Use of global stress response element ddaa in moorellaceae

Abstract

The application discloses application of global stress response element DdaA in Moraxellaceae. NCBI protein sequence of DdaA is WP_000117148.1, which is used as a target site of a drug or stress to prevent and treat Moraxellaceae bacteria, including but not limited to Acinetobacter baumannii. By disturbing the DdaA regulatory network, the genomic stability of Moraxellaceae bacteria is reduced. By disturbing the DdaA regulatory network, the sensitivity of Moraxellaceae bacteria to phage infection is increased. DdaA pathway targeting drugs prevent and treat by regulating the response of Moraxellaceae bacteria to environmental stress. DdaA pathway targeting drugs are combined with phage therapy to prevent and treat. The environmental stress includes but is not limited to ultraviolet irradiation and mitomycin treatment. The drug targets the network regulated by DdaA. The drug includes a DNA fragment or a plasmid containing multiple DdaA binding sequences "CGWCANNNNNTGWCG". The drug is suitable for preventing and treating multi-drug resistant Acinetobacter baumannii, and can be used in combination to produce a superimposed effect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to the application of a transcriptional regulatory factor in the prevention and control of Acinetobacter baumannii. Background Technology

[0002] Bacteria are tiny and have simple structures, resulting in frequent exchanges of substances between the external environment and their cells. Stresses such as radiation, drought, high temperature, heavy metals, reactive oxygen species, and high osmotic pressure can all cause abnormal bacterial cell metabolism, resulting in varying degrees of damage to genomic DNA, affecting microbial life processes, and threatening their survival and reproduction.

[0003] Rapid and accurate repair of damaged DNA molecules is crucial for bacterial survival, and this process is primarily accomplished by DNA damage repair proteins. These pathways are regulated by a global DNA damage response system.

[0004] Acinetobacter baumannii is a bacterium belonging to the genus Acinetobacter in the family Moraxellaceae of the phylum Proteobacteria. It is one of the most common Gram-negative bacilli in the genus Acinetobacter, widely distributed in water and soil, hospital environments, and on human skin, respiratory tract, digestive tract, and genitourinary tract. It is an opportunistic pathogen. This bacterium is widely distributed in hospital environments and can survive for extended periods, making it an important pathogen causing hospital-acquired infections. Therefore, it is often isolated from specimens such as blood, urine, pus, and respiratory secretions from infected patients. Its strong stress response and increasing resistance to commonly used antibiotics have attracted widespread attention from clinicians and microbiologists. Given reports that multidrug-resistant Acinetobacter baumannii in the Moraxellaceae family can cause a mortality rate as high as 70%, current main measures to combat infection include the use of antibiotics, phage therapy, and vaccine development.

[0005] Because *Acinetobacter baumannii* possesses a remarkable ability to acquire drug-resistant genes and withstand harsh living conditions, it has become resistant to most first-line antibiotics. Therefore, there is an urgent need to identify novel antimicrobial drug action sites targeting these resistant strains. Previous research has suggested that the DNA stress system of pathogenic microorganisms affects the antimicrobial efficiency of drugs, and research on their DNA damage response systems can provide a theoretical basis for the development of targeted drugs. Summary of the Invention

[0006] Addressing the urgent need to control multidrug-resistant Acinetobacter baumannii, this invention delves into the global stress response system within Acinetobacter baumannii, discovering a novel transcriptional regulator, DdaA, and providing an application for the global stress response element DdaA in the Moraxellaceae family. DdaA specifically targets the DdaA regulatory network in Acinetobacter baumannii, exhibiting high specificity, unlike traditional antibiotics that damage the normal flora in the human body, and without inducing drug resistance in the strain within a short period.

[0007] Application of DdaA, a global stress-response element in the Morales family, wherein the NCBI protein sequence of DdaA is WP_000117148.1, is used as a target site for drugs or stress to control opportunistic pathogenic environmental microorganisms of the Morales family, including but not limited to Acinetobacter baumannii. The DdaA protein is derived from Acinetobacter baumannii. Acinetobacter baumannii ATCC 17978, purchased from the U.S. Type Culture Collection, DNA sequence ID: HKO16_RS19275. We are the first to experimentally demonstrate the existence of this protein and elucidate its function. DdaA is a WYL family transcription activator, and the DNA sequence that specifically interacts with it is a first-time discovery in this invention. The specific binding sequence is “CGWCANNNNNTGWCG”, SEQ ID No. 5, where A and T can be represented as W, and N represents any base.

