CN111040026A

CN111040026A - Amyloid hexapeptide and application thereof in broad-spectrum inhibition of bacterial and fungal biofilms

Info

Publication number: CN111040026A
Application number: CN201911258909.6A
Authority: CN
Inventors: 林焕彩; 陈冬茹
Original assignee: ORAL SUBSIDIARY SUN YAT-SEN UNIVERSITY HOSPITAL
Current assignee: ORAL SUBSIDIARY SUN YAT-SEN UNIVERSITY HOSPITAL
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-04-21
Anticipated expiration: 2039-12-10
Also published as: CN111040026B

Abstract

The invention relates to an amyloid hexapeptide and application thereof in broad-spectrum inhibition of bacterial and fungal biofilms, belonging to the field of biological pharmacy. The amyloid hexapeptide provided by the invention can be polymerized into amyloid fibers in vitro. The invention also provides application of the amyloid hexapeptide in preparation of a medicament for inhibiting biofilm formation. The amyloid hexapeptide can be polymerized into amyloid fiber to wrap bacteria or fungi, and plays a role in broad-spectrum inhibition of bacterial and fungal biofilm formation, including gram-positive bacteria, gram-negative bacteria and fungi, but the amyloid hexapeptide is not bactericidal, has low cytotoxicity or no toxicity, cannot cause bacterial resistance, and is expected to become a novel efficient cheap medicament for resisting biofilm formation.

Description

Amyloid hexapeptide and application thereof in broad-spectrum inhibition of bacterial and fungal biofilms

Technical Field

The invention relates to an amyloid hexapeptide and application thereof in broad-spectrum inhibition of bacterial and fungal biofilms, belonging to the field of biological pharmacy.

Background

About 60% of human bacterial diseases are caused by biofilms, and the resistance of the bacteria to the external environment is greatly enhanced after the bacteria form the biofilms. At present, medicines which can be clinically used for controlling the biological membrane are bactericides, including chlorhexidine, antibiotics and the like, and long-term use of the bactericides can cause dysbacteriosis in vivo, so that bacteria generate drug resistance, and systemic diseases are caused. However, at present, there is no effective drug which is purely resistant to biofilm and is not bactericidal, so that a novel drug which is resistant to biofilm and is not bactericidal is to be searched.

The polypeptides have the characteristics of simple synthesis, low toxicity, quick drug effect, low immunogenicity and the like, and are more and more widely concerned in recent years. However, many of the currently studied peptide inhibitors, including antibacterial peptides, have bactericidal effects, and decrease the formation of bacterial biofilms by decreasing the number of bacteria, and have disadvantages such as low efficiency, inability to kill "dormant bacteria" inside biofilms, and possible bacterial resistance. Thus, current antimicrobial peptides are not ideal membrane preparations for antibiotics.

Chu et al first reported on Science 2012 that protective peptide 6 (32 amino acids) secreted by Pan cells in the small intestine of a human is capable of inhibiting the formation of biofilm by forming amyloid fiber-coated bacteria and preventing peptide 6 from having no bactericidal effect.Kumar et al subsequently found that amyloid polypeptide A β monomer (42 amino acids) can be bound to the bacterial membrane and gradually polymerized into amyloid fiber-coated bacteria in 2016 to prevent bacterial invasion.

Studies have shown that short peptides consisting of 4-7 amino acids are the smallest structures that form amyloid fibrils. Bednarska et al believe that finding an amyloid peptide inhibitor from the bacterial proteome may have a greater effect. Mutans streptococci are well-known cariogenic bacteria, the surface of which contains amyloid fiber structures, and the C123 segment (consisting of 487 amino acids) of Pac protein is a constituent protein of mutans streptococci amyloid fibers. Therefore, in order to find more efficient amyloid polypeptide that is resistant to biofilm but not bactericidal, we started from the C123 segment of the amyloid component protein-Pac protein of Streptococcus mutans and sought amyloid short peptide inhibitors that can inhibit bacterial biofilm formation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the amyloid hexapeptide with the effects of inhibiting the formation of bacterial biofilms and not killing bacteria.

