Application of global stress response element DdaA in Murrazidae

Abstract

The invention discloses an application of a global stress response element DdaA in Murraziaceae. The NCBI protein sequence of the DdaA is WP000117148.1, the DdaA is used as a target site of drugs or stress and is used for preventing and treating conditional pathogen environmental microorganism moraliaceae bacteria, and the moraliaceae bacteria include but are not limited to acinetobacter baumannii. By disturbing the DdaA regulatory network, the stability of the bacterial genome of the Murraziaceae is reduced. By disturbing the DdaA regulatory network, the sensitivity of the Murraziaceae bacteria to bacteriophage infection is improved. The DdaA pathway targeted drug is used for preventing and treating by adjusting the response of moraxaceae bacteria to environmental stress. The DdaA pathway targeted drug is combined with phage therapy for prevention and treatment. Environmental stress includes and is not limited to ultraviolet irradiation and mitomycin treatment. And the drug target is a network regulated and controlled by DdaA. The medicine comprises a DNA (Deoxyribose Nucleic Acid) fragment or plasmid containing a multi-repeat DdaA binding sequence 'CGWCANNNNNTGWCG'. The composition is suitable for preventing and treating multi-drug-resistant acinetobacter baumannii And combined use can be realized, and a superposition effect is generated.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to the application of a transcription regulatory factor in the prevention and treatment of Acinetobacter baumannii. Background Art

[0002] Bacteria are tiny and simple in structure, and they frequently exchange substances between the external environment and cells. Stress such as radiation, drought, high temperature, heavy metals, reactive oxygen species and high osmotic pressure can cause abnormal bacterial cell metabolism, causing varying degrees of damage to genomic DNA, affecting the life process of microorganisms and threatening their survival and reproduction.

[0003] Rapid and accurate repair of damaged DNA molecules is essential for bacterial survival, and this process is mainly completed by DNA damage repair proteins. These pathways are regulated by the global DNA damage response system.

[0004] Acinetobacter baumannii is a type of bacteria in the genus Acinetobacter, class γ-Proteobacteria, and family Moraxella. It is the most common Gram-negative bacillus in the genus Acinetobacter. It is widely found in water and soil in nature, hospital environment, human skin, respiratory tract, digestive tract and urogenital tract, and is a conditional pathogen. The bacteria are widely distributed in hospital environments and can survive for a long time. They are important pathogens of hospital infections, so they are often isolated from specimens such as blood, urine, pus and respiratory secretions of infected patients. It has a strong stress response ability and a trend of increasing resistance to commonly used antibiotics year by year, which has attracted widespread attention from clinicians and microbiologists. Since it has been reported that the mortality rate caused by multidrug-resistant Acinetobacter baumannii in the family Moraxella is as high as 70%, the main measures to deal with infection now include the use of antibiotics, phage therapy and the development of vaccines.

[0005] Since Acinetobacter baumannii has an outstanding ability to acquire drug-resistant genes and resist harsh living conditions, it is resistant to most first-line antibiotics. Therefore, there is an urgent need to find the action sites of new antimicrobial drugs against these drug-resistant strains. Studies have suggested that the DNA stress system of pathogenic microorganisms affects the antibacterial efficiency of drugs, and the study of their DNA damage response system can provide a theoretical basis for the development of target drugs. Summary of the invention

[0006] In response to the urgent need to prevent and treat multi-drug resistant Acinetobacter baumannii, the present invention deeply explores the global stress response system in Acinetobacter baumannii, discovers a new transcriptional regulatory factor DdaA, and provides the application of the global stress response element DdaA in the Moraxellaceae. DdaA specifically targets the DdaA regulatory network in Acinetobacter baumannii, has high specificity, and is different from traditional antibiotics that damage normal flora in the human body, and will not cause the strain to develop drug resistance in a short period of time.

