Construction method and application of pseudomonas aeruginosa engineering bacteria duec

Abstract

The present application relates to a kind of pseudomonas aeruginosa engineering bacteria DUEC construction method and application.The DUEC engineering strain construction method of the present application is: response to kinase coding gene retS and sheath protein coding gene tssB2, tssB3 Knockout, H1-T6SS secretory effector protein Tse1-Tse4, Tse6-Tse8 catalytic site mutation, Tse5 Coding gene knockout.DUEC engineering strain can not only constitutively express and assemble H1-T6SS, but also can respond to adjacent sister cell source T6SS attack, assemble H1-T6SS at the position of being attacked.The present application constructs a series of effector protein combination inactivation mutant by gene knockout or catalytic site mutation method.Microscope observation and statistical analysis results show that no single effector protein is necessary for T6SS attack and assembly.By bacterial competition experiment, it is verified that all effector protein inactivation mutant 8eff c Lose T6SS + Vibrio cholerae and T6SS + Aeromonas dhaka's bactericidal activity, but 8eff c Can respond to T6SS physical attack, Tse6 N -Cre is specifically delivered to T6SS + In Vibrio cholerae.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing and applying an engineered strain of Pseudomonas aeruginosa, DUEC, specifically an engineered strain that can serve as a physical contact sensor and a protein delivery platform. Background Technology

[0002] Pseudomonas aeruginosa is a very important Gram-negative pathogen and a member of the highly clinically significant ESCAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacters pp.). It can respond to membrane interference signals caused by heterologous or sister cell-derived T6SS attack, RP4 conjugation transfer, or extracellular DNA, and activate H1-T6SS assembly through the threonine phosphorylation pathway (TPP) consisting of TagQRST-PpkA-Fha1-PppA. When *Pseudomonas aeruginosa* cells are challenged by T6SS from sister cells, they can assemble H1-T6SS at the site of attack, delivering effector proteins to the sister cells. However, due to the presence of homologous immune proteins, this does not lead to sister cell death. This response between *P. aeruginosa* sister cells is called T6SS dueling. When *P. aeruginosa* is co-cultured with *Vibrio cholerae*, *P. aeruginosa* can distinguish T6SS in a mixture of the three strains by responding to membrane damage induced by the *Vibrio cholerae* effector protein TseL, rather than by physical puncture of T6SS. + and T6SS - Vibrio cholerae, and activates Pseudomonas aeruginosa H1-T6SS assembly, delivering antibacterial effector proteins to T6SS. + In Vibrio cholerae, T6SS is caused. + Vibrio cholerae cells undergo lysis and death, a phenomenon that occurs in Pseudomonas aeruginosa and T6SS. + The retaliatory response between exogenous bacteria is known as the tit-for-tat response. This unique response mechanism found in Pseudomonas aeruginosa can be used to selectively regulate microorganisms in complex microbial communities. However, the mechanisms of T6SS duels and interspecies interactions between effector proteins in Pseudomonas aeruginosa sister cells are not fully understood, limiting their application as signal sensing modules.

[0003] T6SS is a molecular weapon widely present in Gram-negative bacteria, found in approximately 25% of sequenced Gram-negative bacteria. T6SS secretes toxic effector proteins that kill competitors in direct contact with it in complex environments. Structurally, T6SS is similar to a bacteriophage tail, consisting of a contractile TssB / C outer sheath, a closed Hcp inner tube, a transmembrane complex, and a peg complex. The apex of the Hcp inner tube has a spike complex composed of a VgrG trimer and a conical PAAR protein, facilitating T6SS penetration of the cell membrane. Contraction of the outer sheath releases a large amount of energy, driving the Hcp inner tube, spike complex, and associated effector proteins into neighboring cells (Gram-negative bacteria, Gram-positive bacteria, and fungi). The length of the outer sheath likely determines the effective range of T6SS, as the delivery of effector proteins to target cells is largely contact-dependent. In normal cells, the T6SS sheath / tube extends from the transmembrane complex and terminates on the opposite side of the cell, forming a straight rod-like structure. However, in cells with cell wall defects or in knockout mutants of the TagA stopper protein gene, long, curved T6SS structures can form. The effector proteins secreted by T6SS can kill recipient cells, but puncture of the T6SS tubular structure cannot kill recipient cells.

[0004] Transforming T6SS into a universal protein delivery tool typically does not require its toxic function. However, effector proteins not only determine T6SS function but also participate in its assembly; in some strains, knockout of multiple effector proteins can abolish T6SS assembly. Some effector proteins, like VgrG and PAAR, can also serve as carriers for fusion with cargo proteins. Therefore, *Vibrio cholerae* and *Aeromonas dhakensis* employ an efficient strategy of maintaining T6SS secretory function while abolishing its toxicity through mutations at the catalytic sites of effector proteins, rather than knockout of the gene encoding the effector protein. This strategy may also work in more complex systems, such as *Pseudomonas aeruginosa* T6SS, but this has not yet been verified.

[0005] The P. aeruginosa PAO1 genome encodes three independent T6SS gene clusters (H1-, H2-, and H3-T6SS), each of which secretes specific effector proteins. The eight effector proteins secreted by H1-T6SS can be divided into two categories based on their sites of action: effector proteins that function in the periplasmic space (Tse1, Tse3-Tse5) and effector proteins that function in the cytoplasm (Tse2, Tse6-Tse8). Currently, although the functions of some effector proteins have been well validated, whether effector proteins in P. aeruginosa participate in T6SS assembly and which effector proteins can serve as more effective carriers remain unverified.

