Defensins derived from human oral actinomycetes, their encoding genes and uses

Abstract

The present invention provides a defensin derived from human oral actinomycetes or a biologically active functional fragment, variant or derivative thereof, wherein the defensin has an amino acid sequence as shown in one of SEQ ID NOs: 1-4. The present invention also provides a method for preparing the defensin. The present invention also provides a pharmaceutical composition for inhibiting or killing bacteria, which comprises the defensin as described in claim 1 and a pharmaceutically acceptable diluent, excipient and / or carrier. The defensin provided by the present invention can selectively target bacterial cell wall synthesis to cause bacterial lysis, has the drug characteristics of high efficiency and low toxicity, and can treat systemic bacterial infections in a mouse live model.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to a defensin derived from human oral actinomycetes, a coding gene thereof, and a use thereof for treating systemic infection caused by bacteria. Background Art

[0002] The widespread use of traditional antibiotics has led to the emergence of bacterial resistance. Studies have found that the spread of this resistance is faster than the development of new antibiotics for treating systemic infections, becoming a huge medical challenge facing mankind in the 21st century. Therefore, it is urgent to find new sources of antibiotics.

[0003] As effector molecules of the innate immunity of multicellular organisms, antimicrobial peptides constitute the first line of defense against microbial infections and have long been considered a promising alternative to traditional antibiotics. Antimicrobial peptides are a class of cationic molecules that typically have less than 100 amino acids. They are widely found in plants, fungi, and animals. The antimicrobial activity of most antimicrobial peptides is related to their electrostatic adsorption to the strongly negatively charged surface of bacterial cell membranes. A few have been found to be able to bind to other bacterial components (such as membrane-borne lipid II or intracellular targets). In addition to being endogenous antibiotics, antimicrobial peptides from vertebrates (also called host defense peptides) can also participate in innate and adaptive immune responses as regulatory molecules.

[0004] Although antimicrobial peptides have been successful as multicellular biological defense weapons over the past 1.8 billion years of evolutionary history, decades of intensive human research have rarely applied such molecules to the treatment of systemic infections. A major endogenous factor is that the detergent-like structure of most antimicrobial peptides often brings about universal cell membrane toxicity. In addition, accumulated data also highlight the shortcomings of antimicrobial peptides from humans and other vertebrates as templates for anti-infective drugs. Because of the inherent multifunctional regulatory properties of these peptides, when administered systemically, they often interfere with the function of the human immune system. Evolutionarily, antimicrobial peptides in most multicellular organisms are designed to protect the microenvironment rather than be delivered into the systemic circulation, resulting in inherent defects in systemic treatment.

[0005] Defensins are a class of cationic antimicrobial peptides stabilized by multiple intramolecular disulfide bonds. Based on the connectivity and orientation of the disulfide bridges, defensins are divided into two different structural types: cis- and trans-defensins. The former includes peptides with cysteine-stabilized α-helix and β-sheet (CSαβ) motifs, which are the only type of antimicrobial peptides shared by plants, fungi, and animals, and can be further divided into three major subclasses, namely classical invertebrate-type defensins, classical insect-type defensins, and plant / insect-type defensins. The latter includes vertebrate-derived α-, β-, θ-defensins and large defensins of invertebrates (molluscs, arthropods, and chordates). Evolutionarily, they all likely originated from a common ancestor.

[0006] However, the existing research on antimicrobial peptides is mostly focused on various insects, amphibians, fish or plants, while there are few reports on the research on antimicrobial peptides or defensins in single-cell protists that are the farthest away from humans in evolution. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a defensin derived from human oral actinomycetes. The inventors have found that the defensin can be used to systemically treat various bacterial infections.

[0008] The present invention is realized by the following inventive concept: the inventors found that the antimicrobial peptides derived from bacteria may exclude their inherent characteristics of interfering with the human immune system because their main function is intermediate antagonism. Based on this, the inventors confirmed that the antimicrobial peptides derived from bacteria can be used for systemic treatment without interfering with the function of the human immune system. After a lot of research, the inventors found a defensin gene in the human oral actinomycete genome, and named its encoded product Actinomycesin (abbreviated as AMSIN). Through chemical synthesis and oxidative folding, the inventors obtained high-purity AMSIN products (i.e., AMSIN, AMSIN-1, AMSIN-2 and AMSIN-3), completed systematic antibacterial experiments, clarified its antibacterial mechanism, and evaluated its therapeutic potential.