[0007] An application of a global stress response element DdaA in Moraxellaceae, wherein the NCBI protein sequence of DdaA is WP_000117148.1, as a target site for drugs or stress, for preventing and treating conditional pathogenic environmental microorganisms Moraxellaceae bacteria, wherein the Moraxellaceae bacteria include but are not limited to Acinetobacter baumannii. The DdaA protein is derived from Acinetobacter baumannii Acinetobacter baumannii ATCC 17978, purchased from the American Type Culture Collection, number: ATCC 17978, the corresponding DNA sequence ID is: HKO16_RS19275. We first experimentally proved the existence of this protein and elucidated its function. DdaA is a WYL family transcription activator, and the DNA sequence that specifically interacts with it is first discovered by the present invention, the specific binding sequence "CGWCANNNNNTGWCG", SEQ ID No.5, where the two bases A and T can be represented as W, and N represents any base.

[0008] The application reduces the genome stability of Moraxellaceae bacteria by disrupting the DdaA regulatory network.

[0009] The application increases the sensitivity of Moraxellaceae bacteria to phage infection by disrupting the DdaA regulatory network.

[0010] In the application, the DdaA pathway targeted drug is used to prevent and treat Moraxellaceae bacteria by regulating their response to environmental stress.

[0011] In the application, DdaA pathway targeted drugs are combined with phage therapy for prevention and treatment.

[0012] The environmental stress includes but is not limited to ultraviolet radiation and mitomycin treatment.

[0013] The drug target is the network regulated by DdaA.

[0014] The drug comprises a DNA fragment or plasmid containing a multi-repeated DdaA binding sequence "CGWCANNNNNTGWCG".

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) As a widely existing transcriptional regulatory factor, it is suitable for the prevention and treatment of most bacteria in the Moraxellaceae family: it is suitable for the prevention and treatment of most pathogenic bacteria in the Moraxellaceae family, including Acinetobacter baumannii; it is suitable for the prevention, treatment and inactivation of Acinetobacter baumannii infections in medical environments, daily environments and in vivo.

[0016] (2) It is not affected by the known drug resistance of Acinetobacter baumannii and is suitable for the prevention and treatment of multidrug-resistant Acinetobacter baumannii. It can be used in combination with existing Acinetobacter baumannii prevention and treatment technologies to produce a cumulative effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the gel retardation experiment of DdaA confirm its specific binding to DNA.

[0018] Figure 2 The structure of DdaA protein, in which the WYL domain is marked in green and the WCX domain is marked in orange.

[0019] Figure 3 Sanger sequencing was used to verify the successful knockout of the DdaA system.

[0020] Figure 4 This is the effect of DdaA knockout on the transcriptome expression of Acinetobacter baumannii.

[0021] Figure 5 DdaA knockout results in reduced resistance to DNA damage.

[0022] Figure 6 DdaA knockout results in reduced phage resistance in Acinetobacter baumannii.

[0023] Figure 7 DdaA targeted drug treatment reduces the UV radiation resistance of Acinetobacter baumannii. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] Example 1: Induced expression and purification of Acinetobacter baumannii DdaA protein (1) Strain construction: Based on the in vitro expression system of the pET series vectors, IN-FUSION DNA recombination technology was used to design the amplification primers of the gene to be inserted according to the sequences on both sides of the vector. The DNA sequence encoding DdaA (NCBI gene: HKO16_RS19275) was cloned into the pET-28a vector and transferred into the Escherichia coli expression strain BL21 (DE3) after sequencing was correct.

[0026] (2) Bacterial lysis: Cultivate the expression strain to OD 600When the pH is about 0.4, add IPTG solution with a final concentration of 50mM, and culture Escherichia coli at 30 degrees for 5 hours to induce the massive expression of DdaA. This condition can be used for DdaA in different species. Use an ultracentrifuge to centrifuge the bacterial solution at 8000g for 5 minutes to collect the bacteria, and then resuspend the bacteria with His-A solution. Repeat the centrifugation and resuspension steps 3 times to completely wash away the culture medium remaining in the bacteria. Then use a pressure crusher to lyse the bacteria, and use an ultrasonic crusher to further lyse the bacteria, and break the DNA molecules in the bacteria to facilitate the subsequent purification process. At this time, the lysate should be a clear and transparent liquid. Then use the bacterial solution in an ultracentrifuge and centrifuge it at 15000g for 15 minutes to completely separate the soluble protein solution from the insoluble matter.

