WO2015065209A1 - Protéine possédant une activité hydrolytique contre les capsules polysaccharidiques bactériennes, polynucléotide codant pour ladite protéine, vecteur biologiquement actif, composition contenant la protéine et son utilisation - Google Patents

Protéine possédant une activité hydrolytique contre les capsules polysaccharidiques bactériennes, polynucléotide codant pour ladite protéine, vecteur biologiquement actif, composition contenant la protéine et son utilisation Download PDF

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WO2015065209A1
WO2015065209A1 PCT/PL2014/000126 PL2014000126W WO2015065209A1 WO 2015065209 A1 WO2015065209 A1 WO 2015065209A1 PL 2014000126 W PL2014000126 W PL 2014000126W WO 2015065209 A1 WO2015065209 A1 WO 2015065209A1
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protein
seq
biofilm
proteins
bacterial
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PCT/PL2014/000126
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Ewa BRZOZOWSKA
Krzysztof Pawlik
Sabina GÓRSKA
Andrzej Gamian
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Wrocławskie Centrum Badań Eit + Sp. Z O.O.
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Publication of WO2015065209A1 publication Critical patent/WO2015065209A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Protein having hydrolytic activity against bacterial polysaccharide capsules polynucleotide encoding this protein, biologically active vector, composition containing the protein and its use.
  • the object of the present invention are recombinant proteins of a tail of Klebsiella pneumoniae phage KP32, capable of bacterial polysaccharide capsules hydrolysis, thus preventing biofilm formation and enhancing the effect of antibiotics activity in a process of bacterial biofilm elimination. Further, the object of the present invention are polynucleotides encoding those proteins, expression vectors, compositions containing those proteins and their use.
  • Klebsiella pneumoniae Bacterial strains of Klebsiella pneumoniae (in other words Klebsiella aerogenes) are G-rods commonly existing in natural environment (vegetation, surface waters, soil), which belong to Enterobacteriaceae family. In the human body, they colonise nasal, throat and intestinal mucous membranes. Pathogenic activity of this strain leads to serious diseases of the respiratory system (e.g. acute pneumonia), wound infections, bacteraemia as well as urinary tract infections. Additionally, in 8% of cases it is responsible for causing nosocomial infections, especially in newborns and patients after surgical operations, suffering from malignant neoplasms, as well as those having decreased level of immune resistance. Mortality caused by infections of Klebsiella pneumoniae strains may reach up to 50% of cases.
  • G-rods commonly existing in natural environment (vegetation, surface waters, soil), which belong to Enterobacteriaceae family. In the human body, they colonise nasal, throat and intestinal mucous membranes. Pathogenic
  • Biofilm is an organised structure, encompassing bacterial cells, which undergo adhesion to different kinds of solid surfaces and to each other, forming extensive aggregates.
  • Adhesive factors are, among others, bacterial capsular polysaccharides secreted outside of the cell - exopolysaccharides (EPS). EPS consolidates the cells and protects them against the impact of external environment as well as biological and chemical factors.
  • Biofilm is responsible for over 60% of chronic and recurrent infections.
  • antibacterial agents i.e. minocycline, rifampicin.
  • Application of bacteriophages for this purpose seems to be a promising alternative. The existence of bacteriophages capable of destruction of biofilm structures together with bacteria has been shown.
  • Those phages are capable of production of depolymerases hydrolysing extracellular saccharic polymers. Those enzymes are a structural part of phage tail or they are secreted as a free enzyme during lytic cycle. Due to their activity, the bacteriophage reaches the bacterial cell surface and infects it. Capsular polysaccharides (CPS) protect bacterial cell against phagocytosis and other factors of host immune system and are an important virulence factor. In case of Klebsiella pneumoniae strains, CPSs form a thick layer build of repetitive subunits usually composed of from four to six saccharide molecules and negatively charged uronic acid.
  • CPS Capsular polysaccharides
  • CPSs There are about 80 serological types of CPSs, taking into account their varied carbohydrate structure and primarily the structure of K antigen. It is probable, that the degree of CPS virulence depends on the amount of mannose subunits that they contain. It has been shown that polysaccharide antigens (K antigens) with repeating sequence of - mannose-a-2/3-mannose and L- rhamnose-a-2/3-L-rhamnose - determine the level of pathogenicity.
