Application of the tsrF gene in Pseudomonas aeruginosa and its preparation method for engineered strain

Abstract

This application relates to a species of Pseudomonas aeruginosa. tsrF Application of genes and methods for preparing engineered bacteria. This invention identifies a novel gene in the genome of *Pseudomonas aeruginosa*. tsrF Genes, through regulation in Pseudomonas aeruginosa tsrF Gene expression can regulate factors such as biofilm production, c-di-GMP production, type 6 secretion system (T6SS) expression, c-di-GMP FleQ receptor production, and c-di-GMP PelD receptor production in Pseudomonas aeruginosa, thereby modulating the virulence of Pseudomonas aeruginosa. Furthermore, TsrF protein can be used as a new target for the development of drugs to prevent and treat Pseudomonas aeruginosa, which is of great significance for the prevention and control of Pseudomonas aeruginosa infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a strain of Pseudomonas aeruginosa. tsrF Genes, TsrF proteins, engineered bacteria and their preparation and application. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa Pseudomonas aeruginosa, also known as "aeruginosa bacillus," is a Gram-negative bacillus and one of the most virulent respiratory pathogens. Current research indicates that its high pathogenicity stems from its ability to release various virulence factors, such as pyocyanin and proteases, after invading the host, and to release multiple toxic proteins through the T6SS (also known as the "type six secretion system"). Studies show that T6SS expression is significantly elevated in Pseudomonas aeruginosa isolated from patients with chronic infections, indicating that T6SS promotes chronic infection. T6SS can inject various types of effector proteins (toxin proteins) into adjacent prokaryotic and eukaryotic cells, damaging their cell membranes, cell walls, and genetic material, thus gaining an advantageous position in intraspecific or interspecific competition.

[0003] Antibiotics are commonly used in clinical practice to treat Pseudomonas aeruginosa infections. Studies have found that Pseudomonas aeruginosa has a strong biofilm-forming ability. In clinical practice, bacteria within the biofilm are encapsulated by a large number of extracellular polymers, making it difficult for antibiotics to penetrate and kill the bacteria. However, repeated treatment with drugs can easily lead to the evolution of drug-resistant strains, which in turn form biofilms of drug-resistant strains, resulting in reduced or even complete ineffectiveness of antibiotic treatment.

[0004] Therefore, it is necessary to find new targets for treating infections caused by Pseudomonas aeruginosa and to provide insights into the drug resistance mechanisms of Pseudomonas aeruginosa. Summary of the Invention

[0005] To address the aforementioned technical challenges, it is necessary to identify novel therapeutic targets for Pseudomonas aeruginosa infections, as well as genes that influence Pseudomonas aeruginosa biofilm production and T6SS expression.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a Pseudomonas aeruginosa strain. tsrF Genes, the ones mentioned tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0008] Secondly, the present invention also provides a method for knocking out Pseudomonas aeruginosa. tsrF The application of genes, wherein the application is any one of F11)-F15):

[0009] Application of F11 in reducing the biofilm production of Pseudomonas aeruginosa;

[0010] Application of F12 in reducing T6SS expression in Pseudomonas aeruginosa;

[0011] Application of F13 in reducing the yield of cyclic diguanylic acid (c-di-GMP) in Pseudomonas aeruginosa;

[0012] Application of F14 in reducing the production of FleQ receptor (also known as "c-di-GMP receptor FleQ") of Pseudomonas aeruginosa cyclic diguanylic acid.

[0013] Application of F15 in reducing the production of PelD receptor (also known as "c-di-GMP receptor PelD") of Pseudomonas aeruginosa cyclic diguanylate.

[0014] The tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0015] Thirdly, the present invention also provides a method for knocking out Pseudomonas aeruginosa. tsrF The application of the gene is in reducing the expression of T6SS in *Pseudomonas aeruginosa*, wherein T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS, and H4-T6SS. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0016] Fourthly, the present invention also provides a strain overexpressing Pseudomonas aeruginosa. tsrF The application of genes, wherein the application is any one of F1)-F6):

[0017] F1) Application in increasing the biofilm yield of Pseudomonas aeruginosa;

[0018] Application of F2 in enhancing T6SS expression in Pseudomonas aeruginosa;

[0019] F3) Application in increasing the production of cyclic diguanylic acid in Pseudomonas aeruginosa;

[0020] Application of F4 in increasing the FleQ receptor yield of cyclic diguanylic acid in Pseudomonas aeruginosa;

[0021] F5) Application in increasing the PelD receptor yield of cyclic diguanylic acid in Pseudomonas aeruginosa;

[0022] F6) Application in the preparation of drugs for inhibiting the growth of Escherichia coli;

[0023] The tsrFThe gene is the nucleotide sequence shown in SEQ ID No. 1.

[0024] Fifthly, the present invention also provides a strain overexpressing Pseudomonas aeruginosa. tsrF The application of the gene is in enhancing the expression of T6SS in *Pseudomonas aeruginosa*, wherein T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS, and H4-T6SS. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0025] Sixthly, the present invention also provides a TsrF protein, said TsrF protein being derived from the protein described in claim 1. tsrF Gene expression, wherein the TsrF protein has the amino acid sequence shown in SEQ ID No. 2; furthermore, the protein is composed of 524 amino acids, has a molecular weight of 57.17 kDa, and a theoretical isoelectric point of 8.77.

[0026] In a seventh aspect, the present invention also provides a drug target protein, the drug target protein being the TsrF protein as described above, and the drug being a drug for treating Pseudomonas aeruginosa infection.

