CN114478723B - Antibacterial peptide for improving outer membrane permeability of gram-negative bacteria - Google Patents

Antibacterial peptide for improving outer membrane permeability of gram-negative bacteria Download PDF

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CN114478723B
CN114478723B CN202210109812.4A CN202210109812A CN114478723B CN 114478723 B CN114478723 B CN 114478723B CN 202210109812 A CN202210109812 A CN 202210109812A CN 114478723 B CN114478723 B CN 114478723B
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CN114478723A (en
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孟凡强
陆兆新
吕凤霞
别小妹
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Nanjing Agricultural University
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Abstract

The invention discloses an antibacterial peptide for improving the outer membrane permeability of gram-negative bacteria. The novel antibacterial peptide is obtained by taking the lactobacillus plantarum A as a template and deleting the amino acid sequence, changing the amino acid composition, changing the space structure, changing the charge, hydrophobicity and the like, has strong antibacterial activity, has the MIC of 3.125 mug/mL for escherichia coli, can obviously improve the permeability of the escherichia coli outer membrane when the MIC is 0.78 mug/mL, and improves the antibacterial effect of hydrophobic antibiotics on negative bacteria. According to the invention, by modifying the lactobacillus plantarum A, particularly the antibacterial peptide OP4 capable of effectively improving the outer membrane permeability of gram-negative bacteria is obtained, and the antibacterial effect of erythromycin can be improved by 62.5 times. The invention provides a new potential way for treating gram-negative bacterial infection.

Description

Antibacterial peptide for improving outer membrane permeability of gram-negative bacteria
Technical Field
The invention belongs to the field of biotechnology and molecular biology, and particularly relates to a design and a bacteriostasis mechanism of antibacterial peptide.
Background
Gram-negative bacteria and E.coli and Salmonella are key factors responsible for food poisoning and outbreaks of food-borne diseases. Are also important causative agents of gastrointestinal diseases and bacterial infections. Gram-negative bacteria have an outer membrane composed of lipopolysaccharide and phospholipid, which is dense in structure. Because the asymmetry of the membrane results in the inability of various hydrophobic antibiotics to enter the cell, a bactericidal effect is exerted. The outer membrane of gram-negative bacteria thus confers its inherent antibiotic resistance. Because of the long-term, widespread use of antibiotics, gram-negative bacteria acquire resistance genes to a variety of antibiotics, and thus fewer antibiotics are available to treat negative bacterial infections effectively. In addition, antibiotic development against gram-negative bacteria has also been a bottleneck, and over 50 tens of thousands of compounds were screened in 2017 by gram-orchid smith, and no effective drugs against negative bacteria were found.
Disclosure of Invention
The invention aims to expand the effective treatment spectrum of negative bacteria infection in order to overcome the inherent tolerance of gram negative bacteria to hydrophobic antibiotics. The invention improves and researches the antibacterial mechanism of lactobacillus plantarum A, designs a series of analogues, and particularly discovers an antibacterial peptide OP4 capable of obviously improving the outer membrane permeability of gram-negative bacteria. The antibacterial peptide can obviously improve the antibacterial potency of erythromycin on escherichia coli through in vitro experiments and in vivo experiments of mice.
The invention is realized by the following technical scheme:
a lactobacillus plantarum A with an amino acid sequence of QFKNISLMYGNNVSRKTLTNFFKSLIKKIN (SEQ ID NO. 1).
An analogue of lactobacillus plantarum a as described above comprising the amino acid sequence of any one of the following antimicrobial peptides:
。
use of any of the above-described plantaricin a or an analogue thereof in a medicament for the treatment of a negative bacterial infection.
The invention establishes a detection method of escherichia coli outer membrane permeability, and the result shows that the mutant OP4 has the highest activity.
Further, artificial liposomes containing outer membrane components were constructed, confirming that OP4 binds to lipopolysaccharide LPS through hydrophobic and electrostatic interactions. Interfere with cross-linking of LPS and thereby increase the permeability of the outer membrane.
Furthermore, the OP4 can effectively enhance the speed of the hydrophobic antibiotics entering cells and improve the antibacterial effect of the antibiotics on negative bacteria by improving the permeability of the outer membrane. For example, OP4 increases the antibacterial effect of erythromycin on E.coli by a factor of 62.5.
