CN117138024B - A vancomycin-antimicrobial peptide conjugate and its application - Google Patents
A vancomycin-antimicrobial peptide conjugate and its application Download PDFInfo
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Abstract
本发明涉及一种万古霉素‑抗菌肽偶合物及其应用，在对抗菌肽M2的N端添加一个半胱氨酸进行修饰后，通过双功能蛋白质交联剂sulfo‑SMCC，将万古霉素和抗菌肽改造体通过两步反应进行偶联。万古霉素‑抗菌肽偶合物与万古霉素，抗菌肽M2，以及万古霉素和抗菌肽M2混合物相比抗菌活性显著提高，克服了革兰氏阳性菌对万古霉素的耐药性，对部分革兰氏阴性菌也产生抗菌活性，增大了万古霉素的抗菌谱，同时还具有更强的抗生物被膜能力，能够杀灭滞留菌，抑制细菌群集运动，还能与抗生素形成协同作用共同杀菌。
The present invention relates to a vancomycin-antimicrobial peptide conjugate and its application. After a cysteine is added to the N-terminus of the antimicrobial peptide M2 for modification, vancomycin and the antimicrobial peptide modified body are coupled through a two-step reaction through a bifunctional protein cross-linking agent sulfo-SMCC. Compared with vancomycin, antimicrobial peptide M2, and a mixture of vancomycin and antimicrobial peptide M2, the vancomycin-antimicrobial peptide conjugate has significantly improved antibacterial activity, overcomes the drug resistance of Gram-positive bacteria to vancomycin, and also produces antibacterial activity against some Gram-negative bacteria, thereby increasing the antibacterial spectrum of vancomycin. At the same time, it also has a stronger anti-biofilm ability, can kill retained bacteria, inhibit bacterial clustering movement, and can also form a synergistic effect with antibiotics to kill bacteria together.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a vancomycin-antibacterial peptide conjugate and application thereof.
Background
Since antibiotics have been found to play the most important role in the field of antibacterial and antiinfective, but in recent years, with the continuous emergence of more and more resistant bacteria from the abuse of traditional antibiotics in medical and animal husbandry, the world has entered the post-antibiotic era, microbial resistance has become a serious challenge for life health of humans,
It has been reported that bacterial infection has become the second leading cause of disease death in humans worldwide, and infections caused by drug-resistant bacteria such as staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and the like are important factors threatening the life health of humans. Currently, over 100 tens of thousands of people die annually from drug-resistant bacterial infection, and by the year 2050, this number will climb to 1000 tens of thousands of people, as predicted by the world health organization. Therefore, in the latter antibiotic age, it has been urgent to seek new and more effective drugs against drug-resistant bacteria.
Vancomycin has been found since 1952 to be widely used in serious drug-resistant gram-positive bacteria such as staphylococcus aureus, wherein the vancomycin is mainly combined with 2D-alanine sequences of cell wall peptidoglycan precursor lipopeptides to inhibit cell wall synthesis so as to cause bacterial death, but continuous abuse causes the generation of a plurality of vancomycin-resistant bacteria, and the main drug-resistant mechanism is that the D-alanine of a binding target point of the vancomycin is replaced by D-lactic acid and D-serine, so that the steric hindrance of the vancomycin binding with the target point is increased, the binding affinity of the vancomycin with the target point is reduced, and bacterial cell membrane permeability is reduced so as to cause the drug resistance of bacteria to the vancomycin.
The antibacterial peptide is a natural active polypeptide mainly derived from animals, plants and microorganisms in nature, and is a short peptide formed by connecting 10-50 amino acids through amide bonds, and has the bactericidal effect mainly by destroying the structure of bacterial cell membranes and combining with partial intracellular targets, inhibiting DNA and protein synthesis. Summarizing, the antibacterial peptide has the advantages of wide sources, easy acquisition, strong antibacterial activity, broad spectrum, unique mechanism, difficult drug resistance, simple structure and easy transformation. In addition, the antibacterial peptide also has a certain immunoregulatory activity. At present, more than 30 antibacterial peptides are in clinical research stage, wherein M2 is derived peptide derived from frog antibacterial peptides, and is applied to treatment of skin infectious diseases such as psoriasis, diabetic foot and the like. In addition, it also shows better killing effect on viruses and tumor cells. In summary, in the latter antibiotic age, antibacterial peptides have become the most promising antibacterial and antiinfective drugs to replace traditional antibiotics.