[0027] Eighthly, the present invention also provides an engineered strain of *Pseudomonas aeruginosa*, wherein the engineered strain is a knockout strain. tsrF Pseudomonas aeruginosa gene or overexpression of the gene described tsrF The gene of Pseudomonas aeruginosa, the tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0028] Ninthly, the present invention also provides a method for preparing the above-mentioned Pseudomonas aeruginosa engineered bacteria, using the M1 method to prepare knockout strains. tsrF Pseudomonas aeruginosa gene, or, using the M2 method to prepare overexpression tsrF Genes of Pseudomonas aeruginosa, including:

[0029] The M1 method includes the following steps: using gene knockout technology to remove *Pseudomonas aeruginosa*... tsrF Gene knockout, resulting in knockout tsrF Pseudomonas aeruginosa with genetic defects;

[0030] The M2 method includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] tsrF The gene expression vector was transformed into Pseudomonas aeruginosa, resulting in overexpression. tsrF Genetically modified Pseudomonas aeruginosa.

[0031] Furthermore, gene knockout technology was used to remove Pseudomonas aeruginosa. tsrFGene knockout can be achieved using the following technical approach: amplification with primer pairs UF and UR. tsrF Upstream homologous arm of gene, using Primer pair DF and DR amplification tsrF Downstream homologous arms of the gene; the upstream and downstream homologous arm fragments were fused by overlap PCR, and after electroporation and introduction into the host bacteria, sucrose sensitivity screening combined with PCR verification was used to obtain... tsrF Gene knockout mutant strain (a type of "engineered Pseudomonas aeruginosa");

[0032] Furthermore, it will contain tsrF The gene expression vector can be transformed into Pseudomonas aeruginosa using the following technical methods: amplification using primer pairs OEF and OER. tsrF The full-length gene sequence was cloned into an expression vector and then transformed into a host bacterium to obtain... tsrF Overexpression strain (or a type of "engineered Pseudomonas aeruginosa");

[0033] Furthermore, the primers UF, UR, DF, DR, OEF, and OER can be selected as nucleotide sequences as shown in SEQ ID NO. 3-8, specifically as follows:

[0034] Primer UF: gagctcggtacccggggatccGCTGACGGCGAAGGCCGT (SEQ ID No. 3);

[0035] Primer UR: agcttcatgcgatggaCATCGCCTCCATCTCGTCC (SEQ ID No. 4);

[0036] Primer DF: gatgTCCATCGCATGAAGCTATTGAG (SEQ ID No. 5);

[0037] Primer DR: acgacggccagtgccaagcttCCAGGGACGGGTGGGCGA (SEQ ID No. 6);

[0038] Primer OEF: gtcgacggtatcgataagcttTCCCGTATCGCTACCTGGACG (SEQ ID No. 7);

[0039] Primer OER: cgctctagaactagtggatccCGCAGCCAATCAGGGCCG (SEQ ID No. 8).

[0040] In a tenth aspect, the present invention also provides a method for reducing biofilm production and / or T6SS expression in engineered Pseudomonas aeruginosa, the method comprising reducing biofilm production and / or T6SS expression in the Pseudomonas aeruginosa genome. tsrF The gene was knocked out using genetic engineering methods. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0041] The applications / methods of the second to fifth aspects, the ninth aspect and the tenth aspect of the present invention are all applications / methods for purposes other than disease diagnosis and treatment.

[0042] Technical effects of the present invention:

[0043] This invention is the first to identify a novel strain in the clinical genome of *Pseudomonas aeruginosa*. tsrF The gene was identified and its application in regulating key bacterial physiological functions has been clarified, specifically in regulating "biofilm production," "c-di-GMP production," "T6SS expression," "FleQ receptor production of c-di-GMP," and "PelD receptor production of c-di-GMP" in *Pseudomonas aeruginosa*. This fills a gap in the research on the regulatory mechanism of class IV T6SS (H4-T6SS) in *Pseudomonas aeruginosa*, providing new molecular targets and theoretical foundations for technological breakthroughs in this field. Furthermore, by constructing [a specific gene] within *Pseudomonas aeruginosa*... tsrF Gene knockout mutants significantly altered T6SS activity, biofilm production, and c-di-GMP synthesis levels. These three factors are key regulatory links in *Pseudomonas aeruginosa*'s competitive survival ability, virulence expression, and drug resistance formation, indicating that this invention can intervene in the core pathogenic pathways of bacteria. This invention provides both an efficient and specific biotechnological means for regulating the pathogenicity of *Pseudomonas aeruginosa* and a new technical direction for solving the infection control challenges caused by the high drug resistance and virulent virulence of *Pseudomonas aeruginosa* in clinical practice (such as...). TsrF As a target protein for drugs used to treat Pseudomonas aeruginosa infection, this protein has significant practical implications and application prospects for improving the treatment efficacy of Pseudomonas aeruginosa infection.

[0044] Other features and aspects of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0045] The accompanying drawings, which are included in and form part of this specification, illustrate exemplary embodiments, features, and aspects of this disclosure together with the specification and serve to explain the principles of this disclosure.

[0046] Figure 1This image shows the gene cluster "H4-T6SS" and its functional activity test data of the *Pseudomonas aeruginosa* strain LYSZa7 in Example 1. Part A shows the structure of H4-T6SS in *Pseudomonas aeruginosa* LYSZa7; Part B shows schematic diagrams of the four types of T6SS in strain LYSZa7; Part C shows the determination of the number of viable *E. coli*; and Part D shows the movement of H4-T6SS through mobile genetic elements. The blue lines represent GC content, and each data point represents the percentage of GC content in the corresponding 2500 bp upstream and downstream sequences. (Based on pairing...) t Tests showed P < 0.05; P < 0.01; P < 0.001. ns, no significant difference. EV, control used empty vector (same for subsequent figures).

