Use of a TrmH protein or trmH gene as a target for an anti-acinetobacter baumannii infection drug, an acinetobacter baumannii inhibitor, a pharmaceutical preparation

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a TrmH protein or trmH Gene as target point of drug for resisting Acinetobacter baumannii infection, Acinetobacter baumannii inhibitor and pharmaceutical preparation. The application provides a tRNA methyltransferase TrmH protein or trmH Gene as target point of drug for resisting Acinetobacter baumannii infection, and the application of the tRNA methyltransferase TrmH protein or trmH Gene as target point of drug for resisting Acinetobacter baumannii infection, and the application of the tRNA methyltransferase TrmH protein or By inhibiting the tRNA methyltransferase TrmH protein activity or inhibiting the expression of the tRNA methyltransferase TrmH protein, the abundance of the TrmH protein product can be reduced from the translation regulation level, and the drug resistance and pathogenic ability of Acinetobacter baumannii can be weakened.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a TrmH protein or trmH Application of genes as targets for drugs against Acinetobacter baumannii infection, Acinetobacter baumannii inhibitors, and pharmaceutical formulations. Background Technology

[0002] Acinetobacter baumannii is a significant pathogen causing hospital-acquired infections, leading to various serious conditions such as pneumonia and sepsis, posing a particularly prominent threat to critically ill patients in intensive care units. The difficulty in controlling this bacterium lies in its high susceptibility to multiple antibiotics, resulting in multidrug-resistant Acinetobacter baumannii and severely limiting the range of available clinical treatments.

[0003] The multidrug resistance of Acinetobacter baumannii poses a significant challenge to anti-infective therapy. Novel tetracycline antibiotics, such as iracycline, are important treatment options for combating drug-resistant bacterial infections. Although the current clinical resistance rate is low, resistant strains can be screened in the laboratory, and their resistance mechanisms are not yet fully elucidated, posing a potential threat to long-term therapeutic efficacy. Existing research on the resistance mechanisms of this bacterium largely focuses on genomic mutation identification and transcriptional regulation, such as the upregulation of efflux pump genes. However, bacterial resistance involves multi-level regulation, and intervention at the transcriptional level alone is often insufficient. Many post-transcriptional and translational resistance mechanisms remain to be explored, creating limitations in current understanding. Among multiple translational regulatory processes, tRNA chemical modification is crucial for controlling translational accuracy and efficiency. While its role in bacterial environmental adaptation and stress response is gradually being recognized, research directly linking it to drug resistance in Acinetobacter baumannii is relatively weak. This results in a lack of potential key targets for intervention at the translational level to combat drug resistance. Furthermore, existing anti-infective strategies often face the challenge of targeting only one specific target, either drug resistance or virulence. Bacteria can rapidly adapt through compensatory mechanisms, leading to short-lived treatment effects. Therefore, there is an urgent need to find a multi-effect intervention target that can simultaneously weaken both drug resistance and pathogenicity in Acinetobacter baumannii. Summary of the Invention

[0004] To address the above problems, the present invention aims to provide a tRNA methyltransferase TrmH protein or trmH Application of genes as targets for drugs against Acinetobacter baumannii infection, Acinetobacter baumannii inhibitors, and pharmaceutical formulations. This invention utilizes the inhibition of tRNA methyltransferase TrmH protein activity or inhibition... trmH The expression of [specific protein name] can reduce the abundance of the TrmH protein product at the translational regulation level, thereby weakening the drug resistance and pathogenicity of Acinetobacter baumannii. This invention inhibits TrmH protein activity or inhibits [specific protein expression]. trmHThe expression of this substance can not only directly weaken the drug resistance barrier of Acinetobacter baumannii, achieving the technical effect of significantly sensitizing existing antibacterial drugs, but also significantly disrupt the bacteria's biofilm formation ability and greatly reduce its survival rate in immune cells, making it easier for the host immune system to clear it and reduce the pathogenicity of Acinetobacter baumannii.

[0005] TrmH is a eubacterial tRNA methyltransferase responsible for forming 2'-O-methylguanosine (Gm18) at position 18 of tRNA. Formerly known as SpoU, it belongs to the SPOUT superfamily.

[0006] The first aspect of this invention provides a TrmH protein or its encoding gene. trmH Application of genes as targets for drugs against Acinetobacter baumannii infection.

