Bacterial drug target based on bacterial DNA gyrase aggregate and drug

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a bacterial drug target and a drug based on a bacterial DNA rotary enzyme agglomerate, the bacterial drug target is an agglomerate structure formed by bacterial DNA rotary enzyme in a bacterial body, and the agglomerate structure is crucial to DNA duplication, repair and transcription processes of bacteria; the bacterial drug comprises a DNA mimic peptide fragment and a truncated peptide which are designed based on a drug target aggregate structure, the bacterial drug is capable of targeting bacterial DNA gyrase agglomerates, including DNA gyrase mutants, and interfering formation or functions of the bacterial DNA gyrase agglomerates, thereby inhibiting growth and reproduction of bacteria. The bacterial drug provided by the invention can induce solidification of a DNA gyrase liquid aggregate, shows inhibition on bacterial growth, and can be used as a therapeutic agent for bacterial diseases.

Description

Technical Field

[0001] The invention relates to the field of biomedicine technology, and more specifically, to a bacterial drug target and a drug based on bacterial DNA gyrase condensate. Background Art

[0002] Bacterial DNA gyrase, as an indispensable key enzyme for maintaining the topological structure of DNA in bacteria, plays a vital role in core life processes such as DNA replication, repair and transcription. DNA gyrase is composed of two GyrA and two GyrB subunits to form a tetrameric complex. The conservation of DNA gyrase is reflected in its structure and function. From an evolutionary perspective, ikB The gene is highly conserved within different bacterial genera or families, making it an effective molecular marker for identifying closely related bacterial species. ikB The gene encodes the B subunit of the gyrase, which is a single-copy housekeeping gene and an important new target in bacterial phylogenetic analysis. In addition, GyrB and ParE, as components of type IIA topoisomerase, are structurally related and are equally essential for DNA replication and repair processes. Their basic roles are conserved in maintaining DNA structure and function. This conservation also makes them prime candidates for the development of dual-targeted antibacterial agents with broad-spectrum antibacterial activity.

[0003] Currently, drug development based on DNA gyrase has become a research hotspot in the field of antibacterial drugs. Its core strategy is to effectively block the DNA replication and repair process of bacteria by interfering with or inhibiting the activity of DNA gyrase, thereby achieving the purpose of antibacterial drugs. For example, the widely used fluoroquinolone antibiotics have successfully achieved the inhibitory effect on a variety of bacteria by precisely acting on DNA gyrase and topoisomerase IV.

[0004] However, with the widespread use of antibiotics, the problem of bacterial resistance is becoming increasingly serious. Bacteria can develop resistance through gene mutation, especially yrA or ikB Gene mutations can rapidly develop resistance mechanisms to existing drugs. This phenomenon not only weakens the efficacy of traditional antibiotics, but also poses a serious threat to public health security. The newly discovered antibiotic Evybactin can target the DNA gyrase of Mycobacterium tuberculosis and act on the DNA gyrase A subunit, but the antibiotic Evybactin still has problems such as drug-resistant mutations, so new research and development strategies are needed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a bacterial drug target and drug based on bacterial DNA gyrase aggregates, specifically provides a new use of DNA gyrase aggregates as bacterial drug targets, and an antibacterial strategy based on DNA mimetic peptides and truncated peptides, which aims to effectively inhibit the growth and reproduction of bacteria by specifically binding to and interfering with the formation or function of bacterial DNA gyrase aggregates, thereby opening up a new avenue for antibacterial treatment.

[0006] In the first aspect, the present invention provides a bacterial drug target based on bacterial DNA gyrase aggregates, using the following technical solution: A bacterial drug target based on bacterial DNA gyrase condensates, wherein the bacterial drug target is a condensate structure formed by bacterial DNA gyrase in bacteria, and the condensate structure is crucial to the bacterial DNA replication, repair and transcription processes.

[0007] The present invention identifies bacterial DNA gyrase aggregates as a novel bacterial drug target, wherein the DNA gyrase is composed of two GyrA and two GyrB subunits to form a tetrameric complex.

[0008] The present invention uses bioinformatics tools to analyze the GyrA and GyrB subunits of bacterial DNA gyrase and identifies the disordered regions (IDRs) that may be involved in the formation of bioaggregates. Then, experimental methods are used to verify that the GyrA and GyrB subunits of bacteria represented by mycobacteria have the ability to form bioaggregates.

[0009] Furthermore, an in vitro condensate system was established using GyrA-YFP and / or GyrB-mCherry proteins purified from Escherichia coli. The purified proteins were mixed under specific experimental conditions, and the formation of condensates was monitored at different time points using a fluorescence microscope. The results showed that DNA gyrase can form condensates in vitro.

[0010] The dynamic properties of the condensates were analyzed using FRAP (fluorescence recovery after photobleaching) experiments. Quantitative analysis of the FRAP experiments revealed the liquid-like properties of the DNA gyrase condensates by fitting the normalized fluorescence intensity to a single exponential function.

[0011] The purified recombinant proteins were mixed, specifically the mixture contained 0.25 pmol of linearized pUC19 plasmid DNA and 50 pmol of DNA gyrase (composed of 50 pmol of GyrA and GyrB in equal amounts) and 50 pmol of DNA gyrase interacting protein (A0QSY0) truncated body, and the 1.5 condensate formation ability detection method was used to show that the liquid condensate of DNA gyrase was converted into a fibrous state. The FRAP technology was used to analyze the dynamic changes of DNA gyrase condensates, and the results showed that these condensates exhibited non-liquid properties.

[0012] In biological research, the formation of solid or gel-like phases is often closely related to the occurrence of diseases, typical examples of which include the deposition of amyloid fibrils in degenerative diseases and cancer. Such abnormal phases not only destroy the normal physiological functions of cells, but may even directly cause cell function loss or death. Based on the inspiration of this phenomenon, the present invention further studies the solidification process of DNA gyrase condensates and explores its possible impact on bacterial growth dynamics.