[0006] Among the many synthetic biology modules, there is a lack of tools capable of specifically responding to physical contact signals. The T6SS confrontation / tease response may fill this gap and be used to regulate specific microorganisms in mixed communities. However, due to the complexity of Pseudomonas aeruginosa components, specific protein interactions, and unexplained mechanisms, developing Pseudomonas aeruginosa into a signal sensing and regulatory module is very challenging. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned technical problems by providing a novel engineered Pseudomonas aeruginosa strain, DUEC, and its application. The main objective of this invention is to construct a strain capable of responding to external signals and specifically distinguishing T6SS within a mixed community. + and T6SS - The strain specifically delivers cargo proteins fused with appropriate secretion tags to T6SS. + Engineered strains within the strain.

[0008] The objective of this invention is mainly achieved through the following technical solutions:

[0009] This invention provides a method for constructing the engineered strain of Pseudomonas aeruginosa, DUEC, the method comprising the following steps:

[0010] In wild-type Pseudomonas aeruginosa PAO1, the sensing kinase encoding gene retS and the sphingomyelin encoding genes tssB2 and tssB3 were first knocked out. Then, catalytic site mutations were performed on the H1-T6SS secretory effector proteins Tse1-Tse4 and Tse6-Tse8, and the Tse5 encoding gene was knocked out; thus, the engineered Pseudomonas aeruginosa strain DUEC was obtained.

[0011] The specific mutation is: Tse1 C30A C30 mutation to alanine; Tse2 VK-AA V109 and K110 are mutated to alanine; Tse3 E250Q E250 mutation to glutamine; Tse4 G176VG176 mutation to leucine; Tse6 D396A D196 is mutated to alanine; Tse7 HH-AA H229 and H230 are mutated to alanine; Tse8 S186A S186 is mutated to alanine.

[0012] Under a microscope, the DUEC engineered strain can constitutively express and assemble H1-T6SS, and it can also assemble H1-T6SS at the site of attack in response to T6SS challenge from adjacent sister cells. While the DUEC engineered strain can assemble T6SS, it has lost the toxicity of all H1-T6SS effector proteins and cannot kill T6SS like the wild-type *Pseudomonas aeruginosa*. + Heterogeneous strains. DUEC can function as a standalone signal sensing module, responding to exogenous T6SS puncture by delivering Cre recombinase fused with a suitable secretion tag to T6SS. + Instead of T6SS - Cre fusion proteins are found in the cytoplasm of Vibrio cholerae and are specifically delivered to T6SS within complex microbial communities. + In Vibrio cholerae, it is transmitted to T6SS + The Cre fusion protein in Vibrio cholerae still possesses recombinase activity and can mediate site-specific recombination.

[0013] A *Pseudomonas aeruginosa* strain DUEC constructed using the aforementioned method. Under a microscope, the *P. aeruginosa* DUEC strain not only constitutively expresses H1-T6SS, but also responds to sister cell T6SS attack, assembling its own H1-T6SS phenotype. All H1-T6SS effector proteins in the *P. aeruginosa* DUEC cells are inactivated, and the strain cannot kill T6SS cells. + Phenotype of heterologous cells.

[0014] The engineered Pseudomonas aeruginosa strain DUEC is capable of delivering cargo proteins fused with secretion tags to the T6SS+ strain. The secretion tag includes Tse6. N One of VgrG1a.

[0015] Pseudomonas aeruginosa VgrG1a / 1b / 1c proteins and VgrG-related effector proteins Tse5-Tse7 containing PAAR domains were used as secretion tags, fused with Cre recombinase, to screen for suitable secretion tags for cargo protein delivery.

[0016] Tse6 N (Containing only amino acid residues 1-281 of Tse6) and VgrG1a can serve as secretion tags to effectively transfer Cre recombinase to T6SS via DUEC engineered strains.+ In the cytoplasm of Vibrio cholerae.

[0017] The *Pseudomonas aeruginosa* DUEC cells are able to respond to T6SS physical puncture and specifically deliver proteins to T6SS within a complex microbial community. + In the cytoplasm of the receptor cell.

[0018] The engineered Pseudomonas aeruginosa strain DUEC can be used to deliver various cargo proteins with different toxicities or functions, specifically regulate downstream output, or regulate the composition of microbial communities.

[0019] Passed to T6SS + Cre reporter factors in recipient cells can mediate site-specific recombination.

[0020] The cargo protein Cre recombinase and recombination-mediated output can be replaced by other enzymes or output modules (such as β-galactosidase and downstream gene regulation), giving the DUEC system a wider range of application potential.

[0021] (1) First, we constructed plasmids for gene knockout or catalytic site mutation and constructed corresponding mutant strains through conjugation transfer. The construction method was as follows: amplify the upstream and downstream homologous arms of the corresponding site, with a size of 800-1,000 bp. After sequencing verification of the constructed plasmids, they were transformed into Escherichia coli SM10 or WM6026 strains as donor strains. The donor and recipient strains cultured overnight were suspended in 100 μl LB broth, and 50 μl of each were mixed and spotted on LB plates. After incubation at 37°C for 3 h, they were suspended in 500 μl LB broth and activated for 1 h. 250 μl of each strain was then spread on LB solid plates containing triclosan (25 μg / ml) and gentamicin (20 μg / ml). After overnight incubation at 37°C, the conjugation transferons were picked and cultured in 500 μl antibiotic-free LB broth for 4 h. They were then spread on LBNS plates containing 6% sucrose and incubated at 22°C for 1.5 days before PCR verification. Strains that were correctly verified by PCR and lost plasmid resistance were stored in a -80°C freezer.

[0022] A series of mutant strains with combined inactivation of effector proteins were constructed using methods such as gene knockout or catalytic site mutation. Microscopic observation and statistical analysis showed that no single effector protein was required for T6SS confrontation and assembly. Through combined mutation, a mutant strain 8eff with inactivation of all effector proteins was constructed. c .

[0023] (2) The bacterial competition experiment verified the 8eff c The mutant strain lost its resistance to T6SS + Vibrio cholerae and T6SS + bactericidal activity of Aeromonas dacca.

