A attenuated and synergistic host chassis widely used for production of pseudomonas aeruginosa phage, construction method and application thereof

Abstract

The application discloses a broad-spectrum attenuated and synergistic host chassis for production of pseudomonas aeruginosa phage, a construction method and application thereof, and belongs to the technical field of microorganisms. 68 phages isolated from a model strain of Pseudomonas aeruginosa PAO1 can cover 97% (238 / 245) of 245 Pseudomonas aeruginosa strains isolated from multiple hospitals, and the target of amplifying clinical Pseudomonas aeruginosa phage by a single host strain is achieved. By knocking out phage resistance systems, prophages, virulence factor effector proteins and antibiotic resistance related genes in PAO1, the production of PAO1 phage is improved, and most of the secreted prophages, virulence factors and antibiotic inactivation enzymes are removed from the source to pollute the phage. Further, key virulence factor synthesis related genes such as O antigen, pilus and core oligosaccharide are knocked out, the pollution of O antigen, pilus and core oligosaccharide is reduced, the lipopolysaccharide is greatly truncated, and the problem of purification is solved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to an attenuated and enhanced host chassis that can be widely used in the production of Pseudomonas aeruginosa bacteriophages, as well as its construction method and application. Background Technology

[0002] *Pseudomonas aeruginosa* exhibits multidrug resistance and opportunistic pathogenicity, making it unsuitable for phage production in conventional fermentation facilities. Current common industrial chassis typically knock out individual virulence genes to construct attenuated strains for use in ordinary environments. For example, Qingdao Nuoan Biotech has constructed the non-toxic and non-pathogenic *Salmonella enteritidis* C1106 strain for the industrial production of *Salmonella* phages. However, during phage production, *Pseudomonas aeruginosa* releases large amounts of virulence factors, genetic material, prophages, and antibiotic-inactivating enzymes, contaminating the phages and posing significant safety risks when used directly. Therefore, phages generally require purification before clinical use through methods such as filtration, ultrafiltration, cesium chloride gradient centrifugation, and dialysis. However, these purification methods cannot remove prophages, and the lipopolysaccharide (LPS) content in phages that use LPS as a receptor is difficult to reduce below standard levels. Furthermore, phages exhibit host specificity; currently, no single bacterial chassis can be used for the production of all clinical *Pseudomonas aeruginosa* phages.

[0003] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a host chassis with reduced virulence and enhanced efficacy that can be widely used in the production of Pseudomonas aeruginosa bacteriophages, as well as its construction method and application. This aims to address the problems that existing bacteriophages are host-specific, currently produced mainly through their host bacteria, and there is no single bacterium that can be widely used for the safe production of Pseudomonas aeruginosa bacteriophages; clinically pathogenic Pseudomonas aeruginosa generally possesses multiple virulence factors, making production in conventional fermentation workshops impossible; furthermore, virulence factors, prophages, and antibiotic inactivating enzymes released by the host bacteria during bacteriophage production contaminate the bacteriophages and are difficult to remove, seriously affecting clinical treatment efficacy.

[0005] The study found that the comprehensive host range of 68 phages isolated from the P. aeruginosa model strain PAO1 could cover approximately 97% (238 / 245) of representative clinical P. aeruginosa isolates, indicating that PAO1 can serve as a general-purpose production platform for clinical P. aeruginosa phages.

[0006] Furthermore, this invention aims to increase the yield of Pseudomonas aeruginosa phages, reduce chassis virulence, and reduce contamination from impurities during phage production. It sequentially knocks out phage resistance system genes, prephage key genes, virulence factor effector protein genes, and antibiotic inactivation enzyme genes in PAO1.

[0007] Furthermore, in order to reduce the contamination of O antigen, fimbriae, and core oligosaccharides in the produced bacteriophages, this invention also knocks out the O antigen assembly gene, fimbriae synthesis gene, and core oligosaccharide assembly gene of lipopolysaccharide.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a host chassis that can be widely used in the production of Pseudomonas aeruginosa phages, wherein the host chassis is obtained by sequentially knocking out the phage resistance system gene, the prophage gene, the virulence factor effector protein gene, and the antibiotic inactivation enzyme gene of the Pseudomonas aeruginosa model strain PAO1.

[0010] The phage resistance system genes include genes encoding the following proteins: MTases, S subunit, REases, PA1371, PA1372, GajB, and GajA.

[0011] The prophage genes are the gene encoding PF phage protein and the key gene encoding YMC phage protein;

[0012] The virulence factor effector protein genes include genes encoding the following proteins: transcription regulator Vfr, TesG, protease-IV, lysophospholipase C precursor, and small protease PasP.

[0013] The antibiotic inactivating enzyme gene includes genes encoding the following proteins: glutathione transferase FosA, VanW family proteins, acetylpolyamine aminohydrolase AphA, chloramphenicol acetyltransferase Cat, and β-lactam hydrolase OXA-50.

[0014] The host chassis has the accession number CGMCC No. 36006.

[0015] Optionally, the host chassis may also have at least one of the following knocked out: the O antigen assembly gene, the fimbriae synthesis gene, and the core oligosaccharide assembly gene.

