Application of histone deacetylase-like amide hydrolase HDAH inhibitor in preparation of antibacterial drugs

Abstract

The invention provides an application of an HDAH inhibitor in preparation of antibiotics. According to the invention, research finds that escherichia coli has Zn < 2 + >-dependent histone deacetylase-like amide hydrolase-HDAH, and the HDAH can directly influence the enzyme activity of metabolic enzyme through acetylation modification so as to regulate and control the metabolic level of bacteria; and the transcription level of metabolism-related genes can be indirectly regulated by acting on a transcription regulation factor (such as histone-like protein HU), and the metabolism of escherichia coli is synergistically regulated from two levels of post-translational modification and transcription. The invention also finds that the vorinostat (SAHA), the trichosaliocin (TSA) and the like can be combined with the HDAH and inhibit the deacetylation activity of the HDAH, are HDAH inhibitors, and inhibit the growth and migration ability of escherichia coli after being administered to the escherichia coli.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a histone deacetylase-like amidohydrolase-HDAH, its inhibitor, and the application of the inhibitor in the preparation of antibacterial drugs. Background Technology

[0002] Post-translational modifications (PTMs) regulate many important biological processes in bacteria. Lysine acetylation is one of the most abundant and important forms of protein PTMs in bacteria. In *E. coli*, the degree of acetylation is higher during the stationary phase than during the logarithmic growth phase, indicating that changes in acetylation levels affect the metabolic state of bacteria. The occurrence of Nε-lysine acetylation is mainly mediated by Nε-lysine acetyltransferase (KAT), while the removal of Nε-lysine acetylation is mainly mediated by deacetylase (KDAC), of which NAD... + CobB-dependent (SIR2-like) is the most common KDAC in bacteria, but proteomics results show that CobB can only act on 10% of acetylated lysine residues. Meanwhile, mass spectrometry-based proteomics methods have identified many novel PTMs, such as succinylation, crotonylation, benzoylation, 2-hydroxyisobutyrylation, β-hydroxybutyrylation, and lactation. For these novel acylation modifications, CobB's deacetylation catalytic activity exhibits selectivity and limitations; for example, it is more effective at "erasing" succinylation, 2-hydroxyisobutyrylation, and lactation modifications, but less effective at "erasing" crotonylation, benzoylation, and β-hydroxybutyrylation modifications. Therefore, it is speculated that other deacetylases may exist in *E. coli*.

[0003] Furthermore, bacteria establish a parasitic relationship with their hosts by infecting them and influencing the host's physiological and pathological states through the secretion of metabolic small molecules or protein factors. This is especially true of gut bacteria, including *Escherichia coli*. Post-translational modifications of bacterial functional proteins can lead to abnormal bacterial metabolism or changes in virulence, which may in turn affect the host's physiological homeostasis. Therefore, identifying deacetylases in bacteria and studying their substrates to reveal the biological functions of substrate post-translational modifications can deepen our understanding of the subtle controls by which microorganisms regulate cellular physiology, providing new directions for unraveling the mechanisms of disease development and bacterial life, and potentially leading to the discovery of new antibacterial targets. Summary of the Invention

[0004] The inventors of this application discovered that it contains Zn in Escherichia coli through sequence alignment (BLAST) and structural alignment. 2+The histone-dependent deacetylase-like amidohydrolase, HDAH, is named EcHDAH in this invention, also known as Escherichia coli HDAH. Database searches revealed that similar HDAHs are also found in Pseudomonas aeruginosa, Alcaligenaceae bacterium, and Legionella pneumophila. In this invention, these enzymes are also abbreviated as deacetylase HDAH.

[0005] Through transcriptomics and bioinformatics analysis, the inventors of this application identified and characterized the gene distribution characteristics and patterns regulated by the E. coli deacetylase EcHDAH (EcHDAH). Among them, the differentially expressed genes are mainly related to metabolism (glycolysis, TCA cycle and pentose phosphate pathway). This result suggests that the E. coli deacetylase EcHDAH may play a regulatory role in bacterial metabolism.

[0006] The inventors of this application further utilized mass spectrometry for proteomics identification, systematically analyzing the potential endogenous substrate proteins regulated by E. coli EcHDAH. They discovered that the protein substrates regulated by EcHDAH are mainly metabolic enzymes (such as fructose-1,6-bisphosphatase, succinate dehydrogenase, malate dehydrogenase, and isocitrate lyase) and transcriptional regulators (such as uracil-DNA glycosylase, histone-like HU, and DNA topoisomerase). Based on this, the applicants hypothesize that EcHDAH can not only directly affect the enzyme activity of metabolic enzymes through acetylation modification, thereby regulating their metabolic levels, but also indirectly regulate the transcriptional levels of metabolism-related genes by acting on transcriptional regulators (such as histone-like HU), thus synergistically regulating the metabolism of E. coli at both the post-translational modification and transcriptional levels.

