A bacterial drug target and drug based on bacterial DNA gyrase condensates

Abstract

The present invention relates to the field of biomedical technologies, and specifically discloses a bacterial drug target and a drug based on a condensate of bacterial DNA gyrase. The bacterial drug target is a condensate structure formed by bacterial DNA gyrase in bacteria, and this condensate structure is crucial for the DNA replication, repair, and transcription processes of bacteria; the bacterial drug contains a DNA mimetic peptide segment and a truncated peptide designed based on the condensate structure of the drug target; the bacterial drug can target the condensate of bacterial DNA gyrase, including DNA gyrase mutants, and interfere with the formation or function of the condensate of bacterial DNA gyrase, thereby inhibiting the growth and reproduction of bacteria. The bacterial drug of the present invention can induce the solidification of the liquid condensate of DNA gyrase, showing inhibition of bacterial growth, and can be used as a therapeutic agent for bacterial diseases.

Description

Technical Field

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

[0002] Bacterial DNA gyrase, as a key enzyme indispensable for maintaining the DNA topological structure in bacteria, plays a crucial role in core life processes such as DNA replication, repair, and transcription. DNA gyrase consists of two GyrA and two GyrB subunits, forming a tetrameric complex. The conservation of DNA gyrase is reflected in its structure and function. From an evolutionary perspective, gyrB genes are highly conserved within different bacterial genera or families, making it an effective molecular marker for differentiating closely related bacterial species. gyrB The gene encodes the B subunit of 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 topoisomerases, are structurally related and are equally crucial for DNA replication and repair processes. Their basic functions are conserved in maintaining DNA structure and function. This conservation also makes them the main candidates for developing dual-target antibacterial agents with broad-spectrum antibacterial activity.

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

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

[0005] To solve the above technical problems, the present invention provides a bacterial drug target based on bacterial DNA gyrase condensates and a drug, specifically providing a new use of DNA gyrase condensates as bacterial drug targets, as well as an antibacterial strategy based on DNA mimetic peptides and truncated peptides, aiming to specifically bind to and interfere with the formation or function of bacterial DNA gyrase condensates, thereby effectively inhibiting the growth and reproduction of bacteria and opening up a new way for antibacterial treatment.

[0006] In a first aspect, the present invention provides a bacterial drug target based on bacterial DNA gyrase condensates, adopting the following technical solution:

[0007] 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 for the DNA replication, repair, and transcription processes of bacteria.

[0008] The present invention identifies bacterial DNA gyrase condensates as a novel bacterial drug target, wherein DNA gyrase consists of two GyrA and two GyrB subunits, forming a tetrameric complex.

[0009] The present invention analyzes the GyrA and GyrB subunits of bacterial DNA gyrase using bioinformatics tools to identify intrinsically disordered regions (IDRs) that may be involved in the formation of biological condensates. Subsequently, experimental methods are used to verify the ability of the GyrA and GyrB subunits of bacteria represented by mycobacteria to form biological condensates.

[0010] Furthermore, an in vitro condensate system is established using GyrA-YFP and / or GyrB-mCherry proteins purified from Escherichia coli. The purified proteins are mixed under specific experimental conditions, and the formation of condensates is monitored at different time points using a fluorescence microscope. The results show that DNA gyrase can form condensates in vitro.

[0011] The dynamic properties of the condensates are analyzed using FRAP (fluorescence recovery after photobleaching) experiments. The quantitative analysis results of the FRAP experiments reveal the liquid properties of DNA gyrase condensates by fitting the normalized fluorescence intensity to a single exponential function.

[0012] Mix the purified recombinant proteins. Specifically, the mixture contains 0.25 pmol of linearized pUC19 plasmid DNA, 50 pmol of DNA gyrase (composed of equal amounts of 50 pmol GyrA and GyrB), and 50 pmol of a truncated form of the DNA gyrase interacting protein (A0QSY0). Using a method for detecting the ability to form condensates with a value of 1.5, it is shown that the liquid condensates of DNA gyrase are converted into fibers. The FRAP technique was used to analyze the dynamic changes of DNA gyrase condensates, and the results showed that these condensates exhibited non-liquid characteristics.

[0013] In biological research, the formation of solid or gel-like phases is often closely related to the occurrence of diseases. Typical examples include the deposition of amyloid fibrils in degenerative diseases and cancer. Such abnormal phases not only disrupt the normal physiological functions of cells but may even directly lead to the loss or death of cell functions. Inspired by this phenomenon, the present invention further studied the solidification process of DNA gyrase condensates and explored their possible effects on bacterial growth kinetics.

[0014] The DNA gyrase condensates of the present invention provide new possibilities for the treatment of bacterial diseases and can be used as drug screening targets.

[0015] In a second aspect, the present invention provides a bacterial drug, adopting the following technical solution:

[0016] A bacterial drug is designed based on the above-mentioned bacterial drug target of bacterial DNA gyrase condensates and can specifically bind to and interfere with the formation or function of bacterial DNA gyrase condensates, thereby inhibiting the growth and reproduction of bacteria.

[0017] Preferably, the bacterial drug is at least one of small molecule compounds, polypeptides, antibodies, or their derivatives.

[0018] Preferably, the bacterial drug is a DNA mimetic peptide segment and a truncated peptide.

[0019] Preferably, the DNA mimetic peptide segment and the truncated peptide are designed and screened through bioinformatics, structural biology, and molecular biology techniques.