[0009] In one aspect, the present invention provides a defensin derived from human oral actinomycetes or a biologically active functional fragment, variant or derivative thereof, wherein the defensin has an amino acid sequence as shown in any one of SEQ ID NOs: 1-4.

[0010] Preferably, the amino acid sequence of the defensin is:

[0011] SEQ ID NO:1GFGCPWNAYECDRHCVSKGYTGGNCRGKIRQTCHCY(AMSIN);

[0012] SEQ ID NO:2CFACPFNEHQCHNHCLSKGYRGGFCGGFAAATCRCH(AMSIN-1);

[0013] SEQ ID NO:3DLSCPWAPSVCNRHCLSHGYRGGYCAGPIKLVCHCY(AMSIN-2); or

[0014] SEQ ID NO: 4YNCPTDESPCDRHCRYSGYRGGYCGGILKTSCRCY(AMSIN-3).

[0015] On the other hand, the present invention provides a nucleic acid sequence encoding the defensin, which has a nucleic acid sequence as shown in any one of SEQ ID NOs: 5-8.

[0016] Preferably, the nucleic acid sequence encoding the defensin is:

[0017] SEQ ID NO:5

[0018] GGCTTCGGCTGCCCCTGGAACGCCTACGAGTGCGACCGCCATTGCGTCAGCAAGGGATACACCGGTGGAAACTGCCGCGGCAAGATCCGCCAGACCTGCCACTGCTAC(AMSIN);

[0019] SEQ ID NO:6

[0020] TGCTTCGCCTGCCCTTTCAACGAACATCAGTGCCACAACCACTGCTTGAGCAAAGGATATCGCGGAGGGTTCTGCGGAGGATTCGCTGCCGCCACCTGCCGCTGCCAC(AMSIN-1);

[0021] SEQ ID NO:7

[0022] OR

[0023] SEQ ID NO:8

[0024] TACAACTGCCCTACTGACGAGTCTCCGTGCGACCGTCACTGCAGGTACTCCGGATACCGGGGCGGCTACTGCGGAGGAATACTCAAGACCTCCTGCCGCTGTTAC(AMSIN-3).

[0025] On the other hand, the present invention also provides a primer for preparing a nucleic acid sequence encoding the defensin, which comprises a forward primer and a reverse primer; wherein the sequence of the forward primer is shown in SEQ ID NO:9, and the sequence of the reverse primer is shown in SEQ ID NO:10.

[0026] The present invention also provides a method for preparing a nucleic acid sequence encoding the defensin, which comprises obtaining the nucleic acid sequence by amplifying from human oral actinomycetes by PCR; wherein the sequences of the forward primer and the reverse primer used in the amplification are shown as SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0027] The present invention also provides a method for preparing the defensin, which comprises the following steps: synthesizing the linear peptide of the defensin, and then separating and purifying it by reverse phase high performance liquid chromatography.

[0028] Preferably, the method for preparing defensins of the present invention further comprises the step of renaturing the synthesized linear peptide of the defensin before separation and purification by reverse phase high performance liquid chromatography.

[0029] Preferably, the renaturation comprises dissolving the linear peptide of the synthetic defensin in water, then adding dimethyl sulfoxide to a final concentration of 5-20% (volume ratio), mixing, adding phosphate buffer and incubating overnight.

[0030] In another aspect, the present invention provides a pharmaceutical composition for inhibiting or killing bacteria, comprising the defensin of the present invention and a pharmaceutically acceptable diluent, excipient and / or carrier.

[0031] Preferably, the lethal concentration of the defensin is 0.01-5 μM.

[0032] In another aspect, the present invention also provides the use of the defensin or the pharmaceutical composition in the preparation of a drug for inhibiting or killing bacteria, or treating systemic infection caused by bacteria.