[0047] Figure 2 As in Example 2 tsrF The graph shows the data from the validation experiment of gene regulation of H4-T6SS. Part A shows the Δ function detected by real-time quantitative PCR (RT-qPCR). retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( tsrF Part B shows the expression levels of four T6SS strains in the strain; Part B is for RT-qPCR detection of wild-type LYSZa7(EV), Δ tsrF (EV) and Δ tsrF ( tsrF The expression levels of four T6SS strains were analyzed in part C by Western blot. retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( tsrF The yield of TssB4-Flag protein in strain A was measured, with the RNA polymerase α subunit (α-RNAP) serving as an internal control; Part D was the determination of the number of viable Escherichia coli.

[0048] Figure 3 This is a phylogenetic analysis diagram of the comprehensive regulatory role of TsrF and its homologous proteins in Example 3, based on RNA-seq data ( ), LYSZa7 and Δ tsrF GO enrichment analysis (as shown in Part A) and KEGG enrichment analysis (as shown in Part B) of differentially expressed genes; as shown in Part C, BLAST analysis of TsrF was performed to screen out the top 26 representative homologous proteins. The right panel shows the operon structures (containing these homologous proteins) in the corresponding species, and the conserved T6SS protein in each operon is marked with a different color.

[0049] Figure 4 This is a graph showing experimental data related to the direct regulation of H4-T6SS and biofilm formation by TsrF in Example 4. Part A shows the predicted TsrF domains; Part B shows the electrophoretic mobility detection of TsrF and... tssB4 and tssH4 Direct binding effect of promoters; algB Gene promoters serve as negative controls; section C shows wild-type LYSZa7 and Δ tsrF Δ retS Δ retS Δ tsrF And the biofilm yield data of their complementary strains after 12 hours of culture in LB medium; Part D shows the RT-qPCR detection of Δ retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( tsrF ) strains pelA - D Gene expression level data.

[0050] Figure 5 This is a graph showing the experimental data related to the role of c-di-GMP in TsrF-mediated H4-T6SS expression and biofilm formation in Example 5. Part A shows the results obtained by detecting... cdrA The mRNA level of the gene was used to determine the intracellular c-di-GMP level of the indicator strain; Part B shows the RT-qPCR detection of Δ retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( tsrF Guanylate cyclase gene in strain gcbA and roeA Expression level; Part C shows the Western blot detection of Δ. retS (EV), Δ retS Δ tsrF (EV), Δ retS Δ tsrF ( bifA ) and Δ retS Δ tsrF ( sadC The expression level of TssB4-Flag protein in the strain was shown, with α-RNAP used as an internal control; Part D shows the determination of the number of viable E. coli; Part E shows the Δ retS (EV), Δ retS Δ tsrF (EV), Δ retS ΔtsrF ( bifA ) and Δ retS Δ tsrF ( sadC Detection of biofilm yield of strain )

[0051] Figure 6 This is a graph showing the experimental data related to TsrF mediating AmrZ regulation of c-di-GMP generation in Example 6. Part A shows the RT-qPCR detection of Δ... retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( tsrF ) strains amrZ mRNA levels of genes; Part B shows the results obtained by detecting... cdrA The mRNA level of the gene was used to determine the intracellular c-di-GMP production in the indicator strain; Part C shows the RT-qPCR detection of Δ retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( amrZ ) strains tssH4 and tssB4 Gene expression levels; Part D shows the determination of viable E. coli counts; Part E shows Δ... retS (EV), Δ retS Δ tsrF (EV) and Δ retS Δ tsrF ( amrZ Biofilm yield of strain 12 hours after culturing in LB medium; F shows the electrophoretic mobility assay of TsrF. amrZ The binding effect of gene promoters.

[0052] Figure 7 This is a graph showing the experimental data related to the response of FleQ to c-di-GMP under TsrF regulation in Example 7. Part A shows the RT-qPCR detection of Δ... retS (EV), Δ retS Δ tsrF (EV) strain fleQ and pelD Gene expression levels; Part B shows the Δ gene expression level detected by RT-qPCR. retS Δ tsrF (EV), Δ retS Δ tsrF ( sadC ), Δ retS Δ tsrF ΔfleQ ( sadC ) and Δ retS Δ tsrF Δ pelD ( sadC ) strains tssH4 and tssB4 Gene expression levels; Part C shows the determination of the number of viable E. coli; Part D shows the Δ... retS Δ tsrF (EV), Δ retS Δ tsrF ( sadC ), Δ retS Δ tsrF Δ fleQ ( sadC ) and Δ retS Δ tsrF Δ pelD ( sadC Biofilm yield of strain after 12 hours of culture in LB medium was detected. Detailed Implementation

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0055] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0056] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0057] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0058] In a first aspect, the present invention provides a Pseudomonas aeruginosa strain. tsrF Genes, the ones mentioned tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0059]

[0060] The nucleotide sequence shown in SEQ ID No. 1 is the transcription factor TsrF, first identified in the genome of clinically isolated *Pseudomonas aeruginosa*. This nucleotide sequence can regulate factors such as biofilm production, c-di-GMP production, T6SS expression, FleQ receptor production of c-di-GMP, and PelD receptor production of c-di-GMP in *P. aeruginosa*. It has great potential as a target for the development of anti-*P. aeruginosa* drugs. It can provide an efficient and specific biotechnological means to regulate the pathogenicity of *P. aeruginosa*, and provide a new technical direction for solving the infection control problems caused by the high drug resistance and strong virulence of *P. aeruginosa* in clinical practice. It has important practical significance and application prospects for improving the treatment effect of *P. aeruginosa* infection.