There has been a focus on the possibility that antibacterial peptides may be a break through to solve the problem of bacterial resistance, and the most commonly used method is a combination of antibiotics and antibacterial peptides. The combination mode can only simply superpose the action effects of the antibiotics and the antibacterial peptide, but cannot truly produce the problem of drug resistance of bacteria to the antibiotics. Therefore, a method is needed to make the antibacterial peptide and the antibiotics play a role together in the sterilization process to the greatest extent.
Disclosure of Invention
In order to solve the problem that the combination effect of antibiotics and antibacterial peptides is not ideal, and to make the antibiotics and the antibacterial peptides act together to the greatest extent, the invention provides a vancomycin-antibacterial peptide conjugate.
The first object of the invention is to provide a vancomycin-antibacterial peptide conjugate, which is obtained by covalent crosslinking of vancomycin and antibacterial peptide with an amino acid sequence shown as SEQ ID NO.1 through a crosslinking agent.
Further, the antibacterial peptide is modified with cysteine at the end.
Further, the terminal lysine of the antibacterial peptide is subjected to acylation modification.
Further, all amino acids in the antimicrobial peptide engineered body are L-type amino acids.
Further, the covalent crosslinking is achieved by the bifunctional protein crosslinking reagent Sulfo-SMCC.
Further, the preparation method of the antibacterial peptide modified body comprises the following steps of adopting a solid phase synthesis technology to carry out chemical synthesis according to the amino acid sequence of the antibacterial peptide modified body, purifying by using preparative reverse high performance liquid chromatography, and identifying the molecular weight by using high resolution mass spectrum.
Further, coupling vancomycin with a bifunctional protein crosslinking agent sulfo-SMCC through an amide bond to obtain a vancomycin modification Vm-SMCC with a molecular weight of 1667.55Da.
Further, the preparation method of the vancomycin modification Vm-SMCC comprises the following steps of purifying by using preparative reverse high performance liquid chromatography and identifying the molecular weight by using high resolution mass spectrometry.
Further, coupling the antibacterial peptide with the vancomycin modification Vm-SMCC to obtain the vancomycin-antibacterial peptide conjugate Vm-M2.
Further, the preparation method of the vancomycin-antibacterial peptide conjugate Vm-M2 comprises the following steps of purifying by using preparative reverse high performance liquid chromatography and identifying the molecular weight by using high resolution mass spectrometry.
A second object of the present invention is to provide the use of vancomycin-antibacterial peptide conjugates for the preparation of antibacterial agents.
Further, the antibacterial agent is used for inhibiting gram-positive bacteria or gram-negative bacteria.
Further, the gram positive bacteria include, but are not limited to, staphylococcus aureus (such as staphylococcus aureus CMCC26003, staphylococcus aureus ATCC29213, methicillin-resistant staphylococcus aureus ATCC43300, staphylococcus aureus 15772, staphylococcus aureus 15192, vancomycin-resistant staphylococcus aureus 52 and vancomycin-resistant staphylococcus aureus 11), enterococcus faecalis (such as vancomycin-resistant enterococcus faecalis ATCC29212 and enterococcus faecalis 2), clostridium perfringens, and bacillus cereus.
Further, the gram-negative bacteria include, but are not limited to, escherichia coli (e.g., escherichia coli ATCC25922 and escherichia coli CMCC 44102), acinetobacter baumannii (e.g., acinetobacter baumannii ATCC19606 and acinetobacter pantopractic 1), klebsiella pneumoniae (klebsiella pneumoniae 9883, klebsiella pneumoniae 2, klebsiella pneumoniae 3, klebsiella pneumoniae 4), pseudomonas aeruginosa (e.g., pseudomonas aeruginosa ATCC27853, pseudomonas aeruginosa CMCC10104, pseudomonas aeruginosa 60357, pseudomonas aeruginosa 52097 and pseudomonas aeruginosa 5), shigella flexneri (e.g., shigella flexneri ATCC12022 and shigella flexneri CMCC 51571), salmonella typhimurium (e.g., salmonella typhimurium ATCC14028 and salmonella typhimurium cic 21483), and vibrio parahaemolyticus (e.g., vibrio ATCC 17802).
A third object of the present invention is to provide the use of vancomycin-antibacterial peptide conjugates in antibacterial biofilms.
A fourth object of the present invention is to provide an antibacterial pharmaceutical composition comprising the vancomycin-antibacterial peptide conjugate and an antibiotic.
Further, the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.