[0033] Preferably, the bacterium is a gram-positive bacterium. More preferably, the gram-positive bacterium is selected from Streptococcus pneumoniae (strains D39, R6, ST556 and TIGR4) and streptomycin-resistant strains thereof; Streptococcus mutans; Streptococcus salivarius; Streptococcus sanguinis; Streptomyces, including Streptomyces griseus and Streptomyces afflumaniensis; Lysinibacillus fusiformis; Staphylococcus strains, including penicillin-resistant and methicillin-resistant Staphylococcus aureus; and Enterococcus faecium.

[0034] In yet another aspect, the present invention also provides the use of the defensin or the pharmaceutical composition in the preparation of a broad-spectrum antibacterial drug.

[0035] In yet another aspect, the present invention also provides the use of the defensin or the pharmaceutical composition in the preparation of a drug for treating bacteremia, endocarditis, peritonitis, pneumonia and central nervous system infection or respiratory tract infection.

[0036] The defensin or pharmaceutical composition of the present invention is suitable for the development of various drugs against methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a major human conditional pathogen that can cause bacteremia, endocarditis, pneumonia and central nervous system infection. It is called a "superbug" and lacks highly selective specific drugs. Since its discovery, MRSA infection has spread almost all over the world. The defensin of the present invention provides the possibility for the clinical development of drugs for treating infections, especially MRSA infections, and has a very broad application prospect.

[0037] In another aspect, the present invention provides use of the defensin or the pharmaceutical composition in preparing a medicament for inhibiting bacterial cell wall synthesis.

[0038] The present invention also provides a method for screening drugs, wherein the drug is a drug that inhibits bacterial cell wall synthesis, and the method uses uracil diphosphate-N-acetylmuramoyl-pentapeptide as a test target.

[0039] Preferably, the drug to be screened is an antibiotic or an antimicrobial peptide.

[0040] In a specific embodiment, the method comprises the following steps:

[0041] 1. Cultivate the bacteria to be tested;

[0042] 2. Add the drug to be screened and / or the control substance to the cultured bacteria and continue culturing;

[0043] 3. After treating the bacteria, collect uracil diphosphate-N-acetylmuramoyl-pentapeptide and detect it.

[0044] The inventors of the present invention have found that for the mechanism research of antibiotics and antimicrobial peptides targeting bacterial cell wall synthesis, most of the existing research technologies use membrane-localized water-insoluble Lipid II as the test target. However, since the artificial synthesis of LipidII requires multiple cumbersome enzymatic reactions, the yield is low, and direct extraction from bacteria requires a large amount of organic solvents, which is highly toxic to the human body. The inventors of the present invention have found that the water-soluble cell wall synthesis precursor uracil diphosphate-N-acetylmuramoyl-pentapeptide can be used to replace the water-insoluble Lipid II anchored to the bacterial cell membrane to study the interaction between bacteria and antibiotics. The rationality lies in that the basic units involved in the binding of most antibiotics in the Lipid II molecule are completely consistent in uracil diphosphate-N-acetylmuramoyl-pentapeptide. Compared with the prior art, the present invention has the following advantages:

[0045] The present invention provides a defensin (AMSIN) derived from human oral actinomycetes, which can selectively target bacterial cell wall synthesis to cause bacterial lysis. AMSIN has the characteristics of high efficiency and low toxicity, and can treat systemic bacterial infections in a mouse live model. Complete in vitro and in vivo data show that AMSIN has a stable structure, a clear target, is non-toxic and has the potential to treat systemic infections. It is an antibiotic drug with therapeutic prospects, and its target is to inhibit the cell wall synthesis of sensitive bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0047] Figure 1 Flow chart for obtaining the defensin AMSIN of the present invention. A. Schematic diagram of the circular genome of oral actinomycetes (Actinomyces sp. oral taxon 171str.F0337) encoding AMSIN and its paralogous genes. Among them, the AMSIN cluster (AMSIN and AMSIN-1 to AMSIN-3) and its adjacent genes are marked. The BioProject database number of this genome is PRJNA43131. B. PCR amplification of the gene cluster containing AMSIN. C. Sequence alignment of actinomycete-derived defensins with defensins from various cell organisms. The six completely conserved cysteines are marked with shadows, and the three pairs of disulfide bonds formed by them are marked with lines. Conserved residues other than cysteine ​​are shaded in gray. The protein secondary structure elements are derived from the NMR structural data of AMSIN (see Figure 3 ).