On this basis, we used E.coli to infect the mouse peritoneal cavity and make a sepsis model. OP4 obviously improves the potency of erythromycin in the treatment of the model, and effectively reduces inflammatory response.
Drawings
FIG. 1 is a diagram showing the binding of OP4 to the outer membrane, indicating that the strain is E.coli ATCC 35218;
FIG. 2 is a graph of E.coli outer membrane permeability;
FIG. 3 is a graph of E.coli cell death caused by PlnA analogs;
FIG. 4 binding diagram of LPS and PlnA analogs;
FIG. 5 is a knockout lpxA, waaC and waaY correlation diagram;
FIG. 6 is a plot of binding sites for OP4 and LPS demonstrated by gene knockout and complementation experiments;
FIG. 7 is a graph showing the effect of OP4 on the promotion of antibiotic absorption by E.coli;
FIG. 8 is a graph showing that OP4 increases the inhibitory activity of erythromycin against E.coli and reduces inflammation in mice;
the specific embodiment is as follows:
example 1
Design and related character characterization of lactobacillus plantarum A mutant
Based on the amino acid sequence of the lactobacillus plantarum A, the number and the composition of amino acids are improved and changed, the characteristics of spatial structure, hydrophobicity, charge and the like are changed, and the mutant and the characteristics are characterized in the following table 1:
TABLE 1 physicochemical Properties of plnA and mutants thereof
Antibacterial peptide Sequence(s) Hydrophobicity of Amphiphilic properties Positive charge of
K43D QFKNISLMYGNNVSRDTLTNFFKSLIKKIN -0.357 Unchanged 4
K50D QFKNISLMYGNNVSRKTLTNFFDSLIKKIN -0.357 Unchanged 4
K55D QFKNISLMYGNNVSRKTLTNFFKSLIKDIN -0.357 Unchanged 4
N38R QFKNISLMYGDNVSRKTLTNFFKSLIKKIN -0.37 Increase in 5
R42A QFKNISLMYGNNVSAKTLTNFFKSLIKKIN -0.16 Increase in 5
R42D QFKNISLMYGNNVSDKTLTNFFKSLIKKIN -0.337 Unchanged 4
T44E QFKNISLMYGNNVSRKELTNFFKSLIKKIN -0.463 Increase in 5
V40S QFKNISLMYGNNSSRKTLTNFFKSLIKKIN -0.537 Lowering 6
L52N QFKNISLMYGNNVSRKTLTNFFKSNIKKIN -0.613 Lowering 6
I32N QFKNNSLMYGNNVSRKTLTNFFKSLIKKIN -0.637 Lowering 6
L34N QFKNISNMYGNNVSRKTLTNFFKSLIKKIN -0.613 Increase in 6
L34F QFKNISFMYGNNVSRKTLTNFFKSLIKKIN -0.403 Unchanged 6
G37W QFKNISLMYWNNVSRKTLTNFFKSLIKKIN -0.387 Unchanged 6
I32F QFKNFSLMYGNNVSRKTLTNFFKSLIKKIN -0.427 Unchanged 6
N47L QFKNISLMYGNNVSRKTLTLFFKSLIKKIN -0.127 Lowering 6
S51I QFKNISLMYGNNVSRKTLTNFFKILIKKIN -0.193 Lowering 6
S33F QFKNIFLMYGNNVSRKTLTNFFKSLIKKIN -0.25 Increase in 6
OP1 QFKNASGMYGDNVSRKELTNFFKSLIKKIN -0.483 Increase in 4
OP2 QFKNASGMYGDNVSAKELTNFFKSLIKKIN -0.693 Increase in 3
OP4 QRKINSLMNRVLRKLTNAFKSLIKKIN -0.496 Increase in 8
Lactobacillus plantarum A QFKNISLMYGNNVSRkTLTNFFKSLIKKIN -0.37 Control 6
The bacteriostatic activity of each mutant was measured by a micro broth dilution method, and the results are shown in table 2 below.
