice daily for signs of infection or distress, including changes in weight, appearance / posture, activity / responsiveness, and respiratory status. Mice were humanely euthanized when their weight dropped to ≤13 g, or when they exhibited severe respiratory distress or a near-death state. Euthanasia was performed using carbon dioxide (CO2) according to institutional guidelines. Mice mortality was recorded within 7 days post-infection, and the median lethal dose (LD50) resulting in 50% mortality was calculated using the Probit method. 50 ) and the lethal dose (LD50) that causes 99% mortality in mice. 99 ) and the corresponding 95% confidence interval (CI).

[0091] Table 6. Comparison of virulence of various bacterial strains in a mouse model of systemic infection.

[0092] As can be seen from the experimental results in Table 6, for the WT strain, LD 50 and LD 99 They are 4.47×10 5 and 3.72×10 6 CFU / mouse. Kpn2146Δ virK LD50 of strain 50 and LD 99 They are 8.51×10 5 and 1.07×10 8 CFU / mouse indicates that in virKGene knockout resulted in a mild but statistically significant decrease in virulence. This was confirmed by pTOPO-Apr- virK After plasmid complementation (C- virK The virulence of this strain was partially restored, and its LD50 was [not specified]. 50 and LD 99 They are 1.00×10 6 and 1.26×10 7 CFU / mouse (Table 1). The Mut-S strain exhibited lower LD50. 50 (1.95×10) 6 The CFU / mouse count indicates high toxicity. Deletion in the Mut-S background reduced LD50. 50 Increased to 6.92×10 6 CFU / mouse suggests weakened virulence. virK After complementation, the toxicity is partially restored, LD 50 1.00×10 6 CFU / mouse. Despite differences in LD50 between different strains. 50 The differences were relatively limited and the confidence intervals partially overlapped, and these results still suggest that even in a genetic background of polymyxin resistance, virK It still contributes to the virulence of the strain.

[0093] Example 12 Antibacterial Activity Assay The determination was performed according to the CLSI guidelines [9]. Freshly cultured bacteria were adjusted to 0.5 McFarland turbidity standard and diluted 1:100 in CAMHB (cationically adjusted Mueller-Hinton broth), and then inoculated into 96-well plates containing different concentrations of polymyxin B, polymyxin S2, colistin, or LL-37

[10] (all serially diluted in CAMHB) to achieve a final inoculum concentration of approximately 5 × 10⁻⁶. 5 CFU / mL. The mixture was then incubated at 37°C for 20-24 hours. MIC is defined as the lowest concentration of antimicrobial drug capable of completely inhibiting bacterial growth. According to the CLSI breakpoint criteria, polymyxin resistance is defined as an in vitro polymyxin resistance level exceeding 2 μg / mL.

[0094] Table 7 Differences virK Genetic status K. pneumoniae Antimicrobial susceptibility

[0095] Note: aThe minimum inhibitory concentration (MIC) is indicated and determined according to the broth microdilution method recommended by CLSI.

[0096] PXB: Polymyxin B; CST: Colistin; PXS2: Polymyxin S2; LL-37: An amphiphilic antimicrobial peptide composed of 37 amino acid residues derived from cathelicidin.

[0097] As can be seen from the experimental results in Table 7, at Kpn2146 and Kpn2146Δ virK and C- virK Among the strains, the MICs of different polymyxins were all between 0.125 and 0.25 μg / mL. Although the polymyxin S2-resistant strain Mut-S... virK The gene expression level is high, and it still exhibits stable resistance to polymyxins (including polymyxin B and colistin), with a MIC of 16-32 μg / mL, which is 128-256 times higher than that of the sensitive wild-type strain Kpn2146. Furthermore, in Mut-S, knockout... virK The gene did not alter its sensitivity to polymyxin.