[0016] A second aspect of the present invention provides a method for constructing an attenuated and enhanced host chassis for the production of Pseudomonas aeruginosa bacteriophages, as described in the present invention, comprising the steps of:

[0017] Using the Pseudomonas aeruginosa model strain PAO1 as the production chassis, the phage resistance system gene, prephage gene, virulence factor effector protein gene, and antibiotic inactivation enzyme gene in the production chassis were knocked out in sequence to construct an attenuated and enhanced host chassis that can be widely used for the production of Pseudomonas aeruginosa phages.

[0018] Optionally, the construction method further includes the step of:

[0019] By knocking out at least one of the O antigen assembly gene, fimbrial synthesis gene, and core oligosaccharide gene, an attenuated and enhanced host chassis that can be widely used in the production of Pseudomonas aeruginosa phages was constructed.

[0020] A third aspect of the present invention provides an application of the attenuated and enhanced host chassis described herein, which can be widely used in the production of Pseudomonas aeruginosa bacteriophages, in the preparation of bacteriophages.

[0021] The present invention has the following beneficial effects:

[0022] The host chassis of this invention can lyse 97% of clinical isolates of Pseudomonas aeruginosa, basically meeting clinical needs. Therefore, the host chassis is a general-purpose chassis for Pseudomonas aeruginosa phages.

[0023] The host chassis in this invention has all known phage resistance system genes knocked out, resulting in a significant increase in phage production. This host chassis serves as a phage production enhancement chassis for *Pseudomonas aeruginosa*. The phage resistance system is a defense mechanism developed by bacteria over a long period of evolution to resist phage infection. It employs various strategies to prevent phage adsorption, invasion, or replication, thus ensuring the survival of the bacteria.

[0024] Compared to existing phage host chassis, the host chassis in this invention is not only non-pathogenic, but also knocks out all known prephage key genes, virulence factor effector protein genes, and antibiotic inactivation enzyme genes as much as possible. For the first time, it reduces the contamination of phages by host components from the source and reduces the safety risks of phages.

[0025] The host chassis in this invention, by knocking out the phage resistance system and prophage sequence, can avoid prophage overinfection immunity and improve the infection efficiency of other prophages, making it very suitable as a prophage integration chassis for studying prophage function. Here, overinfection immunity refers to the phenomenon where prophages prevent reinfection by similar or closely related phages by protecting the host bacteria.

[0026] The host chassis of this invention knocks out all known phage resistance systems in PAO1 and significantly improves the transformation efficiency of exogenous plasmids. It can serve as an efficient gene editing chassis for PAO1 and be used to study the function of the PAO1 gene.

[0027] The host chassis in this invention exhibits significantly reduced virulence and higher plasmid transformation efficiency, making it suitable for the industrial production of other valuable components secreted by Pseudomonas aeruginosa.

[0028] Based on the aforementioned host chassis, this invention further knocks out the genes for synthesizing key virulence factors such as O antigen, fimbriae, and / or core oligosaccharides. It not only possesses the advantages of the aforementioned host chassis but also further reduces the contamination of O antigen, fimbriae, and core oligosaccharides in the produced bacteriophages. In particular, knocking out the O antigen or core oligosaccharide-related genes of lipopolysaccharide results in a significant truncation of lipopolysaccharide, making it easier to reduce through traditional purification methods. This solves the difficulty of reducing the lipopolysaccharide content in bacteriophages that use lipopolysaccharide as a receptor to below the standard. Attached Figure Description

[0029] Figure 1 This represents the host spectrum of the clinical Pseudomonas aeruginosa phage library.

[0030] Figure 2 This is a schematic diagram of the chassis construction process.

[0031] Figure 3 This is a diagram showing the genome alignment results for each chassis.

[0032] Figure 4 The graph shows the phage sensitivity test results for each chassis.

[0033] Figure 5 The figure shows the results of phage yield and plasmid transformation efficiency measurements for a universal chassis.

[0034] Figure 6 The image shows the results of the prephage activity assay for a universal chassis.

[0035] Figure 7 This is a graph showing the toxicity test results for a general-purpose chassis.

[0036] Figure 8 The graph shows the decrease in lipopolysaccharide content after purification of phages produced by each chassis.

[0037] Among them, the **( P <0.01) indicates that the results are statistically significant. Detailed Implementation

