In vitro 3d model of chronic pseudomonas aeruginosa infection and uses thereof

Abstract

The application discloses a Pseudomonas aeruginosa chronic infection 3D model in vitro and application thereof, and belongs to the technical field of constructing a pathogenic bacterium in vitro model in a preclinical stage. The application simulates a Pseudomonas aeruginosa chronic infection environment by taking collagen as a support, screens out optimal conditions, and successfully constructs a 3D model in vitro simulating Pseudomonas aeruginosa in vivo infection. The application also uses the constructed Pseudomonas aeruginosa chronic infection 3D model in vitro for further large-scale screening of key regulation genes at a genome level, and reveals the function and molecular mechanism of a key gene PA3831. The application is helpful to large-scale gene screening and mechanism research in vitro, provides new evidence for discovering new diagnostic markers or taking relevant treatment measures for treating chronic infection patients in a clinic, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of preclinical pathogenic bacterial in vitro model construction technology, specifically involving an in vitro 3D model of chronic infection with Pseudomonas aeruginosa and its applications. Background Technology

[0002] Pseudomonas aeruginosa, an opportunistic pathogen, is a major cause of hospital-acquired infections and chronic infections (such as cystic fibrosis pulmonary infections and chronic wound infections). Its core characteristics of chronic infection include biofilm formation, metabolic adaptation, and high antibiotic resistance, which differ significantly from the planktonic phenotype in acute infection models. The complex nature of chronic Pseudomonas aeruginosa infection presents many challenges to its treatment. Therefore, establishing suitable in vitro models to study the mechanisms by which Pseudomonas aeruginosa induces chronic infection is crucial.

[0003] In traditional 2D cultures (such as agar plates and liquid suspension cultures), bacteria grow in a planktonic state, lacking the physical constraints and chemical signaling guidance of the extracellular matrix (ECM), leading to incomplete biofilm development. The barrier function of the host ECM is neglected: in real infections, the host ECM (such as collagen and fibronectin) restricts bacterial spread through mechanical barriers while providing adhesion sites (such as integrin-binding domains). 2D models fail to integrate ECM components, resulting in distorted simulations of bacterial-host interface interactions and an inability to assess the impact of drugs on biofilm permeability. Chronic infections are mitigated by downregulating the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation, shifting to fermentative metabolism to maintain survival. The microenvironment at the infection site is continuously influenced by host cell metabolism (such as lactate secretion and pH fluctuations) and immune responses (such as protease release). Static cultures cannot simulate these dynamic changes, leading to the decline of bacterial phenotypes (such as alginate synthesis) after short-term culture.

[0004] Therefore, there is an urgent need to develop a long-term stable chronic infection model of Pseudomonas aeruginosa to provide new strategies for the diagnosis and treatment of pathogenic bacteria such as Pseudomonas aeruginosa. Summary of the Invention

[0005] In order to solve the above-mentioned problems in the prior art, the purpose of this invention is to provide an in vitro 3D model of chronic infection with Pseudomonas aeruginosa and its uses.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides an in vitro infection 3D model, which is prepared through the following steps:

[0008] (1) Culturing pathogenic bacterial strains;

[0009] (2) Preparation of collagen solution;

[0010] (3) Mix the pathogenic bacterial strain from step (1) with the collagen solution obtained in step (2) and culture for 1 to 7 days to obtain an in vitro infection 3D model.

[0011] Further, the pathogenic bacteria in step (1) is Pseudomonas aeruginosa; the culture is to culture the pathogenic bacterial strain to the logarithmic phase.

[0012] Further, the pathogenic bacteria is *Pseudomonas aeruginosa*; the culture medium is SCFM2 medium or a modified SCFM2 medium, wherein the modified SCFM2 medium includes the following components: aspartic acid, threonine, serine, glutamate, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, tryptophan, histidine, arginine, magnesium sulfate heptahydrate, citric acid, dipotassium hydrogen phosphate, and sodium hydrogen phosphate tetrahydrate.

[0013] Furthermore, the components and formulation of the modified SCFM2 culture medium are as follows:

[0014]

[0015]

[0016] Further, the preparation method of the collagen solution in step (2) includes the following steps: mixing buffer, collagen and serum to obtain the solution.

[0017] Furthermore, the collagen is type I rat tail collagen.

[0018] Further, the buffer solution is PBS buffer with a pH of 7.0–7.2; the final concentration of collagen in the collagen solution is 1 mg / mL, and the final concentration of serum is 40%; the serum is fetal bovine serum.