The invention also aims to provide application of the amyloid hexapeptide in preparing a medicament for inhibiting biofilm formation.

In order to achieve the purpose, the invention adopts the technical scheme that: an amyloid hexapeptide which is polymerizable into amyloid fibrils in vitro.

Preferably, the amino acid sequence of the amyloid hexapeptide comprises Ala-Ser-Asn-Ile-Val-Ile, Thr-Ser-Phe-Val-Leu-Val, Gly-Ile-Asp-Leu-Lys-Ile, Phe-Ala-Lys-Val-Asp-Ile, Ile-Ala-Val-Gly-Thr-Phe, and the corresponding amino acid abbreviations thereof are currently ASNIVI, TSFVLV, GIDLKI, FAKVDI, IAVGTF, respectively.

The amyloid hexapeptide has the following specific characteristics: hexapeptide, which can be predicted to have the ability of forming amyloid fiber by amyloid fiber prediction software such as ZipperDB, Tango and Waltz; during synthesis, N-terminal acetylation and C-terminal amidation modification are carried out; in vitro confirmation of the formation of rigid amyloid fibrils; can inhibit the formation of biological membranes of bacteria and fungi in a broad spectrum, including gram-positive bacteria, gram-negative bacteria and fungi; preventing biofilm formation by forming amyloid fibrils encapsulating bacteria or fungi; sterilization is not carried out; has low or no toxicity to cells.

On the other hand, the invention also provides application of the amyloid hexapeptide in preparing a medicament for inhibiting biofilm formation.

As a preferred embodiment of the use according to the invention, the amyloid hexapeptide is used for inhibiting the formation of gram-positive, gram-negative and fungal biofilms. The amyloid hexapeptide has a broad-spectrum antibiotic membrane-forming effect and does not have a bactericidal effect.

As a preferred embodiment of the use according to the invention, the amyloid hexapeptide is used for the formation of starch-like fiber-encapsulating bacteria and fungi.

As a preferred embodiment of the use of the present invention, the gram-positive bacteria include mutans bacteria, streptococcus sanguis, and staphylococcus aureus; gram-negative bacteria include escherichia coli; fungi include Candida albicans.

As a preferred embodiment of the use according to the invention, the amyloid hexapeptide is non-bactericidal against gram-positive bacteria, gram-negative bacteria and fungi.

In still another aspect, the present invention provides a medicament for inhibiting biofilm formation, which comprises the amyloid hexapeptide of the present invention.

As a preferred embodiment of the medicament of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the beneficial effects that: the amyloid hexapeptide can be polymerized into amyloid fibers in vitro, can wrap bacteria or fungi by forming the amyloid fibers, has the effect of inhibiting the formation of bacterial and fungal biofilms in a broad spectrum, comprises gram-positive bacteria, gram-negative bacteria and fungi, is not bactericidal, has low or no toxicity to cells, does not cause bacterial resistance, and is expected to become a novel cheap medicament for high-efficiency biofilm formation.

Drawings

FIG. 1 is a graph showing the results of in vitro polymerization of hexapeptides numbered P1-P13 in Table 1, and observation by TEM of the formation of amyloid fibrils.

FIG. 2 is a graph showing the effect of amyloid hexapeptide on biofilm formation and free bacteria proliferation.

FIG. 3 is the transmission electron microscope and scanning electron microscope observation images of amyloidogenic bacteria formed by amyloid hexapeptide.

FIG. 4 is a graph showing the results of inhibition of biofilm formation by amyloid hexapeptide against gram-positive bacteria, gram-negative bacteria and fungi.

FIG. 5 is a graph showing the effect of amyloid hexapeptide on the proliferation of gram-positive bacteria, gram-negative bacteria and fungus-free bacteria.

FIG. 6 is a graph showing the effect of hexapeptide on the morphology and proliferation of human Oral mucosal epithelial keratinocytes (NOK).