The invention has the beneficial effects that:
(1) According to the invention, the vancomycin and the antibacterial peptide M2 are coupled through the bifunctional protein cross-linking agent sulfo-SMCC, and the obtained vancomycin-antibacterial peptide conjugate Vm-M2 greatly improves the antibacterial activity of the vancomycin, and effectively solves the problem of drug resistance of partial gram-positive bacteria to the vancomycin. The conjugate Vm-M2 remarkably expands the inherent antibacterial spectrum of vancomycin and has strong antibacterial activity on main gram-negative pathogenic bacteria (including drug-resistant and pan-drug-resistant clinical isolates). In addition, at the same molar concentration, the conjugate exhibits more efficient bactericidal activity than vancomycin, antibacterial peptide M2, and a mixture of vancomycin and antibacterial peptide M2.
(2) The vancomycin-antibacterial peptide conjugate Vm-M2 provided by the invention is obtained through 2-step coupling reaction, and has the advantages of convenience in synthesis, easiness in obtaining raw materials and low synthesis cost. In addition, the conjugate has the advantages of low hemolytic activity, high stability, strong anti-biofilm activity and the like, can be applied to the fields of medicines, cosmetics, aquaculture and the like, and greatly expands the downstream application value.
Drawings
FIG. 1 is the results of mass spectrometry and HPLC analysis of vancomycin modification Vm-SMCC;
FIG. 2 is a mass spectrum and HPLC analysis result of vancomycin-antibacterial peptide conjugate Vm-M2;
FIG. 3 is a synthetic route for vancomycin-antibacterial peptide conjugate Vm-M2;
FIG. 4 is a graph showing the retention and sterilization effect of vancomycin-antibacterial peptide conjugate Vm-M2;
FIG. 5 is the salt ion stability of the vancomycin-antibacterial peptide conjugate Vm-M2;
FIG. 6 is the effect of vancomycin-antimicrobial peptide conjugate Vm-M2 on bacterial colony movement.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The experimental strains were all collected from the university of su affiliated hospitals.
EXAMPLE 1 chemical Synthesis of vancomycin-antibacterial peptide conjugate Vm-M2
The antibacterial peptide M2 is an engineered body derived from frog antibacterial peptide, and consists of 22 amino acids, wherein the sequence is ：Gly1Ile2 Gly3 Lys4 Phe5 Leu6 Lys7 Lys8 Ala9 Lys10 Lys11 Phe12 Gly13 Lys14 Ala15Phe16Val17 Lys18 Ile19 Leu20 Lys21 Lys22(SEQ ID NO.1),, and C-terminal lysine is subjected to amidation modification.
In order to facilitate coupling, according to the amino acid sequence of the frog antibacterial peptide M2, a modified body C-M2 is designed and obtained by utilizing a molecular modification method, wherein the amino acid sequence is Cys1 Gly2 Ile3 Gly4 Lys5 Phe6 Leu7 Lys8 Lys9Ala10 Lys11Lys12 Phe13 Gly14 Lys15 Ala16 Phe17 Val18 Lys19 Ile20 Leu21 Lys22 Lys23(SEQ ID NO.2),, and the C-terminal lysine is subjected to amidation modification, and the preparation method is as follows:
I. According to the amino acid sequence of the C-M2, an automatic polypeptide synthesizer (433A,Applied Biosystems) is used for synthesizing the complete sequence, and HPLC reversed phase column chromatography is used for desalting;
II, determining molecular weight by adopting matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF);
Purified C-M2 was identified for purity by high performance liquid chromatography HPLC.
The secondary amine of vancomycin and succinimidyl ester on a bifunctional protein crosslinking agent sulfo-SMCC undergo an amide condensation reaction in a weak alkaline environment to form a stable amide bond, and the vancomycin reacts with the equivalent of sulfo-SMCC. Vancomycin is dissolved in PBS buffer solution (pH=8.16), after vancomycin is completely dissolved, the pH value of the solution is measured by a pH meter, if the pH value is lower than 8.16, naOH (0.1 mol/L) is needed to adjust the pH value to 8.16, sulfo-SMCC is dissolved in a proper amount of DMSO, then the two are mixed, stirred at room temperature for 3 hours, reverse high performance liquid chromatography is used for monitoring the reaction, after the reaction is completed, the product is purified by preparative HPLC, molecular weight identification is carried out by high resolution mass spectrometry, and the liquid phase and mass spectrometry analysis result is shown in figure 1, and the reaction product obtained in the step is named Vm-SMCC.
