WO2016146037A1 - 用于抑制/瓦解生物被膜的抑制剂及其应用 - Google Patents

用于抑制/瓦解生物被膜的抑制剂及其应用 Download PDF

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WO2016146037A1
WO2016146037A1 PCT/CN2016/076171 CN2016076171W WO2016146037A1 WO 2016146037 A1 WO2016146037 A1 WO 2016146037A1 CN 2016076171 W CN2016076171 W CN 2016076171W WO 2016146037 A1 WO2016146037 A1 WO 2016146037A1
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pslg
biofilm
protein
pseudomonas
inhibiting
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PCT/CN2016/076171
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English (en)
French (fr)
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马旅雁
于珊
吴慧君
王世伟
王迪
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中国科学院微生物研究所
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Priority claimed from CN201510648750.4A external-priority patent/CN105963680B/zh
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Priority to US15/557,812 priority Critical patent/US10499655B2/en
Publication of WO2016146037A1 publication Critical patent/WO2016146037A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the invention relates to the field of biotechnology of microorganisms.
  • the invention relates to the use of a protein for inhibiting, disintegrating or degrading a biofilm of a microorganism.
  • Biofilm generally refers to a membrane-forming polymer formed by microorganisms attached to a carrier, and is a major mode of survival in nature.
  • the microorganisms in the biofilm have stronger environmental adaptability than the single-cell microorganisms, and its formation brings serious harm to humans.
  • about 65% of human bacterial infections are related to biofilms, and the resistance of microorganisms to antibiotics in biofilms is hundreds or even thousands of times higher than that in planktonic state, greatly increasing clinical treatment. Difficulty.
  • the reason for the resistance of bacteria is to remove the adaptive variation of the original bacteria under the stress of antibiotics, and on the other hand, because under normal circumstances, the pathogenic bacteria will form a bacterial biofilm (BF). , providing a protective barrier. It is a polysaccharide protein matrix such as extracellular polysaccharide matrix, fibrin, lipoprotein, etc. secreted by bacteria after being adsorbed on an inert object (such as a biomedical material or a mucosal surface of a living organism), so that the bacteria adhere to each other and their colonies are gathered and entangled therein.
  • an inert object such as a biomedical material or a mucosal surface of a living organism
  • the formed membrane-like substance allows the bacteria to stay in a suitable microenvironment without being washed away; the extracellular matrix can also adsorb the drug or affect the penetration of the drug into the bacterial community, so most drugs can only kill the biofilm.
  • Microorganisms on the surface, and microorganisms inside the biofilm are the main cause of variation. Therefore, this special structure of the biofilm can act as a barrier to protect bacteria against the killing of antibacterial drugs and escape the elimination of the host immune system, and become a potential source of infection, leading to infections associated with refractory clinical biofilms.
  • Pseudomonas aeruginosa is a conditional pathogen that is easily infected with burn patients and immunodeficiency patients and is one of the typical strains of nosocomial infections. Once infected by the bacteria, it is easy to form a biofilm, which causes difficulty in treatment. Clinically, it can cause blood diseases, ear, eye, skin and soft tissues, bones and joints, endocardium and respiratory system infections. It is one of the three major pathogens of human beings. It is also the leading cause of pneumonia. Because of its multi-drug resistance mechanism such as biofilm formation, the drug resistance rate is high, and it often causes intractable and refractory infections in the clinic, which becomes a thorny problem in clinical treatment.
  • microorganisms capable of effectively inhibiting or disintegrating microorganisms such as Pseudomonas aeruginosa. Formulations and methods for biofilms.
  • a first aspect of the invention provides the use of a PslG protein or a coding sequence thereof for (i) preparation of a preparation for inhibiting, disintegrating or degrading a biofilm; and/or (ii) preparation Antibacterial drugs.
  • the biofilm comprises a Pseudomonas biofilm.
  • the biofilm is a biofilm of Pseudomonas aeruginosa.
  • the PslG protein is a recombinant protein.
  • the PslG protein is an isolated and purified recombinant protein.
  • the PslG protein is a mature form of the PslG protein.
  • the preparation is selected from the group consisting of biofilm removers, medical catheter cleaners, medical cannula cleaners, medical instrument duct cleaners, medical device disinfectants, artificial joint cleaners, bacteriostats , bactericide, medical catheter care agent, medical instrument care agent, or a combination thereof.
  • the formulation comprises a microbial envelope inhibitor.
  • the formulation comprises a catheter bacterial biofilm inhibitor, a biofilm inhibitor of a conditionally associated microorganism.
  • the biofilm is located in one or more items selected from the group consisting of medical instruments, catheters, or medical instruments.
  • the PslG protein is selected from the group consisting of:
  • (iii) a protein whose amino acid sequence has a homology of SEQ ID NO.: 2 of ⁇ 95% (preferably ⁇ 98%), which enhances the ability of the protein to inhibit or degrade the biofilm formation.
  • the coding sequence of the PslG protein is selected from the group consisting of:
  • the coding sequence encodes a PslG protein selected from the group consisting of (i), (ii) or (iii).
  • the psG protein or its coding sequence is derived from Pseudomonas aeruginosa.
  • the PslG protein is produced by recombinant cells.
  • the recombinant cell comprises a prokaryotic cell (such as E. coli) or a eukaryotic cell.
  • the recombinant cell is selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas stutzeri, P. fluorescens, and clove pseudomonad Pseudomonas syringae.
  • the formulation or the antibacterial agent is for one or more uses selected from the group consisting of:
  • the Pseudomonas is a Pseudomonas microorganism.
  • the Pseudomonas is selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas syringae, or a combination thereof.
  • the Pseudomonas is Pseudomonas aeruginosa.
  • the formulation or medicament is applied to a wound or wound of a human or animal.
  • the administration is a topical administration.
  • the concentration is from 5 to 100 nM, preferably from 10 to 50 nM; or the dose is from 0.0001 to 10 g, preferably from 0.001 to 1 g.
  • the method of application comprises: applying, applying, spraying a wound, and dressing the wound with a dressing material containing an effective concentration or amount of PslG.
  • the wound or wound includes wounds and/or wounds caused by burns, wounds, surgery, cannula, etc., and is particularly suitable for application to microorganisms (especially Pseudomonas, more particularly Is patina).
  • microorganisms especially Pseudomonas, more particularly Is patina
  • the infection and/or the surface or interior of Pseudomonas is susceptible to the formation of wounds and/or wounds of biofilms, especially Pseudomonas, more particularly Pseudomonas aeruginosa biofilms.
  • the preparation of the article or medicament is in a solid dosage form, a liquid dosage form or a semi-solid dosage form.
  • the dosage form of the article or medicament is selected from the group consisting of lotions, powders, ointments, tinctures, coatings, film-forming binders.
  • the formulation or drug is a liquid.
  • the formulation or drug is a powder.
  • the formulation or medicament further contains an antibiotic.
  • the antibiotic is selected from the group consisting of azithromycin, floxacin (FLX), ciprofloxacin (CIP), tobramycin (TOB), or a combination thereof.
  • the preparation is an external preparation, and further contains one or more optional ingredients selected from the group consisting of a surfactant, a fragrance, and a disinfectant.
  • a second aspect of the invention provides a biofilm inhibitor comprising:
  • the carrier is a solvent (such as water)
  • the carrier comprises a pharmaceutically acceptable carrier.
  • the adjuvant is selected from the group consisting of surfactants, buffers, pH adjusters, or combinations thereof.
  • the inhibitor further comprises an antibiotic.
  • the antibiotic is selected from the group consisting of azithromycin, flofloxacin, ciprofloxacin, tobramycin, or a combination thereof.
  • the inhibitor is for inhibiting the biofilm of Pseudomonas.
  • the inhibitor is used to treat or adjunctively treat a disease associated with a Pseudomonas infection.
  • the inhibitor is used to non-therapeutic, kill or assist in killing Pseudomonas in an environment.
  • a third aspect of the invention provides a pharmaceutical composition for inhibiting a biofilm, comprising:
  • the pharmaceutical composition has a PslG protein or an active fragment thereof in an amount of 0.0001 to 99% by weight, preferably 0.001 to 90% by weight, more preferably 0.01 to 50% by weight, per composition. Total weight.
  • the pharmaceutical composition further comprises an antibiotic.
  • the antibiotic is selected from the group consisting of azithromycin, flofloxacin, ciprofloxacin, tobramycin, or a combination thereof.
  • the medicament is for inhibiting a biofilm of Pseudomonas.
  • the medicament is for the treatment or adjunctive treatment of a disease associated with a Pseudomonas infection.
  • a fourth aspect of the present invention provides a method for non-therapeutic inhibition and/or disintegration of a biofilm in vitro, where a PslG protein or a biofilm inhibitor of the second aspect of the invention is administered, thereby inhibiting / or disintegrate the biofilm.
  • the application concentration is from 5 to 100 nM, preferably, the application concentration is from 10 to 50 nM.
  • the dosage is from 0.0001 to 10 g per square meter, preferably from 0.001 to 1 g per square meter.
  • the biofilm is a biofilm of a Pseudomonas microorganism.
  • the biofilm is a biofilm of Pseudomonas aeruginosa.
  • the inhibitor contains water and/or an aqueous solvent.