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

[0009] The application described above increases the susceptibility of Moraxella bacteria to bacteriophage infection by disrupting the DdaA regulatory network.

[0010] The aforementioned application describes how DdaA pathway-targeting drugs prevent and treat diseases by modulating the response of Moraxella bacteria to environmental stress.

[0011] The aforementioned application involves using DdaA pathway-targeting drugs combined with phage therapy for prevention and treatment.

[0012] The environmental stresses mentioned include, but are 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 the multiple repeat 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 applicable to the prevention and control of most bacteria in the Moraxellaceae family: it is applicable to the prevention and control of most pathogenic bacteria in the Moraxellaceae family, including Acinetobacter baumannii; it is applicable to the prevention and control and inactivation of Acinetobacter baumannii infection in medical environment, daily environment and in vivo.

[0016] (2) It is not affected by known Acinetobacter baumannii resistance and is suitable for the control of multidrug-resistant Acinetobacter baumannii. It can be used in combination with existing Acinetobacter baumannii control technologies and produces a synergistic effect. Attached Figure Description

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

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

[0019] Figure 3 To verify the successful knockout of the DdaA system using Sanger sequencing.

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

[0021] Figure 5 DdaA knockout causes DNA damage and reduces resistance.

[0022] Figure 6 Knockout of DdaA resulted in decreased phage resistance in Acinetobacter baumannii.

[0023] Figure 7 Treatment with DdaA-targeted drugs reduced the UV resistance of Acinetobacter baumannii. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

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

[0026] (2) Cell lysis: This expression strain was cultured to OD100. 600When the concentration reaches approximately 0.4, add 50 mM IPTG solution to a final concentration and culture *E. coli* at 30°C for 5 hours to induce high expression of DdaA. This condition can be used for DdaA from different species. Collect the bacterial cells by centrifuging at 8000g for 5 minutes using an ultracentrifuge, and then resuspend the cells in His-A solution. Repeat the centrifugation and resuspension steps three times to completely wash away any remaining culture medium from the cells. Then, lyse the cells using a pressure lysator and further lyse them using an ultrasonic lysator to break down the DNA molecules, facilitating subsequent purification. The lysate should be a clear, transparent liquid at this point. Finally, centrifuge the bacterial solution 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 and transferred to the ÄKTA purification system. The expressed protein was then purified using Ni affinity purification resin. The flow rate of the ÄKTA purification system was maintained at 1 mL / min throughout the process. Pump A was automatically filled with His-A solution, and pump B with His-B solution. The loading loop and nickel column were filled with His-A solution, and the lysate was then loaded onto the nickel column to ensure the His-tag DdaA protein to be purified was fully bound to the nickel column. After the UV value and conductivity of the ÄKTA purification system stabilized, a 90% His-A to 10% His-B solution was used to elute the contaminating proteins bound to the nickel column. After the UV value and conductivity stabilized, a 50% His-A to 50% His-B solution was used to elute the His-tag DdaA protein bound to the nickel column. The collected product was the highly purified DdaA protein solution. Protein concentration was determined using a NanoDrop 1000. The time and concentration were marked on the collection tubes, and the collection tubes were finally stored in a 4°C refrigerator.

[0028] Example 2: Prediction and Validation of Specific Binding Sequence of Acinetobacter baumannii DdaA Protein The cis-acting element of the 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 also synthesized. Primers were annealed using appropriate buffer systems. DdaA protein and the FAM-labeled double-stranded substrate were added at a final molar ratio of 20:1, and after incubation for 30 minutes, the binding system was transferred to a 12% Native-PAGE gel for electrophoresis and imaged using a Typhoon FLA 9500. DdaA specifically bound to the predicted target DNA fragment (SEQ ID No. 5), and the conserved bases could not be altered. Figure 1As shown, the first lane contained only double-stranded DNA with a conserved DdaA binding site, without the DdaA protein; the second lane contained both DdaA protein and double-stranded DNA with a conserved DdaA binding site, resulting in significant gel hindrance; the remaining lanes contained DdaA protein, but the specific binding sequence for DdaA protein was mutated, resulting in a significant reduction or even disappearance of gel hindrance. DdaA contains three domains: the N-terminal HTH domain is responsible for double-stranded DNA binding, while the C-terminal WYL and WCX domains may be involved in molecular signal transduction. Simultaneously, DdaA can form a stable dimer structure (…). Figure 2 ), and the palindromic property of the sequence combined with it is mutually corroborated.