The maleimide group at the other end of the bifunctional protein crosslinking agent sulfo-SMCC can react with the sulfhydryl group on the N-terminal cysteine of the antibacterial peptide to form a stable thioether bond under a weak acid environment (pH=5.5). Vm-SMCC reacts with the antibacterial peptide C-M2 in an equivalent amount. Both were dissolved in PBS buffer (ph=5.5), after complete dissolution, the pH of the solution was measured with a pH meter, and if the pH was below 5.5, it was necessary to adjust the pH to 5.5 with NaOH (0.1 mol/L) solution. The reaction was stirred at room temperature for 3 hours, monitored by reverse-phase high performance liquid chromatography, after completion of the reaction, the product was purified by preparative HPLC and molecular weight was identified by high resolution mass spectrometry, the results of which are shown in FIG. 2, and the final product was designated Vm-M2.
The synthetic route is shown in fig. 3.
Example 2 pharmacological experiments on vancomycin-antibacterial peptide conjugate Vm-M2
Determination of vm-M2 antibacterial Activity:
(1) The minimum inhibitory concentration (Minimum Inhibitory Concentration) of Vm-M2 was determined (2-fold dilution).
And selecting 11 gram-positive bacteria and 18 gram-negative bacteria for MIC measurement test, inoculating the test strains into MH liquid culture medium (Meilun organism), culturing at 37 ℃ in a shaking manner to logarithmic phase, and diluting the culture solution cultured to the logarithmic phase to 2X 10 5 cfu/mL with fresh MH liquid culture medium for later use.
In each well of the sterile 96-well plate, 90. Mu.L of MH liquid medium was previously added, then 10. Mu.L of vancomycin, M2 and Vm-M2 sample solutions diluted with MH liquid medium to a certain concentration and filtered through a 0.22 μm well filter were added to the first well, and in addition, a physical mixed group of vancomycin and antimicrobial peptide M2 was set up, the system was 80. Mu.L MH+10. Mu.L of vancomycin+10. Mu. L M2, 50. Mu.L was added to the 2 nd well after mixing, and 50. Mu.L was sequentially diluted in a double ratio (Table 1) and sucked out from the 8 th well.
TABLE 1 dilution method
After the above tubes were mixed, the mixture was left to stand at 37℃and incubated with slow shaking at 110rpm for 16 hours, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible.
TABLE 2 antibacterial Activity of vancomycin-antibacterial peptide conjugate Vm-M2
MIC, minimum inhibitory concentration, the above results are the average of three independent replicates.
As can be seen from Table 2, the conjugate Vm-M2 has a stronger and broader antibacterial activity than vancomycin alone and antibacterial peptide alone. For gram-positive bacteria, vm-M2 overcomes the drug resistance of staphylococcus aureus and enterococcus faecalis to vancomycin, and for numerous gram-negative bacteria insensitive to vancomycin, especially for some pan-drug resistant clinical isolates, the conjugate Vm-M2 also exhibits extremely strong antibacterial activity, which is higher than that of the single addition of the antibacterial peptide M2. Meanwhile, by using the physical mixing group of vancomycin and antibacterial peptide as a comparison, the result shows that the pure physical mixing cannot generate stronger antibacterial activity, which further indicates that the chemical coupling generates better antibacterial effect. The GM value is the arithmetic average of MIC values of the antibacterial drugs against the test bacteria, the lower the GM value, the stronger the antibacterial activity, as shown in table 2, the GM value of vancomycin is 56.48 μm, the GM value of antibacterial peptide M2 is 8.66 μm, the GM value of the physical mixture group is 4.40 μm, and the GM value of the conjugate Vm-M2 is 2.60 μm, so it can be considered that the overall antibacterial activity of the conjugate is improved by 21.72 times relative to vancomycin, 3.33 times relative to the antibacterial peptide M2 alone, and 1.69 times relative to the physical mixture group. This demonstrates that the conjugate Vm-M2 has excellent antibacterial activity and clinical application value.
(2) Dynamics of sterilization of vancomycin-antibacterial peptide conjugate Vm-M2
Coli ATCC25922 and staphylococcus aureus CMCC26003 were selected and incubated with MH broth for 6 hours at 37℃and then diluted with fresh MH broth to a bacterial suspension of 10 6 cfu/mL. Vancomycin, M2 and Vm-M2 samples dissolved in sterilized deionized water were added to the bacterial suspension to a final concentration of 4. Mu.M. The bacteria liquid after administration is placed in a 37 ℃ incubator for shake culture, 10 mu L of bacteria liquid is respectively taken for dilution 1000 times in 0,15, 30, 60, 120 and 180 minutes, then 50 mu L of diluted bacteria liquid is taken and coated on MH solid culture medium, and colony count is carried out after the bacteria liquid is cultured in the 37 ℃ incubator for overnight. Since vancomycin is ineffective against gram-negative bacteria, coliform ATCC25922 was treated with polymyxin B as a positive control and sterilized PBS as a negative control.