[0038] This invention provides an attenuated and enhanced host chassis, its construction method, and its application, which can be widely used in the production of Pseudomonas aeruginosa bacteriophages. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0039] Phage cocktails typically involve mixing multiple phages with different types, host profiles, or adsorption receptors to broaden their host range or delay the development of phage resistance in host bacteria. However, no *Pseudomonas aeruginosa* strain has yet been reported that meets the requirements for efficient phage production in most clinical settings. Furthermore, *P. aeruginosa* phage production generally releases various virulence factors, prophages, and antibiotic-inactivating enzymes, contaminating the phages. Virulence factors may trigger an immune response in humans; prophages may transfer virulence and antibiotic resistance genes, increasing the virulence or resistance of pathogens in humans; antibiotic-inactivating enzymes can hydrolyze or chemically modify the molecular structure of antibiotics, rendering them inactive and affecting the therapeutic efficacy of phage-antibiotic combinations. Conventional purification methods such as filtration, ultrafiltration, cesium chloride gradient centrifugation, and dialysis are insufficient to remove these impurities. For example, lipopolysaccharide (LPS) is an important virulence factor for *P. aeruginosa* and also an adsorption receptor for many phages; conventional methods struggle to reduce LPS in these phages to levels suitable for human consumption. Meanwhile, conventional methods cannot remove prephage contamination from bacteriophages. In short, due to the host-specific nature of bacteriophages, current bacteriophage production primarily relies on their host bacteria, and no single bacterial strain can yet be widely used for the safe production of *Pseudomonas aeruginosa* bacteriophages. Clinically pathogenic *Pseudomonas aeruginosa* generally possesses multiple virulence factors, making production in conventional fermentation facilities impossible. Furthermore, virulence factors, prephages, and antibiotic inactivating enzymes released by the host bacteria during bacteriophage production contaminate the bacteriophages and are difficult to remove, severely impacting clinical treatment efficacy. Therefore, the purpose of this invention is to develop a universal host chassis for *Pseudomonas aeruginosa* bacteriophage production that minimizes or eliminates the production of prephages, virulence factors, and antibiotic inactivating enzymes, thereby removing these safety hazards at the source.

[0040] Studies have found that 68 bacteriophage strains isolated from the P. aeruginosa model strain PAO1 have a comprehensive host range covering 97% of 245 P. aeruginosa strains isolated from multiple hospitals, essentially achieving the goal of amplifying clinically usable P. aeruginosa bacteriophages using a single host bacterium. Furthermore, this invention aims to increase P. aeruginosa bacteriophage yield, reduce chassis virulence, and minimize contamination during phage production by sequentially knocking out genes related to the phage resistance system, key prophage genes, virulence factor effector protein-related genes, and antibiotic inactivation enzyme-related genes in PAO1.

[0041] Based on this, embodiments of the present invention provide a host chassis that can be widely used in the production of Pseudomonas aeruginosa phages with reduced virulence and enhanced efficacy. The host chassis is obtained by sequentially knocking out the phage resistance system gene, the prephage gene, the virulence factor effector protein gene, and the antibiotic inactivation enzyme gene of the Pseudomonas aeruginosa model strain PAO1.

[0042] The selected knockout genes are shown in Table 1:

[0043] The phage resistance system genes include genes encoding the following proteins: MTases, S subunit, REases, PA1371, PA1372, GajB, and GajA.

[0044] The prophage genes are the gene encoding PF phage protein and the key gene encoding YMC phage protein;

[0045] The virulence factor effector protein genes include genes encoding the following proteins: transcription regulator Vfr, TesG, protease-IV, lysophospholipase C precursor, and small protease PasP.

[0046] The antibiotic inactivating enzyme gene includes genes encoding the following proteins: glutathione transferase, VanW family proteins, acetylpolyamine aminohydrolase, chloramphenicol acetyltransferase, and β-lactam hydrolase.

[0047] In this embodiment, CRISPR-Cas12a gene editing technology was used to knock out as many genes related to the phage resistance system (collectively referred to as A), prophage (collectively referred to as B), virulence factor effector proteins (collectively referred to as C), and antibiotic inactivating enzymes (collectively referred to as D) in PAO1 as possible, thereby constructing a host chassis PAO1Δ that can be widely used for the production of clinical Pseudomonas aeruginosa phages. ABCD (Abbreviated as C1, its accession number is CGMCC No.36006, the deposit address is China General Microbiological Culture Collection Center, the deposit date is September 25, 2025, and its classification name is Pseudomonas aeruginosa) Pseudomonas aeruginosa This increased the yield of PAO1 phages and removed most of the contamination of phages by secretory virulence factors, prophages, and antibiotic inactivating enzymes from the source.

[0048] In one embodiment, the host chassis also has at least one of the following knocked out: the O antigen assembly gene, the fimbriae synthesis gene, and the core oligosaccharide gene.

[0049] In one implementation, the selected knockout genes are shown in Table 1:

[0050] When the O antigen ligase gene is knocked out waaL At that time, the host chassis was designated as PAO1Δ ABCD-waaL (Abbreviated as C2), its accession number is CGMCC No.36007, the deposit address is China General Microbiological Culture Collection Center, the deposit date is September 25, 2025, and its classification name is *Pseudomonas aeruginosa*. Pseudomonas aeruginosa ;

[0051] When the fimbrial synthesis gene is knocked out pilAAt that time, the host chassis was designated as PAO1Δ ABCD-pilA (Abbreviated as C3), its accession number is CGMCC No.36005, the deposit address is China General Microbiological Culture Collection Center, the deposit date is September 25, 2025, and its classification name is *Pseudomonas aeruginosa*. Pseudomonas aeruginosa ;