FIG. 7 is a graph showing the effect of other species of amyloid hexapeptide on the formation of mutans bacteria biofilm.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

This example is 5 amyloid hexapeptides ASNIVI, TSFVLV, GIDLKI, FAKVDI, IAVGTF that can be polymerized into amyloid fibers in vitro as described herein. The short peptide fragment capable of forming amyloid fiber in the C123 sequence is predicted by three amyloid fiber prediction software, namely ZipperDB, Tango and Waltz through the prediction of the amyloid fiber prediction software. The screened amyloid short peptide needs to meet the following requirements: at least two pieces of software are commonly oriented and correspond to the binding energy of ZipperDB < -23 kcal/mol. And the observation of the product in vitro by a transmission electron microscope proves that the product can be polymerized into amyloid fiber.

The specific prediction method comprises the following steps: logging in Pubmed, searching protein, inputting streptococcus mutans, Pac, downloading amino acid sequence information, and screening out C123 segment sequence (1000-. C123 sequences are input by logging in ZipperDB (http:// services. mbi. ula. edu/ZipperDB), Tango (http:// tando. crg. es) and Waltz (http:// Waltz. switchchlab. org) websites, and predicted sequences of C123 segments capable of forming amyloid fibers are obtained. And (3) combining the three software prediction characteristics, selecting a short peptide which can intersect Waltz or Tango with ZipperDB, and the ZipperDB shows Rosetta Energy < -23 kcal/mol.

Predicting screening results: 14 amyloid short peptides are screened according to screening conditions, are hexapeptides, are successfully synthesized into 13 in vitro, and are protected by N-terminal acetylation and C-terminal amidation during synthesis to simulate the state of the hexapeptides in a parent body, and specific sequences are shown in table 1.

The hexapeptide is synthesized by a solid phase synthesis method, which comprises the following steps: step 1: selecting 400mg of modified resin to start synthesizing the polypeptide, adding 20% of pip/DMF (dimethyl formamide) into the reactor, and oscillating the reactor for 20 min; step 2: filtering to remove solvent, adding DMF into the system, shaking the reactor for 1min, filtering to remove liquid, and performing the operation for three times; and step 3: adding 150uL of ethanol solution added with phenol, ethanol solution of ninhydrin and a spoon of resin into a detection tube, placing the detection tube at 100 ℃ for 20s, checking whether the resin has color change, if the color changes, indicating that Fmoc removal succeeds, adding the prepared corresponding amino acid solution into a reactor, recording the corresponding amino acid solution in a recording table, adding 1mL of DIC/DMF solution, and oscillating the reactor for 1 h. In step 3, if the color is not changed and the coupling is successful, the step 2 is repeated to wash the resin. Repeating the steps, and adding corresponding amino acid until the polypeptide synthesis is finished.

TABLE 1 Hexapeptides in the Pac protein C123 sequence predicted by ZipperDB, Tango and Waltz software

In Table 1 above, the predicted hexapeptides are ordered by binding energies (Rosetta Energy) as shown by ZipperDB, with Rosetta Energy values < -23 indicating the ability to form amyloid fibrils, and lower values indicating a greater ability to form amyloid fibrils.

Experimental example 1

This experimental example is a test of whether hexapeptide numbered P1-P13 in Table 1 of the present invention can form amyloid fibrils or a test of the effect of hexapeptide that can form amyloid fibrils on inhibiting biofilm formation but not killing bacteria.