  • K antigens polysaccharide antigens
  • Bacterial polysaccharide depolymerases produced by bacteriophages belongs to two groups: lyases and hydrolases (so-called polisaccharases). In most of the case those enzymes are characterised by a high specificity that depends on carbohydrate structure of substrates, wherein a particular role is played by uronic acid, previously called glycuronic. Hydrolytic activity- consist in a cleavage of specific bond in polysaccharide, wherein lyases activity is based on a cleavage of bond between neutral monosaccharide and the fourth carbon (C4) of uronic acid, accompanied by the formation of double bond between C4 and C5 of this acid.
  • C4 fourth carbon
  • Bacteriophages having depolymerase activity form characteristic aureole (so-called halo-zones) on bacterial lawn in spot-test. Those zones are visible as clearances around the plaque and they indicate that bacterial saccharide capsules have been removed due to the activity of depolymerase that is capable of diffusion within the layer of agar (the presence of plaque indicates the lysis of bacterial cell).
  • bacteriophages producing depolymerases are also effective to use bacteriophages producing depolymerases together with antibiotic, particularly against biofilm structures. It allows to increase the capability of bacteria number reduction. Probably, when phage depolymerase hydrolyses the exopolysaccharides and at the same time it breaks up the biofilm structure, aeration and nutrition of bacteria present inside the aggregates occurs and in consequence they start to be metabolically active and slightly more susceptible to antibiotic activity.
  • One example could be a synergistic activity of bacteriophage and cyprofloksacin against Klebsiella pneumoniae strains.
  • phage depolymerases is bacteriophage K1 1 depolymerase specific against Klebsiella 390 strain. This protein is build of two subunits, wherein each subunit consists of at least two different polypeptide chains having molecular mass of 62 kDa and 94 kDa, respectively. The overall mass of the monomer being a part of the enzyme is 156 kDa.
  • Depolymerase of phage K1 1 shows the activity of glycosidase and it hydrolyses mainly p-(1 ⁇ 3)-glycosidic bond between D-glucose and D-glucuronic acid of the external capsular polysaccharide.
  • Depolymerase of phage K11 is not active against polymers included in bacterial cell wall. Probably, depolymerase is located on phage 1 1 tailspikes. This bacterial virus contains in its structure so-called base plate having a shape of six-pointed star and from each of the points their orifices have two tailspikes of the same size and shape as both subunits of the depolymerase molecule.
  • Klebsiella pneumoniae bacteriophage depolymerase is an enzyme of phage ⁇ 19 specific against Klebsiella K19 strain. CPS surrounding the cell of this strain is build of sequences of repeated hexasaccharides. Depolymerase showing activity against it is a rhamnosidase related to phage ⁇ 19, which hydrolyses specific a-(1 ⁇ 2)-glycosidic bond between two rhamnose molecules. Its activity results in oligosaccharide products containing rhamnose at the reducing end.
  • bacteriophages highly specific against Klebsiella K20 and «24 strains. In respect to the structure, they are very similar to previously described phage 1 1 .
  • the enzyme acting against CPS of K20 serotype is galactosydase hydrolysing p-(1 ⁇ 2)-glycosidic bond between D-galactose and D- mannose molecules.
  • the depolymerase decomposing polysaccharide of K24 serotype is glucosidase acting also on ⁇ -(1 ⁇ 2)-glycosidic bond, but between D-glucose and D-glucuronic acid.
  • Biofilm is a physical barrier for antibiotics and therefore it increases the microbial resistance. It has been shown that for incidence of nosocomial infections and chronic infections mainly responsible are strains capable of biofilm formation. Bacteriophages can support antibiotic activity by the degradation of polysaccharide sheath and enabling the contact between the antibiotic and the cell. Even so the effectiveness of bacteriophage activity for the elimination of bacteria has been largely documented, they have not been yet included into the group of commonly used drugs due to the possibility of frequent mutagenesis and as a consequence, the alteration of their biological nature.
  • phage application is the lack of possibility to monitor their presence in the body and the risk of secretion of large amount of bacterial endotoxins. Therefore, the usage of macromolecules, such as proteins, gives the opportunity of greater control over the consequences of activity of the therapeutic in the body. Further, the application of polysaccharide depolymerases reduces the risk of body's response to shock caused by the presence of bacterial toxins.
  • bacteriophages for the eradication of bacterial infections caused by plankton forms, as well as those composing the biofilm, is known inter alia from ⁇ 2570 30 ⁇ 1 , US20090191254A1 documents.