[0048] Figure 2Identification of the oxidative folding products of the chemically synthesized defensin AMSIN of the present invention. A. HP-HPLC purification of the target oxidative folding product (indicated by signal); B. MALDI-TOF mass spectrometry identification. The single-charged and double-charged peaks are indicated.

[0049] Figure 3 Schematic diagram of the inhibition of bacterial cell wall biosynthesis by the defensin AMSIN of the present invention. A. Comparison of the effects of the defensin AMSIN of the present invention and three clinical antibiotics (rifampicin (RIF), kanamycin (KAN) and vancomycin (VAN)) on the growth kinetics of sensitive bacteria; B. Determination of the damaging effect of the defensin AMSIN of the present invention on bacterial cell membrane by fluorescent dye method. Vancomycin and scorpion venom lytic peptide Meucin-18 were used as negative and positive controls, respectively; C. Scanning electron microscopy was used to observe the changes in the morphology of bacterial cells treated with the defensin AMSIN of the present invention; D. Treatment of bacterial cells with the defensin AMSIN of the present invention resulted in the accumulation of soluble cell wall synthesis precursors in the cells; E. Comparison of structural schematic diagrams of water-soluble and water-insoluble cell wall precursors (UMP, lipid I and lipid II); F. Circular dichroism (CD) analysis of the mixture of the defensin AMSIN of the present invention and UMP; G. ESI-MS detection of the formation of non-covalent complexes between the defensin AMSIN of the present invention and UMP. In Figures C and D, vancomycin was used as a positive control. The panel in Figure D shows that increasing the concentration of the defensin AMSIN of the present invention can induce the formation of more complexes.

[0050] Figure 4 To evaluate the therapeutic potential of the defensin AMSIN of the present invention. A. The stability of the defensin AMSIN of the present invention in water and serum was determined by the inhibition zone experiment. The horizontal axis is the treatment time (hours), and the vertical axis is the size of the inhibition zone (mm); B. The hemolytic activity of the defensin AMSIN of the present invention was determined, and the lytic peptide Meucin-18 derived from scorpion venom was used as a positive control; C. The DNA binding properties of the defensin AMSIN of the present invention were determined by the inhibition zone experiment; D. The blocking effect of the defensin AMSIN of the present invention on heterologously expressed ion channels was determined by the oocyte voltage clamp technique. E. The mouse pneumonia model was used to determine the in vivo therapeutic effect of the defensin AMSIN of the present invention on lung infections caused by Streptococcus pneumoniae; F. The mouse peritonitis model was used to determine the in vivo therapeutic effect of the defensin AMSIN of the present invention on systemic infections caused by methicillin-resistant Staphylococcus aureus. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments. The embodiments and test examples of the present invention are intended to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0052] Example 1 Discovery and identification of the antimicrobial defense peptide AMSIN

[0053] The inventors found that the genome of Actinomyces sp. oral taxon 171str. F0337 encodes a tandem defensin gene cluster. The cluster consists of four genes, and the encoded products are named Actinomycesin (abbreviated as AMSIN) and AMSIN-1 to AMSIN-3 ( Figure 1 A) (amino acid sequence as shown in SEQ ID NO: 1-4, nucleic acid sequence as shown in SEQ ID NO: 5-8). The inventors amplified the genomic DNA of the species by PCR and sequenced to confirm the existence of the gene cluster ( Figure 1 B). The forward primer used is shown in SEQ ID NO:9, and the reverse primer is shown in SEQ ID NO:10.

[0054] SEQ ID NO:9 5'-TCCTCACCCCACAGAACT-3';

[0055] SEQ ID NO:10 5'-ACAGGCCCCGTGTTACGTCT-3'.

[0056] These four bacterial defensins, like eukaryotic (fungal and animal) defensins, contain six conserved cysteines, forming three pairs of intramolecular disulfide bonds. In addition, they also share five other conserved amino acids, including three glycines, one histidine, and one proline ( Figure 1 C), in which glycine and proline are common structural residues. Among these four bacterial defensins, AMSIN has a high degree of sequence similarity with a group of so-called classical invertebrate defensins, among which the sequence identity with spider defensin Sm defensin is 64%. The sequence identity with fungal sporomycin is 54% ( Figure 1 C).