[0052] When the fimbrial synthesis gene is knocked out simultaneously pilA and O antigen ligase gene waaL At that time, the host chassis was designated as PAO1Δ ABCD-waaL-pilA (Abbreviated as C4), its accession number is CGMCC No.36003, the deposit address is China General Microbiological Culture Collection Center, the deposit date is September 25, 2025, and its classification name is *Pseudomonas aeruginosa*. Pseudomonas aeruginosa ;

[0053] When the fimbrial synthesis gene is knocked out simultaneously pilA O antigen ligase gene waaL and genes involved in core oligosaccharide assembly pa5001 At that time, the host chassis was designated as PAO1Δ ABCD-waaL-pilA-pa5001 (Abbreviated as C5), its accession number is CGMCC No.36004, the deposit address is China General Microbiological Culture Collection Center, the deposit date is September 25, 2025, and its classification name is *Pseudomonas aeruginosa*. Pseudomonas aeruginosa .

[0054] In this embodiment, based on the different adsorption mechanisms of bacteriophages, phage receptor-related genes were further knocked out on the C1 chassis, constructing a series of personalized chassis C2 and C3 targeting different receptor bacteriophages. 、 C4 and C5 further reduced the virulence factors such as lipopolysaccharide and fimbriae in the relevant bacteriophages.

[0055] This invention also provides a method for constructing an attenuated and enhanced host chassis, as described above, which can be widely used in the production of Pseudomonas aeruginosa bacteriophages, comprising the following steps:

[0056] Using the Pseudomonas aeruginosa model strain PAO1 as the production chassis, the phage resistance system gene, prephage gene, virulence factor effector protein gene, and antibiotic inactivation enzyme gene in the production chassis were knocked out in sequence to construct an attenuated and enhanced host chassis that can be widely used for the production of Pseudomonas aeruginosa phages.

[0057] In one implementation, the construction method further includes the step of:

[0058] By knocking out at least one of the O antigen assembly gene, fimbrial synthesis gene, and core oligosaccharide gene, an attenuated and enhanced host chassis that can be widely used in the production of Pseudomonas aeruginosa phages was constructed.

[0059] In one implementation, when the O antigen ligase gene is knocked out... waaL At that time, a host chassis with accession number CGMCCNo.36007 was constructed;

[0060] When the fimbrial synthesis gene is knocked out pilA At that time, a host chassis with accession number CGMCC No.36005 was constructed;

[0061] When the fimbrial synthesis gene is knocked out simultaneously pilA and O antigen ligase gene waaL At that time, a host chassis with accession number CGMCC No. 36003 was constructed;

[0062] When the fimbrial synthesis gene is knocked out simultaneously pilA O antigen ligase gene waaL and genes involved in core oligosaccharide assembly pa5001 At that time, a host chassis with accession number CGMCC No.36004 was constructed.

[0063] In one implementation, the technique used to knock out the gene is CRISPR-Cas12a gene editing technology.

[0064] This invention also provides an application of the attenuated and enhanced host chassis described above, which can be widely used in the production of Pseudomonas aeruginosa bacteriophages, in the preparation of bacteriophages.

[0065] The embodiments of the present invention have the following technical advantages:

[0066] In this embodiment, the Pseudomonas aeruginosa phage produced by the basic chassis C1 can lyse 97% of clinical isolates of Pseudomonas aeruginosa, which basically meets clinical needs. Therefore, the basic chassis C1 is a general-purpose chassis for Pseudomonas aeruginosa phage.

[0067] In this embodiment, the base chassis C1 has all known phage resistance system-related genes knocked out, and the phage production of this chassis is significantly increased, making it a chassis for increasing Pseudomonas aeruginosa phage production.

[0068] Compared to existing phage host chassis, the basic chassis C1 in this embodiment is not only non-pathogenic, but also has as many known prophage key genes, virulence factor effector protein-related genes, and antibiotic inactivation enzyme-related genes as possible knocked out, reducing the contamination of the phage by host components from the source and reducing the safety risks of the phage.

[0069] In this embodiment, the base chassis C1, due to the knockout of the phage resistance system and the prephage sequence, can avoid the superinfection immunity of the prephage and improve the infection efficiency of other prephages, making it very suitable as a prephage integration chassis for studying prephage function.

[0070] In this embodiment, the basic chassis C1 knocked out all known phage resistance system-related genes in PAO1, and the transformation efficiency of exogenous plasmids was also significantly improved. It can be used as a high-efficiency gene editing chassis for PAO1 to study the function of PAO1 genes.

[0071] The C1 base chassis in this embodiment is significantly less toxic and has a higher plasmid conversion efficiency, making it suitable for the industrial production of components such as pyocyanin and rhamnolipid.

[0072] The personalized chassis C2 in this embodiment 、 C3 、 C4 and C5 also knock out genes related to the synthesis of key virulence factors such as O antigen, fimbriae, and / or core oligosaccharides. They not only have the advantages of the basic chassis C1, but also further reduce the contamination of O antigen, fimbriae, and core oligosaccharides in the produced bacteriophages. In particular, after chassis C2, C4, and C5 knock out some genes related to O antigen or core oligosaccharides of lipopolysaccharide, the lipopolysaccharide is significantly truncated, making it easier to reduce through traditional purification methods. This solves the difficulty of reducing the lipopolysaccharide content in bacteriophages that use lipopolysaccharide as a receptor to below the standard.