  • Documents describing bacteriophage enzymes responsible for bacterial cell lysis are also known, and those are enzymes that belong to the group of lysozyme (US8377431 B2, WO2010141 135A2 document). Solutions are also known in the art, wherein phage depolymerases are used to fight against the bacteria.
  • EP0219009B1 document discloses bacteriophage depolymerase of polysaccharides, which is specific against capsules of strains of Erwinia amylovora, a plant pathogen. This enzyme was obtained by recombination of DNA sequence encoding depolymerase into the expression vector. Phage protein degrades the capsules of Ervinia amylovora strains in a specific manner.
  • the object of the invention is a protein having hydrolytic activity against bacterial polysaccharide capsules comprising an amino acid sequence selected from the group comprising SEQ ID NO. 1 and SEQ ID NO. 2.
  • Another object of the invention is a polynucleotide encoding the protein having hydrolytic activity against bacterial polysaccharide capsules comprising an amino acid sequence selected from the group comprising SEQ ID NO. 1 and SEQ ID NO. 2.
  • the polynucleotide comprises a nucleotide sequence selected from the group comprising SEQ ID No. 7 and SEQ ID NO 8.
  • Another object of the invention is a biologically active vector comprising a polynucleotide containing a nucleotide sequence selected from SEQ ID NO. 7 and SEQ ID NO. 8.
  • Another object of the invention is a composition preventing the formation of bacterial biofilm, characterised in that as an active agent it comprises a protein having hydrolytic activity against bacterial polysaccharide capsules that comprises an amino acid sequence selected from the group comprising SEQ ID NO. 1 and SEQ ID NO. 2.
  • the biofilm is formed by Staphylococcus aureus, Enterobacter cloacae and Klebsiella pneumoniae strains.
  • the biofilm is formed by Klebsiella pneumoniae strains.
  • the composition comprises an additional active agent in a form of antibiotic.
  • Another object of the invention is the use of the protein having hydrolytic activity against bacterial polysaccharide capsules, comprising an amino acid sequence selected from the group comprising SEQ
  • the biofilm is formed by Klebsiella pneumoniae, Staphylococcus aurues, Enterobacter cloacae strains.
  • the biofilm is formed by Klebsiella pneumoniae strain.
  • the protein is used in combination with antibiotic.
  • the object of the present invention are also equivalent solutions comprising proteins having amino acid sequence identity for gp31 and gp32 proteins not less than 55% and the similarity of amino acid sequence not less than 75%.
  • the proteins being the object of the invention are molecules having amino acid sequences SEQ ID NO. 1 (NRID1 , gp31 ) and SEQ ID NO. 2 (NRID2, gp32) and showing antibacterial activity, inter alia against Klebsiella pneumoniae, Staphylococcus aureus and Enterobacter cloacae strains.
  • the proteins being the object of the present invention are macromolecules obtained for the first time as a result of recombination and over-expression in E. coli cells.
  • proteins being the object of the present invention show the activity inhibiting the formation of biofilm.
  • the proteins are structural proteins and they do not have the catalytic domains, but show the hydrolytic activity.
  • the proteins of the invention show also synergistic bactericidal activity against above-mentioned bacterial strains in conjunction with the antibiotic used. Those proteins show substrate specificity against saccharide capsules not only of K. pneumoniae, but also against Enterobacter cloacae strain and Gram- positive S. aureus strain.
  • Fig. 1 shows the list of primers for amplification of genes for NRID1 -NRID6 sequences (SEQ ID NO. 1 - SEQ ID NO. 6).
  • Fig. 2 shows electrophoresis - SDS-PAGE - of bacterial lysates BL21(DE3)pLysS following the expression of phage proteins.
  • gp32 and gp45 proteins having molecular weight of 89.1 kDa nad 85.7kDa, respectively, on the right hand side: proteins gp31 - 22 kDa, gp44 -21.5 kDa, gp49- 34 kDa.
  • proteins gp32, gp45, gp31 proteins are present both in a soluble (supernatant) and precipitated form. In the case of gp44 and gp49 proteins, proteins are present only in the residue.
  • Fig. 3 shows the results of electrophoretic analysis of phage proteins obtained following the purification with the use of affinity chromatography in nickel bed.
  • Fig. 4 shows inhibitory effect of gp31 and gp32 protein for Klebsiella pneumoniae biofilm formation in plate test.
  • Fig. 5 shows the degradation of bacterial biofilm formed by Staphylococcus aureus strain with the use of gp31 and gp32 proteins as well as a synergistic effect of the activity of proteins with the antibiotics against the biofilm.