Materials: (1) and (3) storing and using the hexapeptide, storing the hexapeptide in a freeze-dried powder form for a long time, and subpackaging 1 mg/tube. Each time, 1mg was taken out, dissolved in 100ul of DMSO, added with 900ul of double distilled water, adjusted to a concentration of 1mg/ml, and stored temporarily as a mother liquor in a refrigerator at-20 ℃. When the culture medium is used, the mother liquor is added with the culture medium and diluted to different concentrations (0.1mg/ml, 0.05mg/ml, 0.025mg/ml and 0.0125mg/ml), and the control group is the culture medium containing DMSO with the same concentration and without amyloid hexapeptide, and is used for culturing free bacteria and biological membranes. (2) And (3) bacterial culture: the culture conditions of the streptococcus mutans and the streptococcus sanguis are as follows: brain Heart infusion Medium (BHI), 5% CO at 37 ℃₂，10％H₂，85％N₂16-18 h; the culture conditions of the biological membrane are as follows: BHI + 1% sucrose (BHIs for short), 37 ℃ and 5% CO₂，10％H₂，85％N₂And 24 h. The culture conditions of the escherichia coli free bacteria are as follows: lysogenic broth culture medium (LB), 37 ℃, normoxic, 16-18 h; the culture conditions of the biological membrane are as follows: 0.5g/L yeast extract (yeast extract) +10g/L casamino acids (casamino acids), 37 ℃, normoxic, 24 h. The culture conditions of staphylococcus aureus free bacteria are as follows: trypticase Soy Broth (TSB), at 37 ℃ under normal oxygen for 16-18 h; the culture conditions of the biological membrane are as follows: TSB + 3% NaCl + 0.5% glucose (TSBNG for short), at 37 deg.C, under normal oxygen for 24 h. The culture conditions of the candida albicans free bacteria are as follows: saururus culture medium (SLM), 37 ℃, normoxic, 16-18 h; the culture conditions of the biological membrane are as follows: 90% artificial saliva, 10% fetal calf serum and 1% sucrose at 37 ℃ under normal oxygen for 24 h.

The experimental method comprises the following steps:

(1) validation of in vitro polymerization of hexapeptide into amyloid fibers: taking the hexapeptide mother liquor, diluting the hexapeptide mother liquor to 0.05mg/ml by using double distilled water, and polymerizing 13 hexapeptides in the table 1 in vitro for 24 hours at the temperature of 60 ℃ at the pH value of 3 to ensure that the hexapeptide with the capability of polymerizing into amyloid fibers can be completely polymerized. After the polymerization is finished, 10ul of solution is taken out and dropped on a 200-mesh copper net with a carbon film, the solution is absorbed for 2min, the solution is absorbed, 3 percent phosphotungstic acid is negatively dyed for 2min, and the negative dyeing solution is absorbed. The appearance of the polymerized amyloid fiber of hexapeptide is observed under a transmission electron microscope. Hexapeptides that can polymerize into growing stiff starch-like fibers are called amyloid hexapeptides, and hexapeptides that cannot polymerize into amyloid fibers are called non-amyloid hexapeptides. The results of the experiment are shown in FIG. 1.

(2) Effect of amyloid hexapeptide on free bacteria proliferation and biofilm mass: the monomer solutions of amyloid hexapeptide or non-amyloid hexapeptide at different concentrations (0.1mg/ml, 0.05mg/ml, 0.025mg/ml and 0.0125mg/ml) were added to the culture medium of the above bacteria free bacteria and biofilm, and cultured for the corresponding time points (free bacteria culture for 16-18h, biofilm culture for 24h), and the effects on the proliferation of free bacteria and the biofilm formation were observed. Detecting the proliferation of free bacteria: detection OD of enzyme-linked immunosorbent assay (OD)₆₀₀Counting with dot plate, the experimental results are shown in FIG. 2。

(3) The scanning electron microscope is used for observing the influence of the amyloid hexapeptide/non-starch hexapeptide on the shape of a streptococcus mutans biological membrane: when the streptococcus mutans biofilm is cultured, 0.05mg/ml of amyloid hexapeptide or non-amyloid hexapeptide is added, and the culture is carried out for 6 hours and 24 hours. Fixing 2.5% of glutaraldehyde for 3h, sequentially dehydrating 30% to absolute ethyl alcohol, fixing tert-butyl alcohol for 3 times and 10 min/time, freeze-drying hexamethyl silane for more than 3h, spraying gold, and observing the morphology under a scanning electron microscope. The results are shown in FIG. 3.

(4) Inhibition of biofilm formation by gram-positive, gram-negative and fungal bacteria: randomly selecting 3 amyloid hexapeptides (ASNIVI, TSFVLV and GIDLKI) and 3 non-starch-like hexapeptides (YSSNTV, LIGGII and TYSSSNT) as controls, and studying the inhibition effect on the biofilm formation of gram-positive bacteria, gram-negative bacteria and fungi such as streptococcus sanguis, staphylococcus aureus, escherichia coli and candida albicans, the results are shown in FIG. 4, and the transmission electron microscope picture only shows the results of TSFVLV (P3) and LIGGII (P8).