[0061] Secondly, the present invention also provides a method for knocking out Pseudomonas aeruginosa. tsrF The application of genes, wherein the application is any one of F11)-F15):

[0062] Application of F11 in reducing the biofilm production of Pseudomonas aeruginosa;

[0063] Application of F12 in reducing T6SS expression in Pseudomonas aeruginosa;

[0064] Application of F13 in reducing c-di-GMP production in Pseudomonas aeruginosa;

[0065] Application of F14 in reducing FleQ receptor production of c-di-GMP in Pseudomonas aeruginosa;

[0066] Application of F15 in reducing PelD receptor production in Pseudomonas aeruginosa c-di-GMP;

[0067] The tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0068] Thirdly, the present invention also provides a method for knocking out Pseudomonas aeruginosa. tsrF The application of the gene, specifically its application in reducing the expression of T6SS in *Pseudomonas aeruginosa*, is described below. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1; furthermore, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS.

[0069] Fourthly, the present invention also provides a strain overexpressing Pseudomonas aeruginosa. tsrF The application of genes, wherein the application is any one of F1)-F6):

[0070] F1) Application in increasing the biofilm yield of Pseudomonas aeruginosa;

[0071] Application of F2 in enhancing T6SS expression in Pseudomonas aeruginosa;

[0072] Application of F3 in increasing the yield of c-di-GMP in Pseudomonas aeruginosa;

[0073] Application of F4 in increasing the FleQ receptor yield of c-di-GMP in Pseudomonas aeruginosa;

[0074] F5) Application in increasing the PelD receptor yield of c-di-GMP in Pseudomonas aeruginosa;

[0075] F6) Application in the preparation of drugs for inhibiting the growth of Escherichia coli;

[0076] The tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0077] Fifthly, the present invention also provides a strain overexpressing Pseudomonas aeruginosa. tsrF The application of the gene, specifically its application in enhancing the expression of Pseudomonas aeruginosa T6SS, is described below. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1; furthermore, the T6SS is at least one of H1-T6SS, H2-T6SS, H3-T6SS and H4-T6SS.

[0078] Sixthly, the present invention also provides a TsrF protein, said TsrF protein being derived from the protein described in claim 1. tsrF Gene expression, wherein the TsrF protein has the amino acid sequence shown in SEQ ID No. 2; furthermore, the protein is composed of 524 amino acids, has a molecular weight of 57.17 kDa, and a theoretical isoelectric point of 8.77.

[0079] In this invention, the amino acid sequence shown in SEQ ID No. 2 is:

[0080] In a seventh aspect, the present invention also provides a drug target protein, wherein the drug target protein is the aforementioned TsrF protein, and the drug is a drug for treating Pseudomonas aeruginosa infection.

[0081] Eighthly, the present invention also provides an engineered strain of *Pseudomonas aeruginosa*, wherein the engineered strain is a knockout strain. tsrF Pseudomonas aeruginosa gene or overexpression of the gene described tsrF The gene of Pseudomonas aeruginosa, the tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0082] Ninthly, the present invention also provides a method for preparing the above-mentioned Pseudomonas aeruginosa engineered bacteria, using the M1 method to prepare knockout strains. tsrF Pseudomonas aeruginosa gene, or, using the M2 method to prepare overexpression tsrF Genes of Pseudomonas aeruginosa, including:

[0083] The M1 method includes the following steps: using gene knockout technology to remove *Pseudomonas aeruginosa*... tsrF Gene knockout, resulting in knockout tsrFPseudomonas aeruginosa with genetic defects;

[0084] The M2 method includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] tsrF The gene expression vector was transformed into Pseudomonas aeruginosa, resulting in overexpression. tsrF Genetically modified Pseudomonas aeruginosa.

[0085] Furthermore, gene knockout technology was used to remove Pseudomonas aeruginosa. tsrF Gene knockout can be achieved using the following technical approach: amplification with primer pairs UF and UR. tsrF Upstream homologous arm of gene, using Primer pair DF and DR amplification tsrF Downstream homologous arms of the gene; the upstream and downstream homologous arm fragments were fused by overlap PCR, and after electroporation and introduction into the host bacteria, sucrose sensitivity screening combined with PCR verification was used to obtain... tsrF Gene knockout mutant strain (a type of "engineered Pseudomonas aeruginosa");

[0086] Furthermore, it will contain tsrF The gene expression vector can be transformed into Pseudomonas aeruginosa using the following technical methods: amplification using primer pairs OEF and OER. tsrF The full-length gene sequence was cloned into an expression vector and then transformed into a host bacterium to obtain... tsrF Overexpression strain (or a type of "engineered Pseudomonas aeruginosa");

[0087] Furthermore, the primers UF, UR, DF, DR, OEF, and OER can be selected as nucleotide sequences as shown in SEQ ID NO. 3-8, specifically as follows:

[0088] Primer UF: gagctcggtacccggggatccGCTGACGGCGAAGGCCGT (SEQ ID No. 3);

[0089] Primer UR: agcttcatgcgatggaCATCGCCTCCATCTCGTCC (SEQ ID No. 4);

[0090] Primer DF: gatgTCCATCGCATGAAGCTATTGAG (SEQ ID No. 5);

[0091] Primer DR: acgacggccagtgccaagcttCCAGGGACGGGTGGGCGA (SEQ ID No. 6);

[0092] Primer OEF: gtcgacggtatcgataagcttTCCCGTATCGCTACCTGGACG (SEQ ID No. 7);

[0093] Primer OER: cgctctagaactagtggatccCGCAGCCAATCAGGGCCG (SEQ ID No. 8).

[0094] In a tenth aspect, the present invention also provides a method for reducing biofilm production and / or T6SS expression in engineered Pseudomonas aeruginosa, the method comprising reducing biofilm production and / or T6SS expression in the Pseudomonas aeruginosa genome. tsrF The gene was knocked out using genetic engineering methods. tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1.