[0007] In some alternative implementations, the application includes at least any one of the following a1-a4 applications: a1: using TrmH protein or trmH TrmH inhibitors targeting genes are used as drugs against Acinetobacter baumannii infection. a2: using TrmH protein or trmH Screening of anti-Acinetobacter baumannii infection drugs at the in vitro or cellular level targeting genes; a3: Contains TrmH protein or trmH Combination drugs for treating Acinetobacter baumannii infection, which are formed by combining gene-targeted TrmH inhibitors and antibiotic antibacterial drugs; and combination drug compositions containing TrmH inhibitors and tetracycline antibacterial drugs can be developed, in which TrmH inhibitors are used to weaken the bacterial resistance barrier and tetracycline antibacterial drugs are used to kill bacteria that have regained sensitivity, and the two work together to exert an anti-infective effect. a4: Detection trmH Reagents or kits for gene mutations or TrmH protein expression levels; In some alternative implementations, the detection trmH Reagents or kits for gene mutations or TrmH protein expression levels are used to determine the degree of resistance of Acinetobacter baumannii to antibiotics, or to screen for anti-Acinetobacter baumannii infection drugs that can inhibit TrmH activity.

[0008] In some alternative embodiments, the antibiotic class includes tetracyclines.

[0009] A second aspect of the present invention provides an Acinetobacter baumannii inhibitor, said Acinetobacter baumannii inhibitor having at least any one of the functions described in b1-b2: b1: The Acinetobacter baumannii inhibitor has the effect of inhibiting Acinetobacter baumannii. trmH The function of gene expression; b2: The Acinetobacter baumannii inhibitor has the function of inhibiting the activity of TrmH protein in Acinetobacter baumannii.

[0010] In some alternative embodiments, the Acinetobacter baumannii inhibitor includes knockout. trmH Genes or reduction trmH Reagents for gene expression.

[0011] In some alternative embodiments, the reagent comprises one or more plasmids, the plasmids comprising... trmH Upstream homologous arm insertion fragment of gene, trmH Insertion fragment into the downstream homologous arm of the gene; The trmH The nucleotide sequence of the inserted fragment in the upstream homologous arm of the gene is shown in SEQ ID NO: 1; The trmH The nucleotide sequence of the inserted fragment in the downstream homologous arm of the gene is shown in SEQ ID NO: 2.

[0012] In some optional embodiments, the plasmid further includes a forward selection marker gene and a reverse selection marker gene; In some alternative embodiments, the positive selection marker genes include tellurite resistance genes and / or gentamicin resistance genes; The reverse selection marker gene includes a sugar-sensitive gene. bag B.

[0013] In some alternative embodiments, the Acinetobacter baumannii inhibitor comprises with trmH mRNA complementary antisense RNA or small interfering RNA; In some alternative embodiments, the Acinetobacter baumannii inhibitor comprises an inactivated Cas protein, which can interact with... trmH The Cas protein binds to guide RNA (sgRNA) upstream of the gene promoter region or coding sequence to form a complex; inactivated Cas proteins can bind to... trmH A guide RNA (sgRNA) upstream of the gene promoter region or coding sequence binds to form a complex, which blocks the flow of the gene through steric hindrance. trmH The transcription process of genes is inhibited, thereby suppressing the expression of TrmH protein.

[0014] In some alternative embodiments, the Acinetobacter baumannii inhibitor comprises a small molecule compound capable of specifically binding to the TrmH protein.

[0015] In some optional embodiments, the Acinetobacter baumannii inhibitor includes reagents that knock out the coding gene corresponding to the TrmH protein and / or reagents that cause mutations in the dimer interface residues of the TrmH protein to lose its dimerizing ability and / or reagents that cause mutations in key residues at the SAM binding site of the TrmH protein to lose its activity. In some optional embodiments, the key residues at the SAM binding site in the TrmH protein include at least one residue from the following: T190, S192, L211, G212, N213, E214, G217, T230, I231, Q233, S239, L240, V242, and A245.

[0016] A third aspect of the present invention provides a pharmaceutical formulation comprising the above-mentioned Acinetobacter baumannii inhibitor.

[0017] In some optional embodiments, the pharmaceutical preparation further includes the antibiotic drug; In some alternative embodiments, the antibiotic includes tetracyclines; In some optional embodiments, the dosage form of the pharmaceutical preparation includes: injection, infusion, tablet, capsule, granule, dry suspension, oral solution, oral emulsion, pill, ointment, gel, and patch.

[0018] Compared with the prior art, the present invention has the following advantages: 1. This invention provides a tRNA methyltransferase TrmH protein or trmH The application of genes as targets for drugs against Acinetobacter baumannii infection involves inhibiting the activity of the tRNA methyltransferase TrmH protein or inhibiting... trmH The expression of [specific protein name] can reduce the abundance of the TrmH protein product at the translational regulation level, thereby weakening the drug resistance and pathogenicity of Acinetobacter baumannii. This invention inhibits TrmH protein activity or inhibits [specific protein expression]. trmH The expression of this substance can not only directly weaken the drug resistance barrier of Acinetobacter baumannii, achieving the technical effect of significantly sensitizing existing antibacterial drugs, but also significantly disrupt the bacteria's biofilm formation ability and greatly reduce its survival rate in immune cells, making it easier for the host immune system to clear it and reduce the pathogenicity of Acinetobacter baumannii.