TABLE 3 sterilizing speed of vancomycin, M2 and Vm-M2 (E.coli ATCC 25922)
TABLE 4 sterilizing speed of vancomycin, M2 and Vm-M2 (Staphylococcus aureus CMCC 26003)
As shown in tables 3 and 4, for staphylococcus aureus CMCC26003, conjugate Vm-M2 exhibited extremely fast sterilization speed, and all live bacteria could be killed within 30 minutes, and a small amount of live bacteria still remained in antimicrobial peptide M2 at 120 minutes, and the sterilization speed of vancomycin was very slow, and bacteria could not be killed effectively within 180 minutes, only the effect of inhibiting bacterial growth could be achieved. For E.coli ATCC25922, conjugate Vm-M2 exhibited a sterilization rate comparable to that of the positive control polymyxin B, and was able to kill all bacteria within 30 minutes. Vancomycin does not have any bactericidal or bacteriostatic activity.
(3) Experiment of resident bacteria
Most bacteria die quickly after being impacted by antibacterial drugs, but a small part of sub-populations can not be thoroughly killed, unlike traditional drug-resistant bacteria, the sub-populations of bacteria are not changed in gene sequence, the sub-populations of bacteria are called detaining bacteria, the bacteria are phenotype variant bacteria in a dormant state, the drug can not play a role in killing the bacteria although being combined with a target point, so the detaining bacteria have tolerance to almost all antibacterial drugs, but the drug resistance characteristics of the detaining bacteria can not be inherited to offspring, after being collected and re-cultured, the drug resistance characteristics of the detaining bacteria disappear, and the MIC value of the detaining bacteria is not changed obviously. In recent years, more and more clinical cases show that the detention bacteria are an important cause of chronic infectious diseases, and the symptoms are repeated, and research on prevention and removal of the detention bacteria is also attracting more and more attention. In order to detect the killing effect of the conjugate Vm-M2 on the detaining bacteria, staphylococcus aureus CMCC26003 is selected and placed in a shaking table at 37 ℃ for culture at 200rpm for 6 hours until the logarithmic phase of growth, the culture is diluted to 10 8 CFU/mL by using MH liquid culture medium, 900 mu L is taken and added into a centrifuge tube, ciprofloxacin (CIP) with the final concentration of 20 times MIC is added into bacterial liquid, the culture is carried out for 8 hours at 37 ℃ by using a shaking table, and 10 mu L of bacterial liquid is diluted for 1000 times and 50 mu L of diluted bacterial liquid is coated on a plate. At the 8h time point, 300. Mu.L of bacterial liquid was removed, the medium was centrifuged off, washed 3 times with sterile PBS, ciprofloxacin was removed, resuspended in 300. Mu.L of MH liquid medium, divided into 3 aliquots, 100. Mu.L of each tube was added with vancomycin, M2 and Vm-M2 samples at a final concentration of 4. Mu.M, followed by 1000-fold dilution of samples at the 24h and 48h time points, plating, placing in a 37℃incubator for 16h, and taking out the plates for colony counting. The experiment also sets up a physically mixed group of vancomycin and antimicrobial peptide M2 each incubated at 4 μm.
As shown in the experimental results of fig. 4, after ciprofloxacin is incubated for 8 hours, stable and drug-resistant resident bacteria are formed, the ciprofloxacin is continuously given to be incubated for 48 hours, the number of living bacteria is almost unchanged, the sterilization effect of vancomycin on resident bacteria is not obvious, the number of resident bacteria colonies is only reduced by 1 order of magnitude after the ciprofloxacin is incubated for 48 hours, the sterilization effect of the antibacterial peptide M2 on resident bacteria is stronger than that of vancomycin, the number of resident bacteria colonies after the ciprofloxacin is incubated for 48 hours is reduced by 3 orders of magnitude, the sterilization effect of the resident bacteria of a physical mixed group is similar to that of the single antibacterial peptide M2, the conjugate Vm-M2 shows extremely strong sterilization effect of resident bacteria, the number of resident bacteria colonies after the ciprofloxacin is incubated for 48 hours is reduced by more than 4 orders of magnitude, and statistical analysis shows that when the ciprofloxacin is given to be incubated for 48 hours, obvious differences exist between the resident bacteria colonies of a conjugate group and the single antibacterial peptide group and a physical mixed group. Therefore, the sterilizing effect of the conjugate detention bacteria is obviously stronger than that of the complex group of vancomycin and the single antibacterial peptide M2, and the physical mixing group of the vancomycin and the antibacterial peptide is not better than that of the conjugate group.