[0027] (3) Protein purification: The bacterial lysate was then filtered through a 0.22 μm pore size filter membrane and transferred to the ÄKTA purification system, where the expressed protein was purified using Ni affinity purification resin. The flow rate of the ÄKTA purification system was maintained at 1 mL / min throughout the process, and the A pump was filled with His-A solution and the B pump was filled with His-B solution through an automatic program. The loading loop and the nickel column were filled with His-A solution, and the lysate was then loaded onto the nickel column to allow the His-tag DdaA protein to be purified to fully bind to the nickel column. After the UV value and conductivity of the ÄKTA purification system stabilized, a mixture of 90% His-A and 10% His-B solution was used to elute the impurities bound to the nickel column. After the UV value and conductivity stabilized, a mixture of 50% His-A and 50% His-B solution was used to elute the His-tag DdaA protein bound to the nickel column, and the collected product was a DdaA protein solution with a high purity. The protein concentration was determined using NanoDrop1000, and the time and concentration were marked on the collection tube, which was then stored in a 4°C refrigerator.

[0028] Example 2: Prediction and verification of specific binding sequence of Acinetobacter baumannii DdaA protein The cis-acting element of DdaA protein was predicted, and a FAM-labeled double-stranded DNA substrate was designed and synthesized. Four point mutation substrates (SEQ ID No.1-SEQ ID No.4) were synthesized at the same time, and an appropriate buffer system was selected to anneal the primers. DdaA protein and FAM-labeled double-stranded substrate were added at a final molar concentration ratio of 20:1. After incubation for 30 minutes, the binding system was transferred to 12% Native-PAGE gel for electrophoresis and imaged with Typhoon FLA 9500. DdaA can specifically bind to the predicted target DNA fragment (SEQ ID No.5), and the conservative bases cannot be changed. Figure 1As shown, in the first lane, only double-stranded DNA containing the conserved DdaA binding site was added without DdaA protein; in the second lane, both DdaA protein and double-stranded DNA containing the conserved DdaA binding site were added, resulting in obvious gel retardation; DdaA protein was added to the remaining lanes, but the clip sequence for the specific binding of DdaA protein was mutated, and it can be seen that the gel retardation phenomenon was greatly reduced or even disappeared. DdaA contains three domains, of which the HTH domain at the N-terminus is responsible for double-stranded DNA binding, while the WYL and WCX at the C-terminus may be related to molecular signal transduction. At the same time, DdaA can form a stable dimer structure ( Figure 2 ), which is confirmed by the palindrome property of its combined sequence.

[0029] Example 3: Editing Acinetobacter baumannii using a gene editing system Using the existing CRISPR knockout system for Acinetobacter baumannii, dD The gene was subjected to nonsense mutation to obtain a DdaA system knockout strain. The specific steps are as follows: ① The pCasAb-apr vector was electroporated into Acinetobacter baumannii, and Ab::pCasAb-apr was obtained after apramycin screening. ② The sgRNA sequence was designed according to the DNA sequence to be knocked out, and the DNA sequence encoding the sgRNA was introduced into the pSGAb-kana vector using vector point mutation technology. ③ The pSGAb-kana containing the sgRNA sequence and the long single-stranded DNA designed according to the editing purpose were transferred into the Ab::pCasAb-apr strain induced by IPTG, and dual resistance to apramycin and kanamycin was screened. The obtained colonies were verified by PCR, and the PCR products were sequenced to confirm that the gene had been successfully knocked out. ④ The correctly sequenced strains were transferred to LB medium containing sucrose for culture, and after one day of culture, they were obtained. dD Scarless knockout strains. dD The knockout strains were verified by Sanger sequencing, and the results were as follows Figure 3 At the same time, to verify the following phenotype dD We used the pWH-1266 backbone to insert the DNA shown in NCBI gene: HKO16_RS19275 back into dD Scarless knockout strain, restoring the function of the DdaA system.

