Application of shigella effector protein in regulation and control of intestinal pathogenicity
By screening and identifying the key toxic effector protein IpaJ of Shigella and knocking out the IpaJ gene through gene editing technology, the problem of difficult to understand and control the pathogenic mechanism of Shigella in the existing technology is solved, and the Shigella is weakened The effect of ella bacteria's pathogenic ability provides new ideas for the development of new drugs.
Patent Information
- Application Number
- CN202411710385.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-21
AI Technical Summary
The prior art is difficult to effectively understand and control the pathogenic mechanisms mediated by Shigella through T3SS, especially in the process of intestinal pathogenesis, and there is a lack of targeted therapeutic methods for key toxic effector proteins.
By screening and identifying the key toxic effector protein IpaJ that invades epithelial cells by Shigella infection, the IpaJ gene was obtained by forward screening, and the IpaJ gene was knocked out through gene editing of λ-Red homologous recombination to attenuate Shigella pathogenic ability.
It reveals the toxic function of Shigella effector protein IpaJ during the infection process, provides a theoretical basis for the development of T3SS small molecule inhibitors, weakens Shigella's intestinal pathogenic ability, and has important clinical application value.
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Figure CN119490572A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular biology, and specifically relates to an application of Shigella effector protein in regulating intestinal pathogenicity. Background Art
[0002] Shigella is one of the main causes of infectious diarrheal diseases and is characterized by carrying a large plasmid encoding most of the virulence genes, including the type III secretion system (T3SS). T3SS effectors are key virulence factors that support the infection strategies of many clinically important Gram-negative pathogens. Effector proteins are a class of exosomes secreted by pathogens, which are crucial for important stages such as the tight binding of bacteria to the host, penetration of the host cell membrane, and survival in the host cell. Small molecule specific inhibitors of T3SS have some unique functions that are superior to traditional antibiotics and are expected to be developed into new drugs to control pathogens and effectively curb the development of drug resistance.
[0003] The main approach to understanding T3SS-mediated pathogenesis is to identify the application of one effector at a time, with the goal of piecing together individual functions and revealing the infection mechanism. Based on this, by understanding the pathogenicity of the Gram-negative bacillus Shigella and identifying Shigella toxic effector proteins, we can provide new ideas for understanding T3SS-mediated pathogenesis and provide theoretical support for the development of new drugs. Summary of the invention
[0004] The present invention screened and identified the key toxic effector protein of Shigella infection and invasion of epithelial cells, namely the pathogenic factor of the present invention. Specifically, the pathogenic factor is Shigella effector protein IpaJ, and the amino acid sequence of the effector protein IpaJ is shown in SEQ ID NO.1.
[0005] The present invention reveals through experiments that the effector protein IpaJ and its encoding gene play a toxic function in the process of Shigella infection and invasion of epithelial cells. The nucleotide sequence of the Shigella effector protein IpaJ encoding gene has an open reading frame as shown in SEQ ID NO.2.
[0006] Furthermore, the present invention provides the use of the pathogenic factor of the pathogen in intestinal pathogenesis. The pathogenic factor is Shigella effector protein IpaJ, and the amino acid sequence of the effector protein IpaJ is shown in SEQ ID NO.1.
[0007] The present invention adopts a forward screening method to obtain a Shigella effector protein IpaJ gene, wherein the IpaJ gene encodes the Shigella effector protein IpaJ, and the nucleotide sequence has an open reading frame as shown in SEQ ID NO.2, and the amino acid sequence of the effector protein IpaJ is shown in SEQ ID NO.1.
[0008] Furthermore, the present invention provides the use of the Shigella effector protein IpaJ gene in intestinal pathogenicity.
[0009] A person skilled in the art can reduce the pathogenicity of Shigella by knocking out the IpaJ gene in combination with the existing technology.
[0010] In order to understand the application of pathogenic factors in the intestinal pathogenic process, the present invention also provides a method for knocking out Shigella genes. The method is to knock out the Shigella effector protein IpaJ gene by gene editing using λ-Red homologous recombination to obtain a mutant strain in which the effector protein IpaJ gene is knocked out, and the nucleotide sequence of the IpaJ gene knockout fragment is shown in SEQ ID NO.3.