[0073] The present invention will be further described in detail below through specific embodiments.

[0074] First, the source of the items involved in the following experiments will be explained.

[0075] 245 clinical isolates of Pseudomonas aeruginosa were obtained from sputum samples of patients in multiple hospitals. All isolates underwent next-generation whole-genome sequencing analysis, which confirmed that they contained all mainstream multilocus sequence typing (MLST) and were representative of the strains.

[0076] Pseudomonas aeruginosa model strain PAO1: The model strain was donated by Researcher Jin Fan of Shenzhen Institutes of Advanced Technology. After whole-genome sequencing comparison, it was found to be identical to the genome sequence of PAO1 (accession number: AE004091.2) in the GenBank database.

[0077] 68 bacteriophages: All 68 bacteriophages in the bacteriophage library were isolated and purified from wastewater in hospitals, nursing homes and sewage treatment plants across the country using the Pseudomonas aeruginosa model strain PAO1, and all underwent genome sequencing analysis.

[0078] 1. Identification of the general host chassis of Pseudomonas aeruginosa bacteriophages

[0079] (1) MLST typing: MLST typing was performed on 245 clinical isolates of Pseudomonas aeruginosa using the PubMLST (Public databases for molecular typing and microbial genome diversity) tool. The representativeness of the 245 clinical isolates of Pseudomonas aeruginosa was analyzed by referring to the published international and domestic trends of Pseudomonas aeruginosa.

[0080] (2) Host spectrum determination: 300 μL of fresh bacterial culture (OD600 ≈ 0.6) was mixed with 20 mL of 0.75% LB (Luria-Bertani) semi-solid agar medium and poured into a pre-prepared LB agar plate to obtain a double-layer plate. After the double-layer plate cooled, 5 μL of phage stock solution was added to each double-layer plate and allowed to air dry. The host spectrum of 68 phage strains was determined using this method.

[0081] 2. Development of an attenuated and enhanced host chassis for the production of Pseudomonas aeruginosa bacteriophages

[0082] (1) Prediction and screening of phage resistance system, prophage, virulence factor effector protein, and antibiotic resistance-related genes in PAO1: The Defense Finder tool was used to predict phage resistance system-related genes in the PAO1 genome; the PHASTEST tool was used to predict prophage sequences; the VFanalyzer tool was used to predict virulence factor effector protein-related genes; and the Resistance Gene Identifier tool was used to predict antibiotic resistance genes. Key sequences of phage resistance system and prophage, genes encoding virulence factor effector proteins, and genes encoding antibiotic inactivating enzymes in the prediction results were selected for knockout.

[0083] (2) Construction of the basic chassis: The genes screened in the previous step were knocked out in the following order: genes related to phage resistance system, key sequences of prephage, genes encoding virulence factor effector proteins, and genes encoding antibiotic inactivating enzymes. The knocked-out genes are shown in Table 1. The specific method is as follows: First, the genes carrying... cas12a The gene plasmid pCas12a was electrotransfused into P. aeruginosaPAO1 competent cells were recovered by shaking at 37°C for 2 h, then plated on LB agar plates containing tetracycline (50 μg / mL) and incubated overnight at 37°C. The next day, single colonies were selected for PCR identification of positive bacteria. After the positive bacteria reached the logarithmic growth phase, 0.2% (w / v) arabinose was added for 2 h of induction, followed by washing twice with 10% glycerol to prepare electrotransformation competent cells. Then, the recombinant plasmid pCrRNA-V, carrying the upstream and downstream homologous arms (500 bp each) of the knockout gene and crRNA (23 bp), was electrotransformed into competent cells. After recovery by shaking at 37°C for 2 h, the cells were plated on LB agar plates containing tetracycline (50 μg / mL) and gentamicin (50 μg / mL). After static incubation at 37°C for 24 h, single colonies were selected for site-specific PCR amplification. Smaller PCR products were screened by 1% TAE-agarose gel electrophoresis and then sequenced for first-generation sequencing to determine whether the target gene had been knocked out.

[0084] (3) Personalized chassis construction based on phage adsorption mechanism: Using the method in the previous step, the gene encoding the O antigen ligase in the basic chassis C1 was knocked out. waaL Gene encoding fimbriae protein pilA and genes involved in core oligosaccharide assembly pa5001 (The knocked-out genes are shown in Table 1.) A strain C2 with a synthesis defect of phage adsorption receptor (O antigen, fimbriae, and core oligosaccharide) was constructed. 、 C3 、 C4 and C5.

[0085] (4) Phage susceptibility assessment of the chassis: The susceptibility of each chassis to 68 phage strains was determined using the same host spectrum assay method as described above, and the versatility of the basic chassis (i.e., chassis C1) and the personalized chassis (i.e., chassis C2) were assessed. 、 C3 、 The compatibility of C4 and C5 with various phages.