[0019] 2. This invention provides a novel target and regulatory level for drug resistance intervention. Existing research on the drug resistance mechanisms of *Acinetobacter baumannii* has long focused on genomic mutation identification and transcriptional regulation, with little attention paid to translational and post-transcriptional regulation. This invention is the first to discover and verify the crucial role of the tRNA methyltransferase *TrmH* in the regulation of drug resistance and pathogenicity in *Acinetobacter baumannii*, expanding the perspective of drug resistance intervention from the traditional genomic and transcriptional levels to the translational regulatory level, providing a novel target for overcoming drug resistance in *Acinetobacter baumannii*.

[0020] 3. This invention achieves a more robust sensitization effect through post-transcriptional regulation. Existing transcriptional regulation strategies targeting efflux pumps can lead to drug resistance relapse because bacteria can restore the transcription of efflux pump genes through mutations in regulatory factors. However, the mechanism of action of TrmH revealed in this invention downregulates protein abundance by affecting the translation efficiency or protein stability of the target gene without altering its transcriptional level. Because this regulation occurs after transcription, bypassing the traditional transcriptional circuit, interventions based on this mechanism are less likely to be counteracted by bacterial transcriptional compensation mechanisms, potentially resulting in a more durable sensitization effect.

[0021] 4. This invention provides a single-target approach that simultaneously achieves both sensitization and toxicity reduction. Existing anti-infective strategies typically target only one specific drug, either drug resistance or virulence. This invention, by targeting the single protein TrmH, can simultaneously weaken the resistance of Acinetobacter baumannii to multiple antimicrobial drugs, inhibit its biofilm formation ability, and reduce its survival ability within host immune cells. This pleiotropic effect is not present in existing single-target approaches and holds promise for a more comprehensive anti-infective effect in clinical treatment.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 In Example 1 trmH Gene knockout verification electrophoresis image; Figure 2This is a principal component analysis plot for transcriptomics. Figure 3 This is a principal component analysis diagram for proteomics. Figure 4 Comparison images of biofilm formation observed using a confocal laser scanning microscope; Figure 5 This is a comparison of the results of macrophage phagocytosis and killing experiments; the left image shows macrophages' response to 5661R and 5661R-Δ. trmH The phagocytosis rate detection results are shown in the right figure; the right figure shows 5661R and 5661R-Δ. trmH Results of 2-hour survival rate assay in macrophages. Detailed Implementation

[0025] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0026] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein may also include the plural forms. The terms “comprising,” “including,” “containing,” and “having” are inclusive and therefore indicate the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.

[0027] In the description of the embodiments of this invention, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this invention, "multiple" means two or more, unless otherwise explicitly defined.

[0028] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0029] In the description of the embodiments of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0030] In the description of the embodiments of the present invention, the term "multiple" refers to two or more (including two), similarly, "multiple groups" refers to two or more (including two groups), and "multiple pieces" refers to two or more (including two pieces).

[0031] In the description of the embodiments of the present invention, unless otherwise explicitly specified and limited, the technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention according to the specific circumstances.

[0032] Acinetobacter baumannii is an important opportunistic pathogen in clinical practice, and its multidrug resistance poses a significant challenge to anti-infective therapy. New-generation tetracycline antibiotics, such as tigecycline and eracycline, have been developed, exerting their antibacterial effects by binding to bacterial ribosomes and blocking protein synthesis. However, under the selective pressure of antimicrobial drugs, bacteria can develop resistance through various mechanisms, among which the efflux pump system is one of the most crucial mechanisms mediating multidrug resistance. Taking the AdeABC efflux pump in Acinetobacter baumannii (composed of three proteins: AdeA, AdeB, and AdeC) as an example, these three proteins assemble into an efflux system that spans the bacterial membrane, actively expelling drugs that have entered the cell, preventing them from reaching effective concentrations and rendering them ineffective. Therefore, in-depth research into the regulatory mechanisms of bacterial resistance and the search for new targets to overcome efflux pump resistance are of significant clinical importance.

[0033] In exploring mechanisms of drug resistance regulation, a long-neglected aspect is the chemical modification during protein translation. For a gene to function, it must first be transcribed to produce messenger RNA (mRNA), which is then translated into protein by ribosomes. Transfer RNA (tRNA) is a nucleic acid molecule responsible for recognizing codons on mRNA and transporting the corresponding amino acids during translation. Various chemical modifications naturally exist on tRNA molecules, which can finely regulate the speed, accuracy, and efficiency of translation. Among these modifications, one called 2'-O-methylguanosine (Gm) affects the stability of the tRNA's spatial structure. In *Escherichia coli*, this modification is catalyzed by the TrmH methyltransferase. The TrmH protein in *Acinetobacter baumannii*, the focus of this invention, contains a typical SpoU family RNA methyltransferase domain according to bioinformatics analysis. Its protein structure is highly similar to that of *Escherichia coli* TrmH, and the sequence of this protein is highly conserved within the *Acinetobacter baumannii* community, suggesting that it may have the same catalytic function. From a broader bacterial perspective, analysis of the NCBI COG database shows that the SpoU family, to which TrmH belongs, is present in over 89% of sequenced bacterial strains, covering multiple major bacterial phyla, indicating that this type of protein has a wide distribution and important basic biological functions in bacteria. However, the relationship between TrmH-regulated tRNA modifications and drug resistance and pathogenicity in Acinetobacter baumannii remains undiscovered in current technology.