[0011] Furthermore, as an implementation method for Shigella gene knockout, the method includes constructing a targeting fragment required for knocking out the Shigella effector protein IpaJ gene, electroporating it into a wild-type Shigella containing a Psim17 plasmid, and using λ-Red homologous recombination to knock out the gene to obtain a mutant Shigella strain of the effector protein IpaJ gene.
[0012] Based on the records of the present invention, the present invention also requests protection for a targeting fragment required for knockout, which replaces the Shigella effector protein IpaJ gene with a chloramphenicol resistance gene cassette, the front end is 1000bp upstream of the effector protein IpaJ gene, the middle is a sequence encoding the chloramphenicol resistance gene, and the rear end is 1000bp downstream of the effector protein IpaJ gene. The nucleotide sequence of the targeting fragment required for IpaJ gene knockout is shown in SEQ ID NO.3.
[0013] In order to fully and unambiguously understand the technical solution of the present invention, it is necessary to supplement that the Shigella effector protein of the present invention is represented by a non-italic font "IpaJ", and the Shigella effector protein gene is represented by an italic font "IpaJ". Of course, those skilled in the art can clearly and completely understand the meaning and expression of the relevant genes and their encoded proteins based on the description of the present invention.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] 1. The present invention discloses that the Shigella effector protein IpaJ plays a toxic function in Shigella-infected epithelial cells and is a key toxic effector protein of Shigella. The effector protein IpaJ has an amino acid sequence as shown in SEQ ID NO.1. The present invention lays a theoretical foundation for the development of T3SS small molecule inhibitors using pathogenic factors of pathogens by analyzing its pathogenic mechanism, which is of great significance for the development of new drugs for pathogens and the prevention of the generation of drug resistance.
[0016] 2. The present invention utilizes a transient overexpression method to analyze the effector protein IpaJ gene, which can cause cell death in 293T cells; the IpaJ gene is knocked out by a λ-Red homologous recombination gene knockout method, and it is determined that IpaJ plays an important role in the infection and invasion of Shigella and epithelial cells.
[0017] 3. The present invention is of great significance in the development of T3SS small molecule specific inhibitors. By using genetic engineering technology, the intestinal pathogenicity of Shigella is weakened by knocking out the Shigella effector protein IpaJ gene. The present invention provides a new solution for the development of new drugs for controlling pathogens from the perspective of molecular biology. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings involved in the embodiment of the present invention are briefly described below. Obviously, the listed drawings only present part of the content of the embodiment of the present invention.
[0019] Figure 1 It is the technical roadmap of the present invention.
[0020] Figure 2 It is a schematic diagram of the morphology of the Shigella effector protein IpaJ promoting cell death described in the examples of the present invention.
[0021] Figure 3 a and Figure 3 b is a quantitative graph showing that the Shigella effector protein IpaJ promotes cell death as described in the examples of the present invention.
[0022] Figure 4 It is a schematic diagram of the construction of the targeting fragment required for knocking out the Shigella effector protein IpaJ described in the examples of the present invention.
[0023] Figure 5 a and Figure 5 b is a PCR gel image of the target fragment required for knocking out the Shigella effector protein IpaJ described in the example of the present invention.
[0024] Figure 6 This is a diagram showing the PCR identification results of the Shigella effector protein IpaJ knockout described in the examples of the present invention.
[0025] Figure 7 a and Figure 7 b is a graph showing the comparison of the invasive ability of the wild type and mutant Shigella described in the examples of the present invention. DETAILED DESCRIPTION
[0026] Figure 1 The technical roadmap of the present invention is given. The embodiment of the present invention provides a method for verifying the toxicity function of Shigella effector protein IpaJ, including:
[0027] S101, using the Shigella genome as a template, cloned the IpaJ gene into a eukaryotic expression vector, and transferred it into 293T cells by calcium transfer to make it transiently overexpressed. The cell morphology was observed at 24h and 48h, and the supernatant was collected to detect LDH to determine whether the Shigella effector protein IpaJ gene can induce cell death;
[0028] S102, construct the target fragment required for knocking out the Shigella effector protein IpaJ by Overlap PCR, electroporate it into the wild-type Shigella competent cells containing Psim17, and use the gene editing technology of λ-Red homologous recombination to specifically knock out the effector protein gene IpaJ and clarify its toxic function;
[0029] S103, obtaining a mutant strain with IpaJ gene knockout, and performing molecular detection and verification on the mutant strain;
[0030] S104, infect Hela cells with the wild-type strain and the mutant strain, compare the changes in the invasion ability of the two strains on Hela cells, and determine the changes in the pathogenicity of the mutant strain.