[0013] The DNA gyrase condensate of the invention provides new possibilities for treating bacterial diseases and can be used as a target for drug screening.

[0014] In a second aspect, the present invention provides a bacterial drug, which adopts the following technical solution: A bacterial drug is designed based on the above-mentioned bacterial drug target based on bacterial DNA gyrase condensates, which can specifically bind to and interfere with the formation or function of bacterial DNA gyrase condensates, thereby inhibiting the growth and reproduction of bacteria.

[0015] Preferably, the bacterial drug is at least one of a small molecule compound, a polypeptide, an antibody or a derivative thereof.

[0016] Preferably, the bacterial drug is a DNA mimicking peptide and a truncated peptide.

[0017] Preferably, the DNA mimicking peptide segments and truncated peptides are designed and screened through bioinformatics, structural biology and molecular biology techniques.

[0018] Preferably, the DNA mimicking peptide segment is MfpA, and the primer sequence required for its synthesis is: F:ggaggtggaggaggtggaggtggaatgcgtataggggcaaacgg; R:AGTCCAAGTCCAGCTAATTAAGCTTgccgtggacggccagaccgt.

[0019] Preferably, the bacterial drug has DNA mimicking peptides and truncated peptides as active ingredients, and also includes pharmaceutically acceptable excipients.

[0020] Preferably, the preparation method of the bacterial drug comprises uniformly mixing the DNA mimicking peptide segment and the truncated peptide with a pharmaceutically acceptable excipient.

[0021] Preferably, the bacterial drug is in the form of a tablet, a capsule, a powder, a granule, or a suspension.

[0022] Preferably, the bacterial drug exhibits inhibition of bacterial growth and can be used as a therapeutic agent for bacterial diseases.

[0023] In summary, the present invention has the following beneficial effects: The invention provides a bacterial drug target based on bacterial DNA gyrase condensates, which provides new possibilities for treating bacterial diseases.

[0024] The bacterial drug of the present invention is designed with bacterial DNA gyrase condensates as drug targets, including DNA mimicking peptide segments that transform liquid condensates of DNA gyrase into solid (fibrous structure). By adding MfpA protein to an in vitro condensation system and an overnight culture of Escherichia coli, it is found that it promotes the solidification of liquid condensates of DNA gyrase and has a significant inhibitory effect on bacterial growth, and can be used as a therapeutic agent for bacterial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Multiple sequence alignment of the bacterial GyrA and GyrB protein families.

[0026] Figure 2 The crystal structure of bacterial DNA gyrase.

[0027] Figure 3 IDR prediction results for bacterial GyrA and GyrB subunits.

[0028] Figure 4 An in vitro condensate formation system constructed using purified GyrA-YFP and / or GyrB-mCherry proteins.

[0029] Figure 5 Fluorescence recovery imaging and relative fluorescence intensity changes of DNA gyrase droplets after photobleaching.

[0030] Figure 6 This is the predicted clustering region of DNA gyrase GyrA and GyrB sequences.

[0031] Figure 7 Fluorescence images and relative fluorescence intensity changes of the effects of DNA gyrase interacting proteins on the structure of its liquid condensate.

[0032] Figure 8 The fibrous structure of DNA gyrase under an electron microscope.

[0033] Fig. 9 The effect of DNA gyrase solid aggregates on bacterial growth was evaluated by serial dilution method.

[0034] Fig.10 Effects of targeted DNA gyrase aggregates on host bacterial load in a zebrafish model.

[0035] Fig.11 To analyze the disordered regions of DNA mimicking proteins AlbG (2XT4) and MfpA (6ZT4) using the PONDER algorithm.

[0036] Fig.12 To predict the aggregation regions of DNA mimicking proteins AlbG (2XT4) and MfpA (6ZT4) using the TANGO algorithm.

[0037] Fig.13 Construct configuration and cloning of MfpA truncation fragments.

[0038] Fig.14 The antimicrobial activity of the MfpA truncated genes was evaluated for CFU assay.

[0039] Fig.15 Electron microscopy shows the formation of DNA gyrase condensate fibers promoted by MfpA4-7.

[0040] Fig.16 Shown are the pathogen load and lung pathological damage in the lungs of mice after treatment with MfpA4-7. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0042] Example 1 Identification and verification of the ability of bacterial DNA gyrase GyrA and GyrB subunits to form bioaggregates: 1.1 The protein sequences of bacterial GyrA and GyrB subunits were collected from the National Center for Biotechnology Information and UniProt databases, and the amino acid sequences of GyrA and GyrB subunits of different bacteria were compared (the comparison results are shown in Figure 1 ), Figure 1In the figure, A on the top is the amino acid sequence of the GyrA subunit, and B on the bottom is the amino acid sequence of the GyrB subunit; the results show that the GyrA and GyrB protein families are highly conserved, indicating that some key features and functional regions of these proteins remain highly consistent in different bacteria. At the same time, at the structural level, the GyrA and GyrB subunits of DNA gyrase also have a high degree of similarity (the crystal structure of DNA gyrase is shown in Figure 2 ) Figure 2 In the figure, A is the crystal structure of the DNA gyrase of Mycobacterium tuberculosis, B is the crystal structure of the DNA gyrase of Escherichia coli K12, and C is the crystal structure of the DNA gyrase of Staphylococcus aureus. The results further verify the consistency of the functions of these proteins in multiple bacteria.