[0019] Furthermore, the culture conditions described in step (3) are 37°C and 5% CO2.

[0020] Furthermore, the culture time described in step (3) is 3 days.

[0021] The present invention also provides a method for constructing the above-mentioned in vitro infection 3D model, the method being:

[0022] (1) Culturing pathogenic bacterial strains;

[0023] (2) Preparation of collagen solution;

[0024] (3) Mix the pathogenic bacterial strain from step (1) with the collagen solution obtained in step (2) and culture for 1 to 7 days to obtain an in vitro infection 3D model.

[0025] This invention also provides the use of the above-mentioned in vitro infection 3D model in infection mechanism research, screening, prevention and / or treatment of infection drugs.

[0026] The present invention has achieved the following beneficial effects:

[0027] This invention uses collagen as a scaffold to simulate the chronic infection environment of *Pseudomonas aeruginosa*, screening for optimal conditions and successfully constructing an in vitro 3D model simulating in vivo *P. aeruginosa* infection. This invention also uses the constructed in vitro *P. aeruginosa* chronic infection 3D model for further large-scale genomic screening of key regulatory genes, revealing the function and molecular mechanism of a key gene, PA3831. This invention facilitates large-scale gene screening and mechanistic studies in vitro, providing new evidence for the discovery of new diagnostic biomarkers or the development of relevant treatment measures for patients with chronic infections, and shows promising clinical application prospects.

[0028] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the in vitro 3D model of chronic Pseudomonas aeruginosa infection according to the present invention.

[0031] Figure 2 This invention optimizes the conditions for an in vitro 3D model of chronic Pseudomonas aeruginosa infection. (A) qPCR detection of expression changes of LadS, PA1414, gacA, and gacS genes at four time points (C1, C3, C5, and C7); (B) qPCR detection of expression changes of Psl, pelD, pelE, and AlgD genes at four time points (C1, C3, C5, and C7); (C) Biofilm formation detection and detection of extracellular polysaccharide and protein content.

[0032] Figure 3Joint analysis of transcriptome sequencing data from in vitro 3D models and in vivo acute infection models. (A) Experimental flowchart; (B) Heatmap of joint analysis of transcriptome sequencing data; (C) KEGG enrichment analysis results of highly and poorly expressed genes in C3D.

[0033] Figure 4 Screening for key genes in chronic Pseudomonas aeruginosa infection using transposon sequencing. (A) Experimental flowchart; (BC) Mutant genes with fluorescence values ​​above and below WT identified by transposon sequencing data analysis; (DE) Gene enrichment results for UP and DOWN, respectively.

[0034] Figure 5 PA3831 regulates the expression of virulence genes through the Fis / exsA signaling pathway. (A) Heatmap of differentially expressed genes in the PAXen41 and ΔPA3831 groups. (B) Heatmap of differentially expressed T3SS genes in the PAXen41 and ΔPA3831 groups. (C) SYTOX Green staining and flow cytometry analysis of cytotoxicity in A549 cells infected with PAXen41 or ΔPA3831; the right panel shows the percentage of cell death in the flow cytometry results. (DH) Expression levels of exsA (D) and some T3SS genes (EH) in the PAXen41, ΔPA3831, ΔPA3831+PL2a, and ΔPA3831+Fis groups.

[0035] Figure 6 To investigate how the absence of PA3831 induces antibiotic resistance through enhanced carbon metabolism. (A) KEGG analysis of upregulated genes in the ΔPA3831 group compared to the PAXen41 group. (B) Normalized expression of aceE, aceF, aceA, and glcB genes in PAXen41 and ΔPA3831. (C) Western blotting analysis of the interaction between PA3831 and its binding proteins in LC / MS results. (D) Spot detection of PAXen41, ΔPA3831, ΔPA3831+PL2a, and ΔPA3831+aceE strains grown on LB agar plates with or without ticarcillin. (E) Detection of pyruvate concentrations in PAXen41 and ΔPA3831 using a kit. (FH) qPCR detection of resistance-related genes.

[0036] Figure 7 This is a schematic diagram illustrating the functional mechanism of PA3831 in chronic Pseudomonas aeruginosa infection. Detailed Implementation

[0037] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0038] The following experiments, where no temperature is specified, are reactions conducted under normal temperature conditions, which is room temperature, or 25±5℃.

[0039] The PAXen41 strain was donated by Dr. S. Lory (Harvard Medical School).