[0057] Example 2 Chemical synthesis, oxidative folding and identification of AMSIN

[0058] In order to study the antibacterial activity, mechanism and therapeutic potential of AMSIN, the inventors first chemically synthesized its linear peptide. Using the traditional oxidative refolding method, the inventors obtained a high-purity AMSIN refolding product ( Figure 2A). The oxidative folding method used is as follows: 1 mg of the polypeptide is first dissolved in 0.2 ml of sterile water, and then dimethyl sulfoxide (DMSO) is added to a final concentration of 10% (v / v). After mixing, incubate at room temperature for 30 minutes, add 0.8 mL of phosphate buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.5), and incubate at 25°C overnight. The oxidative renaturation product is purified by RP-HPLC. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the inventors determined that the mass-to-charge ratio (m / z) of AMSIN is 4065.9 ( Figure 2 B), which is highly consistent with the theoretical molecular weight of the oxidized product, 4065.6 Da. MALDI-TOF analysis was performed at the Institute of Biophysics, Chinese Academy of Sciences.

[0059] Example 3 Determination of antibacterial activity of AMSIN

[0060] The classic inhibition zone method was used to determine the antibacterial activity of AMSIN. For different bacterial strains, the inventors used different bacterial culture media, including Luria-Bertani (LB) medium, THY medium (containing 30 g Todd Hewitt liquid medium and 5 g yeast extract per liter), tryptic soy broth (TSB) medium (Oxoid, Basingstoke, UK), ordinary broth medium and brain heart infusion broth (BHI) (Beijing Luqiao Technology Co., Ltd.), potato dextrose (PD) medium. The specific method is as follows:

[0061] 1. Place the bacterial plate at 37℃ or 30℃ / 28℃ for overnight culture, take a single clone and inoculate it into liquid culture medium, and culture it to the late logarithmic growth phase;

[0062] 2. Take 10 μL of fresh bacterial culture solution and add 6 mL of preheated solid medium containing 0.8% (v / v) agar, mix thoroughly and pour into a ф90 mm plate;

[0063] 3. After the culture medium solidifies, use a rubber-tipped dropper to make a 2mm hole, set up a concentration gradient of multiple dilutions, load 2μL of sample into each well, and culture overnight at the corresponding temperature;

[0064] 4. Measure the diameter of the inhibition zone, draw a scatter plot with the logarithm of the peptide dosage as the independent variable and the square of the inhibition zone diameter as the dependent variable, and calculate the lethal concentration C L =2.93 / (ak10 m / k ), m represents the intercept, k represents the slope, and a represents the depth of the hole, which is 1 mm.

[0065] Using this technology, the inventors quantitatively evaluated the antibacterial activity of AMSIN. The results showed that AMSIN had a wide range of antibacterial activity against a variety of Gram-positive bacteria (Table 1). The lethal concentration of Streptococcus pneumoniae (strains D39, R6, ST556 and TIGR4) and its streptomycin-resistant strains was around 0.10 μM; the lethal concentration range for three types of human oral sources, Streptococcus mutans, Streptococcus salivarius and Streptococcus sanguinis, was between 0.5-1.4 μM; the lethal concentration for Staphylococcus strains, including some clinical isolates of penicillin-resistant and methicillin-resistant Staphylococcus aureus, was between 0.6-5.0 μM; the lethal concentration for Enterococcus faecium was 4.65 μM.

[0066] Table 1 Lethal concentration values ​​of AMSIN for various sensitive bacteria

[0067]

[0068]

[0069] Note: StrR refers to streptomycin-resistant.

[0070] Example 4 Study on the antibacterial mechanism of AMSIN

[0071] In order to study the antibacterial mechanism of AMSIN, the inventors first evaluated its effect on bacterial growth dynamics and selected three clinical antibiotics with different antibacterial mechanisms as controls, including vancomycin that inhibits bacterial cell wall synthesis, rifampicin that inhibits bacterial RNA synthesis, and karatomycin that interferes with bacterial protein synthesis. The method used is as follows:

[0072] 1. Inoculate a monoclonal strain of Streptococcus mutans cultured overnight into a normal broth medium and culture at 37°C until the OD is 0.25-0.3. 600 ;

[0073] 2. Dilute 1-fold to 0.125-0.15 OD with culture medium containing AMSIN or antibiotics 600 .