[0020] Preferably, the DNA mimetic peptide segment is MfpA, and the primer sequences required for its synthesis are:

[0021] F:ggaggtggaggaggtggaggtggaatgcgtataggggcaaacgg;

[0022] R: AGTCCAAGCTCAGCTAATTAAGCTT gccgtggacggccagaccgt。

[0023] Preferably, the bacterial drug uses DNA mimetic peptide segments and truncated peptides as active ingredients, and also includes pharmaceutically acceptable excipients.

[0024] Preferably, the preparation method of the bacterial drug comprises mixing the DNA mimetic peptide segments and the truncated peptides uniformly with pharmaceutically acceptable excipients and then preparing the mixture.

[0025] Preferably, the bacterial drug is one of tablets, capsules, powders, granules, suspensions.

[0026] Preferably, the bacterial drug exhibits inhibition of bacterial growth and can be used as a therapeutic agent for bacterial diseases.

[0027] In summary, the present invention has the following beneficial effects:

[0028] The present invention provides a bacterial drug target based on bacterial DNA gyrase condensates, providing new possibilities for the treatment of bacterial diseases.

[0029] The bacterial drug of the present invention is designed with bacterial DNA gyrase condensates as the drug target, including DNA mimetic peptide segments that cause the liquid condensates of DNA gyrase to transform into a solid state (fibrous structure). By adding MfpA protein to the in vitro condensation system and the Escherichia coli overnight culture medium respectively, it is found that it promotes the solidification of the liquid condensates of DNA gyrase and has a significant inhibitory effect on the growth of bacteria, and can be used as a therapeutic agent for bacterial diseases. Description of the Drawings

[0030] Figure 1 Multiple sequence alignment of bacterial GyrA and GyrB protein families.

[0031] Figure 2 Crystal structure of bacterial DNA gyrase.

[0032] Figure 3 Prediction results of IDRs of bacterial GyrA and GyrB subunits.

[0033] Figure 4 In vitro condensate formation system constructed using purified GyrA-YFP and / or GyrB-mCherry proteins.

[0034] Figure 5 Fluorescence recovery after photobleaching imaging and relative fluorescence intensity changes of DNA gyrase droplets.

[0035] Figure 6Predicted aggregation regions for DNA gyrase GyrA and GyrB sequences.

[0036] Figure 7 Fluorescence images and changes in relative fluorescence intensity of the impact of DNA gyrase interacting proteins on its liquid condensate structure.

[0037] Figure 8 Fibrous structure of DNA gyrase under electron microscopy.

[0038] Figure 9 Evaluation of the impact of DNA gyrase solid condensates on bacterial growth by serial dilution method.

[0039] Figure 10 Impact of targeting DNA gyrase condensates on host bacterial load in zebrafish models.

[0040] Figure 11 Analysis of the disordered regions of DNA-mimicking proteins AlbG(2XT4) and MfpA(6ZT4) using the PONDER algorithm.

[0041] Figure 12 Prediction of the aggregation regions of DNA-mimicking proteins AlbG(2XT4) and MfpA(6ZT4) using the TANGO algorithm.

[0042] Figure 13 Structure configuration and cloning of MfpA truncated fragments.

[0043] Figure 14 Evaluation of the antibacterial activity of MfpA truncated genes by CFU assay.

[0044] Figure 15 DNA gyrase condensate fiber formation promoted by MfpA4-7 under electron microscopy.

[0045] Figure 16 Pathogen load and pulmonary pathological damage in the lungs of mice after treatment with MfpA4-7. Detailed implementation mode

[0046] The present invention will be 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 be able to implement it, but the specific embodiments cited shall not be construed as limiting the present invention.

[0047] Example 1 Identification and verification of the ability of bacterial DNA gyrase GyrA and GyrB subunits to form biological condensates:

[0048] 1.1 Collect the protein sequences of bacterial GyrA and GyrB subunits from the National Center for Biotechnology Information (NCBI) in the United States and the UniProt database, and compare the amino acid sequences of GyrA and GyrB subunits of different bacteria (the comparison results are shown in Figure 1 ), Figure 1 in which A above is the amino acid sequence of the GyrA subunit and B below is the amino acid sequence of the GyrB subunit; the results show that the GyrA and GyrB protein families exhibit high conservation, indicating that in different bacteria, some key features and functional regions of these proteins remain highly consistent. 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 which A is the crystal structure of DNA gyrase of Mycobacterium tuberculosis, B is the crystal structure of DNA gyrase of Escherichia coli K12, and C is the crystal structure of DNA gyrase of Staphylococcus aureus, and the results further verify the functional consistency of these proteins in multiple bacteria.

[0049] 1.2 Using Escherichia coli ( Escherichia coli and Mycobacterium smegmatis ( Mycobacterium smegmatis ) as models, use the bioinformatics tool PONDER (Prediction of Naturally Disordered Regions) algorithm to predict the intrinsically disordered regions (IDRs) (the prediction results are shown in Figure 3 ), Figure 3 in which A is the IDR prediction result of the GyrA and GyrB subunits of Escherichia coli, where the left side represents the GyrB subunit and the right side represents the GyrA subunit, and B is the IDR prediction result of the GyrA and GyrB subunits of Mycobacterium smegmatis, where the left side represents the GyrB subunit and the right side represents the GyrA subunit. The results show that the GyrA and GyrB subunits have widely distributed intrinsically disordered regions, indicating their potential to form biomolecular condensates.