[0040] Example 1: Preparation of an in vitro 3D model of chronic Pseudomonas aeruginosa infection

[0041] 1. Activation of PAXen41 strain

[0042] PAXen41 strain was activated by streaking, and single clones were picked and incubated overnight at 37°C and 220 rpm in modified SCFM2 medium (composition shown in Table 1). The next day, the culture was inoculated into fresh modified SCFM2 medium at a ratio of 1:100 and placed in a shaker at 37°C and 220 rpm for 4 hours.

[0043] 2. A 3D system based on type I rat tail collagen as a scaffold is configured.

[0044] For example, in a 10 mL system: first, add 1 mL of 10×PBS to a 15 mL BD tube, then add 1 M NaOH (with a volume ratio of 0.023:1 to type I rat tail collagen) to adjust the pH to 7.0-7.2, add type I rat tail collagen to a final concentration of 1 mg / mL, and then add fetal bovine serum (FBS) (final concentration 40%, v / v) to a total system volume of 10 mL. Mix well and transfer to a 96-well plate on ice, 100 μL / well.

[0045] 3. In vitro 3D model assembly and key indicator detection

[0046] Take 100 μL / well (1*10) of the logarithmic phase bacterial culture obtained in step 1. 6 (1 bacterial culture / well) was added to the 3D system obtained in step 2, using type I rat tail collagen as a scaffold. The 96-well plate was then sealed with sealing film and incubated in a cell culture incubator (37℃, 5% CO2). Bacterial culture was collected from 6 wells / sample at days 1, 3, 5, and 7, lysed with Trizol, and RNA was extracted. The expression levels of LadS, PA1414, gacA, gacS, Psl, pelD, pelE, and AlgD genes were detected by qPCR. The rpsL gene was used as an internal control.

[0047] The remaining samples at days 1, 3, 5, and 7 were analyzed for biofilm formation and the content of extracellular polysaccharides and proteins. Biofilm formation was determined using crystal violet staining, extracellular polysaccharides were detected using the phenol-sulfuric acid method, and protein content was quantified using the BCA method.

[0048] The experimental results are as follows:

[0049] Samples cultured for 1, 3, 5, and 7 days according to the above steps (labeled C1, C3, C5, and C7 respectively) were collected for testing of known key genes controlling the transition from acute to chronic infection (LadS, PA1414, gacA, gacS). Figure 2 A) Key genes for biofilm formation (pelE, pelE, Psl, AlgD) Figure 2 B) Formation of biofilms and detection of their components (extracellular polysaccharides and proteins) Figure 2 C). It can be seen that the model can highly express key genes controlling the transition from acute to chronic infection, as well as key genes controlling biofilm formation, extracellular polysaccharides, and proteins, with the highest expression levels observed in the model obtained after 3 days of culture. Therefore, day 3 is the optimal culture time, and the in vitro Pseudomonas aeruginosa chronic infection 3D model obtained by culturing for 3 days according to the above steps is the preferred model of this invention.

[0050] The key genes controlling the transition from acute to chronic infection (LadS, PA1414, gacA, gacS), the key genes controlling biofilm formation (pelE, pelE, Psl, AlgD), and the components (extracellular polysaccharides and proteins) controlling biofilm formation are all known. References are as follows:

[0051] 1. Broder, UN, Jaeger, T. & Jenal, U. LadS is a calcium-responsive kinase that induces acute-to-chronic virulence switch in Pseudomonas aeruginosa. NatMicrobiol 2, 16184, doi: 10.1038 / nmicrobiol.2016.184 (2016).

[0052] 2.Gooderham, WJ&Hancock, RERegulation of virulence and antibioticresistance by two-component regulatory systems in Pseudomonas aeruginosa. FEMSMicrobiol Rev 33, 279-294, doi:10.1111 / j.1574-6976.2008.00135.x(2009).

[0053] 3.Cao,P.,Fleming,D.,Moustafa,DAet al.A Pseudomonas aeruginosa smallRNA regulates chronic and acute infection.Nature 618,358-364,doi:10.1038 / s41586-023-06111-7(2023).

[0054] 4.Singh, PK, Schaefer, AL, Parsek, MR et al. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407,762-764, doi:10.1038 / 35037627(2000).

[0055] 5. Koo, H., Allan, RN, Howlin, RPet al. Targeting microbial biofilms: current and prospective therapeutic strategies. Nat Rev Microbiol 15, 740-755, doi:10.1038 / nrmicro.2017.99(2017).

[0056] Table 1. Composition and proportions of the modified SCFM2 culture medium in the in vitro 3D model.

[0057]

[0058] The following is the preparation method for the comparative example.