[0024] (3) The 8eff was verified through Cre transfer experiments. c The question was whether the mutant strain could be developed into a non-toxic protein delivery platform. First, Cre recombinase was chosen as the cargo protein because it can mediate site-specific DNA recombination in the cytoplasm, and Cre-mediated recombination can be detected using the recently constructed reporter plasmid pFIGR. The pFIGR plasmid contains loxP sites flanking the ampicillin resistance gene, interrupting the transcription of the gentamicin resistance gene. Before Cre recombination, pFIGR conferred ampicillin resistance but gentamicin sensitivity upon the strain; after Cre-mediated recombination, the transcriptional blockade of the gentamicin resistance gene was lifted, and the gentamicin gene was expressed normally, conferring gentamicin resistance upon the strain. The pFIGR plasmid was then transformed into T6SS. + and T6SS - In Vibrio cholerae, Cre-mediated recombination was detected. Secondly, suitable secretion tags were screened. Three VgrG proteins (VgrG1a / b / c) and three VgrG-related effector proteins (Tse5 / 6 / 7) from Pseudomonas aeruginosa H1-T6SS were selected as secretion tags and fused with Cre recombinase. The constructed fusion plasmids were then transformed into Pseudomonas aeruginosa 8eff. c and T6SS - In the (ΔretS, ΔtssB1, ΔtssB2, ΔtssB3) mutant strains. Finally, through Cre transfer experiments, it was found that 8eff c Able to use Tse6 N -Cre or VgrG1a-Cre fusion protein delivered to T6SS + Instead of T6SS - In Vibrio cholerae, T6SS was conferred. + Gentamicin resistance in Vibrio cholerae, based on 8eff c Based on its characteristics, it was named DUEC.

[0025] (4) The bacterial competition experiment and Cre transfer experiment were used to verify whether DUEC could respond to T6SS physical puncture. First, the bacterial competition experiment was used to verify that the 4eff mutant strain with complete inactivation of Vibrio cholerae V52 effector protein was effective. c (ΔrtxA, ΔhlyA, ΔhapA, VipA-mCherry, TseL D425A TseH H64A VgrG3 D842A VasX ΔC16 The results showed that VC-4 had a killing effect on Escherichia coli. cNeither ΔtssM nor DUEC could kill E. coli MG1655. Subsequently, Cre transfer experiments showed that DUEC could respond to VC-4eff. c The source of T6SS physical puncture specifically removes Tse6 N -Cre fusion protein delivers VC-4eff c In mutant strains.

[0026] (5) Cre transfer experiments verified that DUEC can distinguish T6SS in mixed microbial communities. + and T6SS - Vibrio cholerae. Containing Tse6 N -DUEC and T6SS of Cre fusion protein - Pseudomonas aeruginosa strains, respectively, were compared with T6SS containing the pFIGR plasmid. + and T6SS - A mixture of Vibrio cholerae was tested. Results showed that only the DUEC strain was able to transfer active Cre to Vibrio cholerae, inducing gentamicin resistance. Simultaneously, from hundreds of gentamicin-resistant Vibrio cholerae, 10 single clones were randomly selected for bacterial competition experiments. The results revealed that all gentamicin-resistant Vibrio cholerae were T6SS. + Instead of T6SS - Vibrio cholerae. These results demonstrate that DUEC can specifically distinguish T6SS in mixed microbial communities. + Cells deliver active Cre recombinase to T6SS + In recipient cells, Cre-mediated recombination confers gentamicin resistance.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention constructs a strain capable of normally assembling T6SS and unable to kill T6SS through methods such as gene knockout or catalytic site mutation. + Cells, but capable of functioning as independent signal sensing modules, respond to T6SS physical contact by specifically delivering Cre fusion proteins to T6SS. + Instead of T6SS - DUEC engineered strains in the cytoplasm.

[0029] DUEC, as a unique protein delivery platform, can be used to deliver different cargo proteins. These cargo proteins can specifically regulate downstream pathways in recipient cells, which has very important application value. Attached Figure Description

[0030] 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.

[0031] Figure 1 Figures A and B show the microscopic observation results of the H1-T6SS effector protein Tse-related mutant strain; Figures A and B are respectively from the parental strain H1-T6SS. + Real-time imaging results of TssB1-sfGFP signals in (ΔretS, ΔtssB2, ΔtssB3, TssB1-sfGFP) effector protein knockout mutants (A) or effector protein combination inactivation mutants (B). Images were taken at 10-second intervals for 5 minutes. C, Real-time color scale for sfGFP signal labeling. The large image shows a representative image of the corresponding strain within a 30μm×30μm region, and the small image is a 2x magnified image within a 5μm×5μm region, with the magnified region marked with a small white box. The scale bar in the large image is 5μm, and the scale bar in the small image is 1μm, and this is applied to all small images. The genotype of the strain is labeled below the image.

[0032] Figure 2 This is a schematic diagram showing the statistical analysis results of T6SS assembly and T6SS showdown of the mutant strain obtained in Example 1 according to the method in Example 2; the parent strain H1-T6SS is shown in the 30μm×30μm region. + The proportion of active cells capable of forming T6SS sheaths in mutants with single knockout of effector proteins or mutants with combined inactivation of effector proteins (A) and the proportion of active cells capable of forming T6SS sheaths in pairs (B); eff refers to the number of effector protein mutants that remain active after combined inactivation of effector proteins, 8eff c This refers to mutant strains with simultaneous inactivation of 8 effector proteins. The error bar refers to the mean ± standard deviation of at least three distinct biological replicates and 10 distinct regions; N represents the total number of cells counted for each strain within a 30μm × 30μm region. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