  • a fifth aspect of the present invention provides a method for modifying the ability to form a biofilm of a Pseudomonas strain, comprising the steps of:
  • the phrase "above” means that the expression level of the PslG protein of the recombinant strain after the transformation is 1.5 times or more ( ⁇ ), preferably 2 times or more, of the starting strain before the transformation.
  • the phrase "below” means that the biofilm production amount of the recombinant strain after the transformation is 1/2 or less ( ⁇ ), preferably 1/3 or less of the starting strain before transformation. .
  • a sixth aspect of the invention provides an article for inhibiting or disintegrating a biofilm, the article comprising (a) a medically acceptable substrate, and (b) coated or attached to the substrate PslG protein.
  • the article of manufacture comprises an article for dressing a wound.
  • the wound includes wounds and/or wounds caused by burns, wounds, surgery, cannula, and the like.
  • the article comprises a product for human or veterinary use
  • the article further comprises (c) an antibiotic applied to or attached to the substrate.
  • the antibiotic is selected from the group consisting of azithromycin, flofloxacin, ciprofloxacin, tobramycin, or a combination thereof.
  • the article contains from 0.0001 to 10% by weight, preferably from 0.001 to 5% by weight, more preferably from 0.01 to 2% by weight, based on the total weight of the article.
  • the weight ratio of the PslG protein to the antibiotic is from 1:1000 to 1000:1, preferably from 1:100 to 100:1; more preferably from 1:10 to 10:1. .
  • Figure 1 shows that overexpression of PslG in P. aeruginosa PAO1 inhibits the formation of biofilm of PAO1 strain
  • Figure 2 shows that the addition of PslG inhibits the formation of P. aeruginosa biofilm
  • Figure 3 shows the disintegration of the formed P. aeruginosa biofilm by 1 nM PslG;
  • Figure 4 shows the disintegration of the formed P. aeruginosa biofilm by 50 nM PslG;
  • FIG. 5 shows the results of PslG (WT) and its muteins E156Q and E276Q after treatment with a 6-hour P. aeruginosa biofilm for 30 minutes, wherein E156 and E276 are key active sites of the PslG protein.
  • Figure 6 shows the inhibitory effect of 50 nM PslG on the Pseudomonas syringae biofilm.
  • Figure 7 shows the inhibitory effect of the lyophilized PslG protein on the biofilm.
  • Figure 8 shows a comparison of the effects of PslG-treated bacterial cells and planktonic cells on antibiotic susceptibility.
  • 8(a) and 8(c) are the results of measuring the MIC value of the floating liquid to TOB and CIP;
  • FIG. 8(b), 8(d) is the result of measuring the MIC value of TOB and CIP of the bacterial cells released from the biofilm after PslG treatment.
  • Figure 9 shows the inhibitory effect of PslG in combination with antibiotics on Pseudomonas aeruginosa.
  • Fig. 9(a) is a comparison diagram of the effects of PslG+TOB and TOB alone
  • Fig. 9(b) is a comparison diagram of the effects of PslG+CIP and CIP alone.
  • Figure 10 shows that PslG has no cytotoxic effect on colonic epithelial cells and macrophages.
  • Fig. 10A shows that after 12 hours of PslG treatment, the MTT assay detects its cytotoxicity against epithelial cells HT-29 and Caco2;
  • Fig. 10B shows that PslG has no cytotoxic effect on macrophages.
  • Figure 11 shows the sensitivity of PslG enhanced biofilm to macrophages.
  • the 24-hour PAO1 biofilm cultured on glass coverslips was treated with PslG and co-incubated with macrophages, scraped and resuspended in 1 ml of physiological saline for colony forming unit count (CFU).
  • CFU colony forming unit count
  • Figure 12 shows a mouse implant infection model.
  • the colony forming unit per ml of the PAO1 biofilm on the implant in vivo is mono-treated or co-treated with PslG, tobramycin.
  • the horizontal dashed line represents the detection limit of the experiment, and the average value of the biofilm count in 5 mice is shown (*P ⁇ 0.01).
  • Figure 13 shows the ratio of dead-live nematodes in the same number of free cells of the nematode liquid killing experiment and the biofilm cells under the dissociation of PslG and the four-instar nematode.
  • the graph shows the mean ⁇ SD value of three replicate experiments.
  • FIG. 14 shows the degradation of Psl polysaccharide by PslG in vitro and its degradation rate of Psl polysaccharide at different temperatures.
  • Fig. 14A shows the results of immunological dot hybridization of 4 mg/ml and 2 mg/ml Psl polysaccharide and 50 nM PslG for 1 h before and after incubation at 30 °C, and the Psl polysaccharide concentration is marked above the corresponding point after the corresponding dot calculation
  • Figure 14B shows the difference The efficiency of degradation of Psl polysaccharide by 50nM PslG at temperature. The experiment was repeated three times.
  • PslG protein a protein of unknown function of Pseudomonas
  • PslG protein a protein of unknown function of Pseudomonas
  • the present invention has been completed on this basis.
  • Pseudomonas aeruginosa is a genus of Pseudomonas, a conditional pathogen that is easily infected with burn patients and immunodeficiency patients and is one of the typical strains of nosocomial infections. Once infected by the bacteria, it is easy to form a biofilm, which causes difficulty in treatment.
  • Pseudomonas aeruginosa relies on extracellular polysaccharides, extracellular proteins, and extracellular DNA to maintain its biofilm structure. Therefore, the development of degrading enzymes against these three substrates to inhibit or disintegrate the formation of biofilms has always been a hot topic. .
  • This patent uses a specific PslG protein to degrade the extracellular polysaccharide Psl, which plays a key role in the formation of Pseudomonas biofilms, thereby inhibiting and disintegrating the biofilm of Pseudomonas.
  • biofilm refers to a membrane-forming polymer formed by a microorganism attached to a carrier, which is a major mode of survival in nature.
  • the microorganisms in the biofilm have stronger environmental adaptability than the single-cell microorganisms, and its formation brings serious harm to humans.
  • about 65% of human bacterial infections are related to biofilms, and the resistance of microorganisms to antibiotics in biofilms is hundreds or even thousands of times higher than that in planktonic state, greatly increasing clinical treatment. Difficulty.
  • biofilms can pollute the water supply and drainage pipeline system, causing a series of industrial problems related to biofouling.
  • the biofilm of the inner wall of the pipeline can cause water pollution, and the corrosive effect of the biofilm can shorten the service life of the pipeline facility.
  • the biofilm is a highly organic microbial cell population coated with a protective epipolysaccharide (also known as exopolysaccharide) matrix through which the cells adhere to the inactive membrane surface and live animal and plant cells.
  • the surface is the group that is fixed.
  • protein of the invention protein of the invention
  • PslG protein protein
  • PslG polypeptide PslG enzyme
  • PslG protein protein of the invention
  • PslG polypeptide PslG enzyme
  • PslG protein protein of the invention
  • PslG polypeptide PslG enzyme
  • these wild-type or mutant, full-length or mature forms of the protein, or the active fragment or the derivative protein retain the function of inhibiting and/or disintegrating the biofilm (especially the Pseudomonas biofilm).
  • the term includes not only proteins derived from Pseudomonas aeruginosa but also homologous proteins from Pseudomonas or other genus microorganisms (generally, these homologous proteins from other species)
  • the identity with the amino acid sequence set forth in SEQ ID NO.: 2 is > about 60%, more preferably > 70%, > 80%, > 90%, or optimally > 95%).
  • the term encompasses protein forms with or without a starting amino acid (Met).
  • the nucleotide sequence of the PslG full-length gene is as shown in SEQ ID NO.: 1, and the pslG cloned into the E. coli expression vector is a sequence starting from the 91st nucleotide.
  • the amino acid sequence of the purified PslG protein (31-442) is SEQ ID NO.: 2.
  • the PslG protein is derived from Pseudomonas aeruginosa P. aeruginosa PAO1 strain, and has a total length of 442 amino acids and a signal peptide (positions 1-30).
  • the mature form of PslG after removal of the signal peptide (1-30) is 411 amino acids in length.
  • Both the full-length form and the mature form of PslG can be expressed and purified in a conventional manner.
  • a commonly used expression system is expressed using E. coli.
  • the experiments of the present invention show that PslG can not only disintegrate the biofilm produced by Pseudomonas but also inhibit the formation of biofilm by Pseudomonas.
  • the present invention discloses for the first time that PslG has an inhibitory and disintegrating effect on the biofilm of Pseudomonas.
  • microbial envelope inhibitor and “biofilm inhibitor” are used interchangeably and generally do not inhibit the structure of the biofilm by inhibiting the exchange or interconnection between microorganisms. Extermining, therefore, does not create coercive stress on the survival of microorganisms, and thus does not lead to new "drug resistance”.
  • biological film inhibitor of the present invention and “invention of the present invention” are used interchangeably and both refer to a PslG protein, an active fragment thereof and/or an agonist thereof as an active ingredient, thereby being capable of inhibiting, disintegrating or A composition or mixture that degrades a Pseudomonas biofilm.
  • biofilm inhibitor also referred to as a biofilm disintegrator
  • a biofilm disintegrator containing the protein of the present invention as an active ingredient for inhibiting and/or disintegrating a biofilm.