[0043] 1.2 Escherichia coli ( Escherichia coli ) and Mycobacterium smegmatis ( Mycobacterium smegmatis ) as the model, and the bioinformatics tool PONDER (Prediction of Naturally Disordered Regions) algorithm was used to predict disordered regions (IDR) (prediction results are shown in Figure 3 ), Figure 3 In the figure, A is the IDR prediction result of GyrA and GyrB subunits of Escherichia coli, wherein the left side represents the GyrB subunit and the right side represents the GyrA subunit; B is the IDR prediction result of GyrA and GyrB subunits of Mycobacterium smegmatis, wherein the left side represents the GyrB subunit and the right side represents the GyrA subunit. The results show that GyrA and GyrB subunits have widely distributed disordered regions, indicating that they have the potential to form bioaggregates.

[0044] Example 2 Experimental verification of in vitro formation of DNA gyrase liquid condensates: 2.1 Using mycobacterial DNA gyrase as a model, gene cloning and protein expression techniques were used to obtain recombinant proteins of GyrA and GyrB subunits.

[0045] 2.1.1 PCR amplification of target fragment Mycobacterium smegmatis ( M. smegmatis mc 2 155) genomic DNA was used as a PCR amplification template and pET28a plasmid (containing an N-terminal His tag) was used as an expression vector to amplify the gene fragments encoding the GyrA and GyrB subunits. During primer design, YFP and mCherry fusion tags were constructed for GyrA and GyrB, respectively.

[0046] Primer sequences for pET28a-GyrA-YFP -28a-GyrA-YFP-F: GTATTTCCAGGGCCATATGatgactgatacgacgctgccg -GyrA-LINKER-R: tccacctccacctcctccacctcccgcctcgggtgactcggcgg -LINKER-YFP-F: ggaggtggaggaggtggaggtggaatggtgagcaagggcgaggagctgtt -28a-GyrA-YFP-R: CGAGTGCGGCCGCAAGCTTCTAttgtacagctcgtccatgc Primer sequences for pET28a-GyrB-mCherry -28a-GB-MC-F: GTATTTCCAGGGCCATATGgtggctgcccagaagaacaat -GyrB-LINKER-R: TCCACCTCCTCCACCTCCaacatccaggaagcgaacg -LINKER-mCherry-F: cttcctggatgttGGAGGTGGAGGAGGTGGAGGTGGAATGGTGAGCAAGGGCGA -28a-GB-MC-R: TCGAGTGCGGCCGCAAGCTTTCActtgtacagctcgtccatgc.

[0047] 2.1.2 Plasmid vector preparation The pET28a plasmid (containing an N-terminal His tag) was selected as the expression vector, and the plasmid was double-digested with Nde I and Hind III to linearize the vector.

[0048] 2.1.3 Recombinant plasmid construction The GyrA gene sequence was cloned into the pET28a vector to construct pET28a-YFP-GyrA; The GyrB gene sequence was cloned into the pET28a vector to construct pET28a-GyrB-mCherry.

[0049] 2.1.4 Expression and purification of recombinant proteins The constructed recombinant plasmids were transformed into E. coli BL21(DE3). E. coli The cells were cultured in LB medium containing 100 mg / L kanamycin until OD 600 The p-value reached 0.6, and then 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to induce the protein, and the cells were incubated at 16°C overnight, followed by centrifugation and collection of the cells. The bacteria carrying the recombinant protein were resuspended in buffer A (10 mM sodium phosphate pH 7.6, 150 mM sodium acetate, 0.1 mM magnesium acetate, 0.5 mM DTT, 2.5% glycerol and 10 mM imidazole), and the bacteria were lysed using a Frech Press cell disruptor. The lysate was centrifuged at 12,000 g for 30 minutes at 4°C to obtain the supernatant containing the recombinant protein, and the supernatant was incubated with Ni-NTA 6FF agarose beads (Easybio, catalog number BE6947) equilibrated with buffer A for 2 hours at 4°C. Subsequently, the beads were washed with 15 column volumes (CV) of buffer A containing 20 mM imidazole to remove unbound proteins. After that, the target protein was released by elution with buffer A containing 300 mM imidazole. Further purification was performed using an AKTA Purifier 10 chromatography system. Fractions containing the recombinant protein were collected using Superdex 200 Increase 10 / 300 GL or Superdex 75 Increase 10 / 300 GL. Finally, the purified protein was concentrated using an Amicon Ultra-15 centrifugal filter (30 kDa molecular weight cutoff).

[0050] 2.1.5 Verification of aggregate formation ability Purified recombinant GyrA (50 pmol) and GyrB (50 pmol) proteins and linearized pUC19 plasmid DNA (0.25 pmol, obtained by treatment with the rapid endonuclease BamHI) were mixed and incubated at 4°C in a Grace Bio-Labs CoverWell™ imaging chamber.

[0051] Observation was performed using a Leica TCS SP8 STED confocal microscope with a 100x oil immersion objective. Fluorophores were excited and detected using the following wavelengths: 488 nm for EGFP, 514 nm for YFP, and 550 nm for mCherry. Fluorescence signals were collected using a 6-channel Spectra X LED light source and a Sedat Quad filter set, and photographed (see the in vitro condensate formation system for details). Figure 4 ). Figure 4In the figure, from left to right are the photographic results at 0h, 6h, 12h, and 24h. The yellow fluorescence in the figure represents GyrA-YFP, showing the condensate structure of DNA gyrase.