[0074] 3. Add 200 μL of drug-treated bacterial culture to each well, culture with shaking at 37°C on a Bioscreen C instrument (Lab Systems, Helsinki, Finland) and record the effect of the drug on the bacterial growth curve.

[0075] The results showed that AMSIN and vancomycin had consistent bactericidal kinetics ( Figure 3 A), and its effects are different from those of karaomicin and rifampicin, suggesting that it may be an inhibitor of bacterial cell wall synthesis.

[0076] In order to verify whether AMSIN lacks the same damaging effect on bacterial cell membranes as vancomycin, the inventors used fluorescent dye technology to compare their effects on Bacillus megaterium cell membranes. The method used is as follows:

[0077] 1. Suspend Bacillus megaterium cells (5×10 5 ) were mixed with 1 μM of propidium iodide (PI), a fluorescent dye that specifically binds to DNA molecules, and incubated at room temperature in the dark for 5 minutes.

[0078] 2. The basal fluorescence value of the cells was measured in real time using a F-4500FL fluorescence spectrophotometer (Hitachi High-Technology Company, Japan) (λexc=525nm; λems=595nm). When the fluorescence reached a constant value, AMSIN was added and the effect of drug treatment on the fluorescence intensity was continuously measured. Vancomycin and the cell lytic peptide Meucin-18 from scorpion venom were used as negative and positive controls, respectively. The assay showed that AMSIN and vancomycin did not cause significant damage to the bacterial cell membrane ( Figure 3 B).

[0079] In order to compare the effects of AMSIN and vancomycin on bacterial morphology, the inventors performed scanning electron microscopy observations. The method was as follows: Bacillus megaterium was exposed to 10 times C L The concentration of AMSIN was incubated at 37°C for 1.5 hours, fixed with 2.5% glutaraldehyde for 1 hour, and then washed three times with PBS (pH 7.3). The cells were dehydrated with ethanol gradient, then dried by the fully automatic critical point dryer BAL-TECCPD-030, and then gold-sprayed by the sputtering coater BAL-TEC SCD005. The image was taken by a FEI Quanta 450 scanning electron microscope. The results showed that both can significantly change the morphological characteristics of Bacillus megaterium. Compared with cells that have not been treated with drugs, the cell length after drug treatment has changed significantly ( Figure 3 C).

[0080] The cell wall precursor accumulation assay showed that, similar to vancomycin, AMSIN treatment of bacteria led to a significant increase in the expression of UDP-MurNAc-Pentapeptide (UMP), a soluble cell wall precursor in the cytoplasm. Figure 3 D) further supports that the target of AMSIN is to inhibit cell wall synthesis precursors. The method used is as follows:

[0081] 1) After Staphylococcus aureus (S. aureus) was rejuvenated, it was inoculated into 40 ml of broth liquid culture medium to an OD of 0.5.

[0082] 2) Add chloramphenicol to a final concentration of 130 μg / ml and incubate for 15 min.

[0083] 3) Add AMSIN and vancomycin and an equal volume of culture medium, continue culturing for 30 min, and centrifuge at 12000 rpm for 1 min.

[0084] 4) Resuspend the cells in 500 μl ddH2O, add dropwise to 13 ml 100°C H2O, and stir magnetically for 30 min.

[0085] 5) Continue stirring, bring to room temperature, and centrifuge at 20,000 rpm, 4°C, for 1 h. RVC2-18CD plus CHRIST (Germany) desktop centrifuge concentrator freeze-dried samples.

[0086] 6) Add 450 μl of 50 mM NH4Ac (pH 5.2) to dissolve the complex.

[0087] 7) Add 50 μl of glacial acetic acid, invert and mix, and incubate at 12,000 rpm, 4°C, for 1 h.