  • the "biofilm inhibitor” of the present invention includes:
  • the biofilm inhibitor of the present invention can prevent the pathogenic microorganism from being protected by inhibiting the biofilm of the microorganism, thereby increasing the susceptibility to common drugs, thereby realizing effective control of various pathogenic bacteria, and having the technical advantage unmatched by common antibiotic means.
  • biofilm of the present invention is inhibited and may be used in combination with other substances selected from the group consisting of antibiotics.
  • antibiotics include, but are not limited to, azithromycin, floxacin, ciprofloxacin, tobramycin, or a combination thereof.
  • the biofilm inhibitor of the present invention can effectively and/or specifically inhibit the biofilm of microorganisms, and representative microorganisms include, but are not limited to, Pseudomonas microorganisms, especially Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas syringae.
  • biofilm inhibitor of the present invention can be used in a variety of different applications and applications. Representative examples include, but are not limited to, medical devices, removal and cleaning of tissue conduit biofilms, removal of catheter biofilms, inhibition of microbial envelopes of medical catheters and other conduits.
  • the effective administration concentration or dose of the protein or biofilm inhibitor of the present invention is not particularly limited and may be determined depending on the type of the bacteria, the occasion of administration, and the like.
  • a preferred concentration range is from 5 to 100 nM, preferably from 10 to 50 nM; or the amount of the application is from 0.0001 to 10 g, preferably from 0.001 to 1 g (e.g., for every 1 or 10 square meters).
  • biofilm inhibitor of the present invention can inhibit, disintegrate or degrade the biofilm of Pseudomonas.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount of PslG protein, and An active fragment and/or agonist thereof and (b) a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared as an injection or a powder, for example, by physiological saline or an aqueous solution containing glucose and other adjuvants by a conventional method or by freeze-drying.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, powders, tablets and capsules are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the pharmaceutical compositions of the invention may also be used with antibiotics.
  • a safe and effective amount of a PslG protein of the invention is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the present invention also provides a method of inhibiting, disintegrating or degrading a biofilm by administering the protein of the present invention, the biofilm inhibitor of the present invention or the pharmaceutical composition of the present invention.
  • the biofilm inhibitor is applied at a concentration of from 5 to 100 nM, preferably from 10 to 50 nM.
  • the biofilm is a bacterial biofilm or a microbial capsule.
  • the biofilm comprises a biofilm of a catheter bacterial biofilm and/or an opportunistic infection-associated microorganism.
  • the biofilm is a biofilm of Pseudomonas.
  • the biofilm is a biofilm of Pseudomonas aeruginosa.
  • the method of the present invention can be used for medical purposes such as prevention, treatment, and the like, and can also be used for non-therapeutic non-medical uses (such as for assisting in killing microorganisms in the environment).
  • Representative application methods include (but are not limited to):
  • the present invention provides a use of a PslG protein and its coding sequence for inhibiting, disintegrating or degrading a biofilm of a microorganism.
  • the main advantages of the invention include:
  • the present inventors have found for the first time that the PslG protein has an inhibitory and disintegrating effect on the biofilm of Pseudomonas aeruginosa.
  • the biofilm inhibitor is capable of specifically inhibiting or disintegrating the biofilm of P. aeruginosa.
  • the present invention has potential broad application prospects, for example, it is expected to be developed into an antibacterial drug to eliminate the resistance of P. aeruginosa.
  • the experimental materials used in the examples of the present invention can be obtained from commercially available channels unless otherwise specified. Among them, the Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000, 406: 959-964.)
  • a patina with PslG can be obtained by an arabinose-inducible expression plasmid (pHERD20T-PslG) (pslG cloned into pHERD20T, pHERD20T see reference (Applied and Environmental Microbiology 2008, 74:7422-7426.))
  • the cells were cultured in Jensen liquid medium at 37 ° C overnight, and then inoculated to Jensen medium containing different concentrations of arabinose at 1% inoculation, and cultured in a 96-well plate at 30 ° C for 24 hours.
  • the biofilm of the biofilm was detected by the crystal violet method: after discarding the free cells in the well, it was washed three times. Next, the biofilm was stained with 0.1% crystal violet, and after washing three times, the crystal violet bound to the biofilm was dissolved with 30% acetic acid, and the value of OD 560 (ie, the concentration of crystal violet) was measured by a spectrophotometer. The biomass of the biofilm is measured by its size. Among them, PAO1 and ⁇ psl strains which do not synthesize Psl polysaccharide are negative control, Psl polysaccharide can induce expression (arabinose as inducer) of PAO1, and P BAD- psl strain is a positive control.
  • Example 2 In addition of PslG protein inhibits the formation of Pseudomonas aeruginosa biofilm
  • Example 3 Disintegration of the formed Pseudomonas aeruginosa biofilm by 1nM PslG protein
  • Example 4 Disintegration of the formed Pseudomonas aeruginosa biofilm by 50nM PslG protein
  • E156 and E276 may be their key catalytic sites. Crystal structure analysis of PslG protein suggests that E156 and E276 may be their key active sites.
  • the method was the same as in Example 2, and the PslG concentration was 50 nM.
  • PslG was obtained by freeze-drying to obtain a powder, and after being dissolved in sterile sterile water, the biofilm inhibition test was carried out, and the original PslG solution which was not freeze-dried was used as a control group.
  • the method was the same as in Example 2, and the PslG concentration was 50 nM.
  • Pseudomonas aeruginosa was cultured for 24 hours, and the biofilm of Pseudomonas aeruginosa grown for 24 hours was treated with PslG, and the liquid bacteria dissociated from the bacteria treated by PslG and the bacteria treated by PslG were respectively taken.
  • 0.2 ml was uniformly applied to the LBNS plate, and a test strip for measuring MIC was placed in the center of the plates, and the MIC value was read after incubation at 37 ° C for 16 hours.
  • Control group As shown in Fig. 8 (a) and 8 (c), the MIC value of the floating liquid to TOB and CIP was measured.
  • the results showed that the PslG-treated cells were more sensitive to tobramycin (TOB) and ciprofloxacin (CIP) than the planktonic cells, and the MIC value was also lower.
  • the Pseudomonas aeruginosa solution was cultured for 24 hours at 37 °C, and 0.2 ml of the bacterial solution was applied to the LBNS plate.
  • the differently treated filter papers were placed on the plate, as shown in Fig. 9, and cultured at 37 ° C for 24 hours. Mushroom circle.
  • Fig. 9(a) As shown in Fig. 9(a), four experimental groups were set up, respectively, buffer alone; 50 nM PslG alone; tobramycin (TOB) alone; and TOB+PslG in combination.
  • Example 10 PslG combined with fleroxacin (FLX) inhibited the formation of Pseudomonas aeruginosa biofilm
  • the minimum inhibitory concentration (MIC) of Pseudomonas aeruginosa against FLX was measured, and the IC50 value of PslG was determined by a conventional method.
  • PslG of 1/2 IC50 was used in combination with 1/4 MIC, 1/2 MIC FLX, and their inhibitory effects on P. aeruginosa biofilm were measured.
  • Example 11 PslG is not cytotoxic to mammalian epithelial cells and immune cells
  • the cytotoxicity of PslG was detected using human colonic epithelial cell lines Caco2 and HT-29.
  • the PAO1 biofilm was cultured on a glass coverslip for 24 hours, treated with PslG for 1 hour, and then incubated with macrophages for 2 hours. The remaining biofilm was scraped off with a blade and resuspended in 1 ml of physiological saline for colony count (CFU). .
  • PslG is not cytotoxic to host cells and can enhance the sensitivity of biofilms to macrophages, thus suggesting the potential application value of PslG in the treatment of biofilm-associated infections.
  • Example 13 mouse implant infection model showed that PslG can promote the clearance of biofilm in vivo
  • the implant coated with Pseudomonas aeruginosa biofilm was inserted into the mouse peritoneum and partially treated with the following treatment: 50 nM PslG single treatment; 50 mg kg -1 tobramycin single treatment; 50 nM PslG + 50 mg kg -1 Comycin treatment. After 24 hours of in vivo culture, the mice were sacrificed, and the implants were removed and the bacteria were counted on LB plates after homogenization. The number of Pseudomonas aeruginosa on the implant was tabulated in terms of CFU/ml data.
  • Example 14 The virulence of the biofilm-coated cells under PslG dissociation did not change significantly.
  • Liquid killing experiments were carried out using C. elegans as a host model.
  • experimental group 1 Two experimental groups were set up, namely experimental group 1 and experimental group 2.
  • Experimental group 1 The free bacterial cells dissociated from the bacterial membrane (Pseudomonas aeruginosa biofilm) after enrichment with the same amount of PslG were co-incubated with the four-instar nematode.
  • Experimental group 2 The liquid free bacterial cells grown for 24 hours were incubated with the four-instar nematode.
  • Experimental group 1 and experimental group 2 were placed in a 96-well plate, respectively, and after 48 hours of incubation at 25 ° C, the proportion of dead nematodes was observed with a stereo microscope. Three replicates per group and the results are expressed as mean ⁇ SD values.
  • the nucleotide sequence of PslG shown in SEQ ID NO.: 1 was inserted into the multiple cloning site of pET15b (purchased from Merck Millipore) to obtain recombinant expression plasmid PGL01-pslG, and PGL01-pslG expressed SEQ ID NO.: 2
  • binding buffer 25 mM Tris-HCl pH 8.0, 200 mM NaCl.