[0050] Example 2 Experimental verification of the formation of DNA gyrase liquid condensates in vitro:

[0051] 2.1 Using mycobacterial DNA gyrase as a model, recombinant proteins of GyrA and GyrB subunits were obtained by gene cloning and protein expression techniques.

[0052] 2.1.1 PCR amplification of the target fragment

[0053] Using Mycobacterium smegmatis ( M. smegmatis mc 2155) Genomic DNA was used as the PCR amplification template, and the pET28a plasmid (containing an N-terminal His tag) was used as the expression vector to amplify the gene fragments encoding the GyrA and GyrB subunits. YFP and mCherry fusion tags were constructed for GyrA and GyrB respectively during primer design.

[0054] Primer sequences for pET28a-GyrA-YFP

[0055] -28a-GyrA-YFP-F:

[0056] GTATTTCCAGGGCCATATGatgactgatacgacgctgccg

[0057] -GyrA-LINKER-R:

[0058] tccacctccacctcctccacctcccgcctcgggtgactcggcgg

[0059] -LINKER-YFP-F:

[0060] ggaggtggaggaggtggaggtggaatggtgagcaagggcgaggagctgtt

[0061] -28a-GyrA-YFP-R:

[0062] CGAGTGCGGCCGCAAGCTTCTAttgtacagctcgtccatgc

[0063] Primer sequences for pET28a-GyrB-mCherry

[0064] -28a-GB-MC-F:

[0065] GTATTTCCAGGGCCATATGgtggctgcccagaagaacaat

[0066] -GyrB-LINKER-R:

[0067] TCCACCTCCTCCACCTCCaacatccaggaagcgaacg

[0068] -LINKER-mCherry-F:

[0069] cttcctggatgttGGAGGTGGAGGAGGTGGAGGTGGAATGGTGAGCAAGGGCGA

[0070] -28a-GB-MC-R:

[0071] TCGAGTGCGGCCGCAAGCTTTCActtgtacagctcgtccatgc。

[0072] 2.1.2 Plasmid vector preparation

[0073] The pET28a plasmid (containing an N-terminal His tag) was selected as the expression vector, and the plasmid was digested with Nde I and Hind III to linearize the vector.

[0074] 2.1.3 Construction of recombinant plasmids

[0075] The GyrA gene sequence was cloned into the pET28a vector to construct pET28a-YFP-GyrA;

[0076] The GyrB gene sequence was cloned into the pET28a vector to construct pET28a-GyrB-mCherry.

[0077] 2.1.4 Expression and purification of recombinant proteins

[0078] The constructed recombinant plasmids were separately transformed into E. coli BL21(DE3). E. coli Cultured in LB medium containing 100 mg / L kanamycin until OD 600Reached 0.6, then 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to induce the protein, and it was incubated overnight at 16 °C. Subsequently, centrifugation was carried out to collect the bacterial cells. The bacteria with 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. The supernatant was incubated with Ni-NTA 6FF agarose beads (Easybio, catalog number BE6947) equilibrated with buffer A at 4 °C for 2 hours. Subsequently, the beads were washed with 15 column volumes (CV) of buffer A containing 20 mM imidazole to remove unbound proteins. Then, elution was performed using buffer A containing 300 mM imidazole to release the target protein. Further purification was carried out using an AKTA Purifier 10 chromatography system. Using Superdex 200 Increase 10 / 300 GL or Superdex 75 Increase 10 / 300 GL, the fractions with the recombinant protein were collected. Finally, the purified protein was concentrated using an Amicon Ultra-15 centrifugal filter (30 kDa molecular weight cut-off).

[0079] 2.1.5 Verification of the ability to form condensates

[0080] The purified recombinant GyrA (50 pmol) was mixed with GyrB (50 pmol) protein and linearized pUC19 plasmid DNA (0.25 pmol, obtained by treatment with the rapid restriction enzyme BamHI), and incubated at 4 °C in a Grace Bio-Labs CoverWell™ imaging chamber.

[0081] Observation was carried out using a Leica TCS SP8 STED confocal microscope with a 100× oil immersion objective. The fluorophores were excited and detected at the following wavelengths: EGFP at 488 nm, YFP at 514 nm, and mCherry at 550 nm. The fluorescence signals were collected using a 6-channel Spectra X LED light source and a Sedat Quad filter set, and photographs were taken (for the in vitro condensate formation system, see Figure 4 ). Figure 4 Among them, from left to right are the photographed results at 0 h, 6 h, 12 h, and 24 h. The yellow fluorescence in the figure represents GyrA-YFP, showing the condensate structure of DNA gyrase.

[0082] 2.1.6 Analysis of the dynamic properties of DNA gyrase condensates

[0083] An 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 about 2 μm was photo-bleached using a 514 nm argon ion laser with a power of 20%. Through the fluorescence recovery after photo-bleaching (FRAP) technique, the dynamic properties of DNA gyrase condensates were further evaluated. At t = 0, a specific area was photo-bleached, and the fluorescence recovery of this area was captured at subsequent time points (see Figure 5 ). Figure 5 In, A shows the photo-bleached area and the 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 photo-bleaching, 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. The final results prove that the condensate formed by GyrA and GyrB subunits has liquid-like properties.

[0084] Example 3 Evaluation of the potential of drug targets:

[0085] In biological research, the formation of solid or gel-like phases is often closely related to the occurrence of diseases. Typical examples include the deposition of amyloid fibrils in degenerative diseases and cancers. Such abnormal phases not only disrupt the normal physiological functions of cells but may even directly lead to the loss of cell function or death. Inspired by this phenomenon, the present invention further studied the solidification process of DNA gyrase liquid condensates and explored its possible impact on bacterial growth kinetics.