TABLE 2 antibacterial Activity of plnA and mutants thereof (. Mu.g/mL)
Example 2
E.coli outer and inner membrane permeability assay
The permeability of the outer membrane was detected with N-phenyl-1-naphthylamine (NPN). Coli EHEC O157: h7 was incubated to mid-log phase and OD600 = 0.05 was adjusted using HEPES buffer (5 mm, ph 7.2). Various concentrations of Lactobacillus plantarum A or its analogues were added and incubated for 1 hour at 37 ℃. To the mixture was added 2. Mu.L NPN (final concentration 10. Mu.M). The excitation and emission wavelengths of the NPNs were set to 350 and 420nm.
Small single-flap vesicles (SUVs) were prepared using an ultrasonography method. 100mg of lipid (DOPC: dope=1:1) (sigma in the united states) was dissolved in chloroform and evaporated to form a thin film on the bottom of a round bottom bottle. Multilamellar vesicles (MLVs) were formed by suspension in HEPES buffer (50 mM, pH7.2, 70mM calpain). Subsequently, they were sonicated until the liquid clearly formed SUV. Finally, SUV was purified from SephadexG-50 to remove free calcium. In addition, LPS-liposome SUS (LPS: lipid=1:100) (Sigma in the united states) was prepared using the above-described ultrasonic method. Different concentrations of PlnA were added to liposomes or LPS-liposome SUVs and leakage of the liposomes was monitored at excitation wavelength 490nm and emission wavelength 515 nm. 1% Triton X-100 was used as positive control.
As shown in FIG. 1, the binding of OP4 to the outer membrane is shown, indicating that the strain is E.coli ATCC 35218; in the figure, a is a graph of antimicrobial activity and outer membrane permeability of OP4 when incubated with cation and membrane components; b is an outer membrane permeability graph of E.coli incubated with OP4 and Wild when the outer membrane proteins were removed by trypsin and the polysaccharides by glycosidases; d is a graph of leakage rate of liposomes and LPS-liposomes caused by different concentrations of OP 4; c is a graph of endotoxin levels in the medium after treatment with different concentrations of PlnA, OP4, EDTA and polymyxin B (Pol B); e is an endotoxin level diagram of endotoxin detection kit detected after 20 mug/mL LPS and different concentrations of OP4 are mixed; f is a graph of the binding of LPS and OP4 detected by HPLC; it can be seen that plnA and OP4 significantly improved the permeability of the outer membrane, and that plnA and OP4 did not improve the permeability of the outer membrane after glycosidase treatment, indicating that plnA and OP4 may bind to glycosides. Subsequent binding experiments of OP4 to magnesium ions, calcium ions, lecithin, cholesterol, lipopolysaccharide and peptidoglycan showed that OP4 bound to lipopolysaccharide. And OP4, after binding to LPS, interferes with the integrity of the outer membrane of e.coli, resulting in a significant increase in LPS release into the culture medium. By detecting the binding of the two by HPLC, a significant OP4-LPS peak can be found. The artificial liposome is adopted to simulate the structure of a cell membrane, so that the permeability of the liposome can be remarkably improved when LPS is added into the liposome, and therefore, the binding site of OP4 on the outer membrane is lipopolysaccharide.
As shown in FIG. 2, an E.coli outer membrane permeability graph is shown; in the figure, a is a leakage pattern of liposomes caused by PlnA and its analogues. The specific process is as follows: liposomes (DOPC: dope=1:1) containing 70mm calcein were prepared. PlnA was added at 5min and TritonX-100 was added at 45min to destroy all liposomes. The fluorescence of calcein was monitored at excitation wavelength 490nm and emission wavelength 515 nm. b is PlnA, which results in an increase in the permeability of the outer membrane of the cell. After passing through the outer membrane, the NPN binds to the inner membrane and emits strong fluorescence. The excitation and emission wavelengths of the NPNs were set to 350 and 420nm. c increases the permeability of the intracellular membrane. The intracellular β -galactosidase flows out of the cell and ONPG hydrolyzes to produce a yellow ONP. The ONP was monitored with a spectrophotometer at 420nm. d and e are PlnA resulting in leakage of protein, potassium ions and ATP. The concentration of protein was determined by coomassie-brilliant blue method and the concentration of potassium ions and ATP was measured by kit. f is the effect of different concentrations of OP4 on the permeability of the inner and outer membranes.