  • the resuspension was sonicated and centrifuged at 12000 rpm for 45 minutes to remove the precipitate.
  • the supernatant containing the PslG protein was passed through a nickel affinity column and washed several times with binding buffer to remove non-specifically bound proteins.
  • the protein bound to the nickel column resin was reconstituted with a binding buffer mixed with phosphatase (final concentration 0.12 mg/ml). The mixture was incubated overnight at 4 °C to remove the His tag and the protein sample was eluted with binding buffer.
  • PslG was passed through an ion exchange column (Source 15Q HR 16/10, GE Healthcare) and eluted sequentially with a buffer of linear concentration gradient of 0-1 M NaCl in 25 mM Tris-HCl, pH 8.0. Finally, the protein sample was purified by ion-exchange column and molecular sieve with a 10 mM Tris-HCl pH 8.0 buffer containing 100 mM NaCl (Superdex 20010/300GL, GE Healthcare) to obtain a PslG protein having a molecular weight of 47 kDa.
  • Example 16 Recombinant PslG protein can degrade Psl polysaccharide and PslG protein in vitro at different temperatures
  • Psl polysaccharides Preparation and purification of Psl polysaccharides are referred to published literature (Byrd MS, Sadovskaya I, Vinogradov E et al. Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for beneficial enzymes in Psl and LPS production. Molecular Microbiology 2009; :622-638) proceed.
  • a standard curve was prepared as follows: The Psl polysaccharide prepared by the above method was subjected to 10-fold gradient dilution, and the Psl polysaccharide was detected by immunoblotting.
  • Psl polysaccharide antibody Psl polysaccharide antibody
  • the antibody obtained for the immunogen was tested by immunoblotting for the remaining Psl polysaccharide, and the signal intensity after analysis by Quantity One (Bio-Rad) software was compared with a standard curve to calculate the Psl polysaccharide initiation after PslG protein treatment. The remaining amount of Psl polysaccharide at a concentration of 4 mg/ml.
  • the concentration of Psl polysaccharide was changed from 4 mg/ml to 2 mg/ml, and the other steps were unchanged, and the remaining amount of Psl polysaccharide at a starting concentration of Psl polysaccharide of 2 mg/ml after PslG protein treatment was obtained.
  • the concentration of Psl polysaccharide was changed from 4 mg/ml to 2 mg/ml, and 50 nM PslG protein was replaced with 0 nM PslG protein to obtain PslG protein untreated Psl polysaccharide at a starting concentration of Psl polysaccharide at 2 mg/ml. remaining.

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Abstract

本发明公开了用于抑制/瓦解生物被膜的抑制剂及其应用。具体地，本发明提供了PslG蛋白及其编码序列在抑制/瓦解生物被膜方面的应用。本发明还提供了含有所述的PslG蛋白作为抑制/瓦解生物被膜的活性成分的组合物和制品。实验表明PslG蛋白可有效抑制和/或瓦解假单胞菌的生物被膜。

Description

用于抑制/瓦解生物被膜的抑制剂及其应用 技术领域
本发明涉及微生物的生物技术领域。具体地，本发明涉及一种蛋白在抑制、瓦解或降解微生物的生物被膜(biofilm)中的应用。
背景技术
“生物被膜”(biofilm)一般是指微生物依附某载体形成的膜性聚合物，是其在自然界中的一种主要的生存方式。生物被膜中的微生物比单细胞微生物具有更强的环境适应性，它的形成给人类带来了严重的危害。如在医药领域，据研究表明，约65％的人类细菌性感染疾病与生物被膜有关，生物被膜中微生物对抗生素的耐药性比浮游状态时高成百甚至上千倍，大大增加了临床治疗的难度。
而细菌产生耐药性的原因，除去原菌在抗菌素的胁迫压力下产生的适应性变异之外，另一方面是因为在正常情况下，致病菌会形成细菌生物被膜(bacterial biofilm，BF)，为其提供了保护屏障。实为细菌吸附于惰性物体(如生物医学材料或生物机体黏膜表面)后，分泌出的胞外多糖基质、纤维蛋白、脂蛋白等多糖蛋白复合物，使细菌相互粘连并将自身菌落聚集缠绕其中形成的膜样物，使细菌可以停留在一个适宜的微环境中而不会被冲散；胞外基质还可吸附药物或影响药物渗透到细菌群落中，因而大多数药物只能杀灭生物被膜表面的微生物，而生物被膜内部的微生物则是产生变异的主要原因。因而生物被膜的这种特殊结构可以作为一种屏障保护细菌抵御抗菌药物的杀伤和逃逸宿主免疫系统的清除，而成为潜在的感染源，导致难治性临床生物被膜的相关感染。
铜绿假单胞菌(Pseudomonas aeruginosa，PA)是一种条件致病菌，其很容易感染烧伤患者和免疫缺陷病人，也是医院内感染的典型菌株之一。一旦被该菌感染之后，极易形成生物被膜，从而造成治疗的困难。临床上，它能引起血病、耳、眼、皮肤及软组织、骨及关节、心内膜和呼吸系统等感染，是人类的三大致病菌之一。它也是引起肺炎的首要致病菌。由于其具有形成生物被膜等多重耐药机制，耐药率高，经常在临床上引起顽固性、难治性感染，成为临床治疗的棘手问题。
因此，本领域迫切需要开发能够有效抑制或瓦解铜绿假单胞菌等微生物的 生物被膜的制剂和方法。
发明内容
本发明的目的是提供了有效抑制或瓦解铜绿假单胞菌等微生物的生物被膜的制剂和方法。
本发明的第一方面提供了一种PslG蛋白或其编码序列的用途，所述PslG蛋白或其编码序列用于(i)制备抑制、瓦解或降解生物被膜的制剂；和/或(ii)制备抗菌药物。
在另一优选例中，所述的生物被膜包括假单胞菌属生物被膜。
在另一优选例中，所述的生物被膜为铜绿假单胞菌的生物被膜。
在另一优选例中，所述的PslG蛋白为重组蛋白。
在另一优选例中，所述的PslG蛋白为分离纯化的重组蛋白。
在另一优选例中，所述的PslG蛋白为成熟形式的PslG蛋白。
在另一优选例中，所述制剂选自下组：生物被膜清除剂、医用导管清洁剂、医用插管清洁剂、医用仪器管道清洁剂、医用器具消毒剂、人工关节清洁剂、抑菌剂、杀菌剂、医用导管护理剂、医用仪器护理剂、或其组合。
在另一优选例中，所述制剂包括微生物被膜抑制剂。
在另一优选例中，所述制剂包括导管细菌生物被膜抑制剂、条件性感染相关微生物的生物被膜抑制剂。
在另一优选例中，所述的生物被膜位于一种或多种选自下组的物品：医药仪器、导管、或医用器具。
在另一优选例中，所述的PslG蛋白选自：
(i)具有SEQ ID NO.:2所示氨基酸序列的蛋白；