[0033] Figure 3 The graph shows the results obtained by following the method in Example 3 for all mutant strains obtained in Example 1; parental strain H1-T6SS +Results of competition experiments between *Pseudomonas aeruginosa* mutants with single knockout of effector proteins or mutants with combined inactivation of effector proteins and *Vibrio cholerae* or *Aeromonas daca*; Figure A shows the survival of *Vibrio cholerae* in the competition experiment between *P. aeruginosa* mutants and *Vibrio cholerae*; Figure B shows the survival of *P. aeruginosa* in the competition experiment between *P. aeruginosa* mutants and *Vibrio cholerae*; Figure C shows the survival of *Aeromonas daca* in the competition experiment between *P. aeruginosa* mutants and *Aeromonas daca*; Figure D shows the survival of *P. aeruginosa* in the competition experiment between *P. aeruginosa* mutants and *Aeromonas daca*; killer:prey = 5:1, error bars refer to the mean ± standard deviation of at least three distinct biological replicates; one-way ANOVA - Dunnett's multiple comparison test was used to compare the differences in competitive ability between parental strains and mutants; *P<0.05, ****P<0.0001; ns, not significant.

[0034] Figure 4 Figure 4 shows the results of the *Pseudomonas aeruginosa* DUEC strain being able to deliver active Cre to recipient cells via the VgrG1a secretory tag. Figure A shows the VgrG secretory tag fusion with Cre, with Cre recombinases inserted before the stop codons of VgrG1a, VgrG1b, and VgrG1c, respectively. Figures B-E show the Cre recombination efficiency (B) of *Pseudomonas aeruginosa* DUEC after being challenged by wild-type *Vibrio cholerae* (WT), the survival rate of *Vibrio cholerae* undergoing Cre recombination (C), and the presence of the pFIGR plasmid. Vibrio cholerae recipient cell survival (D) and Pseudomonas aeruginosa donor cell survival (E); DL is the detection limit, and the error bar refers to the mean ± standard deviation of at least three distinct biological replicates. One-way ANOVA - Dunnett's multiple comparison test was used to compare the differences in recombination efficiency (B), recombinant Vibrio cholerae survival (C), recipient cell survival (D), or donor cell survival (E) between other strains and Pseudomonas aeruginosa containing only Cre; *P<0.05; ns, not significant.

[0035] Figure 5 Example 4 showed that the Pseudomonas aeruginosa DUEC strain could transmit Tse6 via H1-T6SS. N - Schematic diagram of Cre delivery to recipient cells; Figure A is a schematic diagram of VgrG-dependent effector protein secretion tag fusion with Cre, Tse5 N (Contains only amino acid residues 1-1145 of Tse5), Tse6 N (Contains only amino acid residues 1-281 of Tse6) and Tse7 N (Containing only amino acid residues 1-225 of Tse7) followed by the insertion of Cre recombinase; Figures B-E show Pseudomonas aeruginosa DUEC or T6SS respectively.- (Δ) Cre recombination efficiency (B) after challenge with wild-type (WT) or ΔtssM strains of Vibrio cholerae, survival of Vibrio cholerae undergoing Cre recombination (C), survival of Vibrio cholerae recipient cells with pFIGR plasmid (D), and survival of Pseudomonas aeruginosa donor cells (E); DL is the detection limit, and the error bar refers to the mean ± standard deviation of at least three distinct biological replicates. One-way ANOVA-Dunnett's multiple comparison test was used to compare the differences in recombination efficiency (B), strain survival (C), recipient cell survival (D), or donor cell survival (E) between other strains and Pseudomonas aeruginosa containing only Cre; **P<0.01, ***P<0.001; ns, not significant.

[0036] Figure 6 As demonstrated in Example 4, Pseudomonas aeruginosa DUEC was found to be able to sense T6SS upon physical contact. Tse6... N -Cre fusion protein delivered to Vibrio cholerae with completely inactivated effector proteins 4eff c Schematic diagram of results in mutant strains; Figures A and B represent Vibrio cholerae wild-type (WT), ΔtssM, and 4eff, respectively. c In the competition experiment between the strain and *E. coli* MG1655, the survival of *E. coli* MG1655 (A) and *Vibrio cholerae* V52 (B) are shown. Figures C-F show the Cre recombination efficiency of *Pseudomonas aeruginosa* DUEC after being challenged by different *Vibrio cholerae* strains (C), the survival of *Vibrio cholerae* undergoing Cre recombination (D), the survival of *Vibrio cholerae* recipient cells with pFIGR plasmid (E), and the survival of *P. aeruginosa* donor cells (F), respectively. DL is the detection limit, and the error bar refers to the mean ± standard deviation of at least three distinct biological replicates. (One-way analysis...) Analysis of difference - Dunnett's multiple comparison test was used to compare the differences in Escherichia coli survival (A), Vibrio cholerae survival (B), or the differences in Cre recombination efficiency (C), Cre recombination survival (D), and survival of Vibrio cholerae with pFIGR plasmid (E) and Pseudomonas aeruginosa donor cell survival (F) in competition experiments between other Vibrio cholerae and wild-type strains; **P<0.01, ****P<0.0001; ns, not significant.