[0095] The applications / methods of the second to fifth aspects, the ninth aspect and the tenth aspect of the present invention are all applications / methods for purposes other than disease diagnosis and treatment.

[0096] The nucleotide sequence shown in SEQ ID No. 1 of this invention (i.e., Pseudomonas aeruginosa) tsrF The TsrF gene has been shown to positively regulate biofilm production in Pseudomonas aeruginosa. By knocking out this gene, biofilm production in Pseudomonas aeruginosa can be reduced, thereby reducing the protective effect of extracellular polymers on Pseudomonas aeruginosa and making it easier for antibiotics to enter the bacteria and kill them. Furthermore, the TsrF protein can also be used as a target for Pseudomonas aeruginosa infection, providing a new direction and idea for the development of drugs against Pseudomonas aeruginosa infection.

[0097] The nucleotide sequence shown in SEQ ID No. 1 of this invention (i.e., Pseudomonas aeruginosa) tsrF Studies have shown that T6SS expression in *Pseudomonas aeruginosa* isolated from patients with chronic infection is significantly elevated, indicating that T6SS promotes chronic infection. By knocking out this gene, the corresponding T6SS expression is inhibited, which can reduce the release of virulence factors, such as effector proteins, from *P. aeruginosa* into patients, thereby reducing its virulence. Furthermore, TsrF protein can also be used as a target for *P. aeruginosa* infection, providing new directions and ideas for the development of anti-*P. aeruginosa* drugs.

[0098] The nucleotide sequence shown in SEQ ID No. 1 of this invention (i.e., Pseudomonas aeruginosa) tsrFThe gene has been shown to regulate c-di-GMP production, c-di-GMP receptor FleQ production, and c-di-GMP receptor PelD production in Pseudomonas aeruginosa. These substances are positively correlated with the virulence of Pseudomonas aeruginosa (including biofilm production and T6SS expression). By knocking out this gene, the production / expression levels of these factors can be reduced, thereby reducing the virulence of Pseudomonas aeruginosa. Furthermore, the TsrF protein can also be used as a target for Pseudomonas aeruginosa infection, providing a new direction and approach for the development of drugs against Pseudomonas aeruginosa infection.

[0099] In addition, by analyzing the nucleotide sequence shown in SEQ ID No. 1 (i.e., Pseudomonas aeruginosa) tsrF Overexpression of genes can also play a role in the development of related drugs, such as in the application of anti-E. coli drugs.

[0100] The present invention will be further illustrated by the following embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0101] Example 1: Functional verification of H4-T6SS in Pseudomonas aeruginosa strain LYSZa7

[0102] A novel T6SS gene cluster was first identified in a *Pseudomonas aeruginosa* strain LYSZa7 isolated from a patient (see [link to relevant documentation]). Figure 1 (As shown in Parts A and B), this invention names it H4-T6SS. Using the genome of wild-type LYSZa7 strain as a template, the coding genes of the core components of H1-T6SS, H2-T6SS, H3-T6SS, and H4-T6SS were knocked out respectively through homologous recombination technology. tssH1 , tssH2 , tssH3 , tssH4 Single mutants ΔT6SS-1, ΔT6SS-2, ΔT6SS-3, and ΔT6SS-4, and four T6SS complete deletion mutants ΔT6SS-1, 2, 3, and 4 were obtained; simultaneously, a variant of ΔT6SS-4 was constructed. tssH4 Gene-complemented strain ΔT6SS-4 (T6SS-4).

[0103] The specific process of constructing the complement in this embodiment is as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] tssH4The gene was cloned into the pUCP20 vector (PCR amplification primers PPF and PPR are shown in SEQ ID No. 9-10), and the complement plasmid was transformed into the ΔT6SS-4 mutant strain via triparental conjugation. The successful construction of the complement was confirmed by resistance screening (clobenzylpenicillin, 150 μg / mL) and DNA sequencing.

[0104] The sequences of the PPF and PPR mentioned above are shown in SEQ ID No. 9-10, as follows:

[0105] Primer PPF: gagctcggtacccggggatccTCCCGTATCGCTACCTGGAC (SEQ ID No. 9)

[0106] Primer PPR: acgacggccagtgccaagcttCGCAGCCAATCAGGGCCG (SEQ ID No. 10)

[0107] Using *Escherichia coli* DH5α as the recipient bacterium, a bacterial competition experiment was conducted to detect the killing ability of various mutant strains. The specific procedure for the bacterial competition experiment in this embodiment was as follows: *Escherichia coli* containing pBBR1-MCS5 was mixed with *Pseudomonas aeruginosa* in equal proportions. A sterile 0.45 μM nylon membrane was placed on LB agar plates, and 20 μl of the mixed bacterial solution was placed on the nylon membrane. The plates were then incubated at 37°C for 16 h. The bacteria on the membrane were washed off with sterile water, the bacterial solution was serially diluted, and then spread onto LB agar plates containing 50 μg / ml gentamicin. The plates were incubated at 37°C, and the colony counts were recorded.

[0108] The results showed that the ΔT6SS-4 mutant strain had a significantly reduced killing ability against Escherichia coli DH5α, and its defective phenotype was similar to that of the ΔT6SS-1 and ΔT6SS-3 mutant strains; while the complement strain ΔT6SS-4 (T6SS-4) could completely restore the killing efficiency against DH5α. Figure 1 (As shown in section C); the killing ability of the four T6SS complete deletion mutants ΔT6SS-1,2,3,4 was significantly reduced compared to each single mutant. Figure 1 As shown in section C). The GC content of the upstream and downstream sequences of the H4-T6SS gene cluster is lower than the average GC content of 66.2% for strain LYSZa7 ( Figure 1 As shown in section D), integrase-related genes and functional genes involved in bacterial virulence and drug resistance were detected in the upstream and downstream regions of this gene cluster. Figure 1 (As shown in section D).

