Raoultella ornithinolytica antibacterial peptide and application thereof

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a raoultella ornithinolyticus antibacterial peptide and application thereof.The raoultella ornithinolyticus antibacterial peptide is a polypeptide (also called antisense peptide) formed by coding of the same reading frame on a complementary chain of a DNA positive-sense chain of a functional domain in truncated CpxA, an amino acid sequence of the raoultella ornithinolyticus antibacterial peptide is shown as SEQ ID NO: 2, the raoultella ornithinolyticus antibacterial peptide is combined with meropenem (MEM) to synergistically inhibit growth of a strain B1645-1B carrying NDM-1, and the raoultella ornithinolyticus antibacterial peptide has a great application prospect and provides a new medicine for treating infectious diseases caused by raoultella ornithinolyticus and solving medicine resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a Raoultia ornithine-lytica antimicrobial peptide and its application. Background Art

[0002] Bacterial resistance has become a global hotspot this century and a serious public relations hazard. Carbapenem antibiotics are hailed as humanity's "last line of defense" against bacterial infections, with their broad antimicrobial spectrum and potent activity. According to 2019 statistics, carbapenem-resistant pathogens, including Acinetobacter baumannii and Klebsiella pneumoniae, were responsible for 50,000 to 100,000 deaths worldwide. Carbapenem-resistant bacteria are resistant to nearly all β-lactam antibiotics. Clinical treatment presents significant challenges, and the development of new inhibitors is currently the primary approach to combating carbapenem-resistant bacteria.

[0003] The two-component signal transduction system (TCS) consists of two basic components: histidine kinase (HK) on the intracellular membrane and response regulator (RR) in the cytoplasm. It is widely present in various prokaryotes. Among them, HK contains a signal-sensing region, which is automatically phosphorylated when stimulated and transfers the phosphate group to RR. The phosphorylated RR will bind to the promoter and regulate gene expression at the transcriptional level. The two-component signal transduction system CpxAR is a TCS that monitors membrane homeostasis. CpxR is RR and CpxA is HK. After CpxR is activated, it induces high expression of the downstream gene CpxP, and its mRNA is processed into small molecules to regulate RNA CpxQ. In addition, studies have shown that CpxAR can regulate bacterial resistance to ceftriaxone and β-lactam drugs. The cytoplasmic response regulator protein CpxR regulates the expression and spread of blaKPC on the IncFII plasmid, thereby promoting Klebsiella pneumoniae ( Klebsiella pneumoniae ) Carbapenem resistance development.

[0004] Raoultia ornithinei Raoultella ornithinolytica It has the ability to convert histidine into histamine, causing severe poisoning in humans, manifested as swelling and redness of the skin, and secondary diabetic foot. R. ornithinolytica Genome sequencing analysis predicted histidine decarboxylase hdc gene (orf00241), which catalyzes the conversion of histidine to histamine, suggesting this could be an important drug target for future treatments R. ornithinolyticaFifty-five genes were predicted to be involved in the two-component transduction system, including BaeR / BaeS, CpxA / CpxR, PhoP / PhoQ, ArcA / Arc, DcuS / DcuR, RcsC / RcsB, YehU / YehT, and PmrA / PmrB systems, which are responsible for R. ornithinolytica The two-component signal transduction system CpxA / CpxR directly regulates the antimicrobial resistance of strain B1645-1B. bla KPC exist K. pneumoniae The expression and spread of zygotic IncFII on the surface of bacteria regulates carbapenem resistance. R. ornithinolytica The two-component signal transduction system CpxAR in B1645-B may regulate bla NDM-1 Expression and propagation on the conjugative IncFII plasmid pCYNDM01 requires further study.

[0005] Therefore, the present invention provides an ornithine-degrading Raoultia antimicrobial peptide, which is a polypeptide (also called an antisense peptide) encoded by the same reading frame on the complementary chain of the DNA sense chain truncated by the functional domains at different sites in CpxA. It is clarified that the antisense peptides at different sites of CpxA target and bind to the functional domains in CpxA of the bacterial intracellular membrane two-component signal transduction system CpxA / CpxR and inhibit their synthesis, thereby reducing bla NDM-1 expression and spread of carbapenem antibiotic resistance. Summary of the Invention

[0006] The present invention first provides a Raoultella ornithinolytica antimicrobial peptide, which is obtained by truncating a functional domain in CpxA.