[0037] Figure 7 Example 4 showed that *Pseudomonas aeruginosa* DUEC could respond to T6SS in a mixed community. + Cells, and specifically deliver Cre to T6SS + Schematic diagram of results in recipient cells; Figures A-D represent Pseudomonas aeruginosa DUEC or T6SS, respectively. -(Δ) Cre recombination efficiency after co-culturing with wild-type Vibrio cholerae and ΔtssM (A), survival of Vibrio cholerae undergoing Cre recombination (B), survival of Vibrio cholerae recipient cells with pFIGR plasmid (C), and survival of Pseudomonas aeruginosa donor cells (D). Figure E (left) shows Cre recombination of Vibrio cholerae obtained by co-culturing Pseudomonas aeruginosa DUEC with wild-type and ΔtssM. Figure E (middle) shows the competition results of different Vibrio cholerae strains mixed with Escherichia coli MG1655 containing the luciferase reporter gene. Figure E (right) shows the colony morphology of different Vibrio cholerae strains mixed with Escherichia coli MG1655 containing the luciferase reporter gene. Ec represents Escherichia coli MG1655 containing pBAD8kan-lux, WT represents wild-type Vibrio cholerae, and ΔtssM represents inactivated Vibrio cholerae T6SS strains and gentamicin-resistant Vibrio cholerae clones (1-10). DL represents the detection limit, the error bar refers to the mean ± standard deviation of at least three distinct biological replicates, and the two-tailed Student's t-test is used to compare Pseudomonas aeruginosa DUEC or T6SS. - Differences in Cre recombination after mixing with two different types of Vibrio cholerae (A), differences in survival of Vibrio cholerae with Cre recombination (B), differences in survival of Vibrio cholerae with pFIGR plasmid (C), and differences in survival of Pseudomonas aeruginosa donor cells (D); **P<0.01, ****P<0.0001; ns, not significant.

[0038] Figure 8 A diagram illustrating the precise cargo transfer pattern of DUEC in a mixed community; Left image: Wild-type Pseudomonas aeruginosa (PA) sensing adjacent T6SS. + Following cell attack, H1-T6SS assembles, delivering toxic effector proteins to the initially attacking cells, leading to lysis of the initial attacking cells (gray lysed cells); Middle image: *Pseudomonas aeruginosa* DUEC, capable of assembling active T6SS but with inactivated effector proteins, does not kill adjacent T6SS cells. + Cells, but can sense T6SS physical contact (Vibrio cholerae 4eff) in mixed communities. c ), precisely for T6SS +Cells respond; Right figure: The tit-for-tat signal transduction module consists of a signal-sensing TagQRST, an autophosphorylated PpkA kinase, and Fha1, which can be phosphorylated by PpkA. PppA phosphatase antagonizes the effects of PpkA, and phosphorylated Fha1 leads to the assembly of T6SS in Pseudomonas aeruginosa DUEC cells, subsequently delivering Cre to recipient cells. Cre fused with the secretory tag remains active and can mediate specific recombination in recipient cells. Therefore, the post-translational tit-for-tat sensing module now possesses a powerful gene switch function, which can be widely used to regulate downstream outputs. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0040] The specific culture medium formulation and culture conditions involved in the embodiments of this invention are as follows:

[0041] All strains used in this invention were cultured in LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride) at 37°C. LBNS medium (10 g / L tryptone, 5 g / L yeast extract) was used to screen strains that had lost plasmid resistance; sucrose was added to a final concentration of 6% before use, and the medium was incubated at 22°C. The antibiotic concentrations used were as follows: 100 μg / mL streptomycin, 50 μg / mL kanamycin, 100 μg / mL carbenicillin, 20 μg / mL gentamicin, and 25 μg / mL triclosan. The 0.5×PBS used in the experiment was obtained by diluting 10×PBS (NaCl: 1.37M, KCl: 26.8mM, NaH2PO4: 81.0mM, KH2PO4: 17.6mM) 20 times.

[0042] The plasmids used in this invention are shown in Table 1:

[0043] Table 1. Plasmids and their characteristics

[0044]

[0045] The primers used in this invention are shown in Table 2:

[0046] Table 2 Primers and Sequences

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] The strains used in this invention are shown in Table 3:

[0053] Table 3. Strains and descriptions

[0054]

[0055]

[0056] Example 1: Construction of Pseudomonas aeruginosa mutant strain

[0057] Obtaining the background strain:

[0058] In wild-type Pseudomonas aeruginosa PAO1, the gene encoding sense kinase retS and the genes encoding sphingomyelin tssB2 and tssB3 were first knocked out.

[0059] Construction of Tse effector protein knockout or point mutation-related mutants: The upstream homologous arm was amplified using the KO1 and KO2 primers in Table 2, and the downstream homologous arm was amplified using the KO3 and KO4 primers in Table 2. The upstream and downstream homologous arms were overlapped using KO1 and KO4 primers in Table 2. The overlapped homologous arm fragments were cloned into the suicide plasmid pEXG2.0 using the Gibson assembly method. The correctness of the plasmid was verified by PCR and Sanger sequencing. The verified plasmids were transformed into donor cells, *E. coli* WM6026 or SM10, as donor strains. The overnight cultured donor and recipient strains were resuspended in 100 μl of LB broth, mixed 1:1, and co-cultured at 37°C for 3 h. The culture was then scraped into 500 μl of LB broth, activated at 37°C and 950 rpm for 1 h, and then plated onto LB agar plates containing selective antibiotics. After overnight culture at 37°C, conjugation transferons were picked and cultured in 500 μl of antibiotic-free LB broth for 4 h. These were then plated onto LBNS plates containing 6% sucrose (tryptone: 10 g / L, yeast extract: 5 g / L) and cultured at 22°C for 1.5 days. PCR verification was performed using the KO5 and KO6 primers listed in Table 2. For mutant strains with catalytic site mutations, the corresponding fragments were amplified using KO5 and KO6 primers, followed by Sanger sequencing to verify the correctness of the mutant strains.

[0060] The Tse-encoding gene was knocked out individually through two homologous recombinations. The plasmids used are shown in Table 1. Specifically, tse1 knockout used the suicide plasmid pEXG2.0 tse1, tse2 knockout used pEXG2.0 tse2, tse3 knockout used pEXG2.0 tse3, tse4 knockout used the suicide plasmid pEXG2.0 tse4 constructed from a mutant strain, tse5 knockout used pEXG2.0 tse5, tse6 knockout used pEXG2.0 tse6, tse7 knockout used pEXG2.0 tse7, and tse8 knockout used pEXG2.0 tse8. Using this gene knockout method, Tse effector protein single-knockout strains were constructed, named Δtse1-Δtse8, respectively. The obtained mutant strains were observed under a microscope according to the method described in Example 2, and the results are as follows: Figure 1 As shown.