[0031] Primer table
[0032]
[0033]
[0034] The verification method and identification method of the toxic function of the Shigella effector protein IpaJ are now described in detail in conjunction with Examples 1-4.
[0035] Example 1
[0036] This example describes the isolation and cloning of the Shigella effector protein gene IpaJ
[0037] Will contain Psim17 +The Shigella were streaked on LB plates and cultured overnight in a static shaker at 30°C; on the second day, single clones were picked out and cultured in 50 ml of LB liquid at 30°C, 220 rpm overnight; on the third day, the overnight bacteria were centrifuged at room temperature, 5000 rpm, for 10 min, the supernatant was discarded, the colony precipitate was transferred to a ceramic dish, and it was ground into powder with liquid nitrogen, and the genomic DNA was extracted using the TRlzol method.
[0038] Using this as a template, PCR amplification was performed using IpaJ gene-specific primers IpaJ-F and IpaJ-R to amplify a DNA fragment containing the full-length coding frame of the IpaJ gene. PCR reaction conditions: 94°C pre-denaturation for 2 min; 94°C for 15 s, 52°C for 30 s, 68°C for 40 s, 30 cycles; PCR products were identified by 1% agarose gel electrophoresis, then cut and purified, and dissolved in deionized water for later use.
[0039] The plasmid PHAGE-BSD-nFlag was double-digested with SphI and XbaI enzymes (NEB) to linearize the vector. The double-digestion reaction conditions of the vector were: 37°C, 2h; the digestion product was identified by 1% agarose gel electrophoresis, then the gel was cut, recovered, purified, and dissolved in deionized water for later use.
[0040] The obtained PCR amplification product was connected to the linearized vector by seamless cloning (Yesen Company), transformed into Escherichia coli Stable3 competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), positive clones were screened and detected, and the cells were shaken overnight. The extracted plasmid was named PHAGE-IpaJ, and Qingke Biotechnology Co., Ltd. was commissioned for sequencing to obtain the nucleotide sequence shown in SEQ ID NO.2.
[0041] The nucleotide sequence obtained above was searched by Blastx on the NCBI website (http: / / www.ncbi.nlm.nlm.nih.gov / ) to obtain the amino acid sequence shown as SEQ ID NO.1.
[0042] Example 2
[0043] This example describes that the Shigella effector protein IpaJ causes cell death.
[0044] The correctly sequenced PHAGE-IpaJ plasmid was transferred into 293T cells by calcium transfection. The medium was changed 8 hours after transfection, and the cell morphology was observed at 24 and 48 hours. The cells were collected to detect whether the protein was expressed and the supernatant was collected to detect LDH to measure the cell mortality rate.
[0045] like Figure 2 , Figure 3 a and Figure 3As shown in Figure b, after transient overexpression of the effector protein IpaJ in 293T cells, it was observed that the effector protein IpaJ can cause obvious cell morphological changes, mainly manifested as cells becoming rounded and suspended in pieces, and releasing a certain amount of lactate dehydrogenase LDH. It can be seen that the Shigella effector protein IpaJ can clearly cause cell morphological changes and lead to cell death.
[0046] Example 3
[0047] This example describes the knockout of Shigella effector protein IpaJ by gene editing through λ-Red homologous recombination and molecular detection verification. Figure 4 Shown is a flowchart for constructing targeting fragments.