(ii)将如SEQ ID NO.:2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而形成的、具有对生物被膜形成产生抑制和/或降解能力的由(i)衍生的蛋白；
(iii)氨基酸序列与SEQ ID NO.:2所示序列的同源性≥95％(较佳地≥98％)，增强蛋白对生物被膜形成产生抑制或降解能力的蛋白。
在另一优选例中，所述的PslG蛋白的编码序列选自：
(a)编码如SEQ ID NO.:2或所示的氨基酸序列的核苷酸序列；
(b)序列如SEQ ID NO.:1所示的核苷酸序列；
(c)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥95％(较佳地≥98％)的核苷酸序列；
(d)如SEQ ID NO.:1所示多核苷酸的5’端和/或3’端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的核苷酸序列；
(e)与(a)-(d)任一所述的核苷酸序列互补的核苷酸序列。
在另一优选例中，所述的编码序列编码上述选自(i)、(ii)或(iii)的PslG蛋白。
在另一优选例中，所述的pslG蛋白或其编码序列来源于铜绿假单胞菌。
在另一优选例中，所述的PslG蛋白是由重组细胞产生的。
在另一优选例中，所述的重组细胞包括原核细胞(如大肠杆菌)或真核细胞。
在另一优选例中，所述重组细胞选自：铜绿假单胞菌(Pseudomonas aeruginosa)、斯氏假单胞菌(Pseudomonas stutzeri)、荧光假单胞菌(P.fluorescens)、丁香假单胞菌(Pseudomonas syringae)。
在另一优选例中，所述的制剂或所述的抗菌药物用于选自下组的一种或多种用途：
(1)抑制假单胞菌生物被膜的形成；
(2)瓦解和/或降解假单胞菌已经形成的生物被膜；
(3)抑制假单胞菌的生长。
在另一优选例中，所述的假单胞菌为假单胞属(Pseudomonas)微生物。
在另一优选例中，所述的假单胞菌选自下组：铜绿假单胞菌、斯氏假单胞菌、荧光假单胞菌、丁香假单胞菌、或其组合。
在另一优选例中，所述假单胞菌为铜绿假单胞菌。
在另一优选例中，所述的制剂或药物被施用于人或动物的伤口或创面。
在另一优选例中，所述的施用为体表施用。
在另一优选例中，所述施用的浓度为5-100nM，较佳地10-50nM；或者所述施用的剂量为0.0001-10g,较佳地为0.001-1g。
在另一优选中，所述的施用方式包括：涂抹、涂覆、喷洒伤口，使用含有效浓度或数量的PslG的包扎材料对伤口进行包扎。
在另一优选例中，所述的伤口或创面包括烧伤、创伤、手术、插管等引起的创面和/或伤口，尤其适用于施用于易受微生物(尤其是假单胞菌属、更尤其是铜绿 假单胞菌)的感染和/或表面或内部易形成生物被膜(尤其是假单胞菌属、更尤其是铜绿假单胞菌生物被膜)的创面和/或伤口。
在另一优选例中，所述的制品或药物的剂型为固体剂型、液体剂型或半固体剂型。
在另一优选例中，所述的制品或药物的剂型选自下组：洗剂、粉剂、膏剂、汀剂、涂覆料、成膜粘合剂。
在另一优选例中，所述制剂或药物为液体。
在另一优选例中，所述制剂或药物为粉剂。
在另一优选例中，所述的制剂或药物还含有抗生素。
在另一优选例中，所述的抗生素选自下组：阿奇霉素、弗洛沙星(FLX)、环丙沙星(CIP)、妥布霉素(TOB)、或其组合。
在另一优选例中，所述的制剂为外用制剂，并且还含有选自下组的一种或多种任选成分：表面活性剂、芳香剂、消毒剂。
本发明第二方面提供了一种生物被膜抑制剂，包括：
(a)抑制有效量的PslG蛋白、其活性片段和/或其激动剂；
(b)载体；
(c)任选的助剂。
在另一优选例中，所述的载体为溶剂(如水)
在另一优选例中，所述的载体包括药学上可接受的载体。
在另一优选例中，所述的助剂选自下组：表面活性剂、缓冲剂、pH调节剂、或其组合。
在另一优选例中，所述抑制剂还包括抗生素。
在另一优选例中，所述的抗生素选自下组：阿奇霉素、弗洛沙星、环丙沙星、妥布霉素、或其组合。
在另一优选例中，所述的抑制剂用于抑制假单胞菌的生物被膜。
在另一优选例中，所述的抑制剂用于治疗或辅助治疗与假单胞菌感染相关的疾病。
在另一优选例中，所述的抑制剂用于非治疗性的、杀灭或辅助杀灭环境中假单胞菌。
本发明第三方面提供了一种用于抑制生物被膜的药物组合物，包括：
(a)安全有效量的PslG蛋白、其活性片段和/或其激动剂；
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物中，PslG蛋白或其活性片段的含量为0.0001-99wt％，较佳地为0.001-90wt％，更佳地0.01-50wt％，按组合物的总重量计。
在另一优选例中，所述药物组合物还包括抗生素。
在另一优选例中，所述的抗生素选自下组：阿奇霉素、弗洛沙星、环丙沙星、妥布霉素、或其组合。
在另一优选例中，所述的药物用于抑制假单胞菌的生物被膜。
在另一优选例中，所述的药物用于治疗或辅助治疗与假单胞菌感染相关的疾病。
本发明第四方面提供了一种体外非治疗性的抑制和/或瓦解生物被膜的方法，在需要施用的场合，施用PslG蛋白或本发明第二方面所述的生物被膜抑制剂，从而抑制和/或瓦解生物被膜。
在另一优选例中，所述的施用浓度为5-100nM，较佳地，施用浓度为10-50nM。
在另一优选例中，所述施用的剂量为0.0001-10g/每平方米,较佳地为0.001-1g/每平方米。
在另一优选例中，所述生物被膜为假单胞属(Pseudomonas)微生物的生物被膜。
在另一优选例中，所述生物被膜为铜绿假单胞菌的生物被膜。
在另一优选例中，所述抑制剂含有水和/或水性溶剂。
本发明第五方面提供了一种对假单胞菌属菌株生物被膜的形成能力进行改造的方法，包括步骤：
(a)提供一假单胞菌属的出发菌株，
(b)向所述出发菌株中导入外源的PslG蛋白编码序列，从而获得含有所述PslG蛋白编码序列的重组菌株；
(c)测定上一步骤的所述重组菌株的生物被膜形成能力和/或产生PslG蛋白的能力，从而选出生物被膜形成能力低于和/或所述产生PslG蛋白能力高于所述出发菌株的重组菌株。
在另一优选例中，所述的“高于”是指，所述改造后重组菌株的PslG蛋白的表达量是改造前的出发菌株的1.5倍以上(≥)，较佳地2倍以上。
在另一优选例中，所述的“低于”是指，所述改造后重组菌株的生物被膜的产生量是改造前出发菌株的1/2以下(≤)，较佳地1/3以下。
本发明第六方面提供了一种用于抑制或瓦解生物被膜的制品，所述制品含有(a)医学上可接受的基材，和(b)涂覆于或附着于所述基材上的PslG蛋白。
在另一优选例中，所述的制品包括用于包扎伤口的制品。
在另一优选例中，所述的伤口包括烧伤、创伤、手术、插管等引起的创面和/或伤口等。
在另一优选例中，所述制品包括人用或兽用的产品
在另一优选例中，所述制品还包括(c)涂覆于或附着于所述基材上的抗生素。
在另一优选例中，所述的抗生素选自下组：阿奇霉素、弗洛沙星、环丙沙星、妥布霉素、或其组合。
在另一优选例中，所述的制品中含有为0.0001-10wt％，较佳地为0.001-5wt％，更佳地0.01-2wt％，按制品的总重量计。
在另一优选例中，所述PslG蛋白与所述抗生素的重量比为1:1000至1000：1，较佳地为1:100至100：1；更佳地为1:10至10：1。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了铜绿假单胞菌PAO1中过表达PslG抑制PAO1菌株生物被膜的形成；
图2显示了外加PslG抑制铜绿假单胞菌生物被膜的形成；
图3显示了1nM PslG对已形成的铜绿假单胞菌生物被膜的瓦解；
图4显示了50nM PslG对已形成的铜绿假单胞菌生物被膜的瓦解；
图5显示了PslG(WT)及其突变蛋白E156Q和E276Q对生长6个小时的铜绿假单胞菌生物被膜处理30分钟后的结果，其中，E156和E276 是PslG蛋白的关键活性位点。
图6显示了50nM PslG对丁香假单胞菌生物被膜的抑制效果。
图7显示了冻干的PslG蛋白对生物被膜的抑制效果。
图8显示了PslG处理的菌细胞与浮游菌细胞对抗生素敏感性的效果比较图。其中，图8(a)、8(c)为浮游菌液对TOB、CIP的MIC值测定结果；图8(b)、 8(d)为经PslG处理后从生物被膜上游离出的菌细胞对TOB、CIP的MIC值测定结果。
图9显示了PslG与抗生素联合使用对铜绿假单胞菌的抑制效果。其中，图9(a)为PslG+TOB与单独使用TOB的效果对比图；图9(b)为PslG+CIP与单独使用CIP的效果对比图。
图10显示了PslG对结肠上皮细胞和巨噬细胞均没有细胞毒性作用。
其中，图10A显示，PslG处理12小时后，MTT实验检测其对上皮细胞HT-29和Caco2的细胞毒性；图10B显示，PslG对巨噬细胞无细胞毒性作用。
图11显示了PslG增强生物被膜对巨噬细胞的敏感性。
将培养于玻璃盖玻片上的24小时PAO1生物被膜经PslG处理并与巨噬细胞共孵育后，刮下并重悬在1毫升生理盐水中做菌落形成单位计数(CFU)。图中所示为三次重复实验所得CFU的平均值及SD值并对不同实验组进行了T检验(*,P<0.01)。
图12显示了小鼠植入物感染模型。体内植入物上的PAO1生物被膜经PslG、妥布霉素单处理或共处理后的每毫升菌落形成单位。水平虚线代表实验的检测极限，图示为5只小鼠体内生物被膜计数的平均值(*P<0.01)。
图13显示了线虫液体杀伤实验等量游离菌细胞和PslG解离下的生物被膜细胞与四龄线虫共孵育后的死活线虫比例。图示为三次重复实验的平均值±SD值。
图14显示了PslG体外降解Psl多糖及其在不同温度下的对Psl多糖的降解速率。其中，图14A为4mg/ml及2mg/ml Psl多糖与50nM PslG于30℃共孵育1h前后的免疫点杂交结果，相应点迹计算后Psl多糖浓度标记于相应点上方；图14B显示的是不同温度下50nM PslG降解Psl多糖的效率。实验重复三次。