[0059] Comparative Example 1: Preparation of an in vitro 3D model using Matrigel as a scaffold

[0060] The preparation method of the C3D sample in Example 1 was followed, except that the type I rat tail collagen scaffold was replaced with a Matrigel scaffold, thus obtaining an in vitro 3D model (i.e., M3D) using Matrigel as the scaffold. Comparative Example 2: Preparation of an acute infection model in mice.

[0061] Eight-week-old female C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were intratracheally injected with Pseudomonas aeruginosa (logarithmic growth phase PAXen41 strain) to adjust OD. 600 The infection dose per mouse was 1*10-17 A mouse acute infection model was obtained by mixing 50 μL of the sample. Lung tissue was collected 24 hours later for transcriptome sequencing.

[0062] The following experimental examples demonstrate the beneficial effects of the present invention.

[0063] Experimental Example 1: Validation of the in vitro 3D model of chronic Pseudomonas aeruginosa infection according to the present invention

[0064] Lung tissue from the acute in vivo infection model (AI) obtained in Comparative Example 2 was collected and subjected to transcriptome sequencing together with two in vitro infection models using different scaffolds obtained in Example 1 and Comparative Example 1. Figure 3 A) After obtaining the transcriptome sequencing data, it was analyzed in conjunction with publicly available datasets (Pseudomonas aeruginosa transcriptome sequencing data from clinical wound and sputum samples, i.e., clinical chronic infection samples, named burn wound, chronic wound, and sputum, respectively). The results are as follows: Figure 3 As shown in Figure B, the sequencing data of the in vitro 3D model (M3D) sample constructed using Matrigel as a scaffold are more similar to the sequencing data of the in vivo acute infection model (AI) sample, while the sequencing data of the C3D sample of this invention are more similar to the sequencing data of the clinical chronic infection sample. This indicates that the in vitro 3D model (M3D) constructed using Matrigel as a scaffold is more similar to the acute infection model, while the in vitro 3D model (C3D) constructed using the type I rat tail collagen scaffold of this invention is more similar to the chronic infection model.

[0065] Further enrichment analysis revealed that genes with high and low expression were enriched in the "ABC transporter" and "Flagellar assembly" pathways, respectively. Figure 3 C).

[0066] The above results indicate that the present invention has successfully constructed a collagen-based 3D in vitro culture model of Pseudomonas aeruginosa that can simulate a chronic infection environment.

[0067] Experimental Example 2: Large-scale in vitro gene screening and identification of key genes regulating chronic infection

[0068] A Tn5 transposon mutant library (27,648 single clones) was further established, and a large-scale screening was conducted using the in vitro Pseudomonas aeruginosa chronic infection 3D model of this invention. A series of candidate genes playing a role in chronic Pseudomonas aeruginosa infection were identified by Tn-seq. Figure 4 ).

[0069] The specific experimental method was as follows: *Escherichia coli* S17-lpir containing the tagged plasmid pUTminiTn5 was used as the donor, and the plasmid was transformed into the recipient strain *Pseudomonas aeruginosa* PAXen41 at a ratio of 1 donor cell to 10 recipient cells. Bacterial cells were mixed into 50 ml and dropped onto a sterile nylon membrane, which was then placed on a non-selective LB agar plate. The plate was incubated at 30°C for 18 h. The filter was washed with 1 ml of sterile LB to restore the bacteria. These transzygotes were then placed on 30 agar plates and incubated at 37°C for 18 h. Next, 27,648 mutants were generated and cultured overnight in SCFM2 at 37°C with shaking (220 rpm), with 40% glycerol added for preservation and propagation. For further screening, these mutants were incubated overnight in SCFM2 at 37°C with shaking (220 rpm), then inoculated 1:100 into fresh medium for 4 hours, followed by inoculation into 3D in vitro models for 3 days. Finally, their fluorescence values ​​were measured using a fluorescence microplate system (SuPerMax 3100). After screening for fluorescence values, the selected mutants were inoculated onto LB agar plates and incubated at 37°C for 18 hours, then used to generate transposon sequencing results (performed by Watson Biotechnology Co., Ltd.).