[0007] In certain embodiments, the Raoultella ornithine-lytica antimicrobial peptide has less than 30 amino acids.

[0008] In certain embodiments, the Raoultella ornithine-lytica antimicrobial peptide has less than 25 amino acids.

[0009] In certain embodiments, the number of amino acids in the Raoultella ornithine-lytica antimicrobial peptide is between 20 and 25.

[0010] In certain embodiments, the amino acid sequence of the Raoultella ornithinelyticus antimicrobial peptide comprises SEQ ID NO: 2.

[0011] In certain embodiments, the amino acid sequence of the Raoultella ornithinelyticus antimicrobial peptide is shown in SEQ ID NO: 2.

[0012] In certain embodiments, the Raoultia ornithine antimicrobial peptide CpxAF1 combined with MEM synergistically inhibits the growth of the NDM-1-carrying strain B1645-1B.

[0013] The present invention also provides a method for preparing the Raoultia ornithine-lytica antimicrobial peptide as described above, wherein the method adopts artificial synthesis or engineered bacterial expression.

[0014] The present invention also provides a pharmaceutical composition comprising the above-mentioned Raoultella ornithinelyticus antimicrobial peptide and optionally one or more antibiotics.

[0015] The present invention also provides a use of the above-mentioned Raoultia ornithinelyticus antimicrobial peptide, wherein the use is one or more of a) to f); a) For the treatment of infectious diseases caused by Raoultia ornithinei; b) Preparation of medicaments for the treatment of infectious diseases caused by Raoultia ornithinei; c) for inhibiting Raoultia ornithinei; d) Preparation of medicaments for inhibiting Raoultia ornithinei; e) for mitigating drug resistance; f) preparing pharmaceutical compositions for alleviating drug resistance; Optionally, the method is optionally used for non-diagnostic or non-therapeutic purposes.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention provides an antimicrobial peptide from Raoultia ornithinolytica, which is obtained by truncation of the functional domain in CpxA. The antimicrobial peptide is combined with MEM to synergistically inhibit the growth of NDM-1-carrying strain B1645-1B, has very broad application prospects, and provides a new drug for treating infectious diseases caused by Raoultia ornithinolytica and resolving drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 shows the MIC results of peptide CpxAF2 combined with MEM against strain B1645-1B. 1-8: 2-fold horizontal gradient dilution of MEM, concentration from 1024 to 8ug / ml, 9: negative control, meropenem concentration of 4ug / ml, no bacteria added, 10: positive control, added bacteria concentration of 5×10 5 CFU / ml, without the addition of meropenem and peptide CpxAF2; AH: vertical 2-fold gradient dilution of peptide CpxAF2, concentration from 1024-8ug / ml; arrows point to MIC results. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1: Activation and separation and purification of bacteria Remove the glycerol-stored strain from a -70°C freezer and, after thawing in an ice bath, inoculate the culture into sterile Mueller-Hinton (MH) medium. MH medium consists of: 1.75 g of acid-hydrolyzed casein, 0.2 g of beef powder, and 0.15 g of soluble starch. Dissolve in 80 mL of deionized water, then add deionized water to 100 mL. Incubate in a 37°C incubator for 24 hours. Pick a single colony from the MH medium and inoculate it into 5 mL of freshly sterilized LB liquid medium. Incubate in a 37°C shaker for 16 hours. For a 1:100 dilution, transfer 50 μL of the culture into a freshly sterilized 5 mL MH tube and shake in a 37°C shaker for 4.5 hours to ensure the bacteria are in the logarithmic growth phase. The strain of Raoultella ornithine-lysingensis B1645-1B (from the blood of an inpatient at the Sinopharm Dongfeng General Hospital affiliated to Hubei Medical College) carrying NDM-1 was diluted to an OD600 nm value of 0.01 using sterilized MH liquid medium, which resulted in a bacterial concentration of 1×10 6 CFU / ml of bacterial solution for future use.