[0061] The plasmids used for inactivating the catalytic sites of effector proteins are shown in Table 1. The suicide plasmid pEXG2.0 was used for mutations at the Tse1 catalytic site. C30A The Tse2 catalytic site mutation was performed using the suicide plasmid pEXG2.0 Tse2. VK-AA The Tse3 catalytic site mutation was performed using the suicide plasmid pEXG2.0 Tse3. E250Q The Tse4 catalytic site mutation was performed using the suicide plasmid pEXG2.0Tse4. G176V The Tse6 catalytic site mutation was performed using the suicide plasmid pEXG2.0 Tse6. D396A The Tse7 catalytic site mutation was performed using the suicide plasmid pEXG2.0 Tse7. HH-AA The Tse8 catalytic site mutation was performed using the suicide plasmid pEXG2.0Tse8. S186A By mutating the catalytic site, a combined mutant strain of the Tse effector protein was constructed. All primers used are shown in Table 3.

[0062] The effector proteins secreted by H1-T6SS were divided into three groups: Tse1 and Tse3 act on peptidoglycan, Tse4 and Tse5 may act on the cell membrane, and Tse2, Tse6, Tse7 and Tse8 act in the cytoplasm.

[0063] Catalytic site mutations were performed on the H1-T6SS secretory effector proteins Tse1-Tse4 and Tse6-Tse8 (Tse1). C30A C30 mutation to alanine; Tse2 VK-AA V109 and K110 are mutated to alanine; Tse3 E250Q E250 mutation to glutamine; Tse4G176V G176 mutant leucine; Tse6 D396A D396 is mutated to alanine; Tse7 HH-AA H229 and H230 are mutated to alanine; Tse8 S186A (S186 was mutated to alanine), and the Tse5 encoding gene was knocked out.

[0064] Three groups of mutant strains with individual inactivation of effector proteins were constructed using gene knockout and catalytic site mutation:

[0065] (1) Strain eff245678: Tse1 and Tse3 catalytic site inactivation mutant; in addition, there is intermediate construct strain eff2345678: Tse1 catalytic site inactivation mutant.

[0066] (2) Strain eff123678: Tse4 catalytic site inactivation and Tse5 knockout mutant.

[0067] (3) Strain eff13458: a mutant strain with inactivated Tse2, Tse6 and Tse7 catalytic sites. In addition, there are intermediate construct strains eff1234568 (a mutant strain with inactivated Tse7 catalytic site) and eff134568 (a mutant strain with inactivated Tse2 and Tse7 catalytic sites).

[0068] Then, the three groups of effector proteins were combined in pairs to construct effector protein combination mutants:

[0069] (4) Strain eff2678: Tse1, Tse3, Tse4 catalytic site mutations and Tse5 knockout mutant. In addition, there is intermediate construct strain eff24678 (Tse1, Tse3 catalytic site mutations and Tse5 knockout mutant).

[0070] (5) Strain eff458: a mutant strain with mutations at the catalytic sites of Tse1, Tse2, Tse3, Tse6 and Tse7. In addition, there is an intermediate construct strain eff1458 (a mutant strain with mutations at the catalytic sites of Tse2, Tse3, Tse6 and Tse7).

[0071] (6) Strain eff138: Tse2, Tse4, Tse6, and Tse7 catalytic site mutations and Tse5 knockout mutant; in addition, there is an intermediate constructed mutant strain eff1348 (Tse2, Tse6, and Tse7 catalytic site mutations and Tse5 knockout mutant). Subsequently, based on eff138, we constructed three groups of mutant strains in which the effector proteins simultaneously lost their activity:

[0072] (7) Strain eff8: Tse1, Tse2, Tse3, Tse4, Tse6 and Tse7 catalytic site mutations and Tse5 knockout mutant. In addition, there is an intermediate construct strain eff38 (Tse1, Tse2, Tse4, Tse6 and Tse7 catalytic site mutations and Tse5 knockout mutant).

[0073] Finally, since Tse8 was recently reported, we investigated the possible active sites of Tse8 (Tse8) based on strain eff8. S186A The catalytic site was inactivated by mutating S186 to alanine, ultimately resulting in a mutant strain 8eff with simultaneous inactivation of 8 effector proteins. c Information on all constructed strains and strains for other uses is shown in Table 3.

[0074] Example 2: Microscopic observation and statistical analysis of Tse effector protein-related mutants

[0075] The effector protein single knockout mutant, the three groups of effector protein individually inactivated mutants, the three groups of effector protein pairwise inactivated mutants, the three groups of effector protein simultaneously inactivated mutants, and the 8eff mutants constructed in Example 1 c Microscopic observation and statistical analysis were performed. *Pseudomonas aeruginosa* strains were streaked onto plates containing suitable antibiotics and incubated overnight at 37°C. The next day, single colonies were picked and transferred to 500 μl of LB agar containing suitable antibiotics and incubated overnight at 37°C and 950 rpm. On the third day, the overnight cultured strains were transferred at a 1:50 ratio to 3 ml of LB agar containing suitable antibiotics and incubated for 3-4 hours. OD was then measured. 600 =0.8-1.0. Take an appropriate amount of mutant culture, centrifuge at 10,000×g for 30-60s, remove the supernatant, and add an appropriate volume of 0.5×PBS to adjust the bacterial OD. 600 =10.0. Prepare a colloid containing 0.5×PBS and 1% agarose. Place two clean coverslips on a glass slide. Add 100 μl of the melted colloid to the gap between the two coverslips. Cover with a clean coverslip and let it stand for about 1 minute until the colloid solidifies. Trim off the edges and cut the remaining colloid into 0.5 cm × 0.5 cm pieces. Add 0.5–1.0 μl of OD... 600 =10.0 bacterial cells were spotted on the colloid, covered with a coverslip, and observed using a Nikon Ti-E inverted microscope. A 100× oil immersion microscope with a Perfect Focus System (PFS) was used. An ET-GFP (Chroma 49002) filter was used to observe the GFP fluorescence signal, and an ET-mCherry (Chroma 49008) filter was used to observe the mCherry fluorescence signal. The edge areas were selected for observation.