2. Determination of the haemolytic Activity of the vancomycin-antibacterial peptide conjugate Vm-M2
The collected human blood was mixed with an Abstract solution for anticoagulation, washed 2 times with physiological saline and resuspended to a suspension of 10 7-108 cells/mL. The diluted erythrocyte suspension is mixed with M2 and Vm-M2 samples dissolved in physiological saline, the temperature is kept at 37 ℃ for 30min, the centrifugation is carried out at 1000rpm for 5min, and the absorption value of the supernatant is measured at 540 nm. The negative control used physiological saline, the positive control used Triton X-100 and the percent hemolysis was calculated as percent hemolysis H% = a Sample of -A Negative control /A Positive control X100%. The results are shown in Table 5.
TABLE 5 hemolytic Activity of M2 and Vm-M2
The results show that when the sample concentration is close to 8 times of GM (arithmetic mean of MIC), the hemolysis percentage of Vm-M2 is 8.16%, and at the same concentration, the hemolysis percentage of M2 is 13.45%, which indicates that the conjugate Vm-M2 has lower hemolysis activity than the single antimicrobial peptide M2 and is not easy to cause rupture and dissolution of mammalian erythrocytes. In particular, the safety is high in the antibacterial activity range.
3. Salt ion stability experimental study of vancomycin-antibacterial peptide conjugate Vm-M2
In humans, there are a number of common salt ions, which are indispensable for maintaining normal vital activities of the human body, most of which are positively charged, and the conjugate Vm-M2 is positively charged due to the coupling of the antibacterial peptide, while the lipid component of the bacterial cell membrane is negatively charged, and studies have shown that the positively charged salt ions can competitively bind to the bacterial cell membrane with the antibacterial peptide, resulting in weakening or disappearance of the antibacterial activity of the antibacterial peptide, so that it is necessary to detect the salt ion stability of the conjugate Vm-M2, and to select escherichia coli ATCC25922, vancomycin-resistant staphylococcus aureus VRSA52, and culture it in MH liquid medium (su biotechnology limited, inc.) at 37 ℃ for 12 hours, respectively. Then diluted to 10 5 CFU/ml with MH broth (NaCl 150mM,KCl 4.5mM,CaCl2 2mM,MgCl2 1mM,ZnCl2 8μM,FeCl34μM,NH4Cl 6μM) containing physiological salt ion concentration, respectively. Samples of Vm-M2 were prepared at different concentration gradients using MH liquid medium containing the corresponding salt ion concentrations. MIC values of Vm-M2 for escherichia coli ATCC25922 and vancomycin-resistant staphylococcus aureus VRSA52 were determined by a 2-fold dilution method, so as to determine the influence of different salt ions on the antibacterial activity of vancomycin-antibacterial peptide conjugate.
As shown in FIG. 5, the conjugate Vm-M2 has strong salt ion tolerance. The antimicrobial activity of the (NaCl 150mM,KCl 4.5mM,CaCl2 2mM,MgCl2 1mM,ZnCl28μM,FeCl3 4μM,NH4Cl 6μM), conjugate Vm-M2 remains substantially unchanged (MIC value increases no more than one fold) at the reference human physiological salt ion concentration.
Example 3 Effect of vancomycin-antibacterial peptide conjugate Vm-M2 on bacterial Cluster Effect
The bacterial colony movement is that bacteria perform migration movement dependent on flagella from an inoculation point to the periphery on the surface of a culture medium in a colony manner, is the reaction behavior of the bacteria to self-adapt to the environment, and has important significance on bacterial drug resistance and biofilm formation process. To investigate the effect of conjugate Vm-M2 on bacterial colony effect, we performed experiments with medium tryptone (10 g/L), naCl (10 g/L), yeast extract (5 g/L), agar (0.3%), each of which was dissolved in deionized water, sterilized at high temperature, added to final concentration of 0.5. Mu.M, 1. Mu.M, 2. Mu.M vancomycin, M2 and Vm-M2, and the same final concentration of vancomycin and antibacterial peptide in a physically mixed group, added with an equal volume of PBS buffer as a blank, cooled in six well plates for standby, E.coli ATCC25922, staphylococcus aureus CMCC26003 were cultured with MH liquid medium (Suzhou alpha Biotechnology Co., ltd.) at 37℃for 6 hours to the log phase of growth, diluted with MH liquid medium to 10 6 CFU/mL, pipetted with 5. Mu.L, dropped onto cooled solid medium, then placed in 37℃incubator for 24 hours, photographed, and the colony area was measured with software by J.