具体实施方式
本发明人经过广泛而深入的研究，意外地发现，一种假单胞菌属的功能未知的蛋白(即PslG蛋白)居然能够非常有效地抑制和瓦解铜绿假单胞菌等微生物的生物被膜。在此基础上完成本发明。
具体地，实验表明，来自铜绿假单胞菌PAO1中的PslG蛋白居然可有效抑制铜绿假单胞菌形成生物被膜。内源性的或外源添加的PslG蛋白，都能够抑 制和瓦解铜绿假单胞菌形成的生物被膜。经本发明生物被膜抑制剂处理后，生物被膜的生物量至少下降50％，最优条件下可达80％。
本发明的进一步实验获得了PslG蛋白的三维晶体结构，揭示了与生物被膜瓦解功能相关的PslG蛋白的活性位点，并且还初步揭示了PslG蛋白可以降解纯化的胞外多糖Psl，说明它是通过降解胞外多糖来抑制和瓦解生物被膜的。
本发明进一步实验表明，将PslG与抗生素联用，不仅不影响抗生素的抗菌活性，还具有显著优于单用抗生素的协同杀菌效果。
假单胞菌属和铜绿假单胞菌
铜绿假单胞菌(Pseudomonas aeruginosa)是假单胞菌属的一种，为条件致病菌，其很容易感染烧伤患者和免疫缺陷病人，是医院内感染的典型菌株之一。一旦被该菌感染之后，极易形成生物被膜，从而造成治疗的困难。
铜绿假单胞菌依赖胞外多糖、胞外蛋白以及胞外DNA来维持其生物被膜结构，因此，开发针对这三种基质的降解酶从而抑制或者瓦解生物被膜的形成一直都是人们研究的热点。
本专利采用特异性的PslG蛋白降解在假单胞菌属生物被膜形成中起关键作用的胞外多糖Psl，从而抑制并瓦解假单胞菌属的生物被膜。
生物被膜
如本文所用，术语“生物被膜”指微生物依附某载体形成的膜性聚合物，是其在自然界中的一种主要的生存方式。
生物被膜中的微生物比单细胞微生物具有更强的环境适应性，它的形成给人类带来了严重的危害。如在医药领域，据研究表明，约65％的人类细菌性感染疾病与生物被膜有关，生物被膜中微生物对抗生素的耐药性比浮游状态时高成百甚至上千倍，大大增加了临床治疗的难度。又如在工业领域，生物被膜会污染给排水管道系统，造成一系列与生物污垢有关的工业问题，如管道内壁的生物被膜可造成水质污染、生物被膜的腐蚀作用缩短管道设施的使用寿命等。
医学上，生物被膜是由保护性表多糖(也称为胞外多糖)基质包被的的高度有机的微生物细胞群，菌细胞经所述的基质附着至无活性膜表面和活的动植物细胞表面，即为固着的群。
PslG蛋白和PslG编码基因
如本文所用，术语“本发明蛋白”、“PslG蛋白”、“PslG多肽”、“PslG酶”之间可以互换使用，均指来源于铜绿假单胞菌(Pseudomonas aeruginosa)PslG基因编码的功能未知的蛋白或其他类似微生物的同源蛋白。应理解，该术语还包括该PslG蛋白的野生型和突变型，包括该PslG蛋白的全长形式(如SEQ ID NO.:2中的第1-442位)或成熟形式(如SEQ ID NO.:2中的第31-442位)，以及包括该PslG蛋白的活性片段或衍生蛋白。在本发明中，这些野生型或突变型的、全长或成熟形式的蛋白、或活性片段或衍生蛋白都保留抑制和/或瓦解生物被膜(尤其是假单胞菌属生物被膜)的功能。此外，应理解，该术语不仅包括来源于铜绿假单胞菌(Pseudomonas aeruginosa)的蛋白，还包括来自假单胞菌属或其他属微生物的同源蛋白(通常，这些来自其他物种的同源蛋白与SEQ ID NO.:2所示氨基酸序列的相同性(identity)≥约60％，更佳地≥70％，≥80％，≥90％，或最佳地≥95％)。此外，应理解，该术语包括了含有或不含有起始氨基酸(Met)的蛋白形式。
在本发明的优选例中，PslG全长基因核苷酸序列如SEQ ID NO.:1所示，克隆到大肠杆菌表达载体的pslG为从第91个核苷酸开始的序列。
纯化的PslG蛋白(31-442)的氨基酸序列如SEQ ID NO.:2。在本发明的一个优选例中，所述的PslG蛋白源自铜绿假胞单菌P.aeruginosa PAO1菌株，全长含有442个氨基酸，含有一信号肽(第1-30位)。去除了信号肽(1-30)后的PslG的成熟形式的长度为411个氨基酸。
无论是全长形式还是成熟形式的PslG，都可常规方法进行表达和纯化。一种常用的表达体系是采用大肠杆菌(E.coli)进行表达。
本发明实验表明，PslG不仅能够瓦解假单胞菌所产生的生物被膜，还能抑制假单胞菌形成生物被膜。
本发明首次揭示了PslG对假单胞菌的生物被膜具有抑制、瓦解作用。
生物被膜抑制剂
如本文所用，“微生物被膜抑制剂”和“生物被膜抑制剂”可互换使用，一般其主要通过抑制微生物之间的交流或相互连接从而抑制生物膜结构的目的，而并不对微生物本身进行杀灭，因此，不会对微生物的生存产生胁迫压力，因而并不导致新的“耐药性”产生。
如本文所用，术语“本发明的生物被膜抑制剂”、“本发明制剂”可互换使用，均指含有PslG蛋白、其活性片段和/或其激动剂作为活性成分，从而能够抑制、瓦解或降解假单胞菌生物被膜的组合物或混合物。
在本发明中，提供了一种生物被膜抑制剂(也可称为生物被膜瓦解剂)，该抑制剂含有本发明蛋白作为抑制和/或瓦解生物被膜的有效成分。
本发明的“生物被膜抑制剂”包括：
(a)PslG蛋白、其活性片段和/或其激动剂；
(b)载体和/或助剂。
本发明的生物被膜抑制剂通过抑制微生物的生物被膜，使病原微生物丧失保护，增加对普通药物的易感性，从而实现对多种致病菌的有效控制，有着普通抗生素手段无法比拟的技术优势。
此外，本发明的生物被膜抑制，还可同选自下组其他物质(如抗生素)联用。代表性的抗生素例子包括(但并不限于)：阿奇霉素、弗洛沙星、环丙沙星、妥布霉素、或其组合。
实验表明，将PslG蛋白与抗生素联用，不仅不影响抗生素的抗菌活性，其杀菌效果比单独使用抗生素更好。
本发明的生物被膜抑制剂，可有效地和/或特异性地抑制微生物的生物被膜，代表性的微生物包括(但并不限于)：假单胞菌属微生物，尤其是铜绿假单胞菌、斯氏假单胞菌、荧光假单胞菌、丁香假单胞菌。
本发明的生物被膜抑制剂，可用于各种不同应用和场合。代表性的例子包括(但并不限于)：医疗设备、机构管道生物被膜的清除和清洁、导管生物被膜的清除、医用导管和其他管道的微生物被膜的抑制。
在本发明中，本发明蛋白或生物被膜抑制剂的有效施用浓度或剂量没有特别限制，可以根据菌种类、施用场合等情况而确定。优选的浓度范围为5-100nM，较佳地为10-50nM；或者所述施用的(剂)量为0.0001-10g,较佳地为0.001-1g(如对于每1或10平方米)。
本发明的实验表明，本发明的生物被膜抑制剂可抑制、瓦解或降解假单胞菌的生物被膜。
药物组合物和施用方法
本发明提供了一种药物组合物，它含有(a)安全有效量的PslG蛋白、、其 活性片段和/或其激动剂以及(b)药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成注射剂或粉剂，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法或用冷冻干燥的方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如注射剂、粉剂、片剂和胶囊宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的药物组合物还可与抗生素一起使用。
使用药物组合物时，是将安全有效量的本发明PslG蛋白施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地，该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
抑制和/或瓦解生物被膜的方法
本发明还提供了一种通过施用本发明蛋白、本发明的生物被膜抑制剂或本发明的药物组合物，从而抑制、瓦解或降解生物被膜的方法。
在另一优选例中，所述生物被膜抑制剂的施用浓度为5-100nM，较佳地，为10-50nM。
在另一优选例中，所述的生物被膜为细菌生物被膜或微生物被膜。
在另一优选例中，所述的生物被膜包括导管细菌生物被膜和/或机会性感染相关微生物的生物被膜。
在另一优选例中，所述生物被膜为假单胞属(Pseudomonas)的生物被膜。
在另一优选例中，所述生物被膜为铜绿假单胞菌的生物被膜。
本发明的方法可以用于预防、治疗等医药用途，还可用于非治疗性的非医药用途(如用于辅助杀灭环境中的微生物)。代表性的应用方法包括(但并不限于)：
(1)治疗不同人种、不同年龄或不同地区的感染铜绿假单胞菌的烧伤患者和免疫缺陷病人。
(2)医疗设备、机构管道生物被膜的清除和清洁、导管生物被膜的清除、医用导管和其他管道的微生物被膜的抑制。
本发明提供了一种PslG蛋白及其编码序列的抑制、瓦解或降解微生物的生物被膜(biofilm)中的应用。本发明的主要优点包括：
(a)本发明首次发现PslG蛋白对铜绿假单胞菌的生物被膜具有抑制、瓦解作用。所述的生物被膜抑制剂能够特异性抑制或瓦解铜绿假单胞菌的生物被膜。
(b)本发明首次解析了PslG蛋白的晶体结构，揭示了PslG蛋白的功能。
(c)本发明首次发现PslG蛋白的2个关键活性位点。
(d)与单独使用抗生素相比，PslG蛋白与抗生素联用不仅可以抑制铜绿假单胞菌生物被膜的形成，而且有望彻底清除铜绿假单胞菌。
因此，本发明具有潜在的广阔应用前景，例如有望开发成抗菌药物，消除铜绿假单胞菌的耐药性。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得，其中，铜绿假单胞菌PAO1 见参考文献(Complete genome sequence of Pseudomonas aeruginosa PAO1,an opportunistic pathogen.Nature 2000,406:959-964.)
