CN117138024A - Vancomycin-antibacterial peptide conjugate and application thereof - Google Patents
Vancomycin-antibacterial peptide conjugate and application thereof Download PDFInfo
- Publication number
- CN117138024A CN117138024A CN202311047826.9A CN202311047826A CN117138024A CN 117138024 A CN117138024 A CN 117138024A CN 202311047826 A CN202311047826 A CN 202311047826A CN 117138024 A CN117138024 A CN 117138024A
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- Prior art keywords
- vancomycin
- antibacterial
- antibacterial peptide
- bacteria
- peptide conjugate
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- A61P31/04—Antibacterial agents
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Abstract
The invention relates to a vancomycin-antibacterial peptide conjugate and application thereof, wherein after cysteine is added to the N end of antibacterial peptide M2 for modification, the vancomycin and an antibacterial peptide modified body are coupled through two-step reaction through a bifunctional protein cross-linking agent sulfo-SMCC. Compared with vancomycin, the vancomycin-antibacterial peptide conjugate, the vancomycin, the antibacterial peptide M2 and the mixture of the vancomycin and the antibacterial peptide M2 have the advantages that the antibacterial activity is obviously improved, the drug resistance of gram positive bacteria to the vancomycin is overcome, the antibacterial activity is also generated for part of gram negative bacteria, the antibacterial spectrum of the vancomycin is increased, meanwhile, the vancomycin-antibacterial peptide conjugate also has stronger anti-biofilm capability, can kill resident bacteria, inhibit bacterial colony movement, and can form synergistic action with antibiotics to jointly sterilize.
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 antibacterial agent has the advantages of wide source, 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: according to the amino acid sequence of the antibacterial peptide modified body, chemical synthesis is carried out by adopting a solid phase synthesis technology, purification is carried out by using preparative reverse high performance liquid chromatography, and molecular weight identification is carried out 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 modifier Vm-SMCC comprises the following steps: purification was performed using preparative reverse-phase high performance liquid chromatography and molecular weight identification was performed 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: purification was performed using preparative reverse-phase high performance liquid chromatography and molecular weight identification was performed 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.
Experimental strains: clinical isolates 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, and has the sequence as follows: gly (Gly) 1 Ile 2 Gly 3 Lys 4 Phe 5 Leu 6 Lys 7 Lys 8 Ala 9 Lys 10 Lys 11 Phe 12 Gly 13 Lys 14 Ala 15 Phe 16 Val 17 Lys 18 Ile 19 Leu 20 Lys 21 Lys 22 (SEQ ID NO. 1) wherein the C-terminal lysine is amidated.
In order to facilitate the coupling, according to the amino acid sequence of the frog antibacterial peptide M2, a molecular modification method is used for designing and obtaining a modification body C-M2, wherein the amino acid sequence is Cys 1 Gly 2 Ile 3 Gly 4 Lys 5 Phe 6 Leu 7 Lys 8 Lys 9 Ala 10 Lys 11 Lys 12 Phe 13 Gly 14 Lys 15 Ala 16 Phe 17 Val 18 Lys 19 Ile 20 Leu 21 Lys 22 Lys 23 (SEQ ID NO. 2), wherein the C-terminal lysine is amidated and modified, and the preparation method is as follows:
according to the amino acid sequence of the C-M2, synthesizing the complete sequence by an automatic polypeptide synthesizer (433A,Applied Biosystems), and desalting by HPLC reversed phase column chromatography;
II, molecular weight determination is carried out by adopting matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF);
III. The purity of the purified C-M2 was determined 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 be used for adjusting the pH value to 8.16, sulfo-SMCC is dissolved in a proper amount of DMSO, then the two are mixed, the reaction is stirred at room temperature for 3 hours, reverse high performance liquid chromatography is used for monitoring the reaction, after the reaction is complete, the product is purified by preparative HPLC, molecular weight identification is carried out by high resolution mass spectrometry, the liquid phase and mass spectrometry analysis results are shown in figure 1, and the reaction product obtained in the step is named as: 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 identified by high resolution mass spectrometry, the liquid phase and mass spectrometry analysis results are shown in fig. 2, and the final product was named: 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) Vm-M2 minimum inhibitory concentration (Minimum Inhibitory Concentration) assay (2-fold dilution method).