[0052] 2.1.6 Analysis of dynamic characteristics of DNA gyrase aggregates The in vitro condensate system was established using GyrA and GyrB proteins purified from Escherichia coli (including their fluorescent fusion forms GyrA-YFP and / or GyrB-mCherry). After the purified GyrA and GyrB proteins were mixed in vitro, a circular area with a diameter of approximately 2 μm was photobleached using a 514 nm argon-ion laser at 20% power. The dynamic properties of DNA gyrase condensates were further evaluated using the fluorescence recovery after photobleaching (FRAP) technique. At t = 0, a specific area was photobleached, and the fluorescence recovery of the area was captured at subsequent time points (see Figure 5 ), Figure 5 In the figure, A shows the photobleaching region and recovery dynamics, and B is the quantitative analysis of the FRAP experiment, based on fitting the fluorescence recovery curve to a single exponential function. The results show that the DNA gyrase condensate recovers dynamically after photobleaching, demonstrating its liquid-like properties. The quantitative analysis results of the FRAP experiment reveal the recovery rate and fluidity of the condensate by fitting the normalized fluorescence intensity to a single exponential function. Finally, the results prove that the condensate formed by the GyrA and GyrB subunits has liquid-like properties.

[0053] Example 3 Potential Assessment of Drug Targets: In biological research, the formation of solid or gel-like phases is often closely related to the occurrence of diseases, typical examples of which include the deposition of amyloid fibrils in degenerative diseases and cancer. Such abnormal phases not only destroy the normal physiological functions of cells, but may even directly cause cell function loss or death. Based on the inspiration of this phenomenon, the present invention further studies the solidification process of DNA gyrase liquid condensates and explores its possible impact on bacterial growth dynamics.

[0054] 3.1 Bioinformatics prediction The aggregation tendency of proteins was analyzed using the TANGO algorithm. The version of the TANGO algorithm used was tango2_3. The parameters were set as follows: C-terminal and N-terminal protection was enabled, the temperature was set to 298 Kelvin, the pH was 7.6, and the salt concentration was 150 mM. These conditions were consistent with the experimental environment of the purified DNA gyrase protein. Specifically, fragments containing more than 5 consecutive amino acid residues and with a score higher than 5% were identified as potential aggregation regions. The prediction results of the TANGO algorithm showed that there were regions with higher aggregation risk in the sequences of the GyrA and GyrB subunits of DNA gyrase (see Figure 6 ), Figure 6 In the figure, A is the aggregation tendency of the GyrA subunit sequence of DNA gyrase, and B is the aggregation tendency of the GyrB subunit sequence of DNA gyrase. The figure shows the predicted results of the aggregation tendency of each region after analyzing the variant sequence of the DNA gyrase protein sequence by the TANGO algorithm.

[0055] 3.2 Experimental Verification The purified recombinant protein was mixed with a mixture containing 0.25 pmol of linearized pUC19 plasmid DNA and 50 pmol of DNA gyrase (composed of 50 pmol of GyrA and GyrB in equal amounts) and 50 pmol of a truncated form of the DNA gyrase interacting protein (A0QSY0). To examine the morphological characteristics of the protein / peptide aggregates, transmission electron microscopy (TEM) was used, and the mixture was diluted 100 times, then the diluted sample was applied to a carbon-coated Formvar grid (Beijing Zhongjing Technology, catalog number BZ11022) and incubated at room temperature for 1 minute. To ensure optimal visualization, the sample was rinsed twice with autoclaved distilled water to remove any excess or unbound material. It was then stained with a 1% (w / v) uranyl acetate solution for 1 minute to enhance contrast and allowed to dry completely.

[0056] First, for imaging, the prepared grids were placed in a Hitachi HT7800 transmission electron microscope (Hitachi, Japan) and observed at different magnifications. Images were captured using the HT7800 system software, and the microscope was operated at an accelerating voltage of 80 kV to obtain a detailed view of the aggregate morphology (see Figure 8 ). Figure 8 In the study, electron microscopy was used to demonstrate the fibrous structure formed by DNA gyrase under specific interaction conditions. The dynamic properties of DNA gyrase aggregates were analyzed using FRAP technology, and the results showed that these aggregates have non-liquid properties (see Figure 7 ), Figure 7In the figure, A shows the effect of DNA gyrase interacting proteins on the structure of its liquid condensate, and B is the dynamic characteristics analysis of DNA gyrase molecular condensates using FRAP technology.

[0057] The above results show that DNA liquid condensates can be induced to transform into fibers and exhibit non-liquid properties.

[0058] Example 4 Effect of DNA gyrase solid aggregates on bacterial growth: 4.1 Effects of inducing solid-state DNA gyrase aggregates on bacterial growth 4.1.1 PCR amplification of gene fragments (using DNA gyrase interacting protein (A0QSY0) as template) The genomic DNA of Mycobacterium smegmatis mc2155 was used as a PCR amplification template to amplify the gene fragment F47 encoding the DNA gyrase interacting protein (A0QSY0).

[0059] The primer sequences are: 80L-MfpA-4-F: GCATCACCATCACCATCACGGATCCagcacgttcaccaactgcag 80L-MfpA-7-R: AGTCCAAGTCCAGCTAATTAAGCTTctcctccaggcgtgcgtcct 4.1.2 Plasmid vector preparation The pQE80L plasmid (containing an N-terminal His tag) was selected as the expression vector, and the plasmid was double-digested with EcoRI and HindIII to linearize the vector.

[0060] 4.1.3 Recombinant plasmid construction The gene sequence of DNA gyrase interacting protein (A0QSY0) was cloned into the pQE80L vector to construct pQE80L-F47.

[0061] 4.1.4 Evaluation of the point plate experiment To detect the inhibitory effect of DNA gyrase interacting protein (A0QSY0) F47 protein expression on bacterial growth, an overnight culture of E. coli containing the indicator plasmid pQE80L-F47 (1.5 ml) was adjusted to the same optical density at 600 nm (OD 600 The value is about 0.8, which is in the stable phase), and then serial dilutions are performed in sterile LB liquid medium. LB medium is used to gradually perform 10-fold dilutions until the desired dilution (10 -7). Then, add 3 µL of the dilution onto LB agar plates containing IPTG (0.5 mM, to induce protein expression) or without IPTG. Take a certain volume (e.g., 1 mL) from the original culture, spread it onto the LB agar plate, and incubate it at 37°C overnight, then observe the growth of the bacteria (see Fig. 9 ), Fig. 9 In the figure, the left side is the uninduced group without IPTG, and the right side is the induced group with IPTG. The results showed that the number of bacterial colonies on the plate of the IPTG-induced group was significantly less than that of the uninduced group, and there were differences in the morphology and size of the colonies, indicating that IPTG-induced DNA gyrase interacting protein (A0QSY0) truncated protein F47 has a significant inhibitory effect on bacterial growth.