[0086] 3.1 Bioinformatics prediction

[0087] The TANGO algorithm was used to analyze the aggregation propensity of proteins. The version of the TANGO algorithm used was tango2_3. The parameter settings were as follows: C-terminal and N-terminal protection were enabled, the temperature was set to 298 Kelvin, the pH value was 7.6, and the salt concentration was 150 mM, which were consistent with the experimental environment for purifying DNA gyrase proteins. Specifically, fragments containing more than 5 consecutive amino acid residues with a score higher than 5% were identified as potential aggregation regions. The prediction results by the TANGO algorithm showed that there were high-risk aggregation regions in the GyrA and GyrB subunit sequences of DNA gyrase (see Figure 6 ). Figure 6Among them, 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 mutant sequences of the DNA gyrase protein sequence by the TANGO algorithm.

[0088] 3.2 Experimental verification

[0089] Mix the purified recombinant proteins. The mixture contains 0.25 pmol of linearized pUC19 plasmid DNA, 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. The mixture was diluted 100-fold, and then the diluted sample was spread on a carbon-coated Formvar grid (Beijing Zhongjing Technology, catalog number BZ11022) and incubated at room temperature for 1 minute. To ensure the best visual effect, the sample was rinsed twice with autoclaved distilled water to remove any excess or unbound substances. Then it was stained with a 1% (w / v) uranyl solution for 1 minute to enhance the contrast and completely air-dried.

[0090] First, in terms of imaging, the prepared grid was placed in a Hitachi HT7800 transmission electron microscope (Hitachi brand, 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 The fibrous structures formed by DNA gyrase under specific interaction conditions were shown by electron microscopy techniques in Figure 7 ). Figure 7 In

[0091] A shows the effect of the DNA gyrase interacting protein on the structure of its liquid condensates, and B shows the analysis of the dynamic properties of the DNA gyrase molecular condensates by the FRAP technique.

[0092] Example 4 Effect of DNA gyrase solid condensates on bacterial growth:

[0093] 4.1 Effect of induced solid DNA gyrase condensates on bacterial growth

[0094] 4.1.1 PCR Amplification of Gene Fragment (Using DNA Gyrase Interaction Protein (A0QSY0) as Template)

[0095] Using the genomic DNA of Mycobacterium smegmatis mc2155 as the PCR amplification template, the gene fragment F47 encoding DNA gyrase interaction protein (A0QSY0) was amplified.

[0096] The primer sequences were as follows:

[0097] 80L-MfpA-4-F:

[0098] GCATCACCATCACCATCACGGATCCagcacgttcaccaactgcag

[0099] 80L-MfpA-7-R:

[0100] AGTCCAAGCTCAGCTAATTAAGCTTctcctccaggcgtgcgtcct

[0101] 4.1.2 Preparation of Plasmid Vector

[0102] The pQE80L plasmid (containing an N-terminal His tag) was selected as the expression vector, and the plasmid was digested with EcoRI and HindIII to linearize the vector.

[0103] 4.1.3 Construction of Recombinant Plasmid

[0104] The gene sequence of DNA gyrase interaction protein (A0QSY0) was cloned into the pQE80L vector to construct pQE80L-F47.

[0105] 4.1.4 Evaluation by Spot Plate Experiment

[0106] To detect the inhibitory effect of the expression of DNA gyrase interaction protein (A0QSY0) F47 protein on bacterial growth, the overnight culture (1.5 ml) of Escherichia coli containing the indicator plasmid pQE80L-F47 was adjusted to the same optical density at 600 nm (OD 600 value of approximately 0.8, in the stationary phase), and then serially diluted in sterile LB liquid medium. Serial 10-fold dilutions were performed stepwise with LB medium until the desired dilution (10 -7 ). Subsequently, 3 μL of the diluted solution was dropped onto LB agar plates containing IPTG (0.5 mM, to induce protein expression) or without IPTG. A certain volume (such as 1 mL) was taken from the original culture and spread on an LB agar plate, and incubated overnight at 37°C, and then the growth of bacteria was observed (see Figure 9), Figure 9 Among them, the left side is the uninduced group without IPTG, and the right side is the induced group with IPTG. The results show that the number of bacterial colonies on the IPTG-induced group plate is significantly less than that of the uninduced group, and there are differences in colony morphology, size and other characteristics, indicating that the truncated protein F47 of IPTG-induced DNA gyrase interacting protein (A0QSY0) has a significant inhibitory effect on the growth of bacteria.

[0107] Example 5 Evaluation of the effect of solidification of target DNA gyrase liquid condensate on the host bacterial load by zebrafish model:

[0108] The effect of target DNA gyrase condensate on the host bacterial load was evaluated by zebrafish model. AB-type wild-type zebrafish were used in the experiment, and the feeding and management were referred to the standards of the Chinese Zebrafish Resource Center. The experimental subjects were zebrafish embryos, and at the 1-4 cell stage, they were respectively injected with M. marinum strains expressing EGFP. The experimental group embryos were inoculated with M. marinum strains mixed with 0.5 mg / mL truncated form F47 of DNA gyrase interacting protein (A0QSY0), while the control group embryos were only inoculated with M. marinum strains mixed with PBS (phosphate buffer), and 200-300 CFU were inoculated into each embryo.