[0034] This invention provides a novel target for anti-Acinetobacter baumannii infection, as well as a method for applying drugs against Acinetobacter baumannii infection. Details are as follows: The first aspect of this invention provides a TrmH protein or its encoding gene. trmH The application of genes as targets for drugs against Acinetobacter baumannii infection. Targeting the tRNA methyltransferase TrmH, inhibiting its expression aims to weaken the drug resistance and pathogenicity of Acinetobacter baumannii.

[0035] In some alternative implementations, the application includes at least any one of the following a1-a4 applications: a1: using TrmH protein or trmH TrmH inhibitors targeting genes are used as drugs against Acinetobacter baumannii infection. a2: using TrmH protein or trmHScreening for anti-Acinetobacter baumannii infection drugs at the in vitro or cellular level with genes as targets; for example, in some alternative embodiments, a methyl transfer reaction system is reconstructed in vitro using purified recombinant TrmH protein, tRNA substrate and SAM donor, and TrmH enzyme activity inhibitors are screened by detecting the amount of Gm-modified products generated; or an engineered strain containing a Gm-modification-dependent reporter gene is constructed, and TrmH activity is indirectly reflected by detecting changes in the expression of the reporter gene.

[0036] a3: Contains TrmH protein or trmH Combination drugs for treating Acinetobacter baumannii infection, which are formed by combining gene-targeted TrmH inhibitors and antibiotic antibacterial drugs; and combination drug compositions containing TrmH inhibitors and tetracycline antibacterial drugs can be developed, in which TrmH inhibitors are used to weaken the bacterial resistance barrier and tetracycline antibacterial drugs are used to kill bacteria that have regained sensitivity, and the two work together to exert an anti-infective effect. a4: Detection trmH Reagents or kits for gene mutations or TrmH protein expression levels; In some alternative implementations, the detection trmH Reagents or kits for gene mutations or TrmH protein expression levels are used to determine the degree of resistance of Acinetobacter baumannii to antibiotics, or to screen for anti-Acinetobacter baumannii infection drugs that can inhibit TrmH activity.

[0037] In some alternative embodiments, the antibiotic class includes tetracyclines.

[0038] A second aspect of the present invention provides an Acinetobacter baumannii inhibitor, said Acinetobacter baumannii inhibitor having at least any one of the functions described in b1-b2: b1: The Acinetobacter baumannii inhibitor has the effect of inhibiting Acinetobacter baumannii. trmH The function of gene expression; b2: The Acinetobacter baumannii inhibitor has the function of inhibiting the activity of TrmH protein in Acinetobacter baumannii.

[0039] In some alternative embodiments, the Acinetobacter baumannii inhibitor includes knockout. trmH Genes or reduction trmH Reagents for gene expression.

[0040] In some alternative embodiments, the reagent comprises one or more plasmids, the plasmids comprising... trmH Upstream homologous arm insertion fragment of gene, trmH Insertion fragment into the downstream homologous arm of the gene; The trmHThe nucleotide sequence of the inserted fragment in the upstream homologous arm of the gene is shown in SEQ ID NO: 1; The trmH The nucleotide sequence of the inserted fragment in the downstream homologous arm of the gene is shown in SEQ ID NO: 2.

[0041] trmH trmH In some optional embodiments, the plasmid further includes a forward selection marker gene and a reverse selection marker gene; In some alternative embodiments, the positive selection marker genes include tellurite resistance genes and / or gentamicin resistance genes; The reverse selection marker gene includes a sugar-sensitive gene. bag B.

[0042] In some alternative embodiments, the Acinetobacter baumannii inhibitor comprises with trmH mRNA-complementary antisense RNA or small interfering RNA; through base pairing with... trmH The mRNA binds, blocking its translation process or promoting its degradation, thereby reducing the expression level of TrmH protein.

[0043] In some alternative embodiments, the Acinetobacter baumannii inhibitor comprises an inactivated Cas protein, which can interact with... trmH The Cas protein binds to guide RNA (sgRNA) upstream of the gene promoter region or coding sequence to form a complex; inactivated Cas proteins can bind to... trmH A guide RNA (sgRNA) upstream of the gene promoter region or coding sequence binds to form a complex, which blocks the flow of the gene through steric hindrance. trmH The transcription process of genes is inhibited, thereby suppressing the expression of TrmH protein.