[0062] Example 5 Zebrafish model to evaluate the effect of target DNA gyrase liquid aggregate solidification on host bacterial load: The effect of target DNA gyrase aggregates on host bacterial load was evaluated using a zebrafish model. AB wild-type zebrafish were used for the experiment, and the breeding and management were in accordance with the standards of the Chinese Zebrafish Resource Center. The experimental subjects were zebrafish embryos, which were injected with EGFP-expressing M. marinum The embryos in the experimental group were inoculated with 0.5 mg / mL DNA gyrase interacting protein (A0QSY0) truncated F47. M. marinum strains, while control embryos were inoculated with only PBS (phosphate buffered saline) M. marinum Each embryo was inoculated with 200-300 CFU of each strain.

[0063] Embryos were incubated at 28.5°C with 0.003% 1-phenyl-2-thiourea (PTU) added to the culture medium to inhibit pigment production. 72 hours after infection, embryos were imaged using Z-stack scanning (see Fig.10 ), Fig.10 In the figure, the upper part is the MM / PBS control group, and the lower part is the MM / F47 protein experimental group. The results show the fluorescence microscopic imaging of zebrafish embryos in the experimental group and the control group after 72 hours of infection. The contrast of color depth directly reflects the difference in bacterial load. The evaluation results show that the solidification effect of DNA gyrase aggregates as targeted drug targets significantly reduces the host's bacterial load.

[0064] Through bioinformatics analysis and experimental verification, it was revealed that the DN gyrase GyrA and GyrB subunits have the potential to form bioaggregates, and their potential application value as drug targets was demonstrated.

[0065] Example 6 Screening and Characterization of DNA Mimicking Proteins: 6.1 Bioinformatics analysis and prediction 6.1.1 Obtaining protein sequences In order to further study the characteristics of DNA mimicking proteins, we first collected protein sequences of bacterial DNA mimicking proteins from the National Center for Biotechnology Information and UniProt databases. The specific protein sequences are shown below: >M.smegmatis MRIGANGDETVWADEEFAGRDFRDEDLSRIRTERVVFTECDFSGVDLSESEHHGSAFRNCTFRRSTIWHSTFTNCSLLGSVFTECRIRPVTFVECDFTLAVLGGCDLRAVDLSDCRLREVSLVGADLRKAVLRRADLTGSRVQDARLEEADLRGTRVDPTFWTTAKVRGAKIDIEQALAYAAAHGLAVHGG----- >M.tuberculosis --------MQQWVDCEFTGRDFRDEDLSRLHTERAMFSECDFSGVNLAESQHRGSAFRNCTFERTTLWHSTFAQCSMLGSVFVACRLRPLTLDDVDFTLAVLGGNDLRGLNLTGCRLRETSLVDTDLRKCVLRGADLSGARTTGARLDDADLRGATVDPVLWRTASLVGARVDVDQAVAFAAAHGLCLAGG----- >M.marinum --------MEHWVDCEFTDRDFRDEDLSRLRTERVVFSECNFGGVNLTESEHRGSAFRNCSFERTTLWHSTFAQCSMLGSVFVSCRMRPLVLDEVDFTLAVLGGNDLRGVDLSGCRLREASLVETDLRKSVLRGADLRGARTNGTKLDDADLRGANLDPSLWRSASLAGARIDVPQALSFALAHGLRLDS------ >M.fortuitum ----MVADETAWADREFTGHDFREEDLSRLTTERVVFTECDFSGVDLSESQHSGSAFRNCTFRRATLWHSTFTNCSLLGSVFTECRLRPIKIVESDLTLAVLGGCDLRGVDLSDCRLREASLVGVDLRKAILRQADLTGARVQDAKLDEADLRGARVDPTFWTTAKLRGAKIDIAQALAYSAAHGLDVHGA----- >M.ulcerans --------MEHWVDCEFTGRDFRDEDLSRLRTERVVFSECNFGGVNLTESEHRGSAFRNCSFERTTLWHSTFAQCSMLGSVFVSCRMRPLVDEVDFTLAVLGGNDLRDVDLSGCRLREASLVETDLRKSVLRGADLRGARTNGTKLDDADLRGANDPSLWRSASLAGARDVPQALSFALAHGLRLAS------ >M.chelonae ------------------ >M.avium --------MTAWVDREFERHDFTDEDLVGLSTERVVFTECNFSGANLAESRHRASAFRNCTFRRTSLWHSTFEQCTMLGSVFEQCRLRPVTFDEVDFTLAVLGGNDLRGVDLSGCRLRETSLVEADLRKAVLRGADLRGARTAGTRLDDADLRGGAADQALWTTASLAGARVDVDQAVAFALGHGLRLDG------ >M.abscessus -------MAEHWTDREITAETFYDEDFRELHTERVVFTECDFSGANLTESLHVGSAFRNCTFRRTSLWHSEFRQCSLLGSTFTDCRVRPSKFTETDFTLSSLAGLDLREMDLSDCRFREANLVGADMRKANLHGADFTGARTQNLKLDGADLRGARIDPTLWTTAALITAKVDLPQAIAFAAAHGLDVHGG----- >M.intracellulare -------MSEEWVDREFDGHDFTDEDLSRLRTERTVFTECNFSGANLAESQHRGSAFRNCSFQRTSLWHSSFAQCSMLGSVFVQCRLRPITFDEVDFTLAVLAGIDLRGVDFSGCRLREASLVEADLRKAVLRGADLRGARTAGARLDGADLRGTTADPGLWTTASLAGARVDVDQAVAFALAHGLRLDGGPHDDG > M.haemophilum --------MQQWTDCEIAGRDFTDDDLSRLRTERVVFTECNFSGANLAESHHRASAFRNCTFKRTLLWHSTFTHCSMLGSVFVQCRLRPLTFDEVDFTLAVLGGNDLRGVDLSGCRLREASLVETDLRTAVLRGADLSGARTTGTRLDDADLRGATVEPSLWRTASLAGARIDVPQAVAFALAHGLCLNGGLDA--.