[0086] (5) Toxicity assessment of the chassis: SPF-grade CD-1 mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Experimental Animal Production License No.: SCXK (Lu) 2019 0003). Mouse feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. (Proposed batch number 22123213). Animals were quarantined and acclimatized for at least 5 days after arriving at the experimental facility. The acclimatized healthy mice were divided into three groups: phosphate buffered saline (PBS) group, PAO1 group, and C1 group, with 8 mice in each group. On the first and fourth days, cyclophosphamide solution of 150 mg / kg and 100 mg / kg was injected subcutaneously to establish an immunosuppressed mouse model. On D5, mice in each group were anesthetized and administered the drug via tracheal route. The PAO1 group was administered 5 × 10 3PAO1 bacterial suspension at CFU dose (150 µl), C1 group administered 5 × 10 3 CFU-dose C1 bacterial suspension (150 µl) was administered to the PBS group, while the PBS group received 150 µl of PBS buffer. Animal survival rates were observed at D5-0.5h (0.5h after administration on day 5), D6, D7, D8, D9, D10, D11, and D12. The frequency of clinical observations could be increased as needed based on animal toxicity during the observation period, and records should be kept promptly. All surviving animals received an intraperitoneal injection of cyclophosphamide 12 days after cyclophosphamide administration. ® Euthanasia was performed on patients with cervical dislocation after anesthesia with a mixture of 50 and xylazine hydrochloride injection. Relevant experimental data were statistically analyzed using GraphPad Prism software, and corresponding survival curves were generated. Based on the survival curve results, the virulence intensity of each strain was compared and analyzed. The Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests were used to statistically verify the differences between groups.

[0087] (6) Phage amplification and counting: Add 1 mL of phage stock solution (10 7 PFU) and 1 mL of host bacterial culture (10 8 CFU was mixed at a multiplicity of infection (MOU) of 0.1 and added to an Erlenmeyer flask containing 50 mL of LB medium. The flask was incubated at 37°C with shaking for 6 h. The culture was then transferred to a 50 mL centrifuge tube and centrifuged at 13400 g for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane to obtain the phage stock solution. The phage stock solution was serially diluted 10-fold eight times with PBS buffer. 10 μL of each dilution was added to a double-layer plate prepared with the host bacterium PAO1. After air-drying in a clean bench, the plates were transferred to a 37°C incubator and incubated for 6 h. The number of phages at each dilution was counted, and the phage titer was calculated.

[0088] (7) Prephage induction in the chassis: OD values ​​of fresh PAO1, PAO1ΔPF1, and C1 bacterial cultures in the logarithmic growth phase were expressed in LB liquid medium. 600 After adjusting the concentration to 0.2, prophage induction was performed using the following method: Add 3 mL of each bacterial culture (OD2000) to the culture medium. 600Mitomycin C was added to the solution (0.2) to a final concentration of 0.2 μg / mL, and the solution was induced at 37℃ / 220 rpm for 16 h. The induced bacterial culture was centrifuged at 5000g for 3 minutes to precipitate the bacteria, while the phage remained in the supernatant. The supernatant was filtered through a 0.22 μm microporous membrane to thoroughly remove bacterial residue, and the filtrate was serially diluted 10-fold seven times. 10 μL of each serial dilution was added to a double-layer plate prepared with C1 bacterial culture. After air-drying in a clean bench, the plate was transferred to a 37℃ incubator and incubated for 6 h to observe the prephage induction.

[0089] (8) Lipopolysaccharide determination of bacteriophages: Bacteriophages Φ49, ΦL9, and ΦY were amplified using PAO1 and personalized chassis C2, C4, and C5, respectively, as described above. The amplified bacteriophage stock solution was purified by sequentially filtering through a 0.22 μm microporous membrane, ultrafiltration through a 100 kda ultrafiltration membrane, and ultracentrifugation with cesium chloride. The lipopolysaccharide content of the purified bacteriophages was determined using the Lipopolysaccharide Detection Kit (Microplate Quantitative Chromogenic Matrix Method) from Xiamen Liposome Biotechnology Co., Ltd.

[0090] The experimental results are as follows:

[0091] Figure 1 This study analyzed the typing of clinical *Pseudomonas aeruginosa* and the host spectrum of the phage library. 'a' shows the MLST typing of 245 clinical isolates of *Pseudomonas aeruginosa*, containing 58 typing types, similar to the mainstream typing of *Pseudomonas aeruginosa* reported globally in the literature, indicating that the strain library has a certain degree of richness and representativeness. Figure 1 In figure a, the bacterium RPA02B corresponds to the abbreviation R02 in figure b, SPA79B corresponds to the abbreviation S79, and so on. Figure b represents the host spectrum of 68 bacteriophages. The horizontal axis represents 245 clinical isolates of Pseudomonas aeruginosa, and the vertical axis represents 68 bacteriophages; the shades of red represent the different degrees of lysis of bacteria by the bacteriophages. As can be seen from the figure, the comprehensive host range of the 68 bacteriophages isolated from P. aeruginosa PAO1 can cover approximately 97% of the clinical isolates of P. aeruginosa.