[0007] The nucleoid structure of bacteria differs from that of eukaryotic chromosomes. Lacking ordered nucleosomes, bacteria are composed of compressed DNA and histone-like proteins involved in this compression, such as HU and H-NS. HU, the most conserved histone-like protein ubiquitous in bacteria, is crucial for bacterial nucleoid condensation and can directly regulate the global transcriptional profile. Studies have confirmed that mutations in specific amino acids of the HU protein in *E. coli* alter bacterial morphology and transcriptional profiles. For example, mutations in HU protein E38K and V42L can lead to transcriptional reprogramming and activate factors related to invasion of mammalian cells, transforming the bacteria into invasive strains. This indicates that HU mutations alter transcriptional regulation patterns and key processes (such as invasion pathogenesis) in cells by changing the topological structure of the DNA-protein complex. Numerous studies have shown that lysine residues in bacterial histone HU undergo acetylation. Based on this, the applicant hypothesizes that acetylation of lysine residues in HU can neutralize the positive charge on the ε-amino group of lysine residues and increase the size of the side chain, thereby affecting the interaction between HU protein and DNA, influencing the nucleoid structure, and ultimately regulating bacterial gene transcription and expression profiles. The E. coli deacetylase EcHDAH may affect the transcription of genes related to bacterial glycolysis (pyruvate kinase, lactate dehydrogenase) by removing lysine acetylation modification on histone HU, thus affecting glucose metabolism and life activities. Therefore, inhibiting HDAH may influence bacterial metabolism and life activities, thereby achieving antibacterial effects.

[0008] The inventors of this application discovered that vorinostat (SAHA) and trichosmin (TSA) can bind to EcHDAH and inhibit its deacetylation activity, thus acting as inhibitors of EcHDAH. When this inhibitor was administered to E. coli, it indeed inhibited the growth and migration ability of the bacteria.

[0009] Based on the above research, this invention was completed, and the following technical solution is proposed:

[0010] Application of HDAH inhibitors in the preparation of antibiotics.

[0011] The antibiotics can be used to kill or inhibit the growth or migration ability of bacteria such as Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium, and Legionella pneumophila.

[0012] The HDAH inhibitor may be an inhibitor that inhibits the expression of the HDAH gene or inhibits the HDAH protein.

[0013] The inhibitor that inhibits HDAH gene expression can be an inhibitor that inhibits the mRNA level of the HDAH gene, including but not limited to: antisense nucleic acid sequences, siRNA, miRNA, shRNA, dsRNA targeting the HDAH gene, or proteins, peptides, enzymes, small molecule compounds (e.g., natural compounds, synthetic compounds, etc.) that inhibit the mRNA level of the HDAH gene.

[0014] The inhibitor of HDAH protein can be an inhibitor of the protein activity or protein level of HDAH, including but not limited to: antibodies against HDAH, proteins, peptides, enzymes, and small molecule compounds (e.g., natural compounds, synthetic compounds, etc.).

[0015] In one embodiment of the present invention, the HDAH is EcHDAH, whose amino acid sequence is shown in SEQ No. 1. SEQ No. 1: MPLPLIYHEDYSPEFPAEHRFPMDKFRLLHDHLIDSGLTTDQALLRPDICPNDILALAHDRSYIERYMNGDLSREDQRRLGLPWSEALARRTVRAVGGSLLSAEMALQHGIACHLAGGTHHAHYDHPAGFCIFNDLAVISRYLLEAGRVHRVLIFDCDVHQGDGTARILHETPDAITVSLHCEQNFPARKAQSDWDIPLPRGMGDMAYLKVVDDALNYLLPLYRPDLVLYDAGVDVHKDDALGYLQLTDAGVAARDEAVLRHCMGRDIPVVGVIGGGYSKDRAALARRHGILHHSAARVIGCSQ.

[0016] In one embodiment of the present invention, the HDAH is a homolog of EcHDAH, and its amino acid sequence has at least 20% identity with the amino acid sequence shown in SEQ No. 1, for example, 22.6%, 20.6%, 21.1%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, such as the HDAH of bacteria such as Pseudomonas aeruginosa, Alcaligenaceae bacterium, or Legionella pneumophila.

[0017] In one embodiment of the invention, the inhibitor is a compound that inhibits HDAH activity, such as vorinostat (SAHA). Trichosanthecin (TSA) APHA 3c wait.

[0018] The use of HDAH or its encoding gene as a drug target in screening antibiotics.

[0019] As mentioned above, the HDAH is EcHDAH or its homologs. The amino acid sequence of EcHDAH is shown in SEQ No. 1. The amino acid sequence of the EcHDAH homolog has at least 20% identity with the amino acid sequence shown in SEQ No. 1, for example, 22.6%, 20.6%, 21.1%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity, such as the HDAH of bacteria such as Pseudomonas aeruginosa, Alcaligenaceae bacterium, or Legionella pneumophila.

[0020] The antibiotics can be used to kill or inhibit the growth or migration ability of bacteria such as Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium, and Legionella pneumophila.