[0029] Example 3: Editing Acinetobacter baumannii using a gene editing system Using the existing Acinetobacter baumannii CRISPR knockout system, for ddaA The gene was subjected to nonsense mutation to obtain the 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 selection with apopramine. ② An 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 a long single-stranded DNA designed according to the editing purpose were transformed into the IPTG-induced Ab::pCasAb-apr strain, and double resistance selection with apopramine and kanamycin was performed. The obtained colonies were verified by PCR, and the PCR products were sequenced to confirm that the gene knockout was successful. ④ The correctly sequenced strains were transferred into LB medium containing sucrose and cultured for one day to obtain... ddaA Strain knockout without leaving a trace. ddaA The knockout strain was validated by Sanger sequencing, and the results are as follows: Figure 3 As shown. At the same time, to verify that the following phenotype is... ddaA Knockout caused this, so we used the pWH-1266 backbone to insert the DNA shown in NCBI gene: HKO16_RS19275. ddaA The strain was knocked out without leaving a trace, restoring the function of the DdaA system.

[0030] Example 4: DdaA-regulated network perturbation causes DNA damage and decreased resistance in Acinetobacter baumannii. Take wild-type Acinetobacter baumannii cultured overnight. ddaA Knockout and replacement strains were transferred to LB medium and cultured until the logarithmic growth phase (OD value between 0.6 and 0.8).

[0031] Transcriptome analysis: Acinetobacter baumannii wild-type, knockout ddaA The strain was revived by streaking LB plates. Fresh colonies were collected in liquid LB broth until OD was reached. 600 Once the concentration reaches 0.6, 2 mL of each bacterial culture is placed on a clean glass surface. The UV irradiation group will receive 15 J / m². 2 The bacterial cultures were exposed to ultraviolet light, while the control group received no treatment at room temperature. Each group of bacterial cultures was incubated at 37°C for 15 minutes to allow for DNA damage. Immediately afterward, the cells were centrifuged to the bottom of EP tubes, rapidly frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted using the RNAprep Pure Cell / BacteriaKit RNAprep Pure DP430 and purified using the RNAClean XP kit (Beckman Coulter) and an RNase-free DNase Set (Qiagen). Libraries were constructed using the U-mRNAseq library preparation kit AT4221, KAITAI-BIO, and a ribonucleic acid consumption kit (Bacteria, Novizan). Sequencing was performed using an Illumina NovaSeq machine, yielding 150 bp paired-end sequencing data. The representative genome of Acinetobacter baumannii ATCC 17978 was then indexed using hisat2 and converted to a suitable format using samtools. FastQ files were cleaned using TrimmaticPE and mapped to the genome using hisat2. The expression matrix was calculated using stringtie, and differentially expressed genes were identified using Deseq2.

[0032] like Figure 4 As shown, the left part shows the transcriptomic changes of wild-type Acinetobacter baumannii before and after radiation, and the right part shows... ddaA Transcriptome changes in knockout strains before and after radiation ddaA Gene knockout significantly reduced the transcriptional DNA damage response in Acinetobacter baumannii under ultraviolet irradiation.

[0033] Ultraviolet irradiation phenotype: Take 400 μL of bacterial suspension from each of the above strains and irradiate them with 10, 10 2 10 3 10 4 10 5 10 6 Serially dilute the bacterial suspension and transfer it to solid LB medium, allowing the bacterial suspension to be completely absorbed by the solid medium. Use a UV crosslinker to crosslink the plates at 0J / m². 2 5 J / m 2 10 J / m 2 , or 20 J / m 2The plates were irradiated with ultraviolet light. They were then incubated overnight at 37°C, and the number of colonies was observed to compare the differences in survival rates among the strains.