[0044] In some alternative embodiments, the Acinetobacter baumannii inhibitor comprises a small molecule compound capable of specifically binding to the TrmH protein. This small molecule compound, obtained through high-throughput or virtual screening, inhibits the catalytic activity of the methyltransferase TrmH or interferes with its dimer formation, thereby losing its function in catalyzing tRNA Gm modification.

[0045] In some optional embodiments, the Acinetobacter baumannii inhibitor includes reagents that knock out the coding gene corresponding to the TrmH protein and / or reagents that cause mutations in the dimer interface residues of the TrmH protein to lose its dimerizing ability and / or reagents that cause mutations in key residues at the SAM binding site in the TrmH protein to lose its catalytic activity. In some optional embodiments, the key residues at the SAM binding site in the TrmH protein include at least one residue from the following: T190, S192, L211, G212, N213, E214, G217, T230, I231, Q233, S239, L240, V242, and A245.

[0046] The amino acid sequence of the TrmH protein (SEQ ID NO: 3) is as follows: MAVIFLESKDNAKIKHLRGLIELNSARKKHQQTVLEGTHLCLAWLQQQKKLFSLFTTEQALEHPDLKKIIELHQGHVFIISEVLYKDLSTLGNTLPCLAIIDLPKTASTIDYSVDTLILENVQDPGNVG TLLRSAAAANIKQIICTQGSASLWSPRVLRAGMGAHFSLSCFENFQLTDILPKFDIPVFVTSHRSTSLYSKDLSKPCVWILGNEGQGASDYALEHAQSVTIPQPGGQESLNVAIAGSICFFEMVRQRQ A third aspect of the present invention provides a pharmaceutical formulation comprising the above-mentioned Acinetobacter baumannii inhibitor.

[0047] In some optional embodiments, the pharmaceutical preparation further includes the antibiotic drug; In some alternative embodiments, the antibiotic includes tetracyclines; In some optional embodiments, the dosage form of the pharmaceutical preparation includes: injection, infusion, tablet, capsule, granule, dry suspension, oral solution, oral emulsion, pill, ointment, gel, and patch.

[0048] Because the TrmH protein is highly conserved in the Acinetobacter baumannii flora and its homologous protein is distributed in more than 89% of sequenced bacterial strains, the technical solution of this invention is applicable to all clinical strains of Acinetobacter baumannii carrying the TrmH homologous protein, as well as other pathogens containing the TrmH homologous protein. In an optional embodiment, the technical solution of this invention is applicable to the Acinetobacter baumannii 5661R-induced drug-resistant strain.

[0049] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or through existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are conventional methods. It should be further noted that the following descriptions are merely exemplary and not intended to limit the specific scope of the invention. Moreover, the comparative examples below are selected to compare with the technical solutions of the present invention to demonstrate the advancement of the present invention, and do not necessarily represent prior art in this technical field.

[0050] Example 1 trmH Methods for constructing gene knockout mutants This embodiment provides a method for knocking out Acinetobacter baumannii using homologous recombination technology. trmH The specific method for gene manipulation. The core principle of this method is: using suicide plasmids as vectors to... trmH The upstream homologous arm sequence (SEQ ID NO: 1) and the downstream homologous arm sequence (SEQ ID NO: 2) of the gene were introduced into the target strain. Through two homologous recombination events, the complete chromosome was transformed. trmH The gene is replaced with a deleted sequence, thereby causing it to lose its function. This method includes the following steps: Step 1: Amplification of homologous arm fragments Using the genomic DNA of Acinetobacter baumannii strain 5661R (an induced drug-resistant strain) as a template, two pairs of specific primers were designed and synthesized for amplification. trmH Approximately 1 kb DNA fragments upstream and downstream of the gene are used as the left and right homologous arms for homologous recombination. During primer design, a 19 bp overlapping sequence suitable for In-Fusion cloning technology is introduced at the 5' end of the primers to facilitate subsequent ligation with the linearized plasmid vector. The specific primer sequences used in this embodiment are detailed in Table 1.

[0051] Table 1 Construction trmH Primer sequences used for knockout strains

[0052] PCR amplification was performed using a standard system. After the amplification products were identified as correct by agarose gel electrophoresis, they were excised and purified to obtain upstream and downstream homologous arm fragments.

[0053] Step 2: Construction of Recombinant Knockout Plasmid The suicide plasmid pMo130-TelR was linearized. Using in-fusion cloning technology, the upstream and downstream homologous arm fragments obtained in step one were ligated to the linearized pMo130-TelR plasmid to construct the recombinant plasmid pMo130- trmH The structural characteristics of this plasmid are: it contains... trmH Upstream and downstream homologous arm sequences of the gene, tellurite resistance gene and gentamicin resistance gene were used as positive selection markers, and sucrose-sensitive gene sacB was used as a negative selection marker.