[0021] A method for screening antibiotics in vitro, the method comprising the following steps:

[0022] 1) Under appropriate environmental conditions, mix the test chemical with HDAH, or mix the test chemical with cells expressing HDAH;

[0023] 2) After an appropriate period of time, detect the binding of the test chemical to HDAH, or detect changes in HDAH deacetylase activity, or detect changes in HDAH expression levels in the cells, or detect cell growth or migration.

[0024] According to the present invention, if the test compound can bind to HDAH, or can inhibit the deacetylase activity of HDAH, or reduce the expression of HDAH in cells, or can inhibit cell growth or migration, then the test compound has HDAH inhibitory activity and can be further developed as a potential antibiotic.

[0025] As mentioned above, the HDAH is EcHDAH or its homologs. The amino acid sequence of EcHDAH is shown in SEQ No. 1. The amino acid sequence of the EcHDAH homolog has at least 20% identity with the amino acid sequence shown in SEQ No. 1, for example, 22.6%, 20.6%, 21.1%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity, such as the HDAH of bacteria such as Pseudomonas aeruginosa, Alcaligenaceae bacterium, or Legionella pneumophila.

[0026] In some embodiments of the present invention, the cells are bacteria with HDAH. In one embodiment of the present invention, the bacteria are Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium, or Legionella pneumophila.

[0027] The antibiotics can be used to kill or inhibit the growth or migration ability of bacteria such as Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium, and Legionella pneumophila.

[0028] According to the present invention, this drug screening method can be used in the new drug development process in the pharmaceutical industry.

[0029] Those skilled in the art will understand that "appropriate environmental conditions" in step 1) of the method refers to the reaction environment and conditions that enable the test chemical substance to mix with HDAH and potentially interact, or the reaction environment and conditions that enable the test chemical substance to mix with cells expressing HDAH and potentially interact, including solution systems and temperature, for example, the experimental operating environment and conditions involved in the embodiments of the present invention.

[0030] Those skilled in the art will also understand that the “appropriate time” in “after an appropriate time” in step 2) of the method refers to the time that allows the test chemical substance to interact with HDAH and carry out subsequent reactions; or the time that allows the test chemical substance to interact with cells expressing HDAH and carry out subsequent biological or chemical reactions.

[0031] According to the present invention, the detection in step 2) of the screening method can be performed using various detection methods known in the art, including but not limited to: flow cytometry, enzyme-linked immunosorbent assay, MTT assay, real-time PCR, Western blot, bacterial culture, ITC analysis, etc.

[0032] In a preferred embodiment of the present invention, the present invention provides the following technical solution:

[0033] Application of EcHDAH inhibitors in the preparation of antibiotics that kill or inhibit Escherichia coli.

[0034] The EcHDAH inhibitor may be an inhibitor that inhibits the expression of the EcHDAH gene or the EcHDAH protein.

[0035] The inhibitors that suppress EcHDAH gene expression include, but are not limited to: antisense nucleic acid sequences, siRNA, miRNA, shRNA, dsRNA, or proteins, peptides, enzymes, and small molecule compounds (e.g., natural compounds, synthetic compounds, etc.) that target the EcHDAH gene, or that inhibit the mRNA level of the EcHDAH gene.

[0036] The inhibitors that inhibit EcHDAH protein include, but are not limited to: antibodies against EcHDAH, proteins, peptides, enzymes, and small molecule compounds (e.g., natural compounds, synthetic compounds, etc.) that inhibit EcHDAH activity or protein levels.

[0037] The amino acid sequence of EcHDAH is shown in SEQ No. 1.

[0038] In one embodiment of the present invention, the EcHDAH inhibitor is a compound that inhibits EcHDAH activity, such as vorinostat (SAHA). Trichosanthecin (TSA) APHA 3c wait.

[0039] The use of EcHDAH or its encoding gene as a drug target in screening for antibiotics that kill or inhibit Escherichia coli.

[0040] As mentioned above, the amino acid sequence of EcHDAH is shown in SEQ No. 1.

[0041] A method for screening antibiotics that kill or inhibit Escherichia coli in vitro, the method comprising the following steps:

[0042] 1) Under appropriate environmental conditions, mix the test chemical with EcHDAH, or mix the test chemical with E. coli expressing EcHDAH;

[0043] 2) After an appropriate period of time, detect the binding of the test chemical substance to EcHDAH, or detect changes in EcHDAH deacetylase activity, or detect changes in the expression level of EcHDAH in the E. coli, or detect the growth or migration of E. coli.

[0044] According to the present invention, if the test compound can bind to EcHDAH, or can inhibit the deacetylase activity of EcHDAH, or reduce the expression of EcHDAH in Escherichia coli, or can inhibit the growth or migration of Escherichia coli, then the test compound has EcHDAH inhibitory activity and can be further developed as a potential antibiotic for killing or inhibiting Escherichia coli.

[0045] As mentioned above, the amino acid sequence of EcHDAH is shown in SEQ No. 1.