The permeability of the endomembrane was detected with O-nitrobenzene-beta-D-galactoside (ONPG) and was degraded to O-nitrophenol by beta-galactosidase. O-nitrophenol was monitored with a spectrophotometer at 420nm. OP4 also has a destructive effect on the inner membrane in addition to the outer membrane of escherichia coli, addition of the PlnA analog can significantly increase leakage of intracellular substances such as proteins, nucleic acids, potassium ions and ATP, and the destructive effect of the PlnA analog on the inner membrane is positively correlated with its hydrophobicity.
Flow cytometry
The permeability of the E.coli inner membrane was monitored by flow cytometry as follows: coli was prepared as described above and treated with PlnA and its analogues (at a concentration of 1 MIC) for 1 hour. The cells were then harvested and washed twice with PBS (50 mm, pH 7.2) and then 1% (1 mg/mL, v/v) sodium iodide (PI) was added. Fluorescence was measured by flow cytometry at excitation 535nm and emission 615 nm. After treatment with PlnA and its analogues, permeability was significantly enhanced. As shown in fig. 3: the results showed that the mortality of E.coli caused by PlnA was 11.9%, with mutants S33F, K43A and OP4 having significantly higher bactericidal efficiency than PlnA and OP4 having a maximum bactericidal efficiency of 33.9%. Other mutants have bactericidal efficiency similar to or lower than that of PlnA.
Example 3
In vitro binding assay
HPLC was used to monitor the interaction of LPS and OP4 as follows: agilent bio-sec5 column (5 μm,7.8 mm. Times.300 mm); the mobile phase is acetonitrile: water = 95:5 (containing 0.1% (v/v) TFA); the flow rate is 1.5mL/min; the detection wavelength is 220nm. The interaction of LPS and OP4 was monitored by HPLC as follows: alignment Bio Sec-5 column (5 μm,7.8 mm. Times.300 mm); the mobile phase is acetonitrile: water=95:5; a flow rate of 1.5ml/min; the detection wavelength is 220nm.
The results are shown in FIG. 4: the peak time for PlnA was 11.9 minutes. After the antibacterial peptide is combined with lipopolysaccharide, the peak time is about 9.08 minutes. The results indicate that PlnA is able to bind to lipopolysaccharide, with a relatively large molecular mass, resulting in a reduced retention time. Among them, the binding efficiency of PlnA to lipopolysaccharide was 42.67%. The mutants OP1, K50D, I32W bound lipopolysaccharide at a significantly lower efficiency than plnA, and mutants N31A, R42A, S F and OP4 bound lipopolysaccharide at a significantly higher efficiency than plnA. The relative molecular mass of PlnA is about 3500Da, the relative molecular mass after combining with LPS is about 73kDa, and the relative molecular mass is increased by 70kDa.
Example 5
LPS modification
Reducing negative charge: mcr1 (phosphotidyl A transferase, gene ID: 39727008) was synthesized by Genscript (Nanjing, china) and cloned into vector pUC57. Subsequently, it is transformed into E.coli CICC21530. After induction with isopropyl β -D-sulfur Gan Tanggan (IPTG, 0.1 mm), the outer membrane permeability of e.coli was examined.
LPS modified strain: the gene lpxA (UDP-N-acetylglucosamine acylase) related to lipopolysaccharide synthesis (primers used are shown in table 3), waaC (ADP-heptasaccharide-LPS heptyl transferase) and waaY (lipopolysaccharide riboheptasaccharide (II) kinase) were knocked out using CRISPR-Cas9 technology. Then, lpxA, waaC and waaY were cloned into pUC57 and supplemented into escherichia coli Δlpxa, escherichia coli Δwaac and escherichia coli Δwaay. Finally, the outer membrane permeabilities of E.coli-. DELTA.lpxA, deltawaaC, E.coli-. DELTA.waaY, deltalpxA+lpxA, deltawaaC+waaC and E.coli-. DELTA.waaY+waaY were monitored as described above.