[0088] 8) RP-HPLC detection and collection of uracil diphosphate-N-acetylmuramoyl-pentapeptide (peak time is about 11 minutes). The column is Thermoscientific Hypersil ODS (4.6×150mm, 5μm), mobile phase A: 50mM NH4Ac (pH5.2), B: methanol. The protein is collected by linear gradient elution method, with a flow rate of 1ml / min. After the sample is eluted, it is freeze-dried and detected by MALDI-TOF.

[0089] Previously, some peptide antibiotics known to inhibit bacterial cell wall synthesis mainly target the pyrophosphate group of membrane-anchored phospholipid II (Lipid II) molecules and the pentapeptide residues linked to N-acetylmuramoyl. Because these components come from UMP ( Figure 3E), the inventors analyzed the interaction between AMSIN and UMP. First, using circular dichroism (CD) technology, the inventors found that the CD spectrum of the mixture of AMSIN and UMP was changed, and a new absorption peak appeared ( Figure 3 F), suggesting that a non-covalent bond complex may be formed between them. Using electrospray ionization mass spectrometry (ESI-MS) technology, the inventors confirmed the existence of the complex ( Figure 3 G) confirmed that the antibacterial activity of AMSIN lies in its binding to cell wall precursors and inhibiting the efficiency of cell wall synthesis.

[0090] Example 5 Evaluation of the therapeutic potential of AMSIN

[0091] Because peptide antibiotics targeting cell wall synthesis can significantly delay the development of drug resistance, the inventors evaluated the therapeutic potential of AMSIN as an anti-infective drug. The inventors found that the antibacterial activity of AMSIN was completely maintained when it was warmed in water at 37°C for 48 hours or in mouse serum for 24 hours ( Figure 4 A), indicating its high stability. Unlike classic hemolytic peptides, AMSIN exhibits very low hemolytic activity at doses far higher than the concentrations used for antibacterial activities ( Figure 4 B). In the presence of high doses of salmon sperm DNA, AMSIN maintained its full antimicrobial activity ( Figure 4 C), suggesting that it lacks the ability to bind to DNA and may not affect the stability of human genetic material. In addition, although AMSIN has a similar spatial structure to scorpion toxins, both are CSαβ structural types, at high concentrations (100 μM) it has no significant effects on a variety of ion channels (such as Na+) present in the human central nervous system and heart. v 1.5 K v 1.4 K v 1.5 K v 2.1 K v 4.3 and hERG) had no effect ( Figure 4 D) Because Na v 1.5 K v 1.5 K v 4.3 and hERG are mainly responsible for the generation and conduction of action potentials in myocardial cells. The results obtained by the inventors show that AMSIN lacks toxicity to the human heart. Using a mouse pneumonia model established with Streptococcus pneumoniae D39, the inventors found that AMSIN significantly reduced the bacterial load in the lungs of infected mice, with a decrease of 4.6-6.7 log10, which is comparable to penicillin in efficiency ( Figure 4E). In a peritonitis model induced by Methicillin-resistant Staphylococcus aureus P1374, AMSIN was able to cure live infected mice ( Figure 4 F).