[0046] According to the present invention, the detection in step 2) of the screening method can be performed using various detection methods known in the art, including but not limited to: flow cytometry, enzyme-linked immunosorbent assay, MTT assay, real-time PCR, Western blot, bacterial culture, ITC analysis, etc.

[0047] Terminology definition:

[0048] "And / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0049] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed.

[0050] Homology: As used herein, the term "homology" refers to the overall correlation between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Therefore, two homologous molecules will share a common evolutionary ancestor. In the context of this disclosure, the term homology includes both identity and similarity.

[0051] In some embodiments, polymer molecules are considered “homological” if at least 20%, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the monomers in the molecule are identical (completely identical monomers) or similar (conservative substitutions). The term “homological” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0052] Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules.

[0053] "Homologous protein" and "homogeneous protein" have the same meaning: proteins with obvious similarity in amino acid sequence that perform the same or similar functions in different organisms or in the same organism.

[0054] Suitable software programs are available from various sources and can be used for the alignment of both protein and nucleotide sequences. Examples include Bl2seq, Needle, Stretcher, Water, or Matcher. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX, or Clustal Omega), MUSCLE, etc.

[0055] The term "inhibition" refers to a reduction in the activity of a protein or cell compared to the absence of an inhibitor. In some embodiments, the term "inhibition" means a reduction in activity of at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In other embodiments, inhibition means a reduction in activity of about 25% to about 50%, about 50% to about 75%, or about 75% to 100%. In some embodiments, inhibition means a reduction in activity of about 95% to 100%, for example, a reduction in activity of 95%, 96%, 97%, 98%, 99%, or 100%. Such a reduction can be measured using a variety of techniques known to those skilled in the art.

[0056] The terms "expression" and "gene expression" have the same meaning, referring to the process by which cells, during their life process, transcribe and translate the genetic information stored in the DNA sequence into biologically active protein molecules.

[0057] The terms "increased expression" and "high expression" have the same meaning, referring to an increase in the copy number of gene transcription and / or translation compared to normal levels. According to the present invention, the increase in the copy number of gene transcription and / or translation is at least 1.1 times the normal level, for example, at least 1.5, 2, 3, 4, 5, 10, 20, or more times.

[0058] The terms "reduced expression" and "low expression" have the same meaning, referring to a decrease in the copy number of gene transcription and / or a decrease in translation compared to normal levels. According to the present invention, the decrease in the copy number of gene transcription and / or the decrease in translation is no more than 0.9 times the normal level, for example, no more than 0.8, 0.7, 0.6, 0.5, 0.33, 0.25, 0.1, or less. Attached Figure Description

[0059] Figure 1 Preliminary transcriptomic analysis of the biological function of the deacetylase HDAH in *E. coli*. This includes: A. Transcriptomic volcano plot showing upregulated (red) and downregulated (green) genes; B. Analysis of the biological processes involved in HDAH regulation of genes; and C. Analysis of HDAH-regulated genes in the TCA cycle (red portion).

[0060] Figure 2 Analysis of in vivo deacetylation activity of HDAH in *E. coli* and its interaction with inhibitors. Specifically: A and B. Analysis of in vivo deacetylation activity of HDAH; C and D. Analysis of the inhibitory effect of acetylation using SAHA or TSA; E. ITC analysis of wild-type HDAH and the inhibitor TSA; F. ITC analysis of wild-type HDAH and the inhibitor SAHA; G. ITC analysis of mutant HDAH and the inhibitor SAHA.

[0061] Figure 3 Analysis of endogenous acetylated protein substrates regulated by HDAH in *E. coli*. This included: A. SILAC quantitative proteomics differential analysis, where red and blue dots indicate significantly different acetylation sites; B. Analysis and comparison of endogenous substrate differences between the two deacetylases, CobB and HDAH; C. Venn diagram analysis of endogenous substrates of the two *E. coli* deacetylases, CobB and HDAH; D. String analysis of endogenous substrates regulated by HDAH; E. GO enrichment analysis for functional attribution of HDAH-regulated endogenous substrates; and F. KEGG pathway enrichment analysis for pathway attribution of HDAH-regulated endogenous substrates.

[0062] Figure 4The effects of HDAH inhibitors on the growth and migration of *E. coli*. The line graph in the upper figure represents the growth of *E. coli* as determined by OD600 measurement using a microplate reader after adding SAHA and DMSO, respectively. The lower figure shows the migration of *E. coli* in solid culture media with added SAHA and TSA compared to the control group with added DMSO. Detailed Implementation

[0063] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0064] Unless otherwise stated, the conventional raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods; the operations performed are known in the art or are performed in accordance with the user manual of commercially available products.

[0065] Example 1: Construction of various recombinant plasmids

[0066] Molecular cloning methods were used to modify plasmids for plasmid construction, which were then used in subsequent experiments.