[0076] All microscopic images were analyzed using Fiji software. Images taken at 10-second intervals and over a 5-minute timeframe were first standardized to correct for signal quenching. Cell counts were performed using phase contrast images. Cell outlines were clarified by adjusting the threshold, and the "analyze particles" option was used for counting, excluding edge cells. Cell counts were manually verified. To ensure accuracy and reproducibility, at least 10 different regions from three replicates were selected for analysis, including the proportion of active cells capable of forming T6SS sheaths and the proportion of paired active cells capable of forming T6SS sheaths. The Fiji plugin "Temporal-Color code" was used to assess active T6SS cells over 5 minutes, and cells with active sfGFP signals were manually counted. All experimental results were performed in at least three independent biological replicates.

[0077] The mutant strains obtained in Example 1 underwent statistical analysis of T6SS assembly and T6SS matchups using the method described in Example 2. The results are as follows: Figure 2 As shown.

[0078] Example 3: Bacterial Competition Experiment

[0079] Killer monoclonal antibodies were cultured overnight at 37°C on LB agar plates containing suitable antibiotics. The next day, an appropriate amount of bacteria was scraped off and the bacterial concentration was standardized to OD. 600 =1.0, activated at 37℃ for 1 hour, Pseudomonas aeruginosa cells were transferred to 3 ml LB at a ratio of 1 / 50, and other cells were transferred to 3 ml LB at a ratio of 1 / 100, and cultured until OD. 600 =0.8-1.0. Alternatively, inoculate a single killer clone into 500 μl of LB broth containing suitable antibiotics, incubate overnight, and then inoculate directly the next day according to the above inoculation ratio, culturing until OD... 600 =0.8-1.0. Centrifuge the obtained killer cells at 10,000×g for 30-60s, remove the supernatant, and resuspend in fresh LB lysate to allow their OD to adjust. 600 =10.0. Overnight cultured Prey cells were suspended in LB broth and uniformly concentrated to OD. 600=1.0 or 2.0. In the competition experiment between Vibrio cholerae and Escherichia coli, the killer:prey ratio was 10:1; in the competition experiment between Pseudomonas aeruginosa and Vibrio cholerae or Aeromonas dacarba, the killer:prey ratio was 5:1. The mixture was spotted onto antibiotic-free LB agar and co-cultured at 37°C for 3 hours. The mixture was then removed from the plate and added to 500 μl of LB agar. After shaking for 3 minutes, a 10-fold serial dilution was performed. 3 μl samples were then spotted onto plates containing appropriate antibiotics to distinguish between killer and prey. All bacterial competition experiments were repeated at least three times. The cells were revived and log... 10 The CFU mean is used to plot the bar chart, with the error bars representing the mean ± standard deviation of three biological parallels. If no revived cells are observed, the sample is counted as 0.5 clones and marked as the detection limit (DL). One-way ANOVA – Dunnett's multiple comparison test or a two-tailed Student's t-test is used to calculate the p-value.

[0080] The results of obtaining all mutant strains in Example 1 according to the method in Example 3 are as follows: Figure 3 As shown.

[0081] Example 4: Cre transfer experiment

[0082] Plasmid construction: First, the Cre recombinase fragment was amplified using Cre-pPSV-F and Cre-pPSV-R from Table 2; the VgrG1a fragment was amplified using VgrG1a-pPSV-RBS-F and VgrG1a-pPSV-R from Table 2; the VgrG1b fragment was amplified using VgrG1b-pPSV-RBS-F and VgrG1b-pPSV-R from Table 2; the Vgr1c fragment was amplified using VgrG1c-pPSV-RBS-F and VgrG1c-pPSV-R from Table 2; and the Tse6 fragment was amplified using Tse6-281-pPSV-RBS-F and Tse6-281-pPSV-R from Table 2. N Fragments; Tse7 was amplified using Tse7-225-pPSV-RBS-F and Tse7-225-pPSV-R from Table 2. N Fragments; Tse5 was amplified using Tse5-1145-pPSV-RBS-F and Tse5-1145-pPSV-R from Table 2. N Fragments; pPSV37 vectors containing the FLAG tag were amplified using pPSV37-RBS-hifi-R and pPSV37-FLAG-hifi-F from Table 2, digested with DpnI for 4 hours, and then recovered for later use. Cre, VgrG1a, VgrG1b, VgrG1c, and Tse5 were assembled using Gibson. NTse6 N and Tse7 N The plasmid was ligated into the pPSV37 vector, and its correctness was verified using PCR and Sanger sequencing. The verified plasmid was then transformed into DUEC or T6SS. - (ΔretS, ΔtssB1, ΔtssB2, ΔtssB3).