[0070] Further experimental verification confirmed that the PA3831 gene is an important gene regulating virulence and drug resistance, and that the deletion of this gene showed lower cytotoxicity (cytotoxicity assay method: A549 cells were infected with Pseudomonas aeruginosa strain PAXen41 and PA3831-deleted strain, with an infection fold (MOI) of 20. After infection at 37°C for 3 hours, the cells were analyzed using SYTOX according to the manufacturer's instructions). TM Green staining (Invitrogen) TM (S7020, USA) to detect cell death in A549 cells. The study also examined the Fis / exsA axis-mediated mechanism (determined by detecting changes in exsA and T3SS-related gene expression using Q-PCR after Fis reinjection in PA3831-deficient strains). Figure 5 ), and its absence favors the drug-resistant phenotype (different Pseudomonas aeruginosa PAXen41 strains were serially diluted 7 or 8 times in LB medium. The diluted bacteria were spotted on LB agar plates with or without antibiotics (16 μg / mL ticarcillin). Incubated overnight at 37°C. Then photographed), by combining pyruvate dehydrogenase (activity detection method: bacterial cells were collected by centrifugation, resuspended in the extract, and then sonicated. The supernatant was used for further detection. Pyruvate was detected using a pyruvate concentration assay kit (Beyotime, SO299S) according to the instructions. Pyruvate was measured at 570 nm using a spectrophotometer, and the pyruvate concentration was calculated.) and carbon metabolism were achieved ( Figure 6Therefore, PA3831 regulates colonization in chronic lung infections by modulating virulence and drug resistance. Figure 7 PA3831 positively regulates the type III secretion system (T3SS) via Fis, while simultaneously downregulating antibiotic resistance through pyruvate dehydrogenase (aceE)-mediated carbon metabolism. These dual mechanisms inhibit the chronic infection process of Pseudomonas aeruginosa. This gene holds promise as a diagnostic biomarker for clinical Pseudomonas aeruginosa infections.

[0071] In summary, this invention, using collagen as a scaffold, successfully constructed an in vitro 3D model simulating in vivo Pseudomonas aeruginosa infection by simulating the chronic infection environment of Pseudomonas aeruginosa and screening for optimal conditions. This invention also used the constructed in vitro 3D model of chronic Pseudomonas aeruginosa infection for further large-scale genomic screening of key regulatory genes, revealing the function and molecular mechanism of a key gene, PA3831. This invention facilitates large-scale gene screening and mechanistic studies in vitro, providing new evidence for the discovery of new diagnostic biomarkers or the development of relevant treatments for patients with chronic infections, and shows promising clinical application prospects.

Claims

1. A 3D model of in vitro infection, characterized in that, The in vitro infection 3D model was prepared through the following steps: (1) Culturing pathogenic bacterial strains; (2) Preparation of collagen solution; (3) Mix the pathogenic bacterial strain from step (1) with the collagen solution obtained in step (2) and culture for 1 to 7 days to obtain an in vitro infection 3D model.

2. The in vitro infection 3D model according to claim 1, characterized in that, The pathogenic bacteria in step (1) is Pseudomonas aeruginosa; the culture is to culture the pathogenic bacterial strain to the logarithmic phase.

3. The in vitro infection 3D model according to claim 2, characterized in that, The pathogenic bacteria is *Pseudomonas aeruginosa*; the culture medium is SCFM2 medium or a modified SCFM2 medium, wherein the modified SCFM2 medium includes the following components: aspartic acid, threonine, serine, glutamate, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, tryptophan, histidine, arginine, magnesium sulfate heptahydrate, citric acid, dipotassium hydrogen phosphate, and sodium hydrogen phosphate tetrahydrate.

4. The in vitro infection 3D model according to claim 1, characterized in that, The preparation method of the collagen solution in step (2) includes the following steps: mixing buffer, collagen and serum to obtain the solution.

5. The in vitro infection 3D model according to claim 4, characterized in that, The collagen is type I rat tail collagen.

6. The in vitro infection 3D model according to claim 4, characterized in that, The buffer solution is PBS buffer with a pH of 7.0–7.2; the collagen solution has a final collagen concentration of 1 mg / mL and a final serum concentration of 40%; the serum is fetal bovine serum.

7. The in vitro infection 3D model according to claim 1, characterized in that, The culture conditions described in step (3) are 37°C and 5% CO2.

8. The in vitro infection 3D model according to claim 1, characterized in that, The cultivation time described in step (3) is 3 days.

9. A method for constructing an in vitro infection 3D model according to any one of claims 1 to 8, characterized in that, The method is as follows: (1) Culturing pathogenic bacterial strains; (2) Preparation of collagen solution; (3) Mix the pathogenic bacterial strain from step (1) with the collagen solution obtained in step (2) and culture for 1 to 7 days to obtain an in vitro infection 3D model.

10. Use of the in vitro infection 3D model according to any one of claims 1 to 8 in the study of infection mechanisms, screening for prevention and / or treatment of infections.

Images