[0092] Figure 2This is a schematic diagram of the chassis construction process. As shown, firstly, genes related to the phage resistance system are knocked out to improve phage yield and plasmid transformation efficiency; then, genes related to the prephage are knocked out to prevent the release of contaminated phages from the prephage; next, genes encoding or regulating effector proteins of various virulence factors are knocked out to reduce chassis virulence and prevent host-secreted effector proteins from contaminating the phages; finally, genes encoding antibiotic inactivating enzymes are knocked out to prevent antibiotic inactivating enzymes from contaminating the phages and affecting the phage-antibiotic combination effect. Thus, the general-purpose host chassis C1 for clinical Pseudomonas aeruginosa phages is completed. In addition, based on the general-purpose host chassis C1, genes related to lipopolysaccharide (LPS) and / or fimbriae synthesis are knocked out to construct personalized chassis C2, C3, C4, and C5 suitable for different receptors, further reducing key virulence factors in the chassis and to some extent avoiding cross-contamination when producing different phages using the same chassis.

[0093] Figure 3 Genome alignment was performed on each chassis. Third-generation sequencing and genome alignment analysis of each chassis revealed that, compared to the wild-type PAO1 genome, the target gene was knocked out in each chassis, and no other spontaneous mutations were found in the genome. All chassis strains have been deposited with the China General Microbiological Culture Collection Center on September 25, 2025, and are classified and named *Pseudomonas aeruginosa*. Pseudomonas aeruginosa The accession numbers are CGMCC No. 36006 (PAO1ΔABCD, C1) and CGMCC No. 36007 (PAO1ΔABCD-). waaL , C2), CGMCC No.36005 (PAO1ΔABCD- pilA , C3), CGMCC No.36003 (PAO1ΔABCD- waaL - pilA , C4), CGMCC No.36004 (PAO1ΔABCD- waaL - pilA -pa5001,C5).

[0094] Figure 4 Phage susceptibility was determined for each chassis. The basal chassis C1 exhibited phage susceptibility consistent with wild-type PAO1, and could produce all phages; chassis C2 lacked the lipopolysaccharide O antigen assembly-related gene. waaL It can be used to produce non-O antigen-dependent bacteriophages, such as those from the genera *Bruynoghevirus*, *Pakpurnavirus*, *Phikmvvirus*, and *Yuavirus*; the C3 chassis lacks genes related to fimbrial synthesis. pilAIt can be used to produce non-fibril-dependent bacteriophages, such as those from the genera *Bruynoghevirus*, *Pakpurnavirus*, and *Pbunavirus*; the chassis C4 lacks... pilA and waaL Genes that can be used to produce non-pillary and O antigen-dependent phages, such as those from the genera *Bruynoghevirus* and *Pakpurnavirus*; chassis C5 lacks... pilA , waaL Genes related to the synthesis of lipopolysaccharides and core oligosaccharides in the outer nucleus pa5001 This technology can be used to produce non-pillary, O-antigen, and core oligosaccharide-dependent nuclear-dependent phage Φ49, avoiding contamination by other phages during production. The top horizontal axis represents each phage; the left vertical axis represents each chassis; the bottom color bars correspond to the genera of the top phages, with different colors representing different genera; the different shades of green in the right-hand diagram represent the different lysis effects of the phages on the chassis bacteria.

[0095] Figure 5 The phage yield and plasmid transformation efficiency of the universal chassis were determined. a) Compared to wild-type PAO1, the yields of low-yielding phages Φ26, Φ30, and Φ36 were significantly increased in the universal chassis C1, with the highest increase being approximately 1000%. b) Compared to wild-type PAO1, the pCAS12a plasmid transformation efficiency of the universal chassis C1 was significantly improved.

[0096] Figure 6 The prephage activity of the universal chassis was measured. Both PAO1 and PAO1ΔPF (PAO1 with only PF phage knocked out) could be induced to produce prephages by mitomycin C. However, no prephages were induced in the universal chassis C1 (which simultaneously knocked out the key gene sequences of PF and YMC phages). This indicates that the basic chassis C1 cannot release prephages, thus demonstrating that knocking out these two phage genes can effectively prevent the release of prephages from the chassis.

[0097] Figure 7 The toxicity of the universal chassis was determined. Mouse challenge experiments showed that, at the same dose, the survival rate of mice challenged with wild-type PAO1 was only 25%, while the survival rate of mice challenged with the universal chassis C1 and the PBS (phosphate-buffered saline) control group was 100%, indicating that the toxicity of chassis C1 was significantly reduced.

[0098] Figure 8This indicates that the lipopolysaccharide (LPS) content in phages produced by personalized chassis is more easily reduced through purification. Lipopolysaccharide synthesis-related genes were knocked out in chassis C2, C4, and C5, resulting in varying degrees of lipopolysaccharide truncation. After purification by filtration, ultrafiltration, and cesium chloride density gradient centrifugation, the LPS content in phages produced by chassis C2, C4, and C5 was significantly lower than that of wild-type PAO1 and the general-purpose chassis C1, indicating that the truncated LPS in chassis C2, C4, and C5 is more easily removed through purification.