[0067] The molecular cloning experiments involved included sequence amplification / enzyme digestion / ligation, seamless cloning / point mutagenesis. ① PCR was performed using high-fidelity DNA polymerase to obtain the wild-type HDAH gene fragment from *E. coli*; ② Restriction endonucleases and DNA ligases were used to digest / ligate the pET-28a vector, with the restriction sites being BamHI and XhoHI, respectively; ③ DH5α competent cells were prepared and transformed; ④ Plasmid miniatures were identified through double enzyme digestion; ⑤ Sequencing was used for identification. Point mutagenesis was performed using a homologous recombination kit.

[0068]

[0069] Example 2

[0070] Transcriptomics methods: ① pET-28a-HDAH plasmid and empty pET-28a plasmid were transformed into BL21(DE3) strain, respectively; ② The transformed strains were cultured in LB medium with kanamycin resistance overnight at 37°C and 220 rpm; ③ IPTG was added to the culture medium to a final concentration of 0.25 mM and cultured at 16°C and 220 rpm for 12 h; ④ The bacterial cells were collected by centrifugation at 5000g for 15 min, then flash-frozen in liquid nitrogen and sent to Novogene for transcriptome sequencing and preliminary analysis.

[0071] Overexpression and purification of HDAH protein: ① The previously constructed recombinant plasmids WT-HDAH (pET-28a-HDAH) and mut-HDAH (pET-28a-HDAH H121A) were transformed into BL21(DE3) competent cells and cultured overnight at 37°C and 220 rpm in 5 mL of kanamycin-containing medium; ② The bacterial culture was transferred at a ratio of 1:100 to a large volume of LB medium containing kanamycin resistance and incubated at 37°C and 220 rpm for approximately 4-6 hours until OD500 was reached. At approximately 600 nm (around 0.6), add IPTG to the bacterial culture to a final concentration of 0.25 mM to induce protein expression, while simultaneously adjusting the temperature to 16℃; ③ After approximately 16 hours of induction, collect the bacterial cells by centrifugation at 5000 rpm for 15 minutes at room temperature; ④ Wash with pre-cooled PBS, and remove the PBS by centrifugation at 5000 rpm for 15 minutes again, repeating three times; ⑤ Add 100 mL of suspension buffer containing 100× PMSF, 100× deacetylase inhibitor, and 100× lysozyme, and incubate on ice for 20 minutes; ⑥ Place the bottle containing the liquid in an ice bath. In the process, the bacterial cells were disrupted using an ultrasonic disruptor at 75% intensity, with sonication for 5 seconds followed by a 5-second pause, for approximately 30 minutes until the bacterial cells were relatively clear. ⑦ A pre-cooled centrifuge was used to centrifuge the sonicated liquid at 16000g, 4℃, for 30 minutes. The supernatant was collected and the precipitate was discarded. ⑧ In a 4℃ refrigerator, the supernatant was added to a nickel column, gently shaken to mix, ensuring the beads were in full contact with the supernatant. The liquid after passing through the column was collected, and this step was repeated three times. ⑨ The liquid after passing through the column was discarded, and the nickel column was filled with washing buffer. The buffer was gently shaken to mix, and allowed to stand until the washing buffer had completely flowed out. This process was repeated five times. Collect the remaining 10 μL of liquid and add it to 100 μL of Coomassie Brilliant Blue solution. If the liquid color changes, it indicates that the impurities have not been washed sufficiently, and washing buffer needs to be added for further washing. After thoroughly washing away the impurities, place a centrifuge tube with a 10000MW filter membrane under the column for subsequent collection. Add 10 mL of elution buffer to the column, mix gently, and let stand until the liquid is completely transferred to the centrifuge tube. Pour the collected liquid back into the column and repeat the previous step for thorough elution. Pre-cool the centrifuge and concentrate the collected eluent. Centrifuge at 3400 rpm and 4°C, pausing the centrifuge every 30 minutes to check the liquid level until approximately 1 mL remains above the filter membrane. Discard the centrifuged liquid, fill the filter membrane with storage buffer, and continue centrifuging under the same conditions until approximately 1 mL remains above the filter membrane. Use Nanodrop to roughly determine the protein concentration. If it is sufficient for subsequent experiments, stop centrifugation, collect the remaining liquid above the filter membrane, and take 12.5 μL to accurately measure the protein concentration using the BCA method. After flash freezing in liquid nitrogen, store the remaining liquid at -80°C.

[0072] Transcriptomic analysis revealed that overexpression of HDAH resulted in the upregulation of 421 genes and the downregulation of 12 genes in E. coli. Figure 1 A); and GO analysis was used to preliminarily determine that HDAH protein in E. coli is mainly related to transcription and translation (RNA degradation, ribosomes, protein transport, etc.) or metabolism (carbohydrate metabolism, amino acid metabolism, TCA cycle, etc.). Figure 1 B and 1C).