  • Fig. 6 shows the degradation of bacterial biofilm formed by Enferobacfer cloacae strain with the use of gp31 and gp32 proteins as well as a synergistic effect of the activity of proteins with the antibiotics against the biofilm.
  • Fig. 7 shows the degradation of bacterial biofilm formed by Klebsiella pneumoniae strain with the use of gp31 and gp32 proteins as well as a synergistic effect of the activity of proteins with the antibiotics against the biofilm.
  • Fig. 8 shows the hydrolytic activity against Klebsiella pneumoniae (PC 2713 PC 2715) determined with the use of an assay for the amount of reducing sugars by Nelson's method against glucose as a reference.
  • Example 1 The invention is illustrated by the following embodiments.
  • Example 1 The invention is illustrated by the following embodiments.
  • Bioinformatic analysis of genomes of bacteriophages P32 and KP34 deposited in GenBankdatabase, having GQ413937 and GQ413938 accession numbers was made.
  • 6 proteins of the tail were chosen: gp31 , gp32, gp37 (phage KP32) and gp44, gp45, gp49 (phage KP34).
  • Primers were designed for the amplification of genes chosen for amino acid sequences of the proteins of the tail: NRID1 (SEQ ID NO. 1), NRID2 (SEQ ID NO. 2), NRID3 (SEQ ID NO. 3), NRID4 (SEQ ID NO. 4), NRID5 (SEQ ID NO.
  • PCR reaction gene sequences for chosen proteins were obtained (designated on the list of sequences as SEQ ID NO. 7- SEQ ID NO. 12).
  • PCR reactions were conducted in two-phases programme, wherein in the phase 1 - 7 cycles, in the phase 2 - 23 cycles with the use of Taq polymerase (Fermentas), the annealing time (annealing) suitable for the length of the gene from 30 seconds to 2 minutes, the annealing temperature range in the phase no. 1 from 48 to 52°C, in the phase no. 2 from 55 to 65°C.
  • Taq polymerase Fermentas
  • annealing time suitable for the length of the gene from 30 seconds to 2 minutes
  • the annealing temperature range in the phase no. 1 from 48 to 52°C, in the phase no. 2 from 55 to 65°C.
  • Cloning into the pGEM-T- easy vector was done, the correctness of the cloning was verified in the system for the selection of colonies, differentiating white and blue ones.
  • the selected clones were multiplicated, the DNA was isolated (mini-prep), digested with the use of restriction enzymes, preferably EcoRI enzyme, additionally the correctness of obtained sequences was verified for each gene by sequencing (Genomed company). The sequencing was conducted based on T7 and SP6 primers and specifically designed primers.
  • the genes for phage proteins were cleaved out of the T vector with the use of restriction enzymes and transferred into the expression vectors: Strep-tag at the C-terminus Ndel/Hindlll , pTB40, HIS-tag at the C-terminus pET21 a - Ndel/Hindlll cloning, GST at the C-terminus with the site for precise protease pGEX-6p-1 digestion BamHI/End-EcoRI preferably pET2Ba with His-tag at the N-terminus- Ndel/End-EcoRI cloning and transformed into expression strains E. coli JM109 (DE3), preferably in BL21(DE3)pLysS. The transformation was conducted with the use of the heat-shock, after the transformation bacteria were plated onto selective medium containing antibiotic kanamycin.
  • strains were obtained capable of phage protein expression: gp31 , gp32, gp44, gp45 and gp49.
  • tail proteins were expressed, together with their purification with the use of affinity chromatography on the nickel bed (the affinity for histidine tail).
  • NRID1 SEQ ID NO. 1
  • NRID2 SEQ ID NO. 2 sequences
  • the inoculum was transferred into flasks with LB medium (6x 200ml) and the antibiotic, bacteria were cultured until OD 600nm (optical density) of 0.6, cooled down and bacteria were induced with 0.1 mM IPTG. Cultivation was performed for 16 hours at 12°C.
  • Bacteria were centrifuged at 5000 g for 20 minutes, resuspended in 10mM Tris/HCI buffer of pH 8.5 with 200mM NaCI, preferably 500m and 1 M NaCI, additionally, for the protein of NRID2 sequence, preferably from 2% to 10% of glycerol.