(5) Effect of amyloid hexapeptide on proliferation of gram-positive, gram-negative and fungal free bacteria: among them, 3 amyloid hexapeptides (ASNIVI, TSFVLV and GIDLKI) and 3 non-starch-like hexapeptides (YSSNTV, LIGGII and TYSSSNT) were selected as controls, and the effects on the proliferation of gram-positive bacteria, gram-negative bacteria and fungus-free bacteria such as Streptococcus sanguis, Staphylococcus aureus, Escherichia coli and Candida albicans were investigated, and the results are shown in FIG. 5, and transmission electron microscopy pictures show only the results of TSFVLV (P3) and LIGGII (P8).

(6) Effect of amyloid hexapeptide or non-amyloid hexapeptide on cytotoxicity: culturing human oral mucosa keratinized epithelial cell, inoculating 8000 cells/well in 96-well plate, adding amyloid hexapeptide or non-amyloid hexapeptide into culture medium, and culturing at 37 deg.C under normal oxygen for 48 h. Observing the morphology of the cells by an inverted microscope; the Cell Counting Kit-8 Kit detects the adherent proliferation of cells, and the result is shown in FIG. 6.

(7) The effect of hexapeptides from other species on the variable chain bacteria biofilm and free bacteria: to investigate whether other species-derived amyloid hexapeptides also have the property of inhibiting biofilm formation but not killing bacteria, ZipperDB was used to search for hexapeptides derived from human, murine and HIV viruses, and the hexapeptide with the strongest ability to form amyloid fibrils was selected from the first predicted protein sequences of the above species shown on ZipperDB, to obtain three hexapeptides (GQSIAI, SSHMCM, NQSVSI), and the specific sequence information is shown in Table 2. When hexapeptide is synthesized, N-terminal acetylation and C-terminal amidation modification are carried out, whether the three hexapeptides can form amyloid fiber or not is verified in vitro, and observation is carried out through a transmission electron microscope. The hexapeptide was added to the medium of the free bacteria of mutans bacteria and the biofilm at a final concentration of 0.05mg/ml, and the effect on the proliferation of the free bacteria and the amount of the biofilm was observed, and the results are shown in FIG. 7.

TABLE 2 hexapeptides from other species

The experimental results are as follows:

(1) validation of in vitro polymerization of hexapeptide into amyloid fibers: of the 13 hexapeptides in table 1, 5 hexapeptides P1(ASNIVI), P3(TSFVLV), P6(GIDLKI), P7(FAKVDI) and P13(IAVGTF) were found to form rigid amyloid fibrils in vitro, called amyloid hexapeptides, whose amyloid fibrils have a pattern diagram as shown in fig. 1. As can be seen from fig. 1: FIG. 1A: the pattern of ZipperDB predicted polymerization of hexapeptide into amyloid fibrils (coronal plane), P3(TSFVLV) in table 1; FIG. 1B: ZipperDB predicted a pattern (cross-section) of the polymerization of hexapeptide into amyloid fibers, P3(TSFVLV) in Table 1; FIGS. 1C-1O show the in vitro polymerization of P1-P13 in Table 1, and TEM observation of the polymerization revealed that P1(ASNIVI), P3(TSFVLV), P6(GIDLKI), P7(FAKVDI) and P13(IAVGTF) polymerized in vitro into amyloid fibrils, and other hexapeptides did not form amyloid fibrils.