[0109] The embryos were incubated at 28.5 °C, and 0.003% 1-phenyl-2-thiourea (PTU) was added to the culture medium to inhibit pigment formation. After 72 hours of infection, the embryos were microscopically imaged using Z-axis stack scanning technology (see Figure 10 ). Figure 10 Among them, 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 images of zebrafish embryos in the experimental group and the control group 72 hours after infection, and the difference in bacterial load is directly reflected by the contrast of color depth. The evaluation results show that the solidification effect of DNA gyrase condensate as a targeted drug target significantly reduces the bacterial load of the host.

[0110] Through bioinformatics analysis and experimental verification, it was revealed that the GyrA and GyrB subunits of DN gyrase have the potential ability to form biological condensates, and their potential application value as drug targets was demonstrated.

[0111] Example 6 Screening and characterization of DNA mimic proteins:

[0112] 6.1 Bioinformatics analysis and prediction

[0113] 6.1.1 Acquisition of protein sequences

[0114] To deeply study the characteristics of DNA mimic proteins, first collect the protein sequences of bacterial DNA mimic proteins from the National Center for Biotechnology Information and the UniProt database in the United States; the specific protein sequences are shown as follows:

[0115] >M.smegmatis

[0116] MRIGANGDETVWADEEFAGRDFRDEDLSRIRTERVVFTECDFSGVDLSESEHHGSAFRNCTFRRSTIWHSTFTNCSLLGSVFTECRIRPVTFVECDFTLAVLGGCDLRAVDLSDCRLREVSLVGADLRKAVLRRADLTGSRVQDARLEEADLRGTRVDPTFWTTAKVRGAKIDIEQALAYAAAHGLAVHGG-----

[0117] >M.tuberculosis

[0118] --------MQQWVDCEFTGRDFRDEDLSRLHTERAMFSECDFSGVNLAESQHRGSAFRNCTFERTTLWHSTFAQCSMLGSVFVACRLRPLTLDDVDFTLAVLGGNDLRGLNLTGCRLRETSLVDTDLRKCVLRGADLSGARTTGARLDDADLRGATVDPVLWRTASLVGARVDVDQAVAFAAAHGLCLAGG-----

[0119] >M.marinum

[0120] --------MEHWVDCEFTDRDFRDEDLSRLRTERVVFSECNFGGVNLTESEHRGSAFRNCSFERTTLWHSTFAQCSMLGSVFVSCRMRPLVLDEVDFTLAVLGGNDLRGVDLSGCRLREASLVETDLRKSVLRGADLRGARTNGTKLDDADLRGANLDPSLWRSASLAGARIDVPQALSFALAHGLRLDS------

[0121] >M.fortuitum

[0122] ----MVADETAWADREFTGHDFREEDLSRLTTERVVFTECDFSGVDLSESQHSGSAFRNCTFRRATLWHSTFTNCSLLGSVFTECRLRPIKIVESDLTLAVLGGCDLRGVDLSDCRLREASLVGVDLRKAILRQADLTGARVQDAKLDEADLRGARVDPTFWTTAKLRGAKIDIAQALAYSAAHGLDVHGA-----

[0123] >M.ulcerans

[0124] --------MEHWVDCEFTGRDFRDEDLSRLRTERVVFSECNFGGVNLTESEHRGSAFRNCSFERTTLWHSTFAQCSMLGSVFVSCRMRPLVLDEVDFTLAVLGGNDLRDVDLSGCRLREASLVETDLRKSVLRGADLRGARTNGTKLDDADLRGANLDPSLWRSASLAGARIDVPQALSFALAHGLRLAS------

[0125] >M.chelonae

[0126] -------MAEHWIDREITGETFYDEDFRELHTERVVFTECDFSGANLTESLHIGSAFRNCTFRRTSLWHSEFRQCSLLGSTLTDCRVRPSKFTETDFTLSSLGGLDLREMDLSDCRFREANLVGTDMRKANLHGADFTGARTQNLKLDGADLRGARIDPTLWTTATLMAAKVDLPQAVAYAAGHGLDVHGG-----

[0127] >M.avium

[0128] --------MTAWVDREFERHDFTDEDLVGLSTERVVFTECNFSGANLAESRHRASAFRNCTFRRTSLWHSTFEQCTMLGSVFEQCRLRPVTFDEVDFTLAVLGGNDLRGVDLSGCRLRETSLVEADLRKAVLRGADLRGARTAGTRLDDADLRGGAADQALWTTASLAGARVDVDQAVAFALGHGLRLDG------

[0129] >M. abscessus

[0130] -------MAEHWTDREITAETFYDEDFRELHTERVVFTECDFSGANLTESLHVGSAFRNCTFRRTSLWHSEFRQCSLLGSTFTDCRVRPSKFTETDFTLSSLAGLDLREMDLSDCRFREANLVGADMRKANLHGADFTGARTQNLKLDGADLRGARIDPTLWTTAALITAKVDLPQAIAFAAAHGLDVHGG-----

[0131] >M. intracellulare

[0132] -------MSEEWVDREFDGHDFTDEDLSRLRTERTVFTECNFSGANLAESQHRGSAFRNCSFQRTSLWHSSFAQCSMLGSVFVQCRLRPITFDEVDFTLAVLAGIDLRGVDFSGCRLREASLVEADLRKAVLRGADLRGARTAGARLDGADLRGTTADPGLWTTASLAGARVDVDQAVAFALAHGLRLDGGPHDDG