[0054] The ligation product was transformed into *Escherichia coli* DH5α competent cells and plated on LB agar plates containing tellurite (20 mg / L) and gentamicin (25 mg / L). Colonies exhibiting the first homologous recombination (plasmid integration into the chromosome) were selected using PCR. Positive colonies were inoculated into LB broth containing 20% ​​sucrose for reverse selection, inducing the second homologous recombination event, which resulted in the excision of the plasmid backbone from the chromosome. Colonies obtained from reverse selection were identified by PCR amplification, and the amplification products were confirmed by Sanger sequencing to confirm the correct insertion of the homologous arm sequence into the plasmid and verify the deletion of the trmH gene fragment. After verification, the pMo130-trmH recombinant plasmid was extracted and purified from the positive clones.

[0055] Step 3: Electroporation transformation and first homologous recombination screening Verify the correct pMo130- trmH The plasmid was transformed into Acinetobacter baumannii strain 5661R via electroporation. The electroporated bacterial culture was plated on LB agar plates containing tellurite (20 mg / L) and gentamicin (25 mg / L) to screen for resistant colonies. PCR identification of the colonies using primers pMo130Tel-F and pMo130Tel-R confirmed that the plasmid had been integrated into the chromosome in the first homologous recombination positive clone.

[0056] Step 4: Second homologous recombination and reverse screening The first recombinant positive clones were inoculated into LB medium containing 20% ​​sucrose and cultured. bagB The sucrose lethal effect of genes was used for reverse selection of colonies that had undergone a second homologous recombination and had their plasmid backbone removed. For colonies grown on sucrose plates, primers were used... trmH ver-F and trmH ver-R was used for PCR identification and sequencing verification to confirm... trmH The gene coding region has been deleted as expected. trmH Gene knockout verification electrophoresis image as shown Figure 1 As shown, the correctly verified mutant strain was named 5661R-Δ trmH .

[0057] Step 5: Drug susceptibility testing of knockout strains The 5661R-Δ was determined using the micro-broth dilution method. trmH The minimum inhibitory concentrations (MICs) of the knockout strain 5661R against multiple antibacterial drugs were determined. Results showed that the knockout strain 5661R-Δ... trmH The minimum inhibitory concentration (MIC) of iracycline was reduced by 8-fold, the overall sensitivity to other tetracycline drugs such as tigecycline was increased by 2 to 4 times, and the sensitivity to levofloxacin was also significantly improved, indicating that the loss of TrmH function can effectively sensitize a variety of antibacterial drugs.

[0058] Example 2: trmH Multi-omics analysis and phenotypic validation after deletion This embodiment systematically reveals, through transcriptomics, proteomics, and combined analysis, that... trmH The molecular-level changes in gene knockout strains were investigated to elucidate the mechanisms by which they affect drug resistance and pathogenicity through post-transcriptional regulation, and to provide corresponding phenotypic verification methods.

[0059] Step 1: Transcriptomics Analysis Extract 5661R-Δ respectively trmH Total RNA from the parent strain 5661R was used for transcriptome sequencing and analysis. Data were selected based on a fold change greater than 2 and corrected for variation. p value( q A value less than 0.05 was used as the criterion for screening differentially expressed genes. The results of principal component analysis of transcriptomics are as follows: Figure 2 As shown, the results indicate that the number of differentially expressed genes between the two groups is limited, and no significant separation was observed between the two groups in the principal component analysis, suggesting that... trmH The deletion had little impact on the overall transcriptional expression pattern of the strain. GO functional annotation and KEGG pathway enrichment analysis were performed on all differentially expressed genes, and only one pathway (fluorobenzoic acid degradation pathway) reached a significant enrichment level. q <0.05), with no entries reaching significant enrichment in any branch of GO functional annotations, further confirming this. trmH The deletion did not cause large-scale transcriptional reprogramming.

[0060] Step 2: Proteomics Analysis Extract 5661R-Δ respectively trmH Total protein from the parent strain 5661R was analyzed using data-independent acquisition (DIA) proteomics. Proteins with a fold change greater than 2 were analyzed. q A value less than 0.05 was used as the standard for screening differentially expressed proteins. The results showed that, in addition to the TrmH protein itself, a total of 15 differentially expressed proteins were identified, of which 5 were upregulated and 10 were downregulated. The principal component analysis diagram of the proteomics is shown below. Figure 3 As shown, principal component analysis revealed a clear separation in protein expression patterns between the two groups of samples, which contrasted sharply with the transcriptome results.