[0066] 6.1.2 Prediction of IDRs (disordered regions) In order to evaluate the structural characteristics of bacterial DNA mimicking proteins (in this example, MfpA protein is used as an example), we used the PONDER algorithm to predict IDRs. The algorithm accurately analyzes the disordered regions in the DNA mimicking protein sequence by setting the parameters VL-X1 and XL1-XT. The prediction results show that the two ends of the DNA mimicking protein contain significant disordered regions (see Fig.11 ), Fig.11 In the figure, A is the disordered region of the DNA mimic protein AlbG (2XT4), and B is the disordered region of the DNA mimic protein MfpA (6ZT4), which reveals that DNA mimic proteins may have the potential to form condensates.

[0067] 6.1.3 Prediction of Aggregation Tendency To further verify the aggregation characteristics of DNA mimicking proteins, we introduced the TANGO algorithm to predict aggregation tendency. Under the specific parameter settings of the TANGO algorithm (C-terminal protection, N-terminal protection, temperature 298 Kelvin, pH 7, salt concentration 100mM), potential high aggregation regions in the sequences of DNA mimicking proteins (such as AlbG, MfpA) were successfully identified. The algorithm marked the potential aggregation regions of fragments containing more than 5 consecutive amino acid residues and with a score of more than 5% (see Fig.12 ), Fig.12 In the figure, A is the aggregation region of the DNA mimic protein AlbG (2XT4), and B is the aggregation region of the DNA mimic protein MfpA (6ZT4). This result shows the ability of DNA mimic proteins to form aggregates, providing an important basis for their application in drug development.

[0068] Example 7 High-throughput screening and verification: 7.1 Construction of MfpA truncation library Taking MfpA protein as an example, we studied the functional properties of different fragments of DNA mimicking proteins in depth. We divided the MfpA protein sequence into 9 structural parts and successfully constructed 45 truncated fragments through the combination of different regions (see Fig.13 ), Fig.13 In the figure, A is the bioinformatics analysis of the MfpA sequence, and B is the cloning and structural configuration of MfpA truncations. These truncations were cloned into the pQE80L vector using EcoRI and HindIII restriction endonucleases (provided by Fermentas, product numbers FD0074 and FD0504, respectively). Subsequently, 1 ng of plasmid DNA was mixed with 50 µl of electrocompetent medium. E. coli The cells were mixed and then transformed.

[0069] The specific sequences of the primers used are as follows: 80L-MfpA-1-F: GCATCACCATCACCATCACGGATCCacggtatgggcggacgaagagttcgccg 80L-MfpA-2-F: GCATCACCATCACCATCACGGATCCatccgcaccgaacgcgtcgt 80L-MfpA-3-F: GCATCACCATCACCATCACGGATCCtccgagcatcatggatcggc 80L-MfpA-4-F: GCATCACCATCACCATCACGGATCCagcacgttcaccaactgcag 80L-MfpA-5-F: GCATCACCATCACCATCACGGATCCgtcacattcgtcgagtgcga 80L-MfpA-6-F: GCATCACCATCACCATCACGGATCCgtcgacctgtcggactgccg 80L-MfpA-7-F: GCATCACCATCACCATCACGGATCCgcagtgctgcggcgggccga 80L-MfpA-8-F: GCATCACCATCACCATCACGGATCCgccgatctgcgcggtacgcg 80L-MfpA-9-F: GCATCACCATCACCATCACGGATCCgccaagatcgacatcgaaca 80L-MfpA-1-R: AGTCCAAGCTCAGCTAATTAAGCTTgcggctcagatcctcgtcgc 80L-MfpA-2-R: AGTCCAAGCTCAGCTAATTAAGCTTctccgagaggtcgacgccgc 80L-MfpA-3-R: AGTCCAAGCTCAGCTAATTAAGCTTgtgccagatggtgctgcgg 80L-MfpA-4-R: AGTCCAAGCTCAGCTAATTAAGCTTagggcggatgcggcattcgg 80L-MfpA-5-R: AGTCCAAGCTCAGCTAATTAAGCTTggcacgcagatcacagccgc 80L-MfpA-6-R: AGTCCAAGCTCAGCTAATTAAGCTTcttacgcagatccgcacc 80L-MfpA-7-R: AGTCCAAGTCCAGCTAATTAAGCTTctcctccaggcgtgcgtcct 80L-MfpA-8-R: AGTCCAAGTCCAGCTAATTAAGCTTgccgcgcaccttggccgtgg 80L-MfpA-9-R: AGTCCAAGTCCAGCTAATTAAGCTTgccgtggacggccagaccg Bioinformatic analysis of MfpA sequences and cloning of MfpA truncation variants. 45 MfpA truncation variant genes with unique structural configurations, each encoding a different structure, were individually cloned into the pQE80L vector with a His6 tag.