(2) Effect of amyloid hexapeptide on free bacteria proliferation and biofilm mass: it was found that 5 amyloid hexapeptides P1(ASNIVI), P3(TSFVLV), P6(GIDLKI), P7(FAKVDI) and P13(IAVGTF) all had the effect of inhibiting the formation of the biofilm of Streptococcus mutans at 0.0125mg/ml to 0.1mg/ml, and did not have the effect of killing Streptococcus mutans. FIG. 2A: effect of 13 hexapeptides on mutans biofilm formation: compared with a control group, P1, P3, P6, P7 and P13 can inhibit the formation of the variable chain bacteria biofilm, the action effect is concentration-dependent (P <0.05), and other hexapeptides have no obvious effect on the variable chain bacteria biofilm formation (P > 0.05). FIG. 2B: the 13 hexapeptides have influence on the proliferation of the free streptomyces variabilis, and the 13 hexapeptides have no influence on the proliferation of the free streptomyces variabilis. FIG. 2C: the dot plate counting result shows that 13 hexapeptides do not influence the viable count of the free mutans.

(3) The scanning electron microscope is used for observing the influence of the amyloid hexapeptide/non-starch hexapeptide on the shape of a streptococcus mutans biological membrane: the transmission electron microscope and the scanning electron microscope can show that the amyloid hexapeptide forms amyloid fiber-wrapped mutans bacteria. Fig. 3 shows, fig. 3A-3C: the control group, the P3 group and the P8 group are observed by naked eyes for 24h biofilm formation, so that the control group and the P8 group form stable adherent biofilms, and the P3 group forms flocculent substances floating in the culture medium. Fig. 3D-fig. 3F: the shapes of the variable chain bacteria biomembranes of the control group, the P3 group and the P8 group after 24 hours are observed by a transmission electron microscope, so that a large amount of amyloid fiber-coated variable chain bacteria appear around the variable chain bacteria of the P3 group. Fig. 3G-fig. 3I: the shapes of the variable-chain bacteria biomembranes of the control group, the P3 group and the P8 group are observed by a scanning electron microscope at 6h, and the P3 forms the amyloid fiber-wrapped variable-chain bacteria biomembrane at 6 h. Fig. 3J-fig. 3L: the shapes of the variable-chain bacteria biomembranes of the control group, the P3 group and the P8 group are observed by a scanning electron microscope for 24 hours, so that variable-chain bacteria biomembrane lumps are formed in the control group and the P8 group, and the variable-chain bacteria of the P3 group are wrapped by a large amount of amyloid fibers.

(4) Inhibition of biofilm formation by gram-positive, gram-negative and fungal bacteria: FIG. 4 shows that amyloid hexapeptide (P3) has a broad spectrum of anti-biofilm activity, as compared to Control (Control) and non-starch-like hexapeptide (P8), and inhibits biofilm formation by forming amyloid fiber-coated bacteria, gram-positive bacteria such as Streptococcus sanguis, Staphylococcus aureus, Escherichia coli, and Candida albicans, gram-negative bacteria, and fungi.

(5) Effect of amyloid hexapeptide on proliferation of gram-positive, gram-negative and fungal free bacteria: FIG. 5A: compared with the Control group (Control) and the non-starch-like hexapeptide group (P8), the amyloid hexapeptide (P3) can wrap free bacteria of bacteria by forming amyloid fiber. FIG. 5B: the amyloid hexapeptide has no bactericidal effect on streptococcus mutans, streptococcus sanguis, staphylococcus aureus, escherichia coli, candida albicans and the like.

(6) Effect of amyloid hexapeptide or non-amyloid hexapeptide on cytotoxicity, fig. 6: effects of amyloid hexapeptide on morphology and proliferation of human Oral mucosal epithelial keratinocytes (NOK). FIG. 6A: the NOK of the control group adheres to the wall and has an antenna; FIG. 6B: group P1, NOK rounded, no antenna; FIG. 6C: group P3, NOK rounded, no antenna; FIG. 6D: group P6, NOK adherent, with tentacles; FIG. 6E: group P5, NOK adherent, with tentacles; FIG. 6F: group P8, NOK adherent, with tentacles; FIG. 6G: group P9, NOK adherent, with tentacles. FIG. 6H: the result of the cell counting experiment (CCK-8) shows that the adherent cell quantity of the P1 and the P3 groups is reduced compared with that of the control group (P <0.05), and the adherent cell quantity of the P6 group is not statistically different compared with that of the control group. Indicating that some amyloid hexapeptides are not cytotoxic.