[0109] The above results indicate that H4-T6SS in Pseudomonas aeruginosa strain LYSZa7 has functional activity and can significantly affect the bacteria's ability to kill other microorganisms. At the same time, H4-T6SS is located in an integrin, which enables the movement and spread of gene clusters, providing a molecular basis for Pseudomonas aeruginosa to adapt to the environment and enhance its competitive ability.

[0110] Example 2: Verification of the regulatory function of TsrF on H4-T6SS

[0111] A predicted transcription factor, TsrF, was identified in the H4-T6SS gene cluster. It was knocked out in the RetS deletion mutant (H4-T6SS activation background) and the wild-type LYSZa7 strain, respectively. tsrF The double mutant Δ was constructed. retS Δ tsrF and single mutant Δ tsrF RT-qPCR results showed that, retS In deletion mutants, tsrF Mutations can inhibit the activity of H1-, H2, H3, and H4-T6SS (e.g., Figure 2 (As shown in Part A of the middle section).

[0112] Similarly, compared to wild-type LYSZa7, Δ tsrF The expression of H1-, H2-, H3-, and H4-T6SS was suppressed in the mutant strain (e.g., Figure 2 (As shown in B).

[0113] Western blot experiments showed that Δ retS Δ tsrF The expression level of TssB4 was significantly reduced in the mutant strain, while the expression level was significantly lower through exogenous expression. tsrF Its expression can be restored ( Figure 2 (As shown in section C).

[0114] By knocking out the membrane protein TssM in H1-, H2-, and H3-T6SS, mutant strains (ΔT6SS-1,2,3) with activity only in H4-T6SS were constructed. The results showed that, compared with Δ... retS When ΔT6SS-1,2,3 mutant strains are co-cultured, Escherichia coli DH5α and Δ retS Δ tsrF The survival rate of ΔT6SS-1,2,3 mutant strains was significantly increased after co-culture; while the survival rate of ΔT6SS-1,2,3 mutant strains was significantly increased. retS Δ tsrF ΔT6SS-1,2,3 mutant strains express tsrF Afterwards, the effect can be reversed. Figure 2 (As shown in section D). Furthermore, when the four T6SS complete deletion mutants were co-cultured with *E. coli* DH5α, the survival rate of *E. coli* further increased, and...tsrF The mutation does not affect the mutant strain's ability to kill Escherichia coli. Figure 2 (As shown in section D); By comparing the ΔT6SS-1,2,3 and ΔT6SS-1,2,3,4 mutants, the antibacterial effect of H4-T6SS can be observed. Figure 2 (As shown in section D). The above results indicate that TsrF is a global activator of T6SS in Pseudomonas aeruginosa LYSZa7, and can positively regulate the expression and antibacterial activity of H4-T6SS.

[0115] The Western Blot experiment procedure in this embodiment is as follows:

[0116] (1) Add 5×SDS loading buffer to the protein sample and boil for 10 min; prepare SDS-PAGE gel according to the protein gel kit.

[0117] (2) Take 15 μL of protein sample and add it to the SDS-PAGE gel loading well. Electrophore at 90V for 30 min, then adjust the voltage to 120V and stop electrophoresis when the SDS loading buffer is 2-3 cm away from the bottom of the protein gel.

[0118] (3) Adjust the PVDF membrane and filter paper to the size of the SDS-PAGE gel. Before the transfer operation, soak the PVDF membrane in methanol solution for at least 15 seconds. After electrophoresis, soak the PVDF membrane, transfer filter paper, and SDS-PAGE gel in the transfer solution. Stack the filter paper-protein gel-PVDF membrane-filter paper neatly from bottom to top (black side of the transfer clamp down), and place it in the electrophoresis tank. Wet transfer at a constant current of 400mA for 1.5 hours (adjust as needed).

[0119] (4) After the transfer is completed, the PVDF membrane is removed and placed in the blocking solution (PBST solution containing 5% skim milk powder) for 1 hour;

[0120] (5) Primary antibody incubation: Dilute the primary antibody with PBST at a ratio of 1:1000, place the blocked PVDF membrane in the primary antibody dilution solution and incubate overnight at 4°C or at room temperature for 2 hours;

[0121] (6) After the primary antibody incubation is completed, the PVDF membrane is washed with PBST 4 times, 15 min each time;

[0122] (7) Secondary antibody incubation: Dilute the secondary antibody with PBST at a ratio of 1:20000, transfer the PVDF membrane to the secondary antibody dilution solution, and incubate at room temperature for 1 hour with shaking at 50 rpm;

[0123] (8) After the secondary antibody incubation, the PVDF membrane was washed with PBST 4 times, 15 min each time;

[0124] (9) Use ECL chemiluminescence solution for development. Generally, the ECL chemiluminescence solution is prepared by mixing the two solutions in the ECL chemiluminescence detection kit at a volume ratio of 1:1. The luminescence solution should be prepared and used immediately.

[0125] Example 3: TsrF is a globally distributed regulatory factor widely distributed in Gram-negative bacteria.

[0126] Extracting wild LYSZa7 strains and Δ tsrF Total RNA from mutant strains was sequenced using RNA-Seq; differential expression analysis was then used for screening. Genes with a p-value of ≤0.05 were identified, and functional and pathway enrichment analyses were performed using the GO and KEGG databases. GO enrichment results indicated that differentially expressed genes are involved in multiple biological functions, including T6SS, metal ion binding, extracellular polysaccharides, pyocyanin synthesis, membrane components, and transport. Figure 3 (As shown in Part A of the middle section).

[0127] Differentially expressed genes were mapped to 13 KEGG pathways, covering secondary metabolism, two-component systems, biofilm formation, etc. (e.g.) Figure 3 (As shown in Part B).