As shown in fig. 6, the colony area of vancomycin group was not significantly different from that of PBS blank control group for escherichia coli ATCC25922, it was not inhibitory to colonisation of test bacteria, and was dose-dependent for staphylococcus aureus CMCC26003, but was weaker than that of antimicrobial peptide M2 and conjugate Vm-M2 alone, and in addition, for the above 2 test bacteria, at the same concentration (2 μm), the colony area of conjugate Vm-M2 group was significantly smaller than that of antimicrobial peptide M2 alone and that of physical mixture group, and it was noted that colony of escherichia coli ATCC25922 was not observed on the culture medium at Vm-M2 administration concentration, so that colony area was 0, probably because escherichia coli was all killed at this concentration, in summary, conjugate Vm-M2 was able to effectively block signal transduction between bacterial cell bodies, effectively inhibit colonial movement of bacteria, and further possessed great value for application of Vm-M2 to drug resistance.
EXAMPLE 4 anti-biofilm Activity study of vancomycin-antibacterial peptide conjugate Vm-M2
1. Biofilm removal Activity assay
Taking out the preserved strain from-80 deg.C refrigerator, quickly thawing in 37 deg.C water bath, dipping a small amount of liquid in inoculating loop, marking on LB solid culture medium in Z-shape, culturing at 37 deg.C until single colony is grown, picking single colony in sterile liquid MHB culture medium, shaking culturing at 37 deg.C and 200rpm to logarithmic phase, detecting bacterial concentration, and diluting to 1X 10 7 CFU/mL. The above-mentioned bacterial liquid was added to a sterile 96-well plate for culturing at 37℃for 48 hours to form a biofilm. The bacterial solution from each well was aspirated and washed three times with PBS. 100. Mu.L of diluted vancomycin, M2 and conjugate Vm-M2 samples were added to each well, the physical mixture group was 50. Mu.L of vancomycin+50. Mu L M2, the final concentrations of the samples were 1. Mu.M, 2. Mu.M, 4. Mu.M and 8. Mu.M, and the samples were incubated at 37℃for 24 hours after addition. Crystal violet dye solution (0.1%) is added to each hole, the dye solution is sucked out after dyeing for 30min, and the solution is washed three times by sterile PBS and air-dried in an ultra clean bench. Adding 100 mu L of absolute ethyl alcohol into each hole, standing for 20min, and dissolving crystal violet. OD values were measured at ultraviolet wavelength 560nm and the experiment was set up in three replicates. The percentage of biofilm formation (Biofilm Retention%, BR%) was calculated as BR (%) =100% - [100% × (F 0-Fdrug)/F0 ], with PBS being selected as the negative control in this experiment and the measurement taken as the maximum biofilm residual amount. Biofilm Retention%, BR% being the percentage of biofilm remaining, F 0 being the absorbance of the PBS treated group and F drug being the absorbance of the dosing treated group.
Table 6 biofilm-clearing Activity of vancomycin, M2 and Vm-M2 (E.coli ATCC 25922)
TABLE 7 biofilm removal Activity of vancomycin, M2 and Vm-M2 (Staphylococcus aureus VRSA 52)
2. Biofilm inhibition activity assay
Taking out the preserved strain from the refrigerator at-80 deg.C, quickly thawing in 37 deg.C water bath, dipping a little liquid on LB solid culture medium, marking in Z shape, culturing at 37 deg.C until single colony is grown, picking single colony in sterile liquid MHB culture medium, shaking culturing at 200rpm to logarithmic phase at 37 deg.C, detecting bacterial liquid concentration, and diluting to 2X 10 7 CFU/mL. 50. Mu.L of the above bacterial solution was added to a sterile 96-well plate, 50. Mu.L of diluted samples of vancomycin, M2 and conjugate Vm-M2 were added to each well, the physical mix group was 25. Mu.L of vancomycin+25. Mu L M2, and the final concentrations of the samples were 1. Mu.M, 2. Mu.M, 4. Mu.M and 8. Mu.M, and incubated at 37℃for 48 hours. Sucking out the bacterial liquid in each hole, washing the bacterial liquid three times by PBS, and placing the plate on an ultra clean bench for ventilation and blow-drying. Crystal violet dye solution (0.1%) is added to each hole, the dye solution is sucked out after dyeing for 30min, and the solution is washed three times by sterile PBS and air-dried in an ultra clean bench. Adding 100 mu L of absolute ethyl alcohol into each hole, standing for 20min, and dissolving crystal violet. OD was measured at uv wavelength 560 nm. The percentage of biofilm formation (Biofilm Formation%, BF%) was calculated as BF (%) =100% - [100% × (F 0-Fdrug)/F0 ], where PBS (F 0) is a negative control and the measurement was taken as the maximum biofilm formation. Biofilm Formation%, BF% as the percentage of biofilm formation and F drug is the absorbance of the dosing treatment group.