Selecting 11 gram-positive bacteria and 18 gram-negative bacteria for MIC assay, inoculating the test strain into MH liquid culture medium (Meinabio), shake culturing at 37deg.C to logarithmic phase, and diluting the culture solution with fresh MH liquid culture medium to logarithmic phase to 2×10 5 cfu/mL for 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 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
Culturing Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003 with MH liquid culture medium at 37deg.C for 6 hr, and diluting with fresh MH liquid culture medium to 10 6 cfu/mL of the bacterial suspension. 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 an incubator at 37 ℃ 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 incubator at 37 ℃ for overnight. Because vancomycin is specific to gramSince the negative bacteria were ineffective, 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 important sources for chronic infectious diseases, such as persistent and repeated symptomsTherefore, studies for preventing and eliminating the resident bacteria have been attracting more and more attention as to the mechanism of the production of the resident bacteria. In order to detect the killing effect of the conjugate Vm-M2 on the detention bacteria, staphylococcus aureus CMCC26003 is selected, placed in a shaking table at 37 ℃ for culturing for 6 hours at 200rpm, and diluted to 10 by MH liquid culture medium until the growth log phase 8 CFU/mL is taken, 900 mu L of the CFU/mL is added into a centrifuge tube, ciprofloxacin (CIP) with the final concentration of 20 times of MIC is added into bacterial liquid, shake cultivation is carried out for 8 hours at 37 ℃, 10 mu L of bacterial liquid is taken for dilution 1000 times, and 50 mu L of bacterial liquid is taken for plating after dilution. At the 8h time point, 300. Mu.L of bacterial liquid was removed, the medium was discarded by centrifugation, 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
Mixing the collected human blood with Alzhi solution for anticoagulation, washing with physiological saline for 2 times, and re-suspending to 10 times 7 -10 8 cell/mL of suspension. 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 follows: percent hemolysis H% = a Sample of -A Negative control /A Positive control X 100%. 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 them in MH liquid medium (su state biotechnology limited) at 37 ℃ for 12 hours, respectively. Then using MH liquid culture medium (NaCl 150mM,KCl 4.5mM,CaCl) containing physiological salt ion concentration respectively 2 2mM，MgCl 2 1mM，ZnCl 2 8μM，FeCl 3 4μM，NH 4 Cl 6. Mu.M) to 10 5 CFU/ml. 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. Under the condition of reference human physiological salt ion concentration (NaCl 150mM,KCl 4.5mM,CaCl) 2 2mM，MgCl 2 1mM，ZnCl 2 8μM，FeCl 3 4μM，NH 4 Cl 6. Mu.M), the antimicrobial activity of the conjugate Vm-M2 remained essentially unchanged (MIC value increased no more than one fold).
Example 3: effect of vancomycin-antibacterial peptide conjugate Vm-M2 on bacterial colonization effects
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 study the effect of the conjugate Vm-M2 on bacterial colonization, we performed the following experiments with medium: tryptone (10 g/L), naCl (10 g/L), yeast extract (5 g/L), agar (0.3%), dissolving each component in deionized water, sterilizing at high temperature, adding vancomycin, M2 and Vm-M2 with final concentration of 0.5 μm,1 μm and 2 μm and vancomycin and antibacterial peptide with the same final concentration into the mixture, adding equal volume of PBS buffer solution as blank, cooling in six-hole plate for standby, collecting Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003, culturing with MH liquid medium (alpha Biotechnology Co., ltd., suzhou) at 37deg.C for 6 hr to growth data period, diluting with MH liquid medium to 10.) 6 CFU/mL, 5. Mu.L was pipetted onto a cooled solid medium, then placed in a 37℃incubator for 24 hours, taken out for photography, and colony area was measured with imageJ software.