(7) The effect of hexapeptides from other species on the variable chain bacteria biofilm and free bacteria: hexapeptide (GQSIAI) with amyloid fibril forming ability was also found to inhibit bacterial biofilm formation without killing bacteria, suggesting that the role of amyloid hexapeptide is independent of species origin. Fig. 7A-7C: the transmission electron microscope is used for observing whether hexapeptides (GQSIAI, SSHMCM and NQSVSI) from other species are polymerized into amyloid fibers, and the GQSIAI can be polymerized into the amyloid fibers. FIG. 7D: the influence of GQSIAI, SSHMCM and NQSVSI on the formation of the variable-chain bacteria biofilm shows that the amyloid hexapeptide GQSIAI can inhibit the formation of the variable-chain bacteria biofilm (P < 0.01). FIG. 7E: and observing the polymerization of the amyloid hexapeptide GQSIAI into the amyloid fiber-coated mutans streptococci by a transmission electron microscope. FIG. 7F: no effect was observed on the proliferation of free mutagens by 3 hexapeptides from other species.

In conclusion, the amyloid hexapeptide has the following specific characteristics: a hexapeptide; amyloid fiber prediction software such as ZipperDB, Tango and Waltz can be used for predicting the ability of forming amyloid fibers; during synthesis, N-terminal acetylation and C-terminal amidation modification are carried out; in vitro confirmation of the formation of rigid amyloid fibrils; the broad-spectrum inhibition of the formation of bacterial and fungal biofilms, including gram-positive bacteria, gram-negative bacteria and fungi, can be realized by forming amyloid fiber to wrap the bacteria or the fungi; sterilization is not carried out; has low or no toxicity to cells. The amyloid hexapeptide has the function of broad-spectrum biofilm formation resistance, does not kill bacteria, does not cause drug resistance, has low cytotoxicity, and is expected to become a novel cheap drug with high-efficiency biofilm formation resistance.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> oral hospital affiliated to Zhongshan university

<120> amyloid hexapeptide and its use for broad-spectrum inhibition of bacterial and fungal biofilms

<130>2019

<160>5

<170>PatentIn version 3.3

<210>1

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<213> Artificial sequence

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Ala Ser Asn Ile Val Ile

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<213> Artificial sequence

<400>2

Thr Ser Phe Val Leu Val

1 5

<210>3

<211>6

<212>PRT

<213> Artificial sequence

<400>3

Gly Ile Asp Leu Lys Ile

1 5

<210>4

<211>6

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<213> Artificial sequence

<400>4

Phe Ala Lys Val Asp Ile

1 5

<210>5

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Ile Ala Val Gly Thr Phe

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Claims

1. An amyloid hexapeptide, wherein the amyloid hexapeptide is polymerizable into amyloid fibrils in vitro.

2. The amyloid hexapeptide according to claim 1, wherein the amino acid sequence of the amyloid hexapeptide comprises Ala-Ser-Asn-Ile-Val-Ile, Thr-Ser-Phe-Val-Leu-Val, Gly-Ile-Asp-Leu-Lys-Ile, Phe-Ala-Lys-Val-Asp-Ile, Ile-Ala-Val-Gly-Thr-Phe.

3. Use of an amyloid hexapeptide according to any one of claims 1-2 in the manufacture of a medicament for inhibiting biofilm formation.

4. The use according to claim 3, wherein the amyloid hexapeptide is used to form amyloid fibril-encapsulating bacteria or fungi.

5. The use according to claim 3, wherein the amyloid hexapeptide is used to inhibit the formation of gram-positive, gram-negative and fungal biofilms.

6. The use of claim 5, wherein the gram-positive bacteria include mutans bacteria, Streptococcus sanguis, and Staphylococcus aureus; the gram-negative bacteria include escherichia coli; the fungi include Candida albicans.

7. The use according to claim 3, wherein the amyloid hexapeptide is non-bactericidal against gram positive bacteria, gram negative bacteria and fungi.

8. A drug for inhibiting biofilm formation, said drug comprising the amyloid hexapeptide according to any one of claims 1 to 2.

9. The medicament of claim 8, further comprising a pharmaceutically acceptable carrier.