[0030] Example 4: DdaA regulatory network disturbance causes decreased DNA damage resistance in Acinetobacter baumannii Take wild-type Acinetobacter baumannii cultured overnight, dD The knockout strain and complemented strain were transferred to LB medium and cultured to the logarithmic growth phase (OD value between 0.6 and 0.8).

[0031] Transcriptome analysis: A. baumannii wild type, knockout dD The strain was streaked on an LB plate to recover. Fresh colonies were collected into liquid LB broth until OD 600 0.6, and then 2 mL of each strain culture was placed on a clean glass surface. The UV irradiation group received 15 J / m 2 UV irradiation, while the control group will not be treated at room temperature. The bacterial solution of each group was placed in an incubator at 37°C to allow the DNA damage reaction to continue for 15 minutes. Then, the bacteria were immediately centrifuged to the bottom of the EP tube, quickly frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted using RNAprep Pure Cell / BacteriaKit RNAprep Pure DP430 and purified by RNAClean XP Kit (Beckman Coulter) and RNase-free DNase Set (Qiagen). Libraries were constructed using U-mRNAseq Library Preparation Kit AT4221, KAITAI-BIO and RNA Depletion Kit (Bacteria, NovaSeq). Sequencing was performed using an Illumina NovaSeq machine, and the results were 150bp paired-end sequencing data. The representative genome of Acinetobacter baumannii ATCC 17978 was subsequently indexed by hisat2 and converted to a suitable format by samtools. The fastq file was cleaned by TrimmaticPE and mapped to the genome by hisat2. The expression matrix was calculated by stringtie, and differentially expressed genes were determined by Deseq2.

[0032] like Figure 4 As shown, the left part shows the transcriptome changes of wild-type Acinetobacter baumannii before and after radiation, and the right part shows dD Transcriptome changes of knockout strains before and after radiation. dD The knockout of the gene significantly reduced the DNA damage response of Acinetobacter baumannii at the transcriptional level under ultraviolet irradiation treatment.

[0033] Ultraviolet irradiation phenotype: Take 400uL of the bacterial solution of each strain and 2 , 10 3 , 10 4 , 10 5 , 10 6 Gradient dilution of bacterial solution was transferred to solid LB medium until the bacterial solution was completely absorbed by the solid medium. 2 , 5 J / m 2 , 10 J / m 2 , or 20 J / m 2The plates were placed in a 37°C incubator overnight, and the number of colonies was observed to compare the survival rates of the strains.

[0034] Mitomycin stress phenotype: Take the above bacterial solution and add mitomycin solution to make the final concentration of mitomycin 0.5, 1.0, 2.0, and 4.0 μg / mL. Incubate the bacterial solution at 37°C with shaking for 30 minutes, then incubate the bacterial solution at 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 Gradient dilution was performed and the culture was transferred to solid LB medium. After the bacterial solution was completely absorbed by the solid medium, the plate was placed in a 37°C incubator for overnight culture, and the number of colonies was observed to compare the survival rate differences among the strains.

[0035] like Figure 5 As shown, dD The knockout of the gene caused the survival rate of Acinetobacter baumannii to drop to 1% to 1% of the original when treated with ultraviolet radiation and mitomycin stress.