TABLE 3 primers for knocking out lpxL and waaC for CRISPR system
Extracting and purifying the LPS of the escherichia coli by using an LPS extraction kit. The method comprises the following steps: cells were collected with a centrifuge and ruptured in lysis buffer. LPS was then extracted with chloroform and purified with purification buffer. Finally, LPS was washed with 70% ethanol and dissolved in mercuric trichloride (10 mm, pH 8.0). Endotoxin detection kit detects endotoxin (south Beijing, china). As shown in fig. 5, lpxA, waaC and waaY have been successfully knocked out; wherein the indicator strain is E.coli ATCC35218. In the figure, a is a schematic diagram of LPS and a synthetic gene. UDP-GlcNac is uridine diphosphate N-acetamido glucose; hep is heptulose; kdo is ketodeoxyoctanoate; PEtN is phosphoethanolamine. b is endotoxin levels when lpxL, waaC and waaY are knocked out or mcr1, lpxL, waaC and waaY are overexpressed. c is the effect of LPS modification on membrane permeability.
As shown in FIG. 6, the binding site diagram of OP4 and LPS was confirmed for the gene knockout and complementation experiments, indicating that the strain was E.coli ATCC35218. In the figure, a is a schematic diagram of LPS and a synthetic gene. UDP-GlcNac is uridine diphosphate N-acetamido glucose; hep is heptulose; kdo is ketodeoxyoctanoate; PEtN is phosphoethanolamine. b is endotoxin levels when lpxL, waaC and waaY are knocked out or mcr1, lpxL, waaC and waaY are overexpressed. c is the effect of LPS modification on membrane permeability. The detection result of the LPS synthesis related gene knockout and the over-expressed escherichia coli surface lipopolysaccharide content shows that the over-expressed phosphoethanolamine-lipid A transferase Mcr1 does not influence the amount of escherichia coli lipopolysaccharide. After knockout of lpxA and waaC, the amount of e.coli lipopolysaccharide was significantly reduced and after overexpression of the gene, the amount of lipopolysaccharide was significantly higher than in the control group. This shows that the expression level of lpxA and waaC genes in the form of plasmids was higher than that of wild type bacteria, and more lipopolysaccharide was synthesized. And then, the outer membrane permeability result of the OP4 on the genetically engineered strain shows that after Mcr1 is over-expressed, the Mcr1 adds phosphatidylethanolamine to the lipopolysaccharide, and the phosphatidylethanolamine has positive charges and can neutralize the negative charges of the lipopolysaccharide lipdA region, so that the binding capacity of the OP4 and the lipopolysaccharide is reduced, and the outer membrane permeability is further reduced. After the lpxA and waaC are knocked out, the content of lipopolysaccharide is reduced, the binding strength with OP4 is reduced, and the outer membrane permeability of OP4 is reduced. Correspondingly, after the gene is over-expressed, the content of lipopolysaccharide is higher than that of wild bacteria, and the lipopolysaccharide is more sensitive to OP4.
Example 6
OP4 in combination with antibiotics
MIC after the combined use of OP4 and antibiotics was determined according to the MIC method for detection of plnA and its analogs (example 1). The synergy index (CI) is calculated as follows: ci=dx/dx+dy/dy. Dx and Dy are MIC when used alone, and Dx and Dy are the corresponding MIC when used in combination. CI is less than or equal to 1.0 and represents synergistic effect; fici=1.0 represents an additive; FICI > 1.0 indicates antagonism.
TABLE 4 MIC of antibiotics with or without OP4 for E.coli (. Mu.g/mL)
The indicator strain was E.coli ATCC35218. CI is a combination index; CI is less than or equal to 0.5 and represents synergistic effect; 0.5< CI <1.0 represents an additive; CI is greater than or equal to 1.0, which indicates antagonism.
TABLE 5 OP4 enhancement of erythromycin efficacy against gram-negative pathogens
Salmonella brucellosis CDC-1 is a multidrug resistant pathogen described in the study of Ju.