[0092] Although specific embodiments of the present invention have been described in detail, it should be appreciated that the present invention is not limited to the specific embodiments. Various improvements, modifications and variations may be made to the present invention without departing from the spirit and scope of the present invention, and these improvements, modifications and variations are within the scope of the present invention. Sequence Listing <110> Institute of Zoology, Chinese Academy of Sciences <120> Defensins derived from human oral actinomycetes, their encoding genes and uses <130> DIC21110019 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 36 <212> PRT <213> Actinomyces sp. oral <400> 1 Gly Phe Gly Cys Pro Trp Asn Ala Tyr Glu Cys Asp Arg His Cys Val 1 5 10 15 Ser Lys Gly Tyr Thr Gly Gly Asn Cys Arg Gly Lys Ile Arg Gln Thr 20 25 30 Cys His Cys Tyr 35 <210> 2 <211> 36 <212> PRT <213> Actinomyces sp. oral <400> 2 Cys Phe Ala Cys Pro Phe Asn Glu His Gln Cys His Asn His Cys Leu 1 5 10 15 Ser Lys Gly Tyr Arg Gly Gly Phe Cys Gly Gly Phe Ala Ala Ala Thr 20 25 30 Cys Arg Cys His 35 <210> 3 <211> 36 <212> PRT <213> Actinomyces sp. oral <400> 3 Asp Leu Ser Cys Pro Trp Ala Pro Ser Val Cys Asn Arg His Cys Leu 1 5 10 15 Ser His Gly Tyr Arg Gly Gly Tyr Cys Ala Gly Pro Ile Lys Leu Val 20 25 30 Cys His Cys Tyr 35 <210> 4 <211> 35 <212> PRT <213> Actinomyces sp. oral <400> 4 Tyr Asn Cys Pro Thr Asp Glu Ser Pro Cys Asp Arg His Cys Arg Tyr 1 5 10 15 Ser Gly Tyr Arg Gly Gly Tyr Cys Gly Gly Ile Leu Lys Thr Ser Cys 20 25 30 Arg Cys Tyr 35 <210> 5 <211> 108 <212> DNA <213> Actinomyces sp. oral <400> 5 ggcttcggct gcccctggaa cgcctacgag tgcgaccgcc attgcgtcag caagggatac 60 accggtggaa actgccgcgg caagatccgc cagacctgcc actgctac 108 <210> 6 <211> 108 <212> DNA <213> Actinomyces sp. oral <400> 6 tgcttcgcct gccctttcaa cgaacatcag tgccacaacc actgcttgag caaaggatat 60 cgcggagggt tctgcggagg attcgctgcc gccacctgcc gctgccac 108 <210> 7 <211> 108 <212> DNA <213> Actinomyces sp. oral <400> 7 gaccttagct gcccctgggc cccatccgta tgtaaccgcc actgccttag tcacggttac 60 aggggaggct attgcgccgg ccccatcaag ctcgtgtgtc actgctac 108 <210> 8 <211> 105 <212> DNA <213> Actinomyces sp. oral <400> 8 tacaactgcc ctactgacga gtctccgtgc gaccgtcact gcaggtactc cggataccgg 60 ggcggctact gcggaggaat actcaagacc tcctgccgct gttac 105 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 tcctcacccc acacagaact 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 acaggccccg tgttacgtct 20

Claims

1. Use of a defensin derived from human oral actinomycetes or a pharmaceutical composition thereof in the preparation of a medicament for treating systemic infection caused by bacteria, wherein the amino acid sequence of the defensin is SEQ ID NO: 1, and the pharmaceutical composition comprises a defensin having an amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable excipient and / or carrier, wherein the bacteria are Gram-positive bacteria, wherein the Gram-positive bacteria are selected from Streptococcus pneumoniae and streptomycin-resistant strains thereof; Streptococcus mutans; Streptococcus salivarius; Streptococcus sanguinis; Streptomyces griseus and Streptomyces afluoromannan; Lysinibacillus fusiformis; penicillin-resistant and methicillin-resistant Staphylococcus aureus; and Enterococcus faecium.

2. Use of a defensin derived from human oral actinomycetes or a pharmaceutical composition thereof in the preparation of a medicament for treating bacteremia, endocarditis, peritonitis, pneumonia and central nervous system infection or respiratory tract infection caused by Gram-positive bacteria, wherein the amino acid sequence of the defensin is SEQ ID NO: 1, and the pharmaceutical composition comprises a defensin having an amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable excipient and / or carrier, wherein the Gram-positive bacteria is selected from Streptococcus pneumoniae and its streptomycin-resistant strain; Streptococcus mutans; Streptococcus salivarius; Streptococcus sanguinis; Streptomyces griseus and Streptomyces aflutolemannis; Lysinibacillus fusiformis; penicillin-resistant and methicillin-resistant Staphylococcus aureus; and Enterococcus faecium.

3. The use according to claim 1 or 2, wherein the method for preparing the defensin comprises the following steps: The linear peptide of the defensin is synthesized and then separated and purified by reverse phase high performance liquid chromatography.

4. The use according to claim 3, wherein the method further comprises the step of renaturing the synthesized defensin linear peptide before separation and purification by reverse phase high performance liquid chromatography.

5. The use according to claim 4, wherein the renaturation comprises dissolving the linear peptide of the synthetic defensin in water, then adding dimethyl sulfoxide to a final concentration of 5-20% by volume, mixing, adding phosphate buffer and incubating overnight.

Images