[0073] Example 3

[0074] ① The constructed WT-HDAH BL21(DE3) strain and the empty vector pET-28a plasmid strain were respectively transferred to 5 mL of LB medium containing kanamycin and cultured overnight. IPTG to a final concentration of 0.5 mM and the corresponding inhibitors SAHA (concentration gradients of 0, 5, 10, 15, 20 μM) and TSA (concentration gradients of 0, 5, 10, 15, 20 nM) were added to the overnight culture and cultured in a shaker at 16℃ for 12 h; ② The same bacterial culture was collected by centrifugation at 12000 rpm for 1 min at room temperature and placed in the same 1.5 mL centrifuge tube, and 1 mL of [unspecified substance] was added. Wash with PBS, repeat three times; discard the supernatant and place on ice, add 1 mL of pre-chilled RIPA, mix well and let stand on ice for 10 min; ③ Pre-chill the centrifuge, centrifuge the product after the previous step at 16000g, 20 min, 4℃; collect the supernatant after centrifugation using a new pre-chilled 1.5 mL centrifuge tube, take 12.5 μL of each protein into a 96-well plate, add one well for each protein, and determine the protein concentration by BCA method; ④ Add 12.5 μL of RIPA to five blank wells, add 12.5 μL of the BCA solution prepared in the Thermo reagent kit to the fifth and sixth blank wells, mix well in the fifth well, transfer 12.5 μL of the liquid to the fourth well, and so on, until the second well, mix well and discard 12.5 μL of the liquid; add 100 μL of working solution to all wells: Cu 2+The reaction solution with a ratio of 100:2 was incubated at 37°C for 30 minutes; protein concentration was detected using a microplate reader; 240 μL of the supernatant collected in the previous step was mixed with 60 μL of 5×SDS-PAGE sample. Mix the buffer thoroughly, heat in a 99°C metal bath for 10 min, and store the heated product at -20°C. ⑤ Perform SDS-PAGE electrophoresis according to the protein concentration determined by the BCA method, loading 7 μL of sample into each well. Load the marker, pET-28a empty vector protein, WT-HDAH protein, and protein samples with added inhibitors, respectively. ⑥ Electrophoresis at 80V for 30 min until the sample passes through the upper gel, then adjust the voltage to 120V and electrophoresis for 60 min until the bromophenol blue band reaches the bottom layer of the gel. Pry open the glass plate, remove the upper gel, transfer the lower gel to an empty box, add Coomassie Brilliant Blue staining solution to cover the gel, and heat in a microwave oven for 1 min. ⑦ Discard the staining solution, add water and rinse quickly in a microwave oven for 10 min. Based on the intensity of the stained bands, observe whether there is a difference in the overall protein expression level. If so, adjust the loading amount until there is no significant difference. Run the gel again with the adjusted loading amount, keeping all other conditions the same. ⑧ Then, perform the transfer in the order of "clamp-sponge-filter paper-NC membrane-gel-filter paper-sponge-clamp", transfer at 200mA for 90min, with ice bath throughout. After transfer, immerse the NC membrane in skim milk and gently shake it on a shaker for 90min. ⑨ Discard the milk and wash the membrane with TBST, repeating three times for 10min. Completely immerse the membrane in acetylated pan-antibody and incubate overnight on a shaker at 4℃, keeping the shaker at the lowest speed. ⑩ Expose in a dark room. Compare the total protein acetylation levels expressed by the empty pET-28a plasmid strain, the WT-HDAH strain, and the strain after adding the corresponding inhibitor.

[0075] To determine the kd values ​​and thermodynamic parameters ΔH and ΔS for the binding of SAHA and TSA to EcHDAH or mutants, ITC experiments were performed. Therefore, 5–20 μM of the corresponding protein was placed in the sample cell of an ITC200 (Microcal, USA) instrument, and 50–200 μM of the corresponding ligand was added to the instrument's syringe. After equilibrating the cells to 30°C, the ligand amount was gradually increased by injection in increments of 1.5–3 μL until the heat no longer changed. The obtained data points were fitted to a unit point binding model using Origin 7 (OriginLab Corporation, USA), and the binding force Kd values ​​were analyzed.

[0076] After HDAH was transformed into E. coli to overexpress HDAH using the above method, the acetylation level of the substrate protein was analyzed, and the results are as follows: Figure 2As shown in A and B, the results indicate that overexpression of HDAH reduces the acetylation level of the substrate protein, suggesting that HDAH has a deacetylation effect. The acetylation level of the substrate protein was analyzed after adding SAHA or TSA, and the results are as follows. Figure 2 As shown in C and D, the results indicate that the addition of SAHA or TSA increased the acetylation level of the substrate protein, exhibiting a dose-response relationship, suggesting that SAHA and TSA inhibited the deacetylation activity of HDAH. ITC analysis was used to analyze the binding of HDAH to the inhibitors SAHA and TSA, and the results are as follows. Figure 2 As shown in E, F, and G, the results indicate that both SAHA and TSA can bind to HDAH, with Kd values ​​of 6.6±1.4 μM and 8.6±2.3 μM, respectively, while the mutated HDAH cannot bind to SAHA.

