Raoultella ornitholytica antibacterial peptide and application thereof

Abstract

The application belongs to the field of biomedicine and particularly relates to an ornithine-degrading Raoultella antibacterial peptide and application. The ornithine-degrading Raoultella antibacterial peptide is formed by a polypeptide (also referred to as antisense peptide) encoded by a complementary strand of a functional domain DNA sense strand in truncated CpxA in the same reading frame. The amino acid sequence of the ornithine-degrading Raoultella antibacterial peptide is shown as SEQ ID NO:2. The ornithine-degrading Raoultella antibacterial peptide and meropenem (MEM) jointly inhibit the growth of NDM-1 strain B1645-1B, has a very large application prospect, and provides a new drug for treating infectious diseases caused by ornithine-degrading Raoultella and solving drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an ornithine-releasing Raoulbacterium antimicrobial peptide and its application. Background Technology

[0002] Antimicrobial resistance is a global concern this century and has become a serious public health hazard. Carbapenems are hailed as the "last line of defense" against bacterial infections due to their broad antibacterial spectrum and high activity. According to 2019 statistics, carbapenem-resistant pathogens, including Acinetobacter baumannii and Klebsiella pneumoniae, caused 50,000 to 100,000 deaths worldwide. Carbapenem-resistant bacteria are resistant to almost all β-lactam antibiotics. Clinical treatment faces enormous challenges, and developing new inhibitors is currently the main means of combating carbapenem-resistant bacteria.

[0003] Two-component signal transduction systems (TCS) consist of two basic components: histidine kinase (HK) on the intracellular membrane and response regulators (RR) in the cytoplasm, and are widely present in various prokaryotes. HK contains a signal sensing region and is automatically phosphorylated upon stimulation, transferring a phosphate group to RR. The phosphorylated RR then binds to a promoter, regulating gene expression at the transcriptional level. The two-component signal transduction system CpxAR is a TCS that monitors intracellular homeostasis; CpxR is RR, and CpxA is HK. Activation of CpxR induces high expression of the downstream gene CpxP, whose mRNA is further processed into small molecules to regulate RNA CpxQ. Furthermore, studies have shown that CpxAR can regulate bacterial resistance to ceftriaxone and β-lactam antibiotics. The cytoplasmic response regulator CpxR regulates the expression and spread of blaKPC on the IncFII plasmid, thereby promoting Klebsiella pneumoniae (…). Klebsiella pneumoniae Carbapenem resistance formation.

[0004] Ornithine-lysinic Rauvolf Raoultella ornithinolytica It has the ability to convert histidine into histamine, leading to severe human poisoning, manifested as skin swelling and redness, and secondary diabetic foot. The inventors... R. ornithinolytica Genome sequencing analysis predicted histidine decarboxylases hdc The gene (orf00241), which catalyzes the conversion of histidine to histamine, suggests that it may be an important drug target for future treatments. R. ornithinolyticaInfection. Fifty-five genes were predicted to be involved in two-component transduction systems 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 Antimicrobial resistance of B1645-1B strain. The two-component signal transduction system CpxA / CpxR modulates bla KPC In K. pneumoniae expression and dissemination on the conjugative IncFII plasmid pCYNDM01 modulates carbapenem antibiotic resistance. Therefore, R. ornithinolytica The two-component signal transduction system CpxAR in B1645-B can modulate bla NDM-1 Expression and dissemination on the conjugative IncFII plasmid pCYNDM01 needs further investigation.

[0005] Therefore, the present application provides an ornithine decarboxylase Raoultella antimicrobial peptide, which is formed by a polypeptide (also known as antisense peptide) encoded by the complementary strand of the DNA sense strand of the functional domain truncated at different sites in CpxA in the same reading frame, and the antisense peptide of different sites in CpxA targets and binds to the functional domain in CpxA of the two-component signal transduction system CpxA / CpxR in the bacterial inner membrane and inhibits its synthesis, thereby reducing bla NDM-1 expression and dissemination, and inhibiting the occurrence of carbapenem antibiotic resistance. SUMMARY

[0006] The present application first provides an ornithine decarboxylase Raoultella antimicrobial peptide, which is obtained by truncating the functional domain in CpxA.

[0007] In certain embodiments, the number of amino acids of the ornithine decarboxylase Raoultella antimicrobial peptide is less than 30.

[0008] In certain embodiments, the number of amino acids of the ornithine decarboxylase Raoultella antimicrobial peptide is less than 25.

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

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

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

[0012] In some embodiments, the Raoultella ornithinolytica antibacterial peptide CpxAF1 in combination with MEM synergistically inhibits the growth of NDM-1 strain B1645-1B.

[0013] The present application also provides a method for preparing the Raoultella ornithinolytica antibacterial peptide as described above, which uses artificial synthesis or engineering bacteria expression.

[0014] The present application also provides a pharmaceutical composition comprising the Raoultella ornithinolytica antibacterial peptide as described above, and optionally one or more antibiotics.

[0015] The present application also provides the use of the Raoultella ornithinolytica antibacterial peptide as described above, which is one or more of a) to f);

[0016] a) for treating infectious diseases caused by Raoultella ornithinolytica;

[0017] b) for preparing a drug for treating infectious diseases caused by Raoultella ornithinolytica;

[0018] c) for inhibiting Raoultella ornithinolytica;

[0019] d) for preparing a drug for inhibiting Raoultella ornithinolytica;

[0020] e) for alleviating drug resistance;

[0021] f) for preparing a pharmaceutical composition for alleviating drug resistance;

[0022] Optionally, the method is optionally used for non-diagnostic purposes or non-therapeutic purposes.