OP4 at 1/3MIC concentration was used in combination with various concentrations of antibiotics to test the sensitivity of E.coli O157 CICC21530 to antibiotics. The results are shown in Table 4. OP4 has no effect on the bacteriostatic effects of β -lactam antibiotics (ampicillin and amoxicillin), aminoglycoside antibiotics (streptomycin, gentamicin and kanamycin), lincomycin, glycopeptide antibiotics (polymyxin B and vancomycin). Has antibacterial effects on erythromycin, tetracycline antibiotics (tetracycline and oxytetracycline), quinolones (ciprofloxacin and norfloxacin), and sulfonamides (trimethoprim). In particular, for erythromycin, the MIC is reduced from 500 mug/mL to 8 mug/mL, and the potency is increased by 62.5 times. The erythromycin which is basically ineffective to the escherichia coli can inhibit the growth of the escherichia coli, and the antibacterial range of the erythromycin is widened. In addition, the OP4 improves the potency of the tetracycline drugs by 8 times and improves the potency of the sulfonamides by 16 times. In addition to E.coli, OP4 enhanced the activity of erythromycin against Yersinia, vibrio parahaemolyticus and Salmonella by a factor of 60-80 (Table 5).
Example 7
Antibiotic intake assay
E.coli WT, E.coli-. DELTA.lpxA and E.coli-. DELTA.waaC, E.coli-. DELTA.waaY, E.coli-. DELTA.lpxA+lpxa and E.coli- +waaC-. DELTA.waaC+waaY and E.coli-. DELTA.waaY+waaY were cultured to mid-log phase. Washed twice with PBS buffer (50 mm, pH 7.2) and incubated with OP 4-antibiotic or antibiotics for 1 hour at 37 ℃. Then, glass frit and PBS were added and the mixture was broken for 30 minutes (pH 2.0). The supernatant was extracted by a PCX solid phase extraction column and the antibiotic eluted with 5% ammoniated methanol. After drying with nitrogen, the antibiotic was dissolved with methanol. Finally, the antibiotic concentration was monitored by HPLC through Agilent XDBC-18 column at 215nm (erythromycin) and 274nm (tetracycline, quinolones, and sulfonamide), respectively.
To quantitatively detect the change of the intake of antibiotics of E.coli before and after OP4 treatment, PCX solid phase extraction column is used to extract antibiotics in cells and to detect the content of antibiotics. The results show that the uptake of erythromycin, tetracycline, ciprofloxacin and trimethoprim by E.coli after OP4 treatment is significantly improved. Taking erythromycin as an example, the uptake of erythromycin by untreated E.coli was 0.25. Mu.g/109 CFU, and the uptake of erythromycin after OP4 treatment reached 1.9. Mu.g/109 CFU, which was increased 7.6-fold, as shown in FIG. 7, indicating that the strain was E.coli ATCC35218. In the figures a, b and c are the survival of E.coli and its mutants incubated in different concentrations of erythromycin. D is antibiotic accumulation in e.coli detected by HPLC with or without OP4. e is erythromycin accumulation in E.coli when LPS is modified. f is the development of erythromycin resistance when E.coli is incubated in sublethal concentrations of erythromycin and ciprofloxacin.
It was demonstrated that the increased E.coli outer membrane permeability caused by OP4 promotes E.coli uptake of hydrophobic antibiotics. The erythromycin intake condition of the genetic engineering strain shows that the erythromycin intake of the engineering strain which overexpresses mcr1 and knocks out lpxL and waaC is obviously reduced compared with the erythromycin intake of the wild type, and the erythromycin intake of the strain which overexpresses lpxL and waaC is obviously improved. This suggests that erythromycin uptake is positively correlated with lipopolysaccharide amount and also positively correlated with outer membrane permeability by OP4.
Example 8
E.coli resistance variation test
Coli was cultured in LB medium with 1/2MIC antibiotics (erythromycin 250. Mu.g/mL and ciprofloxacin 0.03. Mu.g/mL). The concentration of antibiotics was increased every time the bacteria grew normally, and the culture was continued for 30 days. After adaptation, the bacteria were grown for 15 days without antibiotics to observe the sustainability of acquired resistance.
As shown in fig. 7, in the 30-day resistance experiment, the resistance to erythromycin was increased 4-fold and the resistance to ciprofloxacin was increased 16-fold in the control group. However, after addition of sublethal concentrations of OP4, there was no increase in resistance to erythromycin and a 2-fold increase in resistance to ciprofloxacin. This suggests that OP4 may significantly reduce the development of antibiotic resistance.