Jensen培养基见参考文献(Nutritional factors controlling exocellular protease production by Pseudomonas aeruginosa.Journal of Bacteriology 1980,144:844-847.)，由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为：NaCl 5g/L，K2HPO4 3.286g/L，谷氨酸15.56g/L，缬氨酸2.81g/L，苯丙氨酸1.32g/L，葡萄糖12.81g/L，MgSO4·7H2O 0.33g/L，CaCl2·2H2O 0.021g/L，FeSO4 ·7H2O 0.0011g/L，ZnSO4·7H2O 0.0024g/L。
实施例1铜绿假单胞菌PAO1中表达PslG抑制PAO1菌株生物被膜的形成
含有PslG可被阿拉伯糖(arabinose)诱导表达质粒(pHERD20T-PslG)(pslG克隆到pHERD20T上而获得,其中pHERD20T见参考文献(Applied and Environmental Microbiology 2008,74:7422-7426.))的铜绿假单胞菌于Jensen液体培养基中37℃培养过夜后，以1％接种量分别接至含有不同浓度阿拉伯糖的Jensen培养液中，于96孔板中30℃静止培养24小时。
采用结晶紫法检测生物被膜的生物量：弃去孔中游离的菌体后，洗三次。接着用0.1％的结晶紫对生物被膜进行染色，洗三次后，用30％的醋酸溶解结合在生物被膜上的结晶紫，利用分光光度计检测其OD560的值(即结晶紫的浓度)，通过其大小来衡量生物被膜菌的生物量。其中，以不合成Psl多糖的PAO1，Δpsl菌株为负对照，Psl多糖可诱导表达(阿拉伯糖为诱导剂)的PAO1，PBAD-psl菌株为阳性对照。
结果如图1所示，随着阿拉伯糖浓度的增加，即PslG表达的增大，PAO1的生物被膜生物量逐渐降低。0.5％以上的阿拉伯糖浓度使PAO1的生物被膜降低到Δpsl菌株的水平。
结果表明，PslG的过量表达抑制了PAO1菌株生物被膜的形成。
实施例2外加PslG蛋白抑制铜绿假单胞菌生物被膜的形成
接LBNS平板上新鲜单克隆铜绿假单胞菌于LBNS液体培养基中，在37℃下200转/分钟震荡培养12小时。然后以1％接种量接在Jensen培养基中，同时，添加不同浓度的PslG蛋白，于96孔板中在30℃静止培养24小时后，弃去游离的菌体，用结晶紫对生物被膜进行染色并检测生物被膜的生物量(具体操作可参照实施例1中的步骤)。
结果如图2所示。从中可以看出，PslG蛋白在1nM时其抑制率可达50％。10nm-50nm浓度时，可以明显抑制铜绿假单胞菌生物被膜的形成。
实施例3 1nM PslG蛋白对已形成的铜绿假单胞菌生物被膜的瓦解作用
接LBNS平板上新鲜单克隆铜绿假单胞菌于LBNS液体培养基中，在37℃下200转/分钟震荡培养12小时。然后以1％接种量接在Jensen培养液中，于96孔板中30℃静止培养24小时后，弃去游离的菌体，用0.8％生理盐水洗3次后添加100μl含有1nM PslG的Jensen培养液，作用5或30分钟分钟后，用结晶紫染色法检测处理后的生物被膜的生物量(结晶紫染色法具体操作步骤参照 实施例2)。
同时，以未加PslG的Jensen培养液为对照组。
结果如图3所示。从中可以看出，1nM PslG蛋白处理5分钟，对铜绿假单胞菌生物被膜的生物量没有影响。而处理30分钟后生物被膜的生物量降低了50％。
实施例4 50nM PslG蛋白对已形成的铜绿假单胞菌生物被膜的瓦解
接LBNS平板上新鲜单克隆铜绿假单胞菌于LBNS液体培养基中，在37℃下200转/分钟震荡培养12小时。然后以1％接种量接在Jensen培养液中，于96孔板中30℃静止培养24小时后，弃去游离的菌体，用0.8％生理盐水洗3次后添加100μl含有50nM PslG的Jensen培养液，不同时间处理后，用结晶紫染色法检测处理后的生物被膜的生物量(结晶紫染色法具体操作步骤可参照2)。
同时以未加PslG的Jensen培养液为对照组。
结果如图4所示，50nM PslG蛋白处理5分钟，已明显降低铜绿假单胞菌生物被膜的生物量；处理10分钟后生物被膜的生物量降低了50％；处理30分钟后，生物被膜生物量降低至类似不能产Psl多糖菌株的水平。30分钟以上的处理并未有更显著的降低。
以上结果说明，50nM PslG 30分钟处理足以清除依赖于Psl多糖的生物被膜。
实施例5 E156和E276 是PslG蛋白的关键活性位点
发明人经过多次比对分析发现，E156和E276可能是其关键的催化位点。PslG蛋白晶体结构分析提示E156和E276可能是其关键活性位点。
如图5所示，突变蛋白PslG(E156Q)和PslG(E276Q)均丧失了对已形成铜绿假单胞菌生物被膜的瓦解能力。
实施例6 PslG蛋白对丁香假单胞菌生物被膜的抑制作用
方法同实施例2，PslG浓度为50nM。
如图6所示，结果表明，PslG不仅能抑制多株铜绿假单胞菌的生物被膜，对丁香假单胞菌也有同样的抑制效果。
实施例7冻干的PslG蛋白对铜绿假单胞菌生物被膜的抑制作用
将PslG通过冷冻抽干获得粉剂，重溶于灭菌无菌水后进行生物被膜抑制实验，以未经冷冻抽干的原PslG溶液做对照组。方法同实施例2，PslG浓度为50nM。
如图7所示，结果表明，冻干的PslG与PslG溶液均对铜绿假单胞菌的生物被膜有抑制作用。
实施例8 PslG处理的菌细胞与浮游菌细胞对抗生素敏感性的比较
在LBNS液体培养基中，震荡培养铜绿假单胞菌24小时，用PslG处理生长24h的铜绿假单胞菌的生物被膜，分别取浮游菌液和从PslG处理的菌中解离的菌液各0.2毫升，将其均匀涂布于LBNS平板上，在这些平板中央放置测MIC的试纸条，37℃培养16小时后读取MIC值。
对照组：如图8(a)、8(c)所示，浮游菌液对TOB、CIP的MIC值测定。
实验组：如图8(b)、8(d)所示，经PslG处理后从生物被膜游离出的菌细胞对TOB、CIP的MIC值测定。
如图8所示，结果表明，与浮游菌细胞相比，PslG处理的菌细胞对妥布霉素(TOB)和环丙沙星(CIP)更为敏感，其MIC值也更低。
实施例9 PslG蛋白与抗生素联用不影响抗生素的抗菌活性
用37℃震荡培养24h的铜绿假单胞菌液，取0.2毫升菌液涂布于LBNS平板上，将不同处理的滤纸片置于平板中，如图9所示，37℃培养24h后测量抑菌圈。
实验一：
如图9(a)所示，设置4个实验组，分别为单用缓冲液；单用50nM PslG；单用妥布霉素(TOB)；联合用TOB+PslG。
实验二：
如图9(b)所示，设置4个实验组，分别为单用缓冲液；单用50nM PslG；单用环丙沙星(CIP)；联合用CIP+PslG。
结果表明(图9)，与单独使用抗生素的抑菌圈大小相比，PslG与抗生素联用的抑菌圈大小没有显著变化，因此，PslG与抗生素联用不影响抗生素的抗菌活性；单独使用PslG不产生抑菌圈，表明PslG不杀菌。
实施例10 PslG与氟罗沙星(FLX)联合抑制铜绿假单胞菌生物被膜的形成
根据微量稀释法(娄永新、王金良，实用临床细菌学检验与进展，1993:247)测得铜绿假单胞菌对FLX的最低抑菌浓度(MIC)，用常规方法测定PslG的IC50值。
将1/2IC50的PslG分别与1/4MIC、1/2MIC的FLX联用，测定它们对铜绿假单胞菌生物被膜的抑制效果。
结果表明，与单独使用1MIC FLX相比，PslG与FLX联用对铜绿假单胞菌的杀菌效果更为显著。
实施例11 PslG对哺乳动物上皮细胞和免疫细胞没有细胞毒性
采用人类结肠上皮细胞系Caco2及HT-29检测PslG的细胞毒性。
结果如图10A和图10B所示，四唑MTT细胞毒性试验显示，PslG对两种结肠上皮细胞的活力几乎没有影响。并且，PslG对小鼠巨噬细胞(RAW264.7)也未显示任何毒性作用。
实施例12经PslG处理后的生物被膜菌细胞更易被巨噬细胞清除
PAO1生物被膜于玻璃盖玻片上培养24小时，经PslG处理1小时后，再与巨噬细胞一起孵育2小时，剩余生物被膜用刀片刮下并重悬于1毫升生理盐水中做菌落计数(CFU)。
结果如图11所示。结果显示，与未经PslG处理的对照组相比，经PslG与巨噬细胞共处理后的生物被膜剩余菌数最少。结果表明，PslG能够增强生物被膜对巨噬细胞清除作用的敏感性。
因此，PslG对宿主细胞没有细胞毒性且可以增强生物被膜对巨噬细胞的敏感性，由此提示了PslG在治疗生物被膜相关感染中的潜在应用价值。
实施例13小鼠植入感染模型显示PslG可促进体内生物被膜的清除作用
将经铜绿假单胞菌生物被膜包被的植入物插入小鼠腹膜并局部进行下列处理：50nM PslG单处理；50mg kg-1妥布霉素单处理；50nM PslG+50mg kg-1妥布霉素共处理。经24小时体内培养后，将小鼠处死，移出植入物经均化作用后在LB平板上做细菌计数。将植入物上的铜绿假单胞菌数以CFU/ml的数据形式 制表。