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0099] References: [1] Li, J., Rayner, CR, Nation, RL et al.. Heteroresistance tocolistin in multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2006; 50: 2946–50. [2] Chu, J., Koirala, B., Forelli, N. et al.. Synthetic-BioinformaticNatural Product Antibiotics with Diverse Modes of Action. J Am Chem Soc. 2020;142: 14158–68. [3] Wang, Z., Koirala, B., Hernandez, Y. et al.. Bioinformaticprospecting and synthesis of a bifunctional lipopeptide antibiotic thatevades resistance. SCIENCE . 2022; 376: 991–6. [4] Higashihira, S., Simpson, S.J., Collier, C.D. et al.. Halicin IsEffective Against Staphylococcus aureus Biofilms In Vitro. Clin Orthop Relat Res . 2022. [5] Li, H.B., Sun, L., Qiao, H. et al.. Polymyxin resistance causedby large-scale genomic inversion due to IS 26 intramolecular translocationin Klebsiella pneumoniae . Acta Pharm Sin B . 2023; 13: 3678–93. [6] Cheng, Y.H., Lin, T.L., Pan, Y.J. et al.. Colistin resistancemechanisms in Klebsiella pneumoniae strains from Taiwan. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY . 2015; 59: 2909–13. [7] Cheng, Y.H., Lin, T.L., Lin, Y.T. et al.. Amino AcidSubstitutions of CrrB Responsible for Resistance to Colistin through CrrC inKlebsiella pneumoniae. Antimicrob Agents Chemother . 2016; 60: 3709–16. [8] Concordet, J.P. and Haeussler, M. CRISPOR: intuitive guideselection for CRISPR / Cas9 genome editing experiments and screens. Nucleic Acids Res . 2018; 46: W242–W5. [9] CLSI. Performance Standards for Antimicrobial Susceptibility Testing . 36th ed. CLSI supplement M1000. Clinical and Laboratory StandardsInstitute; 2026. https: / / clsi.org / shop / standards / m100 / .

[10] Friberg, C., Haaber, J.K., Vestergaard, M. et al.. Humanantimicrobial peptide, LL-37, induces non-inheritable reduced susceptibilityto vancomycin in Staphylococcus aureus . Scientific Reports . 2020; 10: 13121.

Claims

1. Missing virK Genes in reducing Klebsiella pneumoniae ( Klebsiella pneumoniae Applications in toxicity; The virK The gene is the gene that encodes either (a) or (b) the following protein: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or, (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

2. The application according to claim 1, characterized in that, The term "deficit" refers to knockout or non-expression.

3. virK The application of genes as drug targets in the preparation of drugs or compositions that reduce the virulence of Klebsiella pneumoniae; The virK The gene is as described in claim 1.

4. The application according to any one of claims 1-3, characterized in that, The Klebsiella pneumoniae strain in question is ATCC BAA-2146.

5. virK Application of genes or their encoded proteins in the preparation of products for screening antiviral drugs against Klebsiella pneumoniae; The virK The gene is as described in claim 1.

6. PhoP protein and virK The application of gene promoter binding genomes in the preparation of products for screening antiviral drugs against Klebsiella pneumoniae; The virK The gene is as described in claim 1; The PhoP protein is: (i) A protein consisting of the amino acid sequence shown in SEQ ID NO:3; or, (ii) Proteins derived from (i) with the sequence shown in SEQ ID NO:3 substituted, deleted or added with one or more amino acids and having the same function.

7. The application according to claim 6, characterized in that, The virK The nucleotide targeting sequence in the gene promoter that specifically binds to the PhoP protein is: 5′-ACACCTCAATCAATTTTAA-3′.

8. A method for screening potential antiviral drugs for Klebsiella pneumoniae, characterized in that, Includes the following steps: (1) In the presence of PhoP protein and virK Add candidate substances to the reaction system of gene promoter fragments; (2) Detect the effect of the candidate substance on PhoP protein and virK The influence of gene promoter binding ability; (3) If the candidate substance can inhibit PhoP protein and virK If the gene promoter is bound, it is determined to be a potential antiviral drug for Klebsiella pneumoniae; Among them, the virK The gene promoter fragment contains the nucleotide sequence: 5′-ACACCTCAATCAATTTTAA-3′.

9. Testing virK Application of gene expression level reagents in the preparation of kits for in vitro assessment or prediction of polymyxin-resistant Klebsiella pneumoniae virulence levels; The virK The gene is as described in claim 1.

10. The application according to claim 9, characterized in that, The reagents include those for specific amplification. virK Gene primers; And / or, The assessment or prediction is based on the detection of polymyxin-resistant Klebsiella pneumoniae. virK Upregulation of gene expression levels indicates enhanced virulence of the polymyxin-resistant Klebsiella pneumoniae.

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