[0036] Example 5: DdaA regulatory network disturbance causes decreased phage resistance in Acinetobacter baumannii Take overnight cultured Acinetobacter baumannii ATCC17978 wild type, Acinetobacter baumannii wild type containing restriction endonuclease system, dD The knockout strain and the complemented strain were transferred to LB medium and cultured to the logarithmic growth phase (OD value between 0.6 and 0.8). When mitomycin (MMC) was needed to be added, the final concentration of MMC in the culture medium was 0.5 μg / mL, and the bacterial solution was placed in a 37°C incubator for 30 minutes to activate the DdaA network. The bacteria in 2 mL of the bacterial solution were collected by centrifugation at 7000g and resuspended with 2 mL of fresh LB medium to complete the washing process. Next, 200 μL of the resuspended bacterial solution was added to 5 mL of fresh LB medium, and PhAb24 phage with a multiplicity of infection (MOI) of 0.02 was added. The growth curves of these strains were then measured, and it was found that the knockout of DdaA significantly reduced the phage resistance of Acinetobacter baumannii ( Figure 6 ).

[0037] Example 6: Targeted drugs can inhibit the DdaA regulatory network, resulting in decreased resistance of Acinetobacter baumannii Design a plasmid containing multiple repeats of DdaA binding sites. The plasmid used in this example contains a sequence of multiple repeats of DdaA binding site DNA "CGWCANNNNNTGWCG" (as shown in SEQ ID NO.6), and the plasmid is constructed by synthesis. Take the wild type of Acinetobacter baumannii ATCC17978 cultured overnight and transfer it to LB medium and culture it to the logarithmic growth phase (OD value is between 0.6 and 0.8). Use the electroporation method to transfer the above plasmid into the wild type bacteria of Acinetobacter baumannii. Because these plasmids contain a large number of DdaA binding sequences, which far exceed the number of DdaA binding sequences in the genome, DdaA will be competitively bound to the plasmid DNA, causing the original regulatory function of DdaA to be lost. After resistance screening, a successfully transformed strain is obtained. The successfully transformed strain and the wild type strain are simultaneously subjected to 20 J / m 2 The strains were treated with ultraviolet radiation, and the strains without any treatment were used as the control group. Figure 7 As shown, DdaA pathway targeted drugs reduced the survival rate of Acinetobacter baumannii under ultraviolet irradiation to one-tenth of that without targeted drug treatment.

[0038] The above are only preferred embodiments of the present invention, but the present invention is not limited to the above detailed methods. According to the teachings and inspirations of the present invention, anyone familiar with this technical solution, using the transcriptional regulatory network characteristics of DdaA and its homologs disclosed in the present invention to carry out any prevention and control of bacteria in the Moraxellaceae family, are within the protection scope of the present invention.

Claims

1. An application of a global stress response element DdaA in Moraxellaceae, characterized in that: The NCBI protein sequence of the DdaA is WP_000117148.1, and it is used as a target site of a drug or stress to prevent and treat conditional pathogenic environmental microorganisms, such as Moraxellaceae bacteria, which include but are not limited to Acinetobacter baumannii.

2. The use according to claim 1, characterized in that: The genome stability of Moraxellaceae bacteria was reduced by disrupting the DdaA regulatory network.

3. The use according to claim 1, characterized in that: Disruption of the DdaA regulatory network increases the susceptibility of Moraxellaceae to phage infection.

4. The use according to claim 1, characterized in that: DdaA pathway-targeted drugs can be used to control Moraxella by regulating the response of Moraxellaceae to environmental stress.

5. The use according to claim 3, characterized in that: DdaA pathway targeting drugs combined with phage therapy for prevention and treatment.

6. The use according to claim 1, characterized in that: The environmental stress includes but is not limited to ultraviolet radiation and mitomycin treatment.

7. Use in the preparation of a drug for the prevention and treatment of Moraxellaceae bacteria, characterized in that: The drug target is the network regulated by DdaA.

8. The use according to claim 7, characterized in that The drug comprises a DNA fragment or plasmid containing a multi-repeated DdaA binding sequence "CGWCANNNNNTGWCG", as shown in SEQ ID No.5.

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