结果如图12所示。结果显示，与未经任何处理的对照组相比，经PslG或妥布霉素单处理及PslG和妥布霉素共处理后植入物上的菌数均显著降低。具体地，对照组的菌数(CFU/mL)＞104，经PslG单处理后的菌数(CFU/mL)≈103，经妥布霉素单处理后的菌数(CFU/mL)≈102.8，经PslG和妥布霉素共处理后，有4组样品的菌数(CFU/mL)达到了101，因此，从图中可以看出，PslG与妥布霉素联用的效果最好。
结果表明，PslG和抗生素可同时使用来治疗临床生物被膜(如铜绿假单胞菌生物被膜)的相关感染。
实施例14 PslG解离下的生物被膜菌细胞的毒力没有明显变化
采用秀丽隐杆线虫作为宿主模型进行液体杀伤实验。
设置两个实验组，分别为实验组1和实验组2。
实验组1：富集等量PslG处理后菌膜(铜绿假单胞菌生物被膜)解离下来的游离菌细胞与四龄线虫共孵育。
实验组2：收集生长24小时的液体游离菌细胞与四龄线虫共孵育。
将实验组1和实验组2分别放置于96孔板，在25℃共孵育48小时后，用体视显微镜观察死活线虫的比例。每组三个重复，结果以平均值±SD值表示。
结果如图13所示。结果表明，PslG解离的菌细胞(实验组1)与游离菌细胞(实验组2)相比，其毒力没有明显差异。
实施例15大肠杆菌中表达PslG蛋白
将SEQ ID NO.:1所示的PslG的核苷酸序列插入到pET15b(购自Merck Millipore)的多克隆位点中，得到重组表达质粒PGL01-pslG，PGL01-pslG表达SEQ ID NO.:2所示的PslG蛋白。将表达质粒PGL01-pslG转入大肠杆菌BL21(DE3)，37℃培养至OD600＝0.8后加入0.12mM硫代半乳糖苷于22℃诱导过夜。4200rpm离心菌液15分钟后重悬于结合缓冲液(25mM Tris-HCl pH 8.0,200mM NaCl)中。将重悬液超声破碎，12000rpm离心45分钟去除沉淀。含有PslG蛋白的上清液过镍亲和柱并用结合缓冲液冲洗几次去除非特异性结合的蛋白。结合在镍柱树脂上的蛋白用混有磷酸酯酶(终浓度为0.12mg/ml)的结合缓冲液重溶。将混合液在4℃孵育过夜以去除His标签后将蛋白样品用结合缓冲液洗脱。
为进一步纯化蛋白，将PslG过离子交换柱(Source 15Q HR 16/10,GE Healthcare)并用0-1 M NaCl in 25mM Tris-HCl，pH 8.0线性浓度梯度的缓冲液依次洗脱。最终，将蛋白样品与含100mM NaCl的10mM Tris-HCl pH 8.0缓冲液过分子筛(Superdex 20010/300GL,GE Healthcare)后再用离子交换柱和分子筛纯化得到PslG蛋白，分子量为47kDa。
实施例16 重组PslG蛋白可在体外降解Psl多糖以及PslG蛋白在不同温度下的酶活效果
实验方法：
Psl多糖的制备和纯化参照已发表文献(Byrd MS,Sadovskaya I,Vinogradov E et al.Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production.Molecular Microbiology 2009；73:622-638)进行。
按照如下方法制备标准曲线：上述方法制备得到的Psl多糖进行10倍的梯度稀释，通过免疫印迹法检测Psl多糖。
向溶液1(PBS磷酸缓冲液)中加入Psl多糖与50nM实施例16获得的PslG蛋白，得到反应体系1，反应体系1中Psl多糖的浓度为4mg/ml，PslG蛋白的浓度为50nM。将八个相同的反应体系1分别在20℃、25℃、30℃、37℃、40℃、45℃、50℃和55℃下孵育一小时后，用Psl多糖抗体(Psl多糖为以Psl多糖为免疫原得到的抗体)通过免疫印迹法检测剩余的Psl多糖，通过Quantity One(Bio-Rad)软件进行分析处理后的信号强度并和标准曲线进行对比，以计算PslG蛋白处理后Psl多糖起始浓度为4mg/ml下的Psl多糖的剩余量。
按照上述方法，将Psl多糖的浓度由4mg/ml替换为2mg/ml，其他步骤均不变，得到PslG蛋白处理后Psl多糖起始浓度为2mg/ml下的Psl多糖的剩余量。
按照上述方法，将50nM PslG蛋白替换为0nM PslG蛋白，得到PslG蛋白未处理的Psl多糖起始浓度为4mg/ml下的Psl多糖的剩余量。
按照上述方法，将Psl多糖的浓度由4mg/ml替换为2mg/ml，并将50nM PslG蛋白替换为0nM PslG蛋白，得到PslG蛋白未处理的Psl多糖起始浓度为2mg/ml下的Psl多糖的剩余量。
实验结果：
(1)50nM PslG蛋白与0nM PslG蛋白在30℃下处理Psl多糖1小时后的 结果如图14A所示。结果显示，经过PslG蛋白处理后的Psl多糖较未处理对照的量降低75％。结果表明，外源加入的PslG蛋白能够在体外实验条件下有效降解Psl多糖。
(2)PslG蛋白在不同温度下对Psl多糖的降解速率如图14B所示。结果表明，PslG蛋白在45℃时的降解速率最高，在20-55℃下均有活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种PslG蛋白或其编码序列的用途，其特征在于，用于(i)制备抑制、瓦解或降解生物被膜的制剂；和/或(ii)制备抗菌药物。
  2. 如权利要求1所述的用途，其特征在于，所述的PslG蛋白选自：
    (i)具有SEQ ID NO.:2所示氨基酸序列的蛋白；
    (ii)将如SEQ ID NO.:2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而形成的、具有对生物被膜形成产生抑制和/或降解能力的由(i)衍生的蛋白；
    (iii)氨基酸序列与SEQ ID NO.:2所示序列的同源性≥95％(较佳地≥98％)，增强蛋白对生物被膜形成产生抑制或降解能力的蛋白。
  3. 如权利要求1或2所述的用途，其特征在于，所述的PslG蛋白的编码序列选自：
    (a)编码如SEQ ID NO.:2或所示的氨基酸序列的核苷酸序列；
    (b)序列如SEQ ID NO.:1所示的核苷酸序列；
    (c)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥95％(较佳地≥98％)的核苷酸序列；
    (d)如SEQ ID NO.:1所示多核苷酸的5’端和/或3’端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的核苷酸序列；
    (e)与(a)-(d)任一所述的核苷酸序列互补的核苷酸序列。
  4. 如权利要求1所述的用途，其特征在于，所述的制剂或所述的抗菌药物用于选自下组的一种或多种用途：
    (1)抑制假单胞菌生物被膜的形成；
    (2)瓦解和/或降解假单胞菌已经形成的生物被膜；
    (3)抑制假单胞菌的生长。
  5. 一种生物被膜抑制剂，其特征在于，包括：
    (a)抑制有效量的PslG蛋白、其活性片段和/或其激动剂；
    (b)载体；
    (c)任选的助剂。
  6. 如权利要求5所述的抑制剂，其特征在于，所述抑制剂还包括抗生素。
  7. 一种用于抑制生物被膜的药物组合物，其特征在于，包括：
    (a)安全有效量的PslG蛋白、其活性片段和/或其激动剂；
    (b)药学上可接受的载体。
  8. 一种体外非治疗性的抑制和/或瓦解生物被膜的方法，其特征在于，在需要施用的场合，施用PslG蛋白或权利要求5所述的生物被膜抑制剂，从而抑制和/或瓦解生物被膜。
  9. 一种对假单胞菌属菌株生物被膜的形成能力进行改造的方法，其特征在于，包括步骤：
    (a)提供一假单胞菌属的出发菌株；
    (b)向所述出发菌株中导入外源的PslG蛋白编码序列，从而获得含有所述PslG蛋白编码序列的重组菌株；
    (c)测定上一步骤的所述重组菌株的生物被膜形成能力和/或产生PslG蛋白的能力，从而选出生物被膜形成能力低于和/或所述产生PslG蛋白能力高于所述出发菌株的重组菌株。
  10. 一种用于抑制或瓦解生物被膜的制品，其特征在于，所述制品含有(a)医学上可接受的基材，和(b)涂覆于或附着于所述基材上的PslG蛋白。
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Publication number Priority date Publication date Assignee Title
WO2018185150A1 (en) * 2017-04-04 2018-10-11 Novozymes A/S Polypeptides
CN108660123A (zh) * 2018-02-22 2018-10-16 中国科学院微生物研究所 PslG蛋白或其编码序列在检测微生物数量方面的应用和方法
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