[0070] 7.2 Evaluation of antimicrobial activity of MfpA truncated fragments by CFU assay The growth of E. coli carrying the pQE80L plasmid containing the MfpA truncated gene was assessed by colony forming unit (CFU) assay. The bacterial culture was adjusted to OD 600 =0.1, and then 10-fold serial dilutions were performed. 3 µL of each dilution gradient sample was spotted on LB agar plates containing 100 µg / mL ampicillin, with IPTG-induced groups (+IPTG) and non-induced groups (-IPTG) set up respectively. The plates were incubated at 37°C overnight (see Fig.14 ), Fig.14 In the figure, the top is the non-induced group (-IPTG), and the bottom is the IPTG induced group (+IPTG). Finally, the peptide MfpA4-7 with high antibacterial activity was screened out.

[0071] Example 8 MfpA4-7 protein fragment promotes the fibrillation of DNA gyrase: 8.1 PCR amplification of gene fragments Using the Mycobacterium tuberculosis model strain Mycobacterium smegmatis ( M. smegmatis mc 2 155) as a template to amplify the gene fragment encoding GyrA, GyrB subunits and MfpA.

[0072] Specific sequences of primers: pET28a-GyrA-YFP 28a-GyrA-YFP-F: GTATTTCCAGGGCCATATGatgactgatacgacgctgccg GyrA-LINKER-R: tccacctccacctccacctcccgcgggtgactcggcgg LINKER-YFP-F: ggaggtggaggaggtggaggtggaatggtgagcaagggcgaggagctgtt 28a-GyrA-YFP-R: CGAGTGCGGCCGCAAGCTTCTAttgtacagctcgtccatgc pET28a-GyrB-mCherry 28a-GB-MC-F: GTATTTCCAGGGCCATATGgtggctgcccagaagaacaat GyrB-LINKER-R: TCCACCTCCTCCACCTCCaacatccaggaagcgaacg LINKER-mCherry-F: cttcctggatgttGGAGGTGGAGGAGGTGGAGGTGGAATGGTGAGCAAGGGCGA 28a-GB-MC-R: TCGAGTGCGGCCGCAAGCTTTCActtgtacagctcgtccatgc pQE80L-MfpA4-7 80L-MfpA-4-F: GCATCACCATCACCATCACGGATCCgcagtgctgcggcgggcga 80L-MfpA-7-R: AGTCCAAGCTCAGCTAATTAAGCTTctcctccaggcgtgcgtcct 8.2 Remote Control The pET28a plasmid was selected as the expression vector (this plasmid has a strong promoter and an N-terminal His tag for protein purification). The pET28a plasmid was first double-digested with NdeI and HindIII restriction endonucleases (Fermentas, catalog numbers FD0583 and FD0504) to linearize the vector fragment. The pQE80L plasmid was then double-digested with EcoRI and HindIII (Fermentas, catalog numbers FD0074 and FD0504) to linearize the vector fragment.

[0073] 8.3 Construction of recombinant plasmid The gene sequence of GyrA was inserted into the pET28a plasmid to construct pET28a-YFP-GyrA; the gene sequence of GyrB was inserted into the pET28a plasmid to construct pET28a-GyrB-mCherry; the gene sequence of MfpA was inserted into the pQE80L plasmid to construct pQE80L-EGFP-MfpA4-7.

[0074] 8.4 Expression and purification of recombinant proteins The recombinant plasmids pET28a-YFP-GyrA and pET28a-GyrB-mCherry were transformed into E. coli In BL21(DE3) cells, pQE80L-EGFP-MfpA4-7 was transformed into E. coli E. coli were cultured in LB medium containing 100 mg / L kanamycin and incubated at 37°C in a shaking incubator until OD 600 When the p-value reached about 0.6, 0.5 mM IPTG was added to induce the expression of the recombinant protein. Subsequently, the culture temperature was lowered to 16 °C and incubated overnight to promote the correct folding and accumulation of the protein.

[0075] The bacteria expressing the recombinant protein were resuspended in buffer A (10 mM sodium phosphate pH 7.6, 150 mM sodium acetate, 0.1 mM magnesium acetate, 0.5 mM DTT, 2.5% glycerol and 10 mM imidazole) and lysed using a Frech Press cell disruptor (JNBIO, China). The lysate was centrifuged at 12,000 g for 30 minutes at 4°C to obtain the supernatant, which was then incubated with Ni-NTA 6FF agarose beads (Easybio, catalog number BE6947) equilibrated with buffer A for 2 hours at 4°C. The beads were washed with 15 column volumes of buffer A containing 20 mM imidazole to remove unbound proteins, and the target protein was then eluted with buffer A containing 300 mM imidazole.

[0076] The eluted protein was further purified and buffer exchanged by AKTA Purifier 10 chromatography system (GE AKTA Purifier 10, Germany). The protein was analyzed by molecular weight exclusion method by Superdex 200 Increase 10 / 300 GL (Cytiva, catalog number 28990944) or Superdex 75 Increase 10 / 300 GL (Cytiva, catalog number 29148721) columns. These filtration columns were run in buffer A without imidazole to collect pure, non-aggregated protein for subsequent applications. The protein was concentrated by Amicon Ultra-15 centrifugal filter (30 kDa molecular weight cutoff, Millipore, catalog number UFC903024).

[0077] 8.5 MfpA4-7 protein fragment promotes the fibrillation of DNA gyrase aggregates In the field of biology, the formation of solid or gel-like phases is often closely related to pathological conditions, such as amyloid fibril deposits observed in degenerative diseases and cancer. This abnormal phase not only interferes with normal physiological processes in cells, but may also directly lead to loss of cell function or even cell death. The MfpA4-7 protein fragment can promote the solidification of molecular condensates of DNA gyrase and inhibit bacterial growth.

[0078] 8.5.1 Preparation of MfpA4-7 recombinant protein complex mixture The purified recombinant proteins were mixed in a specific ratio, including 0.25 pmol of linearized pUC19 plasmid DNA, 50 pmol of DNA gyrase (composed of equal amounts of GyrA and GyrB subunits), and 50 pmol of MfpA4-7 protein fragment.