[0077] Example 4

[0078] SILAC enrichment experiment: ① Prepare 4 bottles of M9 restriction medium, 200 mL each, autoclave and set aside; transform pET-28a-HDAH plasmid and pET-28a empty vector plasmid into BL21(DE3) competent cells and culture overnight in 5 mL of LB medium with kanamycin resistance; prepare 100 mL of M9 medium by adding 200 μL of 1M ammonium sulfate, 20 μL of 0.5M calcium chloride, and 800 μL of 25% glucose, filter each solution through a 0.22 μm filter membrane and add it to the M9 medium, then weigh... 13 C-lysine and 12 40 mg of C-lysine was dissolved in M ​​medium, filtered, and then added separately to two different bottles of M9 medium, labeled as relabeled medium. 13 C-lysine) and light standard culture medium ( 12C-lysine); ② Inoculate the overnight cultured bacterial solution into M9 medium supplemented with salt ions at a ratio of 1:40—add the pET-28a empty vector strain to the heavy labeling medium, and add the pET-28a-HDAH strain to the light labeling medium, 400 mL for each strain; after overnight culture, centrifuge at 4°C for 5 min with each 40 mL of bacterial solution per tube at 8000 g. Then, add 25 mL of pre-chilled PBS to each tube, gently mix, and centrifuge at 8000 g for 5 min. Centrifuge for 5 min to collect bacteria, repeat once, and finally combine the bacterial cells into one tube. Add 20 mL of lysis buffer to each tube and mix well; ③ Sonicate the bacterial solution on ice at 65% intensity for 6 seconds, pause for 6 seconds, and continue for 20 min until clear. Then centrifuge the sonicated bacterial solution at 16000 g for 20 min at 4℃. Label the obtained protein as: pET-28a-HDAH()—L, pET-28a empty vector—H; determine the concentration by BCA method; ⑥ Aliquot 7 mg of protein into one tube, aliquot H and L separately, and premix H and L 1:1; slowly add an equal volume of 50% TCA into the tube and precipitate for 2 h; centrifuge at 2000 g for 5 min, discard the supernatant, add 500 μL of ice-cold acetone to wash, incubate on ice for 3 min, centrifuge at 2000 g for 3 min, discard the supernatant, and repeat twice; ⑦ Open the lid and air dry in a clean bench, and rinse with 100 mM acetone. Dissolve the precipitate in NH4HCO3 (final protein concentration 1-2 μg / μL), sonicate at 30% intensity for 3 seconds, pause for 3 seconds, until no large precipitates remain. Add trypsin at 1 μg / μL for enzymatic digestion, simultaneously adding 100 μL of HCl buffer per 100 μg of protein. Incubate overnight at 37°C until clear. Prepare 1M DTT with ddH2O, final concentration 5mM, and add to the digested sample. Shake in a 56°C metal bath for 1 hour. Prepare 0.5M IAA with ddH2O, final concentration 15mM, and add to the sample. React in the dark for 45 minutes. Prepare 0.5M cysteine ​​with ddH2O, final concentration 30mM, and add to the sample. Shake at room temperature for 30 minutes. Add trypsin to the treated protein at a trypsin:protein ratio of 1:100 for a second enzymatic digestion. Incubate in a 37°C metal bath for 4 hours. Desalt using a 10mg C18 column, first filling to 100%. Activate with ACN; after the initial addition, fill the column with 50% ACN. After the initial addition is complete, fill with 0.1% TFA and let stand. Repeat this process 6 times until the final addition is complete; then add the previously treated protein sample to the column.After the sample has been dripped, wash three times with 0.1% TFA; elute with 1 mL of 50% ACN, then elute with 75% ACN, collect the eluent in a 1.5 mL centrifuge tube, and dry under vacuum. Store the sample at -20°C. Add acetylated antibody beads to a 600 μL centrifuge tube (2 mg protein per 15 μL beads), incubate on ice until the beads separate from the supernatant, centrifuge at 1000g, 4°C, for 1 min, and discard the supernatant. Wash with 400 μL pre-chilled PBS, mix well, centrifuge at 1000g, 4°C, for 1 min, and discard the supernatant. Repeat three times. Wash the previously dried sample with 400 μL of IP... Dissolve the sample in buffer, vortex, and centrifuge briefly at 600g. Collect the supernatant. Add the sample to a centrifuge tube containing acetylated antibody beads. Measure the pH and adjust it to match the IP buffer with HCl or NaOH. Incubate overnight at 4°C by rotation, then incubate at room temperature for 30 min. Let the beads precipitate on ice, then centrifuge at 1000g for 1 min and collect the supernatant precipitate. Add 500 μL Wash Buffer I, gently blow up the precipitate, let stand, centrifuge at 1000g for 1 min, and discard the supernatant using a thin pipette tip. Repeat twice. Add 500 μL Wash Buffer II and wash once as above. Wash once with 500 μL dd2H2O as above. Add 60 μL Elution Buffer to wash the precipitate, eluting thoroughly. Centrifuge at 1000g for 1 min and collect the supernatant. Repeat three times. After drying, prepare for desalting and mass spectrometry analysis.