[0034] Mitomycin stress phenotype: Take the above bacterial culture and add mitomycin solution to make the final mitomycin concentrations 0.5, 1.0, 2.0, and 4.0 μg / mL. Incubate the bacterial culture at 37℃ with shaking for 30 minutes, then incubate the culture at 10, 10... 2 10 3 10 4 10 5 10 6 Serial dilutions were performed and transferred to solid LB medium. After the bacterial culture was completely absorbed by the solid medium, the plates were incubated overnight at 37°C. The number of colonies was observed to compare the differences in survival rates among the strains.

[0035] like Figure 5 As shown, ddaA Gene knockout reduced the survival rate of Acinetobacter baumannii to one percent to one ten-thousandth of its original value when subjected to ultraviolet irradiation and mitomycin stress.

[0036] Example 5: DdaA-regulated network perturbation leads to decreased phage resistance in Acinetobacter baumannii. Acinetobacter baumannii ATCC17978 wild-type and Acinetobacter baumannii wild-type containing restriction endonuclease systems were collected from overnight cultures. ddaA Knockout and replacement strains were transferred to LB medium and cultured to the logarithmic growth phase (OD value between 0.6 and 0.8). When mitomycin C (MMC) was required, the final MMC concentration in the medium was 0.5 μg / mL, and the bacterial suspension was incubated at 37°C for 30 minutes to activate the DdaA network. 2 mL of bacterial cells were collected by centrifugation at 7000g and resuspended in 2 mL of fresh LB medium for washing. Next, 200 μL of the resuspended bacterial suspension was added to 5 mL of fresh LB medium, along with PhAb24 phage with a multiplicity of infection (MOI) of 0.02. Subsequent growth curve analysis of these strains revealed that DdaA knockout significantly reduced phage resistance in *Acinetobacter baumannii*. Figure 6 ).

[0037] Example 6: Targeted drugs can inhibit the DdaA regulatory network, resulting in a decrease in Acinetobacter baumannii resistance. A plasmid containing multiple repeating DdaA binding sites was designed. In this example, the design used contained the DNA sequence “CGWCANNNNNTGWCG” (as shown in SEQ ID NO. 6), which contained multiple repeating DdaA binding sites, and the plasmid was constructed using synthetic methods. Wild-type Acinetobacter baumannii ATCC17978, cultured overnight, was transferred to LB medium and cultured to the logarithmic growth phase (OD value between 0.6 and 0.8). The plasmid was then transformed into wild-type Acinetobacter baumannii cells using electroporation. Because these plasmids contain a large number of DdaA binding sequences, far exceeding the number of DdaA binding sites in the genome, DdaA will competitively bind to the plasmid DNA, resulting in the loss of DdaA's original regulatory function. After resistance selection, successfully transformed strains were obtained. The successfully transformed strains and wild-type strains were simultaneously subjected to 20 J / m³... 2 The strain was treated with ultraviolet irradiation, with an untreated strain serving as a control group. For example... Figure 7 As shown, DdaA pathway-targeting drugs reduced the survival rate of Acinetobacter baumannii under ultraviolet irradiation to one-tenth of that without targeted drug treatment.

[0038] The above are merely preferred embodiments of the present invention, but the present invention is not limited to the detailed methods described above. Based on the teachings and inspirations of the present invention, any prevention and control of bacteria in the Moraxellaceae family using the transcriptional regulatory network characteristics of DdaA and its homologs disclosed in the present invention is within the protection scope of the present invention.

Claims

1. The use of a plasmid containing the sequence shown in SEQ ID No. 6 in the preparation of a drug for preventing and controlling opportunistic pathogenic environmental microorganisms of the Moraxellaceae family, characterized in that: The Moraxellaceae bacteria is Acinetobacter baumannii; the SEQ ID No. 6 contains a multiple repeat DdaA binding sequence "CGWCANNNNNTGWCG", the NCBI protein sequence of DdaA is WP_000117148.1, and the drug targets DdaA; The drug reduces the stability of the Moraxellaceae bacterial genome by disrupting the DdaA regulatory network. DdaA pathway-targeted drugs work by modulating the response of Moraxella bacteria to environmental stress. The environmental stress described is ultraviolet radiation; The drug exerts its preventive and therapeutic effects by targeting the transcriptional network regulated by DdaA. The drug interferes with the regulatory network of the DdaA protein by competitively binding to it.

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