[0061] Functional analysis of the differentially expressed proteins revealed significant changes in several proteins with important biological functions. Specifically, the protein abundance of all three components of the AdeABC efflux pump—AdeA, AdeB, and AdeC—was significantly downregulated in the knockout strain. This efflux pump is one of the core mechanisms mediating tetracycline resistance in Acinetobacter baumannii. Simultaneously, PgaC protein, associated with biomembrane synthesis, was significantly downregulated (more than 12-fold), as were PuuP / PlaP transporters associated with putrescine transport. KEGG pathway enrichment analysis showed that the differentially expressed proteins were significantly enriched in multiple aromatic compound metabolic pathways, such as benzoate degradation and fluorobenzoate degradation, as well as molecular functional items such as dioxygenase activity and oxidoreductase activity.

[0062] Step 3: Combined Transcriptome and Proteome Analysis Pearson correlation analysis of genes detected simultaneously in both the transcriptome and proteome revealed a correlation coefficient of only 0.452 between mRNA and protein expression levels, indicating limited overall correlation. Further classification analysis showed that changes in the expression of some proteins could not be explained by changes in their corresponding mRNAs. For example, the three components of the AdeABC efflux pump, PgaC, and CatA were significantly downregulated in knockout strains, but their corresponding genes showed no significant difference in transcriptional levels. Conversely, proteins such as the large subunit of exonuclease VII (XseA) and the bestrophin-like protein (YneE) were significantly upregulated in knockout strains, but their gene transcriptional levels also showed no significant change. These results suggest that... trmH The deletion primarily affects post-transcriptional regulation, that is, it regulates the abundance of the target protein by altering translation efficiency or protein stability, rather than by changing the gene transcription level. TrmH affects the expression of key drug resistance-related proteins such as the AdeABC efflux pump through this post-transcriptional regulatory mechanism.

[0063] Step 4: Phenotypic validation of biofilm formation ability Confocal laser scanning microscopy combined with fluorescence staining was used to analyze 5661R-Δ trmH The biofilm formation ability of the two strains, strain 5661R and its parent strain, was observed. Both strains were inoculated onto cell slides and cultured under suitable conditions until biofilms formed. Fluorescent staining and observation were then performed. Biofilm formation was observed using a confocal laser scanning microscope. Figure 4 As shown, the results indicate that 5661R-Δ trmH The biofilm structure formed on the surface of the climbing tissue was significantly sparse, with both its thickness and coverage area being significantly smaller than that of the parental strain. This phenotype is consistent with the results of significant downregulation of PgaC protein in proteomics analysis. PgaC is a key enzyme in the biosynthesis of poly-N-acetylglucosamine (PNAG), and PNAG is an important component of the biofilm matrix. This validation indicates that... trmHThe absence of PgaC protein weakened the strain's biofilm formation ability by downregulating PgaC protein expression. Step 5: Phenotypic validation of macrophage intracellular survival ability The immortalized mouse bone marrow-derived macrophage line iBMDM was used to conduct experiments on macrophage phagocytosis and bacterial killing. The multiplicity of infection (MOI) was 10 for 5661R-Δ trmH The parent strain 5661R was added to macrophages, and after incubation for 1 hour, extracellular bacteria were removed. Cells were lysed at the initial time point, the number of phagocytosed bacteria was counted, and the phagocytosis rate was calculated. The results are as follows: Figure 5 The left image in the middle (a star (*) represents...) p <0.05, four asterisks (****) represent p <0.0001 indicates statistical significance between the two groups); after culturing for another 2 hours, the cells were lysed again to count the number of surviving bacteria intracellularly and calculate the survival rate. The results are as follows: Figure 5 The right image in the middle (a star (*) represents) p <0.05, four asterisks (****) represent p <0.0001 indicates statistical significance between the two groups. The results show that 5661R-Δ trmH The phagocytosis rate was also significantly lower than that of the parent strain ( p <0.05%, and its intracellular survival rate after 2 hours was also significantly lower than that of the parent strain ( p <0.0001), indicating trmH The deletion not only reduced the extent to which the strain was phagocytosed by macrophages, but also significantly weakened its survival ability within immune cells. This phenotype is consistent with the significant downregulation of the polyamine transport-related proteins PuuP / PlaP in proteomics analysis. Polyamine molecules have been shown to enhance bacterial tolerance to oxidative stress, thereby improving their survival ability in immune cells.

[0064] Based on the results of Example 2, trmH While the deletion did not cause large-scale changes at the transcriptional level, it led to significant alterations in several key proteins at the protein level, including the AdeABC efflux pump, PgaC, and PuuP / PlaP. Phenotypic experiments confirmed reduced biofilm formation and decreased macrophage survival. This indicates that TrmH is a crucial post-transcriptional regulatory node that regulates the expression of drug resistance and pathogenicity-related proteins by affecting translation efficiency or stability. This is the core innovation of this invention, distinguishing it from existing efflux pump transcriptional regulation strategies.