[0079] 8.5.2 Transmission electron microscopy observation of aggregate formation ability To examine the morphological features of protein / peptide aggregates, transmission electron microscopy (TEM) was used. The mixture was diluted 100 times, and the diluted sample was then applied to a carbon-coated Formvar grid (Beijing Zhongjing Technology, catalog number BZ11022) and incubated at room temperature for 1 minute. To ensure optimal visualization, the sample was rinsed twice with autoclaved distilled water to remove any excess or unbound material. It was then stained with 1% (w / v) uranyl acetate solution for 1 minute to enhance contrast and allowed to dry completely.

[0080] For imaging, the prepared grids were placed in a Hitachi HT7800 transmission electron microscope (Hitachi, Japan) and observed at different magnifications. Images were captured using the HT7800 system software and the microscope was operated at an accelerating voltage of 80 kV to obtain detailed views of the aggregate morphology (see Fig.15 ). Electron microscopy technology was used to show the fibrous structure formed by DNA gyrase under specific interaction conditions.

[0081] Example 9 Animal model experimental verification: 9.1 Mouse infection experiment Pathogen-free female C57BL / 6 mice (6-8 weeks old) were provided by Sigenetic (Beijing). The mice were given a 5-7 day adaptation period before the experiment and were then randomly divided into multiple experimental groups. To ensure the reliability of the data, three replicates were set for each time point. The mice were anesthetized by intraperitoneal injection of 250 mg / kg tribromoethanol to ensure the smooth progress of subsequent operations.

[0082] Under anesthesia, mice were inoculated intranasally with the Mycobacterium strain IMMI-114 (approximately 6 × 10 6 CFU dissolved in 25 μL PBS) or a mixture of IMMI-114 and 0.5 mg / mL MfpA4-7 protein. The inoculation drop was dripped into the left nostril of the mouse for infection. At 24 hours and 48 hours after infection, the mice were sacrificed and their intact lung tissues were collected for subsequent analysis.

[0083] 9.2 Lung Tissue Processing and Bacterial Load Detection The collected lung tissue was placed in a 100 μm cell sieve and thoroughly homogenized with PBS to extract the sample. The homogenized lung tissue was diluted proportionally, inoculated on a 7H10 agar plate, and incubated at 37°C for about 3 days to quantitatively evaluate the bacterial load in the lungs. The purpose of this step is to evaluate the degree of infection and the inhibitory effect of MfpA4-7 on mycobacterial infection. The experimental results showed that in the MfpA4-7 intervention group, the bacterial load in the lungs was significantly reduced ( Fig.16 A in ).

[0084] 9.3 Lung Tissue Fixation and Pathological Analysis To further analyze the effects of infection on lung tissue, the collected lung tissue was fixed with 4% paraformaldehyde. The fixed tissue was dehydrated, embedded, and sliced ​​for hematoxylin-eosin (HE) staining using standard procedures. Subsequently, the stained lung tissue was examined in detail using a Leica SCN400 microscope (Wetzlar, Germany) to analyze the pathological changes in the lung ( Fig.16 B in the figure).

[0085] 9.4 Experimental observation and pathological analysis like Fig.16 As shown, the MfpA4-7 protein has a significant inhibitory effect on mouse lung infection. Pathological analysis found that the pathogen load in the lungs of mice treated with MfpA4-7 was significantly reduced, and the degree of pathological damage in the lungs was significantly alleviated. This phenomenon shows that MfpA4-7 has a significant therapeutic effect in anti-mycobacterial infection. Specifically, MfpA4-7 showed a significant inhibitory effect on pathogen load in the mycobacterium-infected mouse model, further verifying its potential as a treatment for mycobacterium infection.

[0086] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A bacterial drug target based on bacterial DNA gyrase aggregates, characterized in that: The bacterial drug target is a condensate structure formed by bacterial DNA gyrase in the bacteria.

2. A bacterial drug, characterized in that The bacterial drug is designed based on the bacterial drug target based on bacterial DNA gyrase aggregates as described in claim 1, and can specifically bind to and interfere with the formation or function of bacterial DNA gyrase aggregates, thereby inhibiting the growth and reproduction of bacteria.

3. The bacterial drug according to claim 2, characterized in that The bacterial drug is at least one of a small molecule compound, a polypeptide, an antibody or a derivative thereof.

4. The bacterial drug according to claim 2, characterized in that The bacterial drugs are DNA mimicking peptide segments and truncated peptides.

5. The bacterial drug according to claim 4, characterized in that The DNA mimicking peptide segment and truncated peptide are designed and screened through bioinformatics, structural biology and molecular biology techniques.

6. The bacterial drug according to claim 4, characterized in that The DNA mimicking peptide segment is MfpA, and the primer sequence required for its synthesis is: F:ggaggtggaggaggtggaggtggaatgcgtataggggcaaacgg; R:AGTCCAAGTCCAGCTAATTAAGCTTgccgtggacggccagaccgt.

7. The bacterial drug according to claim 4, characterized in that The bacterial medicine uses DNA mimicking peptide segments and truncated peptides as active ingredients and also includes pharmaceutically acceptable excipients.

8. The bacterial drug according to claim 7, characterized in that The preparation method of the bacterial drug comprises the steps of uniformly mixing a DNA mimicking peptide segment and a truncated peptide with a pharmaceutically acceptable auxiliary material to obtain the drug.

9. The bacterial drug according to claim 7, characterized in that The bacterial drug is one of tablets, capsules, powders, granules and suspensions.

10. The bacterial drug according to claim 7, characterized in that: The bacterial drug exhibits inhibition of bacterial growth and can be used as a therapeutic agent for treating bacteria.

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