Example 9
Animal experiment
Specific pathogen-free BALB/c mice (male, 8 weeks old, 22-25g body weight) were from the university of Yangzhou comparative medical center (animal Mass certificate number. 202111661, license number. Syxk (su) 2017-0007). Mice were kept 5 animals per cage (individually ventilated cages) for a 12 hour photoperiod (Nanjing university animal center laboratory, license number. Syxk (su) 2011-0036). We performed all animal care and experiments according to the laboratory evaluation and certification association. Animal care guidelines (http:// www.aaalac.org) and approved by the laboratory animal welfare and ethics committee (license, NJAU No. 20210517071).
Coli ATCC35218 was cultured to mid-log phase, and the cells were harvested by centrifugation (5000 g,5 min) and resuspended in 0.9% sterile physiological saline (10 8 CFU/mL). On day 0 by intraperitoneal injection of 0.1mL of E.coli suspension (10 per mouse 7 CFU) to establish a sepsis model. A solution of erythromycin (10 mg/mL) and erythromycin+OP4 (1 mg/mL+1 mg/mL) was prepared in 0.9% physiological saline. 2 hours after inoculation, erythromycin (100 mg/kg BW) was injected into the erythromycin group mice, erythromycin+OP4 (10 mg/kg+10mg/kg BW) was injected into the erythromycin+OP4 group, and physiological saline was injected into the control group. On days 1 and 7, animals were anesthetized with tribromoethanol (200 mg/kgBW). With a 2mL vacuum blood collection tube (containing EDTA-K) 2 ) Blood was collected by cardiac puncture and plasma was collected by centrifugation (5000 g,5 min). 0.1g of organs (heart, liver, spleen, lung, kidney) was weighed and homogenized in 0.9ml of 0.9% physiological saline.
Blood and organ homogenates were serially diluted in sterile 0.9% saline and plated onto agar plates. The agar plates were then incubated overnight at 37℃and the colony count was calculated. In addition, the contents of inflammatory-related cytokines (c-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and nuclear factor κb (NF- κb)) in plasma were detected using ELISA kit.
To further investigate the synergy between OP4 and antibiotics, erythromycin and OP4 were used alone or in combination to treat mouse sepsis caused by e. As a result, FIG. 8 shows that BALB/c mice were intraperitoneally injected with 107CFU of E.coli ATCC35218, and 100mg/kg of erythromycin or 10mg/kg of erythromycin+10 mg/kg of OP4 was intraperitoneally injected 2 hours after infection. a daily survival curves of mice were calculated and plotted with GraphPad. On days 1 and 7, bacterial loads of blood (b), heart (c), liver (d), spleen (e), lung (f) and kidney (g) were counted on LB agar plates. Inflammatory-related cytokines CRP (h), TNF-alpha (i), IL-1β (j), IL-6 (k) and NF- κb (l) were assayed by ELISA. The different lowercase letters indicate significant differences (p < 0.05), and the uppercase letters indicate significant differences (p < 0.05) on day 7 (b-g). After the mice are injected with the escherichia coli intraperitoneally, the death rate reaches 40% within 24 hours, and the death rate reaches 60% on the 2 nd day. Similarly, day 2 mortality in Ery mice reaches 50%, indicating that 100. Mu.g/g erythromycin is not effective in inhibiting E.coli growth. In contrast, mortality in mice from the Ery+OP4 group was significantly reduced to 20% on day 2. These results indicate that 10 μg/g erythromycin+10 μg/g OP4 can be effective in treating abdominal infections. Furthermore, ery+OP4 significantly reduced bacterial load in the blood and heart, liver, spleen, lung and kidney of mice, whereas erythromycin alone was not. For example, bacterial load in the blood, heart, spleen and kidneys of mice decreased more than 10-fold on day 1 and more than 5-fold on day 7 compared to the infected group. Subsequently, inflammatory-related cytokines (CRP, TNF-alpha, IL-1 beta, IL-6 and NF- κb in plasma were tested using ELISA kit) the results indicated that the levels of inflammatory factors were significantly elevated in mice after E.coli infection and erythromycin did not alleviate the occurrence of inflammation, however Ery+OP4 significantly reduced the levels of inflammatory factors, indicating that OP4 increased erythromycin efficacy by at least 10-fold in mice.