[0133] >M. haemophilum

[0134] --------MQQWTDCEIAGRDFTDDDLSRLRTERVVFTECNFSGANLAESHHRASAFRNCTFKRTLLWHSTFTHCSMLGSVFVQCRLRPLTFDEVDFTLAVLGGNDLRGVDLSGCRLREASLVETDLRTAVLRGADLSGARTTGTRLDDADLRGATVEPSLWRTASLAGARIDVPQAVAFALAHGLCLNGGLDA--。

[0135] 6.1.2 Prediction of IDRs (intrinsically disordered regions)

[0136] To evaluate the structural characteristics of the bacterial DNA-mimicking protein (taking the MfpA protein as an example in this embodiment), we used the PONDER algorithm to predict IDRs. This algorithm precisely analyzed the intrinsically disordered regions in the DNA-mimicking protein sequence by setting the parameters VL-X1 and XL1-XT. The prediction results showed that significant intrinsically disordered regions were present at both ends of the DNA-mimicking protein (see Figure 11 ) Figure 11Among them, A is the disordered region of the DNA-mimicking protein AlbG(2XT4), and B is the disordered region of the DNA-mimicking protein MfpA(6ZT4), which reveals the potential ability of DNA-mimicking proteins to form condensates.

[0137] 6.1.3 Prediction of aggregation tendency

[0138] To further verify the aggregation properties of DNA-mimicking proteins, we introduced the TANGO algorithm for predicting aggregation tendency. Under specific parameter settings of the TANGO algorithm (C-terminal protection, N-terminal protection, temperature 298 Kelvin, pH 7, salt concentration 100 mM), 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 exceeding 5% (see Figure 12 ). Figure 12 Among them, A is the aggregation region of the DNA-mimicking protein AlbG(2XT4), and B is the aggregation region of the DNA-mimicking protein MfpA(6ZT4). This result shows the ability of DNA-mimicking proteins to form aggregates, providing an important basis for their application in drug development.

[0139] Example 7 High-throughput screening and verification:

[0140] 7.1 Construction of the MfpA truncation library

[0141] Taking the MfpA protein as an example to deeply study the functional characteristics of different fragments of DNA-mimicking proteins, the MfpA protein sequence was divided into 9 structural parts, and 45 truncated fragments were successfully constructed through combinations of different regions (see Figure 13 ). Figure 13 Among them, A is the bioinformatics analysis of the MfpA sequence, and B is the cloning and structural configuration of the MfpA truncations. These truncated fragments were cloned into the pQE80L vector, and the cloning process used EcoRI and HindIII restriction enzymes (provided by Fermentas, product numbers FD0074 and FD0504 respectively). Subsequently, 1 ng of plasmid DNA was mixed with 50 µl of electrocompetent E. coli cells and then transformed.

[0142] The specific sequences of the primers used are as follows:

[0143] 80L-MfpA-1-F:

[0144] GCATCACCATCACCATCACGGATCCacggtatgggcggacgaagagttcgccg

[0145] 80L-MfpA-2-F:

[0146] GCATCACCATCACCATCACGGATCCatccgcaccgaacgcgtcgt

[0147] 80L-MfpA-3-F:

[0148] GCATCACCATCACCATCACGGATCCtccgagcatcatggatcggc

[0149] 80L-MfpA-4-F:

[0150] GCATCACCATCACCATCACGGATCCagcacgttcaccaactgcag

[0151] 80L-MfpA-5-F:

[0152] GCATCACCATCACCATCACGGATCCgtcacattcgtcgagtgcga

[0153] 80L-MfpA-6-F:

[0154] GCATCACCATCACCATCACGGATCCgtcgacctgtcggactgccg

[0155] 80L-MfpA-7-F:

[0156] GCATCACCATCACCATCACGGATCCgcagtgctgcggcgggccga

[0157] 80L-MfpA-8-F:

[0158] GCATCACCATCACCATCACGGATCCgccgatctgcgcggtacgcg

[0159] 80L-MfpA-9-F:

[0160] GCATCACCATCACCATCACGGATCCgccaagatcgacatcgaaca

[0161] 80L-MfpA-1-R:

[0162] AGTCCAAGCTCAGCTAATTAAGCTTgcggctcagatcctcgtcgc

[0163] 80L-MfpA-2-R:

[0164] AGTCCAAGCTCAGCTAATTAAGCTTctccgagaggtcgacgccgc

[0165] 80L-MfpA-3-R:

[0166] AGTCCAAGCTCAGCTAATTAAGCTTgtgccagatggtgctgcgg

[0167] 80L-MfpA-4-R:

[0168] AGTCCAAGCTCAGCTAATTAAGCTTagggcggatgcggcattcgg

[0169] 80L-MfpA-5-R:

[0170] AGTCCAAGCTCAGCTAATTAAGCTTggcacgcagatcacagccgc

[0171] 80L-MfpA-6-R:

[0172] AGTCCAAGCTCAGCTAATTAAGCTTcttacgcagatccgcacc

[0173] 80L-MfpA-7-R:

[0174] AGTCCAAGCTCAGCTAATTAAGCTTctcctccaggcgtgcgtcct

[0175] 80L-MfpA-8-R:

[0176] AGTCCAAGCTCAGCTAATTAAGCTTgccgcgcaccttggccgtgg

[0177] 80L-MfpA-9-R:

[0178] AGTCCAAGCTCAGCTAATTAAGCTTgccgtggacggccagaccg

[0179] Bioinformatics analysis of the MfpA sequence and cloning of MfpA truncates. Forty-five MfpA truncation variant genes with unique structural configurations were cloned individually, each variant encoding a different structure. These genes were individually cloned into the pQE80L vector with a His6 tag.