[0079] The aforementioned quantitative proteomics (SILAC) combined with mass spectrometry analysis preliminarily identified endogenous acetylated protein substrates regulated by HDAH in *E. coli*, such as... Figure 3 As shown in A, B, C, and D, bioinformatics analysis revealed that these substrate proteins are mainly related to bacterial transcriptional and metabolic regulation, such as... Figure 3 As shown in E and F.

[0080] Example 5

[0081] ① Take 10 μL of the pET-28a-HDAH recombinant protein expression strain constructed in Example 2 into two tubes of LB medium containing kanamycin resistance. Add 15 μM SAHA and DMSO to the two tubes of bacterial solution respectively. Measure the OD600 absorbance value of the bacterial solution every 1 hour using an ELISA reader. Culture for a total of 12 hours. ② Culture the pET-28a-HDAH recombinant protein expression bacteria constructed in Example 2 for about 6-8 hours. Take 200 μL of bacterial culture into a 96-well plate and measure the OD600 value of the bacterial culture with an ELISA reader. Stop the culture when the OD600 value is about 0.6. Take 1 μL of each bacterial culture and drop it into LB culture dishes containing 15 μM SAHA and 0.5 mM IPTG and 15 nM TSA and 0.5 mM IPTG, respectively. Use DMSO as a control. At the same time, ensure that there is a large gap between the colonies. After the colonies are air-dried and fixed, incubate them in a 37℃ incubator for 24 hours. ③ Take pictures to record the morphology, color and migration ability of the colonies when they are just formed and after 24 hours.

[0082] The growth and migration of E. coli after administration of SAHA or TSA are shown in the figure. Figure 4 The line graph in the image above shows the growth of *E. coli* determined by measuring OD600 using a microplate reader after adding SAHA and DMSO, respectively. The results show that the growth of *E. coli* was significantly slowed down after adding SAHA. The image below shows that the colony density of *E. coli* was significantly lower in the solid culture medium after adding SAHA and TSA compared to the control group after adding DMSO, indicating that the migration ability of *E. coli* was inhibited after adding SAHA and TSA. The experiment demonstrates that the growth and migration ability of *E. coli* were significantly reduced after adding the inhibitors SAHA or TSA.

[0083] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of an HDAH inhibitor for the preparation of an antibiotic, characterized in that, The HDAH is EcHDAH or a homologue thereof, the amino acid sequence of EcHDAH is shown as SEQ No. 1; Preferably, the homologue of EcHDAH is HDAH of Pseudomonas aeruginosa, Alcaligenaceae bacterium or Legionella pneumophila.

2. Use according to claim 1, wherein The antibiotic is used to kill or inhibit the growth or migration ability of Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium or Legionella pneumophila.

3. Use according to claim 1 or 2, characterized in that, The HDAH inhibitor is an inhibitor that inhibits the expression of HDAH gene or inhibits HDAH protein; The inhibitor that inhibits the expression of HDAH gene is selected from an antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA against HDAH gene, or a protein, polypeptide, enzyme, small molecule compound that inhibits the mRNA level of HDAH gene; The inhibitor that inhibits HDAH protein is selected from an antibody of HDAH, a protein, polypeptide, enzyme, small molecule compound that inhibits the activity or protein level of HDAH.

4. Use according to any one of claims 1 to 3, wherein The inhibitor is selected from the group consisting of vorinostat Trichostatin A CypX APHA 3c 5. Use of HDAH or its encoding gene as a drug target for the screening of antibacterial agents, characterized in that, The HDAH is EcHDAH or a homologue thereof, the amino acid sequence of EcHDAH is shown as SEQ No. 1; Preferably, the homologue of EcHDAH is HDAH of Pseudomonas aeruginosa, Alcaligenaceae bacterium or Legionella pneumophila.

6. Use according to claim 5, characterized in that, The antibiotic is used to kill or inhibit the growth or migration ability of Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium or Legionella pneumophila.

7. A method of screening for an antibacterial in vitro, characterized in that, The method comprises the following steps: 1) mixing a test chemical with HDAH or mixing the test chemical with a cell expressing HDAH under suitable environmental conditions; 2) after a suitable period of time, detecting the binding of the test chemical with HDAH, or detecting the change of HDAH deacetylase activity, or detecting the change of expression level of HDAH in the cell, or detecting the growth or migration of the cell; The HDAH is EcHDAH or a homologue thereof, the amino acid sequence of EcHDAH is shown as SEQ No. 1; Preferably, the homologue of EcHDAH is HDAH of Pseudomonas aeruginosa, Alcaligenaceae bacterium or Legionella pneumophila.

8. The method of claim 7, wherein, The cell is a bacterium having HDAH; preferably Escherichia coli, Pseudomonas aeruginosa, Alcaligenaceae bacterium or Legionella pneumophila.

Images