Sequence listing
<110> Nanjing agricultural university
<120> an antibacterial peptide for improving the outer membrane permeability of gram-negative bacteria
<130> 20220128
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 2
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Asp
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 3
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Asp Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 4
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Asp Ile Asn
20 25 30
<210> 5
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asp Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 6
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Ala Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 7
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Asp Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 8
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Glu Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 9
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Ser Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 10
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Asn Ile Lys Lys Ile Asn
20 25 30
<210> 11
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Gln Phe Lys Asn Asn Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 12
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Gln Phe Lys Asn Ile Ser Asn Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 13
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Gln Phe Lys Asn Ile Ser Phe Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 14
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Gln Phe Lys Asn Ile Ser Leu Met Tyr Trp Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 15
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Gln Phe Lys Asn Phe Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 16
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Leu Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 17
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Gln Phe Lys Asn Ile Ser Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ile Leu Ile Lys Lys Ile Asn
20 25 30
<210> 18
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Gln Phe Lys Asn Ile Phe Leu Met Tyr Gly Asn Asn Val Ser Arg Lys
1 5 10 15
Thr Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 19
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Gln Phe Lys Asn Ala Ser Gly Met Tyr Gly Asp Asn Val Ser Arg Lys
1 5 10 15
Glu Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 20
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Gln Phe Lys Asn Ala Ser Gly Met Tyr Gly Asp Asn Val Ser Ala Lys
1 5 10 15
Glu Leu Thr Asn Phe Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25 30
<210> 21
<211> 27
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Gln Arg Lys Ile Asn Ser Leu Met Asn Arg Val Leu Arg Lys Leu Thr
1 5 10 15
Asn Ala Phe Lys Ser Leu Ile Lys Lys Ile Asn
20 25
<210> 22
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
tcctaggtat aatactagtg tgatggttgg cgg 33
<210> 23
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ctgctcgttt tagagctaga aatagc 26
<210> 24
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tcctaggtat aatactagtc ccagggatta agtttgactg ttttagagct agaaatagc 59
<210> 25
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
tcctaggtat aatactagta cccttcattt cgtacttttg ttttagagct agaaatagc 59
<210> 26
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
actagtatta tacctaggac tgagctagct 30
<210> 27
<211> 80
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
cgcagtccat cagttctgca tcattggtgc gcacgtgatg ggcgcaggac gtccctcctt 60
atgtcattgc gcagggtaac 80
<210> 28
<211> 80
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
cgttgcccgc actcactgat gcccagcagg caatcccagg tggaagaagg gttcgcacag 60
attccttcct ggcacgctgc 80
<210> 29
<211> 84
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
ccaataaata aaagtcattg agtgtattta acccttcatt tcgtactggg tttgctcaaa 60
aaggcgttcg taataatcac cttt 84

Claims (2)

1. An analogue of plantaricin a, characterized in that it has the amino acid sequence QRKINSLMNRVLRKLTNAFKSLIKKIN.
2. Use of the analogue according to claim 1 for the preparation of a medicament for the treatment of gram negative bacterial infections.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102844039A (en) * 2010-01-12 2012-12-26 吉利亚尼股份公司 Process for the preparation of a biomass comprising plantaricin and uses thereof in medical field
CN105002199A (en) * 2015-06-29 2015-10-28 北京化工大学 Method for inhibiting lactobacillus plantarum by plantaricin A secreted and expressed by saccharomyces cerevisiae

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102844039A (en) * 2010-01-12 2012-12-26 吉利亚尼股份公司 Process for the preparation of a biomass comprising plantaricin and uses thereof in medical field
CN105002199A (en) * 2015-06-29 2015-10-28 北京化工大学 Method for inhibiting lactobacillus plantarum by plantaricin A secreted and expressed by saccharomyces cerevisiae

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Accession No. WP_154766382.1;佚名;Genbank;全文 *
II类细菌素的生物合成及其在食品领域的应用;李萍等;中国食品学报;第21卷(第10期);269-286 *
Plantaricin A, a peptide pheromone produced by Lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells;Sverre L. Sand等;Peptides;第31卷(第7期);1237-1244 *

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