[0180] 7.2 Evaluation of the antibacterial activity of truncated MfpA fragments by CFU assay

[0181] The growth of Escherichia coli carrying the pQE80L plasmid containing the truncated MfpA gene was evaluated by colony-forming unit (CFU) assay. After adjusting the bacterial culture to OD 600 = 0.1, 10-fold serial dilutions were performed. 3 μL of the sample was taken from each dilution and spotted onto LB agar plates containing 100 μg / mL ampicillin, with IPTG induction groups (+IPTG) and non-induction groups (-IPTG) set up respectively. The plates were incubated overnight at 37 °C (see Figure 14 ), Figure 14 . In

[0182] Example 8, the MfpA4-7 protein fragment promotes the fibrosis of DNA gyrase:

[0183] 8.1 PCR amplification of gene fragments

[0184] The genomic DNA of the Mycobacterium tuberculosis model strain Mycobacterium smegmatis ( M. smegmatis mc 2 155) was used as a template. Gene fragments encoding the GyrA and GyrB subunits and MfpA were amplified.

[0185] The specific sequences of the primers:

[0186] pET28a-GyrA-YFP

[0187] 28a-GyrA-YFP-F:

[0188] GTATTTCCAGGGCCATATGatgactgatacgacgctgccg

[0189] GyrA-LINKER-R:

[0190] tccacctccacctcctccacctcccgcctcgggtgactcggcgg

[0191] LINKER-YFP-F:

[0192] ggaggtggaggaggtggaggtggaatggtgagcaagggcgaggagctgtt

[0193] 28a-GyrA-YFP-R:

[0194] CGAGTGCGGCCGCAAGCTTCTAttgtacagctcgtccatgc

[0195] pET28a - GyrB - mCherry

[0196] 28a - GB - MC - F:

[0197] GTATTTCCAGGGCCATATGgtggctgcccagaagaacaat

[0198] GyrB - LINKER - R:

[0199] TCCACCTCCTCCACCTCCaacatccaggaagcgaacg

[0200] LINKER - mCherry - F:

[0201] cttcctggatgttGGAGGTGGAGGAGGTGGAGGTGGAATGGTGAGCAAGGGCGA

[0202] 28a - GB - MC - R:

[0203] TCGAGTGCGGCCGCAAGCTTTCActtgtacagctcgtccatgc

[0204] pQE80L - MfpA4 - 7

[0205] 80L - MfpA - 4 - F:

[0206] GCATCACCATCACCATCACGGATCCgcagtgctgcggcgggccga

[0207] 80L - MfpA - 7 - R:

[0208] AGTCCAAGCTCAGCTAATTAAGCTTctcctccaggcgtgcgtcct

[0209] 8.2 Preparation of plasmid vectors

[0210] The pET28a plasmid was selected as the expression vector (this plasmid has a strong promoter and an N-terminal His tag for protein purification). First, the pET28a plasmid was digested with NdeI and HindIII restriction enzymes (Fermentas, catalog numbers FD0583 and FD0504) to linearize the vector fragment. Then, the pQE80L plasmid was digested with EcoRI and HindIII (Fermentas, catalog numbers FD0074 and FD0504) to linearize the vector fragment.

[0211] 8.3 Construction of recombinant plasmids

[0212] 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.

[0213] 8.4 Expression and purification of recombinant proteins

[0214] The recombinant plasmids pET28a-YFP-GyrA and pET28a-GyrB-mCherry were separately transformed into E. coli BL21(DE3) cells, and pQE80L-EGFP-MfpA4-7 was transformed into E. coli M15(pREP4) cells. E. coli was cultured in LB medium containing 100 mg / L kanamycin at 37 °C in a shaker until the OD 600 reached approximately 0.6, and 0.5 mM IPTG was added to induce the expression of recombinant proteins. Subsequently, the culture temperature was reduced to 16 °C and incubated overnight to promote the correct folding and accumulation of proteins.

[0215] The bacteria expressing the recombinant proteins 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 cells were 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 at 4 °C for 2 hours. The beads were washed with 15 column volumes of buffer A containing 20 mM imidazole to remove unbound proteins, and then the target protein was eluted with buffer A containing 300 mM imidazole.

[0216] The eluted protein was further purified and buffer-exchanged using an AKTA Purifier 10 chromatography system (GE AKTA Purifier 10, Germany). Analysis was performed by size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva, catalog number 28990944) or a Superdex 75 Increase 10 / 300 GL column (Cytiva, catalog number 29148721). These columns were run in buffer A without imidazole to collect pure, non-aggregated protein for subsequent applications. The protein was concentrated using an Amicon Ultra-15 centrifugal filter (30 kDa molecular weight cut-off, Millipore, catalog number UFC903024).

[0217] 8.5 MfpA4-7 protein fragment promotes the fibrillation of DNA gyrase condensates

[0218] In the biological field, the formation of solid or gel-like phases is often closely related to pathological conditions, such as amyloid fiber deposits observed in degenerative diseases and cancers. This abnormal phase not only interferes with normal intracellular physiological processes but may also directly lead to the loss of cell function and even cell death. The MfpA4-7 protein fragment can promote the solidification of DNA gyrase molecular condensates and inhibit bacterial growth.