[0128] Phylogenetic analysis showed that TsrF is not only present in the genus *Pseudomonas* (… Pseudomonas ), and also exists in the genus Vibrio ( Vibrio Burkholderia ( ) Burkholderia ) and Achromobacterium spp. ( Achromobacter In; and among all the above-mentioned bacterial species, tsrF The presence of at least one T6SS structural gene upstream or downstream of the gene indicates that TsrF is primarily a... Regulatory factors related to T6SS (like Figure 3 (As shown in section C).

[0129] Example 4: Transcription factor TsrF directly regulates the expression of H4-T6SS and biofilm-related genes.

[0130] The predicted structure of TsrF protein includes an AAA domain (an ATPase domain associated with various cellular activities) and an HTH domain (a helix-turn-helix DNA-binding domain). Figure 4 As shown in Part A of the genome annotation, the H4-T6SS gene cluster can be divided into two parts based on the transcriptional direction (arrow direction). tssB4 - tssL4 and tssH4 - tssM4 () Figure 1 (As shown in Part A of the middle section).

[0131] Electrophoretic mobility assay (EMSA) showed that TsrF and tssB4 or tssH4After incubation with the promoter probe, hysteresis bands were observed in all samples, indicating that TsrF can bind to the promoter of the H4-T6SS gene cluster. Figure 4 (As shown in Part B). Using TsrF and algB The incubation reaction of the gene promoter probe served as a negative control. [[ID= (As shown in Part B).

[0132] The EMSA experiment in this embodiment is as follows: (1) PCR to obtain the 300-500bp fragment before the start codon of the target gene; (2) Preparation of EMSA electrophoresis gel: The formula is 1ml 5×TBE buffer, 1ml acrylamide / bisacrylamide, 313μL 80% glycerol, 75μL 10% ammonium persulfate (APS), 5μL tetramethylethylenediamine (TEMED), and 7.607ml sterile water; (3) EMSA binding reaction: 1μL binding buffer, 100ng probe, 100μg TsrF, and water to 10μL. After reacting at room temperature for 30min, add 5μL 5×Loading Buffer. Note that the reaction system should be gently aspirated during this process; (4) Electrophoresis: Use 0.5×TBE as buffer and perform pre-electrophoresis at 90V for 30min to remove factors in the gel that may disrupt protein-nucleic acid interactions. After the pre-electrophoresis, load 20 μL of the reaction system onto the gel and electrophore at 90V until the bromophenol blue dye reaches 2 / 3-3 / 4 of the gel and stop electrophoresis; (5) Observe the migration band in the DNA gel imaging system.

[0133] like ​ As shown in section C, the experimental results show that Δ retS Δ tsrF The biofilm formation ability of the double mutant strain was significantly lower than that of the Δ strain. retS Mutant strain, replacement tsrF It can be partially recovered later; Δ retS Δ tsrF Double mutant biofilm synthesis gene pelA - D Gene expression levels were significantly lower than Δ retS mutant strain ( Figure 4 (As shown in section D).

[0134] The process for determining the biofilm yield in this embodiment is as follows:

[0135] (1) Pick a single colony from an LB plate and place it into 3 ml of LB medium. Incubate overnight at 37°C with shaking. Re-inoculate the overnight culture onto fresh medium and incubate at 37°C with shaking until the logarithmic phase.

[0136] (2) Adjust the OD600 value of the test strain to 0.002, add it to a 96-well plate, add 120µL of bacterial solution to each well, make 5 replicates for each test strain, and incubate at 37℃.

[0137] (3) After the appropriate time has been incubated, remove the 96-well plate, use a pipette to extract the bacterial culture, and wash it three times with sterile water to remove the bacteria from the bottom of the well.

[0138] (4) Add 150µL of 0.1% (m / v) crystal violet solution to each well and let stand for 20min;

[0139] (5) Use a pipette to extract the crystal violet and wash off any unadsorbed crystal violet with sterile water;

[0140] (6) Blow dry the water on the 96-well plate, add 200µL of 95% (v / v) ethanol to each well, and let stand for 20 min;

[0141] (7) Use a multi-functional microplate reader to read the absorbance at 570 nm. The experiment should be repeated at least 3 times.

[0142] Example 5: TsrF regulates H4-T6SS and biofilm formation in a c-di-GMP dependent manner.

[0143] RT-qPCR detection Δ retS With Δ retS Δ tsrF c-di-GMP indicator gene in mutant strains cdrA The expression reveals Δ retS Δ tsrF middle cdrA Expression was significantly lower than Δ retS exogenous expression tsrF The expression can be restored ( Figure 5 (As shown in Part A of the diagram); In addition, RT-qPCR also confirmed that TsrF can regulate the c-di-GMP synthesis gene. gcbA , roeA The expression ( Figure 5 (As shown in Part B). Subsequently, in Δ retS Δ tsrF Exogenous expression in mutant strains sadC (Improving c-di-GMP) and bifA (Degradation of c-di-GMP), discovered sadC It can upregulate H4-T6SS expression and bifA No impact ( Figure 5 (as shown in Part C), and sadC Can eliminate Δ retS Δ tsrF The phenomenon of increased E. coli survival rate when ΔT6SS-1,2,3 is co-cultured with E. coli ( Figure 5 (As shown in section D). Finally, through biofilm detection experiments, it was found that increasing Δ retS Δ tsrFThe mutant strain's c-di-GMP level can restore its biofilm formation defect, while in Δ retS Δ tsrF Decreased c-di-GMP levels in mutant strains had no effect on biofilm formation. Figure 5 (As shown in section E).