Table 8 biofilm inhibiting Activity of vancomycin, M2, vm-M2 (E.coli ATCC 25922)
Table 9 biofilm inhibiting Activity of vancomycin, M2 and Vm-M2 (Staphylococcus aureus VRSA 52)
As shown in tables 6,7,8 and 9, for E.coli ATCC25922, vancomycin-resistant Staphylococcus aureus VRSA52, vancomycin had no biofilm removal and inhibition activity, and both the antimicrobial peptide M2 and the conjugate Vm-M2 showed concentration-dependent antimicrobial activity, meaning that at the same dosing concentration, the conjugate Vm-M2 showed stronger biofilm removal and inhibition activity than the antimicrobial peptide M2 alone, and the physical mixture group had no antimicrobial activity than the conjugate group. Therefore, the conjugate Vm-M2 is considered to have good application potential in the aspect of chronic refractory diseases caused by bacterial infection.
Example 5 determination of synergistic antibacterial action of vancomycin-antibacterial peptide conjugate Vm-M2 with conventional antibiotics
Weighing antibiotic (meropenem, polymyxin B, ciprofloxacin and gentamicin) medicines, dissolving into 2mg/mL solution with sterile water, sequentially diluting by a multiple ratio to obtain 8-1/64MIC concentration, adding 11 concentrations of solvent for standby, preparing the coupling compound Vm-M2 into 4 XMIC concentration with sterile water, sequentially diluting by a multiple ratio until 4-1/16MIC, and adding 8 concentrations of solvent for standby. The bacterial liquid is diluted to 5X10 5 CFU/mL for standby. Adding 90 mu L diluted bacterial solution into a 96-well plate, adding antibiotics into the bacterial solution, adding 5 mu L of antibiotics into each hole, adding 11 columns of coupling compound Vm-M2 into the bacterial solution, adding 5 mu L of coupling compound Vm-M2 into each hole, adding 8 rows of coupling compound, finding MICA, MICB, A and B, calculating FIC, FIC= FMICA + FMICB =A/MICA+B/MICB (A and B represent the concentration at the optimal combining point of two drugs), MICA and MICB represent MIC when two drugs are combined, and performing synergism when FIC is less than 0.5, performing antagonism when FIC is more than 4 (including 4), adding two drugs when FIC is less than 0.5 (including 0.5), and performing independent effect when MICA, MICA and MICB represent the optimal combining point of the two drugs, wherein the dosage modes are shown in the following table.
TABLE 10 synergistic antibacterial Effect of Vm-M2 with conventional antibiotics
The synergistic antibacterial effect of the 4 clinical first-line antibiotics and the conjugate Vm-M2 is detected by selecting escherichia coli ATCC25922 and vancomycin-resistant staphylococcus aureus VRSA52 as test bacteria. According to the experimental results, the conjugate has a synergistic effect with other antibiotics except that the conjugate has no synergistic effect with meropenem, which may be related to the inherent action mechanism of the different antibiotics. Overall, the synergistic antibacterial effect of the conjugates with most of the antibiotics used in clinical lines shows great potential in clinical combination.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (4)
1. A vancomycin-antibacterial peptide conjugate is characterized in that the vancomycin-antibacterial peptide conjugate is obtained by covalent crosslinking of vancomycin and an antibacterial peptide with an amino acid sequence shown as SEQ ID NO.2, cysteine is modified at the N-terminal of the antibacterial peptide, and the covalent crosslinking is realized by a bifunctional protein crosslinking agent Sulfo-SMCC.
2. Use of the vancomycin-antibacterial peptide conjugate of claim 1 in the preparation of an antibacterial agent for inhibiting gram-positive or gram-negative bacteria;
the gram positive bacteria are staphylococcus aureus ATCC29213, enterococcus faecalis ATCC29212, clostridium perfringens ATCC13124 and bacillus cereus CMCC63301;
The gram-negative bacteria are E.coli ATCC25922, E.coli CMCC44102, acinetobacter baumannii ATCC19606, and Shigella flexneri ATCC12022.
3. Use of a vancomycin-antibacterial peptide conjugate of claim 1 for inhibiting bacterial biofilm formation.
4. An antibacterial pharmaceutical composition, which is characterized by comprising the vancomycin-antibacterial peptide conjugate of claim 1 and an antibiotic, wherein the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.
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