[0048] First, using the pKD3 plasmid as a template, primers CmR-F and CmR-R were designed to amplify the chloramphenicol resistance gene with FRT sites on both ends of the plasmid. Then, using the Shigella genomic DNA as a template, primers yH1-F and yH1-R were designed to amplify the 1000bp sequence upstream of the IpaJ gene and primers yH2-F and yH2-R were designed to amplify the 1000bp sequence downstream of the IpaJ gene. Reaction conditions: 94°C, 2min; 94°C 15s, 52°C 30s, 68°C 60s, a total of 27 cycles; the PCR product was identified by 1% agarose gel electrophoresis, then excised, recovered, purified, and dissolved in deionized water for later use. Figure 5 a shows the gel image of PCR amplification.
[0049] Using the recovered fragment as a template, the 1000bp sequence upstream of the effector protein IpaJ and the chloramphenicol resistance gene were connected by OverlapPCR amplification, and then the connected fragment and the 1000bp sequence downstream of the effector protein IpaJ were connected again by OverlapPCR amplification. Reaction conditions: 94℃, 2min; 94℃15s, 55℃30s, 68℃60s, a total of 32 cycles; PCR products were identified by 1% agarose gel electrophoresis, excised, recovered, purified, and dissolved in deionized water for later use.
[0050] Plasmid pcDNA.3-cHA was digested with EcoR I (NEB) to linearize the vector. The conditions for the single digestion reaction were as follows: 37°C, 2h; the digestion product was identified by 1% agarose gel electrophoresis, then recovered and purified, and dissolved in deionized water for later use.
[0051] The final ligation product was connected to the linearized vector by seamless cloning (Yesen Company), transformed into Escherichia coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), positive clones were screened and detected, and the cells were shaken overnight. The extracted plasmid was named pcDNA.3-ΔIpaJ and commissioned to Qingke Biotechnology Co., Ltd. for sequencing.
[0052] Using the pcDNA.3-ΔIpaJ plasmid with the correct sequencing results as a template, primers ΔIpaJ-F and ΔIpaJ-R were designed to amplify the target fragment. The reaction conditions were: 94°C, 2 min; 94°C 15 s, 55°C 30 s, 68°C 180 s, a total of 30 cycles; the PCR product was identified by 1% agarose gel electrophoresis, excised, recovered, purified, and dissolved in deionized water for later use. Figure 5 b shows the PCR amplification gel image of the target fragment.
[0053] The Shigella strain containing the pSim17 plasmid (expressing the three enzymes required for λ-Red homologous recombination - exo, bet and gam, temperature-sensitive plasmid, BSD resistance) was placed on a 30°C LB plate (containing BSD antibiotics, 50ug / ml) overnight for 36 hours. The next day, a single clone was picked and cultured in 4ml LB liquid culture medium containing BSD resistance at 30°C, 220rpm in a shaker for 12 hours overnight. On the third day, 30μl of the overnight bacterial solution was taken in 30ml (1:100) LB liquid culture medium containing BSD resistance, cultured at 30°C, 220rpm until OD600 = 0.5, and then transferred to a 42°C water bath for 15 minutes to induce the pSim17 plasmid to express the recombinase. Then cool on ice for 30 minutes, centrifuge at 4°C and 4000g for 5 minutes, discard the supernatant, wash 3 times with ice-cold deionized water, and then wash 3 times with ice-cold 10% sterile glycerol water. After each wash, centrifuge at 4°C and 4000g for 5 minutes, and discard the supernatant. Add 1000μl of ice-cold 10% sterile glycerol water to obtain the competent cells of wild-type Shigella. After quick freezing in liquid nitrogen, store at -80°C for later use.
[0054] Add 5μl (about 300ng) of the gel recovery product of the target fragment to 100μl of competent cells, mix well and place on ice for 30 minutes. Add the mixed solution to a clean pre-cooled electroporation cup and electroporate under the conditions of resistance 200Ω, voltage 1800V, and capacitance 25uF. After the electroporation is completed, quickly add 2ml of LB culture medium and shake the bacteria at 30℃ and 220rpm for 4h. Centrifuge the revived bacterial solution at 12000rpm for 1min, discard the supernatant and resuspend 100μl, take 100μl of the resuspended bacterial solution and spread it on the LB plate containing chloramphenicol resistance, and culture it at 30℃ overnight. Then pick the positive colonies grown on the plate for PCR identification using primers F and R, and send them to Qingke Biotechnology Co., Ltd. for sequencing verification. Figure 6 Shown is a schematic diagram of the PCR identification results of Shigella knockout IpaJ gene.