[0144] The above experimental results indicate that TsrF can regulate the expression activity and biofilm formation of H4-T6SS in a c-di-GMP-dependent manner by activating c-di-GMP synthesis, providing experimental evidence for clarifying the regulatory mechanism of TsrF and subsequent drug target development.

[0145] Example 6: TsrF regulates c-di-GMP synthesis by enhancing AmrZ activity.

[0146] The expression of c-di-GMP metabolic regulatory genes in each strain was detected by RNA-seq and RT-qPCR, and it was found that only amrZ In Δ tsrF Significantly downregulated in mutant strains, and exogenous expression tsrF Its expression can be restored ( Figure 6 (as shown in Part A); then pBBR1-MCS5- amrZ Import Δ retS Δ tsrF The mutant strain was detected by RT-qPCR, which showed the presence of the c-di-GMP indicator gene. cdrA The expression of was significantly increased ( Figure 6 (As described in Part B). Next, the H4-T6SS gene was detected by RT-qPCR ( tssB4 , tssH4 The expression of AmrZ was found to upregulate Δ. retS Δ tsrF Expression of H4-T6SS in mutant strains ( Figure 6 (As shown in Part C); the bacterial competition experiment further showed that Δ retS Δ tsrF The ΔT6SS-1,2,3 mutant strain showed significantly enhanced antibacterial activity against Escherichia coli after expressing AmrZ. Figure 6 (As described in Part D). Biofilm detection experiments showed that Δ retS Δ tsrF After expressing AmrZ, the total biofilm in the mutant strain can be restored to Δ. retS mutant level ( Figure 6 (As described in Part E). Finally, EMSA experiments confirmed that purified TsrF can directly bind to... amrZ The startup sub-region ( Figure 6 (As shown in section F).

[0147] The above experimental results show that TsrF can directly bind amrZ The promoter positively regulates its expression, promotes intracellular c-di-GMP synthesis by increasing AmrZ activity, and thus enhances H4-T6SS activity and biofilm formation. This clarifies the key molecular links in the TsrF regulatory pathway and provides experimental evidence for using the TsrF-AmrZ-c-di-GMP axis as a target for the prevention and control of Pseudomonas aeruginosa infection.

[0148] Example 7: FleQ acts as a c-di-GMP effector in the TsrF-regulated H4-T6SS and biofilm formation process.

[0149] Δ was detected by RT-qPCR. retS With Δ retS Δ tsrF mutant strains fleQ and pelD The expression confirms that the two are in Δ retS Δ tsrF Average down ( Figure 7 (as described in Part A); then construct Δ retS Δ tsrF Δ fleQ Δ retS Δ tsrF Δ pelD mutant strains, and introduced sadC The plasmid increased c-di-GMP levels. RT-qPCR detection showed that only Δ retS Δ tsrF Δ pelD ( sadC The strain can be restored. tssH4 and tssB4 Gene expression, Δ retS Δ tsrF Δ fleQ ( sadC The strain cannot be restored. Figure 7 (As shown in Part B). Bacterial competition experiments show that, at Δ retS Δ tsrF Knocking out FleQ (but not PelD) in ΔT6SS-1,2,3 eliminates the c-di-GMP-induced enhancement of antibacterial activity. Figure 7 (as shown in Part C); Biofilm detection and pelA / pelB Gene expression analysis also showed that sadC Knock out FleQ during overexpression, Δ retS Δ tsrF Total amount of biofilm and pelA / pelB The expression could not be recovered. Figure 7 (As described in Part D).

[0150] The above results confirm that FleQ is a key c-di-GMP effector in the TsrF regulatory pathway, which can mediate the regulation of H4-T6SS expression and antibacterial activity, as well as biofilm formation by c-di-GMP signaling. This provides core experimental evidence for further clarifying the molecular mechanism of the TsrF-c-di-GMP regulatory axis and developing targeted drugs.

[0151] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0152] Unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms "a," "an," "the," and similar words used in this application do not indicate quantity limitation and may indicate singular or plural. The terms "comprising," "including," "having," and any variations thereof used in this application are intended to cover non-exclusive inclusion. The terms "connected," "linked," "coupled," and similar words used in this application are not limited to physical or mechanical connections but include electrical connections, whether direct or indirect. The term "multiple" used in this application refers to two or more, and "and / or" describes the relationship between related objects, indicating that three relationships may exist. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. The terms "first," "second," "third," etc. used in this application are merely to distinguish similar objects and do not represent a specific ordering of objects.

[0153] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications or improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. An application for knocking out a Pseudomonas aeruginosa tsrF gene, characterized in that, The use is any one of F11) - F15): F11) use in reducing production of P. aeruginosa biofilm; F12) use in reducing expression of P. aeruginosa T6SS; F13) use in reducing production of cyclic diguanylate in P. aeruginosa; F14) use in reducing production of FleQ receptor of cyclic diguanylate in P. aeruginosa; F15) use in reducing production of PelD receptor of cyclic diguanylate in P. aeruginosa; The tsrF The gene is the nucleotide sequence shown in SEQ ID No. 1, and the P. aeruginosa is P. aeruginosa in which the retS gene has been knocked out.

2. A method of reducing biofilm production and / or T6SS expression in a P. aeruginosa engineered bacterium, characterized in that, The method comprises knocking out a gene in a Pseudomonas aeruginosa genome by using a genetic engineering method, wherein the gene is a gene shown in a nucleotide sequence of SEQ ID No. 1, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa in which a retS gene has been knocked out. tsrF The method comprises knocking out a gene in a Pseudomonas aeruginosa genome by using a genetic engineering method, wherein the gene is a gene shown in a nucleotide sequence of SEQ ID No. 1, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa in which a retS gene has been knocked out. tsrF The method comprises knocking out a gene in a Pseudomonas aeruginosa genome by using a genetic engineering method, wherein the gene is

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