[0065] In summary, this invention inhibits or suppresses the activity of TrmH protein. TrmHThe expression of this gene not only sensitizes drugs but also significantly disrupts the bacteria's biofilm formation ability and drastically reduces their survival rate within immune cells, making them more susceptible to clearance by the host immune system. The underlying mechanism of this combined effect lies in the fact that TrmH is a key post-transcriptional regulatory node. Inhibiting its function can affect the translation efficiency or stability of proteins without altering the transcriptional levels of key genes such as efflux pumps (e.g., AdeABC) and biofilm synthesis proteins (e.g., PgaC), thereby precisely downregulating the abundance of the target protein. This opens up a completely new and feasible direction for developing novel anti-Acinetobacter baumannii drugs or potentiators that do not rely on traditional transcriptional repression mechanisms.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A TrmH protein or its encoding gene trmH Application of genes as targets for drugs against Acinetobacter baumannii infection.

2. The application according to claim 1, characterized in that, Including at least any one of the following applications a1-a4: a1: using TrmH protein or trmH TrmH inhibitors targeting genes are used as drugs against Acinetobacter baumannii infection. a2: using TrmH protein or trmH Screening of anti-Acinetobacter baumannii infection drugs at the in vitro or cellular level targeting genes; a3: Contains TrmH protein or trmH A combination drug for treating Acinetobacter baumannii infection, consisting of a gene-targeting TrmH inhibitor and an antibiotic. a4: Detection trmH Reagents or kits for gene mutations or TrmH protein expression levels; Optionally, the detection trmH Reagents or kits for gene mutations or TrmH protein expression levels are used to determine the degree of resistance of Acinetobacter baumannii to antibiotics, or to screen for anti-Acinetobacter baumannii infection drugs that can inhibit TrmH activity.

3. An inhibitor of Acinetobacter baumannii, characterized in that, The Acinetobacter baumannii inhibitor has at least one of the functions described in b1-b2: b1: The Acinetobacter baumannii inhibitor has the effect of inhibiting Acinetobacter baumannii. trmH The function of gene expression; b2: The Acinetobacter baumannii inhibitor has the function of inhibiting the activity of TrmH protein in Acinetobacter baumannii.

4. The Acinetobacter baumannii inhibitor according to claim 3, characterized in that, The Acinetobacter baumannii inhibitors include knockout. trmH Genes or reduction trmH Reagents for gene expression.

5. The Acinetobacter baumannii inhibitor according to claim 4, characterized in that, The reagent includes one or more plasmids, the plasmids comprising... trmH Upstream homologous arm insertion fragment of gene, trmH Insertion fragment into the downstream homologous arm of the gene; The trmH The nucleotide sequence of the inserted fragment in the upstream homologous arm of the gene is shown in SEQ ID NO: 1; The trmH The nucleotide sequence of the inserted fragment in the downstream homologous arm of the gene is shown in SEQ ID NO:

2.

6. The Acinetobacter baumannii toxicity inhibitor according to claim 5, characterized in that, The plasmid also includes a forward selection marker gene and a reverse selection marker gene; Optionally, the positive selection marker genes include tellurite resistance genes and / or gentamicin resistance genes; The reverse selection marker gene includes a sugar-sensitive gene. sacB.

7. The Acinetobacter baumannii inhibitor according to claim 3, characterized in that, The Acinetobacter baumannii inhibitor includes […]. trmH mRNA complementary antisense RNA or small interfering RNA; Alternatively, the Acinetobacter baumannii inhibitor may include an inactivated Cas protein, which may be combined with... trmH A complex is formed by the binding of guide RNA upstream of the gene promoter region or coding sequence; Alternatively, the Acinetobacter baumannii inhibitor may comprise a small molecule compound capable of specifically binding to the TrmH protein.

8. The Acinetobacter baumannii inhibitor according to claim 3, characterized in that, The Acinetobacter baumannii inhibitor includes reagents that knock out the coding gene corresponding to the TrmH protein and / or reagents that cause mutations in the dimer interface residues of the TrmH protein to lose its dimerizing ability and / or reagents that cause mutations in key residues at the SAM binding site of the TrmH protein to lose its activity. Optionally, the key residues at the SAM binding site in the TrmH protein include at least one residue from the following: T190, S192, L211, G212, N213, E214, G217, T230, I231, Q233, S239, L240, V242, and A245.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the Acinetobacter baumannii inhibitor according to any one of claims 3-8.

10. The pharmaceutical preparation according to claim 9, characterized in that, The pharmaceutical preparation also includes the antibiotic drug; Optionally, the antibiotics include tetracyclines; And / or, the dosage forms of the pharmaceutical preparations include: injections, infusions, tablets, capsules, granules, dry suspensions, oral solutions, oral emulsions, pills, ointments, gels, and patches.

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