[0023] Compared with the prior art, the present application has at least the following beneficial effects:

[0024] The present application provides a Raoultella ornithinolytica antibacterial peptide, which is obtained by truncating the functional domain in CpxA, and has a synergistic inhibitory effect on the growth of NDM-1 strain B1645-1B in combination with MEM, has a very large application prospect, and provides a new drug for treating infectious diseases caused by Raoultella ornithinolytica and solving drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 2 is a graph of the MIC results of polypeptide CpxAF2 in combination with MEM on strain B1645-1B. 1-8: MEM is diluted horizontally by 2 times, the concentration is from 1024-8 ug / ml, 9: negative control, the concentration of meropenem is 4 ug / ml, no bacteria is added, 10: positive control, the concentration of bacteria added is 5 x 105 CFU / ml, no meropenem and polypeptide CpxAF2 added; A-H: polypeptide CpxAF2 was diluted vertically in 2-fold gradient, concentration from 1024-8 ug / ml; arrow points to MIC result. DETAILED DESCRIPTION

[0026] To make the technical problems, technical solutions and advantages of the present application clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0027] Example 1, activation and separation and purification of bacteria

[0028] The strain stored in glycerol was taken out from -70℃ refrigerator, and after the bacterial solution was dissolved in ice bath, the bacterial solution was inoculated in sterile hydrolyzed casein (Mueller-Hinton, MH) medium, the components of MH medium were as follows: acid hydrolyzed casein 1.75 g, beef powder 0.2 g, soluble starch 0.15 g, 80 mL deionized water was added, after dissolution, deionized water was added to 100 mL. It was placed in a 37℃ constant temperature incubator for 24 h. Single colony was picked from the MH medium and inoculated into fresh sterilized 5 mL LB liquid medium, and shaken in a 37℃ constant temperature incubator for 16 h. Diluted at 1:100: 50 μL of the cultured bacterial solution was taken into fresh sterilized 5 mL MH test tube liquid medium, and shaken in a 37℃ constant temperature incubator for 4.5 h to ensure that the bacteria were in logarithmic growth phase. The OD600 nm value of the strain carrying NDM-1 Raoultella ornithinolytica B1645-1B (from the blood of a patient in Hubei College of Medicine Affiliated National Medicine Dongfeng General Hospital) was diluted to 0.01 with sterilized MH liquid medium, i.e. the bacterial concentration was 1 x 10 6 CFU / ml of bacterial solution was prepared for standby.

[0029] Example 2, determination of the inhibitory effect of polypeptide on Raoultella ornithinolytica strain B1645-1B

[0030]

[0031] First, MEM was dissolved in sterile water at an initial concentration of 2048 μg / ml. It was then serially diluted to adjust the MEP solution to a horizontal concentration gradient of 1024–8 μg / ml, decreasing by 10 units, with 100 μl per well. Next, peptides CpxAF1, CpxAF2, and CpxAF3 were dissolved in sterile water and serially diluted using the microbroth method in 8*12 well plates at an initial peptide concentration of 2048 μg / ml. The peptide solution was then serially diluted to a vertical concentration gradient of 1024–8 μg / ml, decreasing by 8 units, with 100 μl per well. The 9th column well served as a negative control, containing 4 μg / ml MEM and peptides, but no bacteria. The 10th column well served as a positive control, containing 5 × 10⁶ bacteria. 5 CFU / ml, without added MEM and peptides. Carrying NDM-1. K. pneumoniae TH-P12158 and Escherichia coli ATCC25922 strains were used as both positive and negative controls. They carried NDM-1. K. pneumoniae TH-P12158 and Escherichia coli The ATCC25922 strain was obtained from the Department of Laboratory Medicine, Dongfeng General Hospital affiliated with Hubei University of Medicine. Figure 1 As shown, 100 μl of ornithine-lysing Raoultella strain B1645-1B was added to wells 1 through 8, with a final bacterial concentration of 5 × 10⁸ in each well. 5 CFU / ml. Incubate at 37℃ for 16–24 h and read the MIC values. Count data are expressed as number of cases (n), and the mean MIC of the drugs is expressed as the median. The comparison of MICs before and after drug combination was performed using a three-independent-samples t-test in SPSS 27.0 statistical software. P < 0.05 was considered statistically significant.

[0032] The results showed that peptide CpxAF2 had the best inhibitory effect on the growth of *Raoultella ornithine-free* strain B1645-1B, with a MIC greater than 1024 μg / ml. MEM had a MIC of 512 μg / ml for strain B1645-1B. The MIC of peptide CpxAF2 combined with MEM against strain B1645-1B was 128 μg / ml, a statistically significant difference (P<0.05). Peptide CpxAF2 combined with MEM reduced the MIC of meropenem. These results indicate that peptide CpxAF2 combined with MEM synergistically inhibits the growth of strain B1645-1B carrying NDM-1. K. pneumoniae TH-P12158, peptide CpxAF2 combined with MEM synergistic effectEscherichia coli The ATCC 25922 strain had no inhibitory effect.

[0033] In summary, the present application discloses a new polypeptide CpxAF2 which has inhibitory effect on the logarithmic phase growth of the NDM-1 ornithine decarboxylase carrying Raoultella strain B1645-1B.

[0034] The above describes the preferred embodiments of the present application, it should be pointed out that for the ordinary skilled in the art, without departing from the principles of the present application described, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises an ornithin-degrading Raoultella bacteriocin having an amino acid sequence as set forth in SEQ ID NO: 2 and one or more antibiotics.

2. The pharmaceutical composition of claim 1, wherein, The antibiotic comprises MEM.

3. Use of a pharmaceutical composition according to any one of claims 1-2, characterized in that, The use is a) or b); a) the preparation of a medicament for the treatment of an infectious disease caused by Raoultella ornithinolytica; b) the preparation of a medicament for the inhibition of Raoultella ornithinolytica.

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