[0055] Example 4
[0056] This example describes how knocking out the Shigella effector protein gene IpaJ reduces the pathogenicity of Shigella.
[0057] After cervical cancer epithelial cells Hela were inoculated into 12-well plates and cultured for 24 hours, the cells were infected with Shigella wild-type strain and IpaJ mutant strain at an infection multiple of 100. This was repeated for 3 groups, and the blank cell group was used as a negative control. The above cells were placed in a 37°C, 5% CO2 incubator for co-incubation.
[0058] For the invasion experiment, 150 μg / ml gentamicin was added to the cell culture medium 120 minutes after the host cells were infected and incubated for 1 hour to kill extracellular bacteria. After 180 minutes of infection, the cells were rinsed three times with sterile PBS, and 100 μl of 0.1% Titox X-100 was added to lyse the cells for 20 minutes. Each well was repeatedly blown to mix the suspension. The suspension was aspirated and diluted in multiples, then spread on the LB plate, placed in a 30°C incubator for 36 hours, and the colonies were counted. The relative invasion rate was calculated. The invasion rate is calculated by dividing the number of recovered bacteria by the total number of bacterial cells used for infection. Figure 7 a is a plate image of Shigella after multiple dilutions. Figure 7 b is the calculated invasion rate.
[0059] In summary, the present invention discloses a toxic function of Shigella effector protein IpaJ and its application in intestinal pathogenicity. The amino acid sequence of Shigella effector protein IpaJ is shown in SEQ ID NO.1. The present invention reveals the key effector protein gene IpaJ in Shigella and epithelial cell infection and invasion, which has a development reading frame as shown in SEQ ID NO.2. The key effector protein gene IpaJ was knocked out by λ-Red homologous recombination to obtain a mutant Shigella strain with IpaJ gene deletion, and it was determined that the IpaJ gene played a toxic function in the process of Shigella and epithelial cell infection and invasion, laying a theoretical foundation for the development of T3SS small molecule inhibitors using pathogenic factors.
[0060] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered within the protection scope of the present invention.
Claims
1. A pathogenic factor, characterized in that: The pathogenic factor is the Shigella effector protein IpaJ, and its amino acid sequence is shown in SEQ ID NO.
1.
2. The pathogenic factor according to claim 1, characterized in that: The Shigella effector protein IpaJ and its encoding gene play a toxic function in Shigella-infected and invaded epithelial cells.
3. Use of the pathogenic factor according to claim 1 in reducing the intestinal pathogenicity of Shigella.
4. The use according to claim 3, characterized in that: The application is achieved by knocking out the effector protein IpaJ gene.
5. A Shigella effector protein IpaJ gene, characterized in that: The IpaJ gene encodes the Shigella effector protein IpaJ, and the nucleotide sequence has an open reading frame as shown in SEQ ID NO.2; The amino acid sequence of the effector protein IpaJ is shown in SEQ ID NO.
1.
6. A method for reducing the pathogenicity of Shigella, characterized in that: The Shigella effector protein IpaJ gene was knocked out to obtain a mutant Shigella strain lacking the effector protein IpaJ gene. The nucleotide sequence of the IpaJ gene-deficient fragment is shown in SEQ ID NO.
3.
7. The method according to claim 6, characterized in that: The Shigella effector protein IpaJ gene knockout includes: The targeting fragment required for knocking out the Shigella effector protein IpaJ gene was constructed and electroporated into wild-type Shigella containing the Psim17 plasmid. The mutant Shigella strain lacking the effector protein IpaJ gene was obtained by gene knockout using λ-Red homologous recombination.
8. A targeting fragment required for knocking out the effector protein IpaJ gene, characterized in that: The Shigella effector protein IpaJ gene was replaced with a chloramphenicol resistance gene cassette. The front end of the chloramphenicol resistance gene cassette is 1000bp upstream of the effector protein IpaJ gene, the middle is the sequence encoding the chloramphenicol resistance gene, and the back end is 1000bp downstream of the effector protein IpaJ gene. The nucleotide sequence of the targeting fragment is shown in SEQ ID NO.3.