[0219] 8.5.1 Preparation of the MfpA4-7 recombinant protein complex mixture

[0220] The purified recombinant proteins were mixed in specific ratios. The specific mixture included 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 the MfpA4-7 protein fragment.

[0221] 8.5.2 Transmission electron microscopy observation of condensate formation ability

[0222] To examine the morphological characteristics of protein / peptide aggregates, transmission electron microscopy (TEM) was used. The mixture was diluted 100-fold, and then the diluted sample was spread on a carbon-coated Formvar grid (Beijing Zhongjing Technology, catalog number BZ11022) and incubated at room temperature for 1 minute. To ensure the best visual effect, the sample was rinsed twice with autoclaved distilled water to remove any excess or unbound substances. Then it was stained with a 1% (w / v) uranyl acetate solution for 1 minute to enhance the contrast and allowed to dry completely.

[0223] In terms of imaging, the prepared grid was placed in a Hitachi HT7800 transmission electron microscope (Hitachi brand, Japan) and observed at different magnifications. Images were captured using the HT7800 system software. The microscope was operated at an accelerating voltage of 80 kV to obtain a detailed view of the aggregate morphology (see Figure 15 ). The fibrous structures formed by DNA gyrase under specific interaction conditions were demonstrated by electron microscopy techniques.

[0224] Example 9 Animal model experiment verification:

[0225] 9.1 Mouse infection experiment

[0226] Specific pathogen-free female C57BL / 6 mice (6 - 8 weeks old) were provided by Sigenetic (Beijing). Mice were allowed a 5 - 7-day acclimation period before the start of the experiment and were then randomly divided into multiple experimental groups. To ensure the reliability of the data, each experiment at each time point was set up with three replicates. Mice were anesthetized by intraperitoneal injection of 250 mg / kg tribromoethanol to ensure the smooth progress of subsequent operations.

[0227] Under anesthesia, mice were inoculated intranasally with the Mycobacterium strain IMMI-114 (about 6×10 6 CFU dissolved in 25 μL PBS) or a mixture of IMMI-114 and 0.5 mg / mL MfpA4-7 protein. The inoculum droplets were instilled into the left nostril of the mice for infection. At 24 hours and 48 hours after infection, the mice were sacrificed and their whole lung tissues were collected for subsequent analysis.

[0228] 9.2 Lung tissue processing and bacterial load detection

[0229] The collected lung tissues were placed in a 100 μm cell strainer and homogenized thoroughly with PBS to extract samples. After homogenization, the lung tissues were diluted proportionally and inoculated onto 7H10 agar plates 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 Mycobacterium infection. The experimental results showed that in the MfpA4-7 intervention group, the bacterial load in the lungs decreased significantly ( Figure 16 A in

[0230] 9.3 Lung tissue fixation and pathological analysis

[0231] To further analyze the effect of infection on lung tissue, the collected lung tissue was fixed with 4% paraformaldehyde. The fixed tissue was dehydrated, embedded through a standard process, and sectioned for hematoxylin-eosin (HE) staining. Subsequently, a Leica SCN400 microscope (Wetzlar, Germany) was used to examine the stained lung tissue in detail to analyze the pathological changes in the lungs ( Figure 16 in B).

[0232] 9.4 Experimental Observation and Pathological Analysis

[0233] As Figure 16 shown, the MfpA4-7 protein had a significant inhibitory effect on murine lung infection. Through pathological analysis, it was found that the pathogen load in the lungs of mice treated with MfpA4-7 was significantly reduced, and the degree of pathological damage to the lungs was significantly alleviated. This phenomenon indicates that MfpA4-7 has a significant therapeutic effect in anti-mycobacterial infection. Specifically, MfpA4-7 showed a significant inhibitory effect on the pathogen load in a murine model of mycobacterial infection, further verifying its potential as a treatment for mycobacterial infection.

[0234] This specific embodiment is only an interpretation of the present invention and is not a limitation thereto. After reading this specification, those skilled in the art may make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A bacterial drug, characterized in that The bacterial drug is designed with the condensate structure formed by bacterial DNA gyrase in the bacteria as the drug target, including a DNA mimicking peptide segment MfpA4-7 that transforms the liquid condensate of DNA gyrase into a solid state; The nucleic acid sequence of the DNA mimicking peptide segment MfpA4-7 is obtained by the following method: using Mycobacterium smegmatis M. 2 155 genomic DNA was used as a template for PCR amplification. 2 155 amplification is obtained; wherein the amplification primer sequence is as follows: 80L-MfpA-4-F: GCATCACCATCACCATCACGGATCCagcacgttcaccaactgcag 80L-MfpA-7-R: AGTCCAAGTCCAGCTAATTAAGCTTctcctccaggcgtgcgtcct.

2. The bacterial drug according to claim 1, characterized in that The DNA mimetic peptide segment MfpA4-7 is designed and screened through bioinformatics, structural biology and molecular biology techniques.

3. The bacterial drug according to claim 1, characterized in that The bacterial drug uses the DNA mimicking peptide segment MfpA4-7 as an active ingredient and also includes pharmaceutically acceptable excipients.

4. The bacterial drug according to claim 3, characterized in that The preparation method of the bacterial drug comprises the steps of uniformly mixing the DNA mimicking peptide segment MfpA4-7 with pharmaceutically acceptable excipients.

5. The bacterial drug according to claim 3, characterized in that The bacterial drug is one of tablets, capsules, powders, granules and suspensions.

6. The bacterial drug according to claim 3, 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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