[0099] Phage production assays revealed significantly increased yields of phages Φ26, Φ30, and Φ36 produced by the basal chassis C1 of the phage-resistant knockout system, with the highest increase reaching approximately 1000%. Prephage induction experiments showed that both PAO1 and PAO1ΔPF (PF knockout phage in PAO1) could be induced to produce prephages by mitomycin C, while no prephages were induced in the basal chassis C1 (which knocks out key gene sequences of PF and YMC phages), indicating that the basal chassis C1 no longer releases prephages. Mouse challenge experiments showed that the survival rate of mice challenged with PAO1 at the same dose was only 25%, while the survival rate of mice challenged with chassis C1 and the PBS control group was 100%, indicating a significant reduction in the virulence of chassis C1. Lipopolysaccharide (LPS) determination revealed that the LPS content of phages Φ49, ΦL9, and ΦY produced using chassis C2, C4, and C5 was significantly lower than that of Φ49, ΦL9, and ΦY produced and purified using wild-type strain PAO1 after tangential flow ultrafiltration and cesium chloride density gradient centrifugation. This indicates that after the LPS content of the chassis is truncated, the LPS content of the produced phages is more easily reduced using traditional methods.

[0100] Table 1. Complete chassis knockout gene sequence information

[0101]

[0102] In summary, this invention provides a host chassis, its construction method, and its application that can be widely used in the production of Pseudomonas aeruginosa phages with reduced virulence and enhanced efficacy. Chassis C1 is the first clinically applicable general-purpose production chassis for Pseudomonas aeruginosa phages. Chassis C1 knocks out all gene sequences related to effector proteins reported to have cytotoxicity, significantly reducing virulence and reducing contamination of virulence factors during phage production at the source, thus exhibiting good safety. Chassis C1 knocks out key prophage gene sequences, preventing the release of prophages and eliminating prophage contamination at the source. Chassis C1 knocks out multiple genes encoding antibiotic inactivating enzymes, solving the problem of antibiotic inactivating enzymes affecting the efficacy of phage-antibiotic combination therapy at the source. Chassis C1 not only reduces contaminants in phages at the source, saving subsequent purification costs, but also knocks out all known phage resistance systems in PAO1, significantly increasing phage yield and demonstrating strong practicality. Chassis C2 、 C3 、 C4 and C5 have varying degrees of defects in the synthesis of O antigen, fimbriae, and core oligosaccharides. In addition to having the advantages of the C1 chassis, C2, C4, and C5 have also overcome the bottleneck of the difficulty in reducing lipopolysaccharides in bacteriophages to a safe level.

[0103] It should be understood that the design method and application of the Pseudomonas aeruginosa chassis of the present invention are not limited to the above-mentioned standard strain PAO1 chassis. Those skilled in the art can improve the design method or replace it with other Pseudomonas aeruginosa strains based on the above description, but all such improvements and modifications should fall within the protection scope of the appended claims of the present invention.

Claims

1. A host chassis for attenuating and enhancing the efficacy of Pseudomonas aeruginosa bacteriophage production, characterized in that, The host chassis was obtained by sequentially knocking out the phage resistance system gene, the prophage gene, the virulence factor effector protein gene, and the antibiotic inactivation enzyme gene from the Pseudomonas aeruginosa model strain PAO1. The phage resistance system genes include genes encoding the following proteins: MTases, S subunit, REases, PA1371, PA1372, GajB, and GajA. The prophage genes are the gene encoding PF phage protein and the key gene encoding YMC phage protein; The virulence factor effector protein genes include genes encoding the following proteins: transcription regulator Vfr, TesG, protease-IV, lysophospholipase C precursor, and small protease PasP. The antibiotic inactivating enzyme gene includes genes encoding the following proteins: glutathione transferase FosA, VanW family proteins, acetylpolyamine aminohydrolase AphA, chloramphenicol acetyltransferase Cat, and β-lactam hydrolase OXA-50. The host chassis has the accession number CGMCC No. 36006.

2. The attenuated and enhanced host chassis for the production of Pseudomonas aeruginosa bacteriophages as described in claim 1, characterized in that, The host chassis also has at least one of the following knockout genes: O antigen assembly gene, fimbrial synthesis gene, and core oligosaccharide assembly gene. When the O antigen ligase gene is knocked out waaL At that time, the accession number of the host chassis was CGMCC No. 36007; When the fimbrial synthesis gene is knocked out pilA At that time, the accession number of the host chassis was CGMCC No. 36005; When the fimbrial synthesis gene is knocked out simultaneously pilA and O antigen ligase gene waaL At that time, the accession number of the host chassis was CGMCC No. 36003; When the fimbrial synthesis gene is knocked out simultaneously pilA O antigen ligase gene waaL and genes involved in core oligosaccharide assembly pa5001 At that time, the preservation number of the host chassis was CGMCC No. 36004.

3. The application of the attenuated and enhanced host chassis as described in any one of claims 1-2, which can be widely used in the production of Pseudomonas aeruginosa bacteriophages, in the preparation of bacteriophages.

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