[0083] The pPSV37-Cre vector was amplified using Cre-hifi-F and pPSV37-RBS-hifi-R from Table 2, and then recovered after DpnI digestion for 4 hours. The VgrG1a fragment was amplified using VgrG1a-pPSV-RBS-F and VgrG1a-Cre-hifi-R from Table 2; the VgrG1b fragment was amplified using VgrG1b-pPSV-RBS-F and VgrG1b-Cre-hifi-R from Table 2; the Vgr1c fragment was amplified using VgrG1c-pPSV-RBS-F and VgrG1c-Cre-hifi-R from Table 2; and the Tse6 fragment was amplified using Tse6-281-pPSV-RBS-F and Tse6-281-Cre-hifi-R from Table 2. N Fragments; Tse7 was amplified using Tse7-225-pPSV-RBS-F and Tse7-225-Cre-hifi-R from Table 2. N Fragments; Tse5 was amplified using Tse5-1145-pPSV-RBS-F and Tse5-1145-Cre-hifi-R from Table 2. N Fragment. Using Gibson assembly, Cre, VgrG1a, VgrG1b, VgrG1c, and Tse5 are combined. N Tse6 N and Tse7 N The plasmid was ligated into the pPSV37-Cre vector, and its correctness was verified by PCR and Sanger sequencing. Finally, the verified plasmid was transformed into DUEC or T6SS. - (ΔretS, ΔtssB1, ΔtssB2, ΔtssB3).

[0084] Monoclonal strains of *Pseudomonas aeruginosa* containing the Cre fusion plasmid were cultured overnight in LB medium containing the corresponding antibiotics, then transferred at a 1:50 ratio and cultured at 37°C for approximately 3 hours. OD... 600 =0.8-1.0, after centrifuging Pseudomonas aeruginosa cells at 10,000×g for 30-60s, remove the supernatant, add fresh LB, and determine the bacterial concentration to OD. 600 =10.0. After overnight culture of Vibrio cholerae cells containing the pFIGR plasmid, an appropriate amount of bacterial cells was scraped off, and the bacterial cell concentration was determined to OD0.0.600 =1.0. *Pseudomonas aeruginosa* and *Vibrio cholerae* were mixed at a donor-recipient ratio of 10:1. 10 μl of the mixture was spotted onto an LB agar plate containing 1 mM IPTG (isopropyl-β-D-thiogalactoside) and inducing Cre fusion protein expression at 37°C. In the Cre transfer assay of the mixed community of the three bacteria, *Pseudomonas aeruginosa* and *Vibrio cholerae* wild-type WT and ΔtssM were mixed at a ratio of 10:1:1 and co-cultured on an LB agar plate containing 1 mM IPTG at 37°C for 3 h. The mixture was then poured into 500 μl of LB agar, shaken for 3 min, and then diluted 10-fold before spotting. The survival of the recipient and donor bacteria was assessed using LB agar plates containing appropriate antibiotics. Pseudomonas aeruginosa was screened for resistance using 25 μg / ml triclosan and 20 μg / ml gentamicin. Vibrio cholerae containing the pFIGR plasmid was screened using 100 μg / ml streptomycin and 100 μg / ml carbenicillin. Vibrio cholerae undergoing Cre recombination were screened using 100 μg / ml streptomycin and 20 μg / ml gentamicin. From the gentamicin-resistant Vibrio cholerae clones obtained in the Cre transfer experiment of the mixed community of the three strains, 10 single clones were randomly selected and subjected to bacterial competition experiments using Escherichia coli MG1655 containing the luciferase reporter plasmid as a prey to verify whether the obtained clones were wild-type (WT) or ΔtssM Vibrio cholerae. All bacterial competition experiments were performed at least three different biological replicates. Recombination efficiency was calculated as the number of surviving Vibrio cholerae undergoing Cre recombination divided by the number of surviving Vibrio cholerae containing the pFIGR plasmid, and the logarithmic value of revived cells was used. 10 The CFU mean was used to plot a bar chart, with the error bars representing the mean ± standard deviation of three biological parallels. If no revived cells were observed, the sample was counted at 0.5 clones and marked as the detection limit (DL). One-way ANOVA-Dunnett multiple comparison test was used to calculate the p-value. The results obtained in Example 4 are as follows: Figure 7 As shown, the DUEC precision cargo delivery pattern in mixed communities is as follows: Figure 8 As shown.

[0085] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing Pseudomonas aeruginosa engineered bacteria DUEC, characterized in that, The construction method comprises the following steps: In the wild type of Pseudomonas aeruginosa PAO1, the sensing kinase encoding gene retS and the sheath protein encoding gene tssB2 、 tssB3 are first knocked out, then the catalytic site mutations of Tse1-Tse4, Tse6-Tse8, the effectors secreted by H1-T6SS are carried out, and the Tse5 encoding gene is knocked out, and thus the Pseudomonas aeruginosa engineering bacteria DUEC is obtained. The mutation is specifically: Tse1 C30A : C30 mutated to alanine; Tse2 VK-AA : V109 and K110 mutated to alanine; Tse3 E250Q : E250 mutated to glutamine; Tse4 G176V : G176 mutated to valine; Tse6 D396A : D396 mutated to alanine; Tse7 HH-AA : H229 and H230 mutated to alanine; Tse8 S186A : S186 mutated to alanine.

2. The Pseudomonas aeruginosa engineering bacteria DUEC constructed by the construction method of claim 1.

3. The Pseudomonas aeruginosa engineered bacteria DUEC according to claim 2, characterized in that, The P. aeruginosa DUEC cell H1-T6SS effector proteins are all inactivated and cannot kill T6SS + Heterologous strains.

4. The Pseudomonas aeruginosa engineered bacteria DUEC according to claim 2, characterized in that, The engineered Pseudomonas aeruginosa DUEC can deliver cargo proteins fused with secretion tags to T6SS + In Vibrio cholerae; The secretion tag is Tse6 N or VgrG1a; Tse6 N is Tse6 amino acid residues 1-281; The cargo protein is Cre recombinase.

5. The Pseudomonas aeruginosa engineered bacteria DUEC according to claim 4, characterized in that, delivered to the T6SS + The Cre recombinase in V. cholerae is capable of mediating site-specific recombination.

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