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 the refrigerator at-80 ℃, quickly melting in a water bath at 37 ℃, dipping a little liquid by using an inoculating loop, marking lines in four Z-shaped areas on an LB solid culture medium, and culturing at constant temperature at 37 ℃ from the last end until single bacterial colony grows; picking single colony in sterile liquid MHB culture medium, shake culturing at 37deg.C and 200rpm to logarithmic phase; detecting bacterial liquid concentration, diluting to 1×10 7 CFU/mL. 100. Mu.L of the above-mentioned bacterial solution was added to a sterile 96-well plate, and the mixture was cultured 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. Detection of OD at ultraviolet wavelength 560nmThe experiment was set up in three parallels. The percentage of biofilm formation (Biofilm Retention%, BR%) was calculated using the following formula: BR (%) =100% - [100% × (F) 0 -F drug )/F 0 ]PBS was selected as a negative control in this experiment and its measurement was taken as the maximum biofilm residual amount. Biofilm Retention% BR% percent biofilm survival, F 0 Absorbance for PBS treated group, F drug Absorbance values for the treatment group for dosing.
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 ℃, rapidly melting in a water bath at 37 ℃, dipping a little liquid, marking the strain in a Z shape on an LB solid culture medium, and culturing at the constant temperature of 37 ℃ until single colony grows; picking single colony in sterile liquid MHB culture medium, shaking culture at 37deg.C and 200rpm to logarithmic phase; detecting bacterial liquid concentration, diluting to 2×10 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 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 was calculated using the following formula (Biofilm Formation％，BF％)：BF(％)＝100％-[100％×(F 0 -F drug )/F 0 ]Wherein PBS (F 0 ) As a negative control, the measured value was regarded as the maximum biofilm formation amount. Biofilm Formation% and BF% are percentages of biofilm formation, F drug Absorbance values for the treatment group for dosing.
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 effect of vancomycin-antibacterial peptide conjugate Vm-M2 and traditional 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. Diluting the bacterial liquid to 5X 10 5 CFU/mL was ready for use. Add 90. Mu.L of diluted well to 96 well plateBacterial liquid; antibiotics were added to the bacteria at 5 μl per well, one concentration per column, 11 columns total: adding a conjugate Vm-M2 into the bacterial liquid, wherein each hole is 5 mu L, and each row has one concentration, and the total number of the rows is 8; finding MICA, a, B, calculating FIC, fic=fmica+fmicb=a/mica+b/MICB (a, B represents the concentration at the optimal aggregation point for the combination of two drugs, MICA, MICB represents the MIC for the single drug), FIC < 0.5 has synergy, FIC > 4 (including 4) has antagonism, 0.5 < FIC < 2 (including 0.5) has additive effect, 2 < FIC < 4 (including 2) has no effect, and the dosing mode is 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 (10)
1. A vancomycin-antibacterial peptide conjugate, characterized in that: the vancomycin-antibacterial peptide conjugate is obtained by covalent crosslinking of vancomycin and antibacterial peptide with an amino acid sequence shown as SEQ ID NO. 1.
2. The vancomycin-antibacterial peptide conjugate of claim 1, wherein: the end of the antibacterial peptide is modified with cysteine.
3. The vancomycin-antibacterial peptide conjugate of claim 2, wherein: the covalent crosslinking is achieved by the bifunctional protein crosslinking agent Sulfo-SMCC.
4. Use of a vancomycin-antibacterial peptide conjugate according to any one of claims 1-3 for the preparation of an antibacterial agent.
5. The use according to claim 4, characterized in that: the antibacterial agent is used for inhibiting gram-positive bacteria or gram-negative bacteria.
6. The use according to claim 5, characterized in that: the gram positive bacteria comprise at least one of staphylococcus aureus, enterococcus faecalis, clostridium perfringens and bacillus cereus.
7. The use according to claim 5, characterized in that: the gram negative bacteria include at least one of Escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa, shigella flexneri, salmonella typhimurium and Vibrio parahaemolyticus.
8. Use of a vancomycin-antibacterial peptide conjugate according to claims 1-3 for antibacterial biofilms.
9. An antibacterial pharmaceutical composition characterized in that: the antibacterial pharmaceutical composition comprises the vancomycin-antibacterial peptide conjugate of claims 1-3 and an antibiotic.
10. The antimicrobial pharmaceutical composition according to claim 9, wherein: the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.
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