[0020] Example 2: Determination of the inhibitory effect of polypeptides on ornithine-degrading Raoult strain B1645-1B

[0021] First, MEM was dissolved in sterile water, with a starting concentration of 2048 μg / ml. Serial dilutions were performed to adjust the MEP solution to a concentration gradient of 1024-8 μg / ml in a horizontal row with 10 concentration gradients decreasing, with 100 μl per well. Then, the polypeptides CpxAF1, CpxAF2, and CpxAF3 were dissolved in sterile water and microbroth dilution was performed using an 8*12 well culture plate. The starting concentration of the polypeptide was 2048 μg / ml. Serial dilutions were performed to adjust the polypeptide solution to a concentration gradient of 1024-8 μg / ml in a vertical row with 8 concentration gradients decreasing, with 100 μl per well. The 9th vertical column well was a negative control, with a concentration of 4 ug / ml MEM and polypeptide added, and no bacteria were added. The 10th vertical column well was a positive control, with a bacterial concentration of 5×10 5 CFU / ml, without MEM and peptide. Carrying NDM-1 K. pneumoniae TH-P12158 and Escherichia coli ATCC25922 strains were used as positive and negative control strains. K. pneumoniae TH-P12158 and Escherichia coli ATCC25922 strain was obtained from the Laboratory Department of Sinopharm Dongfeng General Hospital affiliated to Hubei Medical College. Figure 1 As shown, 100 μl of ornithine-degrading Raoult strain B1645-1B bacterial solution was added to wells 1 to 8, and the final concentration of bacteria in each well was 5×10 5 CFU / ml. Incubate at 37°C for 16–24 h, and then read the MIC value. Enumeration data are expressed as the number of cases (n), and the mean drug MIC is expressed as the median. Comparison of MICs before and after drug combination was performed using a three-independent sample t-test using SPSS 27.0 statistical software. P < 0.05 was considered statistically significant.

[0022] The results showed that the peptide CpxAF2 had the best inhibitory effect on the growth of the ornithine-lysing Raoul strain B1645-1B. The MIC of the peptide CpxAF2 against the strain B1645-1B was greater than 1024 μg / ml, and the MIC of MEM against the strain B1645-1B was 512 μg / ml. The MIC of the peptide CpxAF2 combined with MEM against the strain B1645-1B was 128 μg / ml, and the difference was statistically significant (P<0.05). The peptide CpxAF2 combined with MEM could reduce the MIC of meropenem. The above results indicate that the peptide CpxAF2 combined with MEM synergistically inhibits the growth of the strain B1645-1B carrying NDM-1, and the peptide CpxAF2 combined with MEM synergistically inhibits the growth of the strain carrying NDM-1. K. pneumoniae TH-P12158, peptide CpxAF2 combined with MEM synergisticallyEscherichia coli ATCC25922 strain had no inhibitory effect.

[0023] In summary, the present invention discovered a new polypeptide CpxAF2, which has an inhibitory effect on the logarithmic growth of the Raoult strain B1645-1B carrying the NDM-1 ornithine-degrading gene.

[0024] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide from Raoultia ornithinei, characterized in that The amino acid sequence of the Raoultia ornithine-lytica antimicrobial peptide is shown in SEQ ID NO:

2.

2. The Raoultia ornithine-lytica antimicrobial peptide according to claim 1, characterized in that The Raoultia ornithine antimicrobial peptide combined with MEM synergistically inhibits the growth of the NDM-1 carrying strain B1645-1B.

3. A method for preparing the Raoultia ornithine-lytica antimicrobial peptide according to any one of claims 1 to 2, characterized in that: The method adopts artificial synthesis or engineering bacteria expression.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Raoultia ornithine antimicrobial peptide according to any one of claims 1-2, and optionally one or more antibiotics.

5. A use of the Raoultia ornithine-lytica antimicrobial peptide according to any one of claims 1-2, characterized in that: The application is one or more of a) to c); a) Preparation of medicaments for the treatment of infectious diseases caused by Raoultia ornithinei; b) the preparation of medicaments for inhibiting Raoultia ornithinei; c) preparing a pharmaceutical composition for alleviating drug resistance.

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