  • the sonication was performed for 5 x 0.5 minute with intervals of 5 minutes on ice. Centrifugation at 15000 g for 30 minutes. The supernatants were subjected to affinity chromatography on the nickel bed, the protin binding to the bed was eluted wit the use of 250 mM imidazole solution in 10 mM Tris/HCI buffer, pH 8.5. Protein concentration was determined with the use of BCA method (Bicinchoninic acid assay) .
  • the electrophoresis in polyacrylamid gel in denaturing conditions, in the presence of SDS, according to Laemmli's method The electrophoretic separation of proteins was performed according to the method described by Laemmli in 5% stacking gel and from 10 to 12.5% resolving gel, in electrode buffer pH 8.6. Before loading onto the gel (maximum volume of 10 ⁇ ) the samples (10 pg) were denatured for 5 minutes in boiling water-bath. The protein separation was performed for about 80 minutes, current 10 mA at the beginning for each plate having the dimensions of 83 x 73 x 0.75 mm, and 20 mA after the entry into the stacking gel.
  • gp31 and gp32 proteins were analysed for antibacterial activity. Spot-test were done on agar plates on bacterial lawn, in screening plates, 96-wells and in glass tubes. The proteins were tested against bacterial strains capable of biofilm formation.
  • Klebsiella pneumoniae bacteria were inoculated into 5 ml LB and the overnight cultivation was performed in 37°C. Bacterial suspension was diluted with the use of LB until optical density OD 600 of 0.3 was obtained, which accounts for 10 8 cfu/ml. Into the glass tubes, 200 ⁇ of bacterial suspension and 200 ⁇ of protein solution of NRID1 (SEQ ID NO. 1) and NRID2 (SEQ ID NO. 2) sequences at the concentration of 0.5 mg/ml were added. The control was performed as a bacterial suspension with 200 ⁇ of LB medium. Stationary cultures were performed for 18 hours at 37°C.
  • Klebsiella pneumoniae bacteria were inoculated into 5 ml LB and the overnight cultivation was performed in 37°C. Bacterial suspension was diluted with the use of LB until optical density OD 600 of 0.3 was obtained, which accounts for 10 8 cfu/ml. Into the wells in the microplate, 100 ⁇ of bacterial suspension and 100 ⁇ of protein solution of NRID1 (SEQ ID NO. 1) and NRID2 (SEQ ID NO. 2) sequences at the concentration of 0.5 mg/ml were added. The control was performed in a form of bacterial suspension with 100 ⁇ of LB medium. Stationary cultures were performed for 18 hours at 37°C.
  • Klebsiella pneumoniae, Staphylococcus aureus, Enterobacter cloacae bacteria were inoculated into 5 ml of LB and the overnight cultivation was performed in 37°C.
  • Bacterial suspension was diluted with the use of LB until the optical density OD 600 of 0.3 was obtained, which accounts for 10 8 cfu/ml.
  • 100 ⁇ of bacterial suspension was added into the wells in microplate.
  • Stationary cultures were performed for 18 hours at 37°C.
  • Bacterial solutions were pooled, washed 3 times with 300 ⁇ of sterile MiliQ water and the bacterial suspension was added once again having OD 600 of 0.3. The above procedure was repeated 3 times for 24-hours.
  • Klebsiella pneumoniae, Staphylococcus aureus, Enterobacter cloacae bacteria were inoculated into 5 ml of LB and the overnight cultivation was performed in 37°C. 1 ml of the overnight culture was poured onto the agar plates and allowed to dry. Onto the formed bacterial lawn 10 ⁇ of gp31 and gp32 protein of 2 mg/ml concentration was spotted, allowed to dry and incubated for 16 hours at 37°C.
  • EPS exopolysaccharide composing bacterial mucous
  • EPS exopolysaccharide composing bacterial mucous
  • the residue was obtained following centrifugation at 14000g for 30 minutes at 4°C and was resuspended in water and dialysed into the water, frozen and liofilized.
  • Capsular EPS was extracted from the dry bacterial mass with the use of 10% TCA, according to the method of Gorska-Fraczek (Gorska-Fraczek S, Sandstrom C, Kenne L, Pasciak M, Brzozowska E, et al.
  • the gp31 protein shows hydrolytic activity against capsular EPS as well as mucous, and it also hydrolyses the starch (a-glycosidase).
  • the gp32 protein does not hydrolyse the starch and it is active only against mucous EPS, but not capsular.

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Abstract

La présente invention concerne des protéines de la queue du bactériophage KP32 de Klebsiella pneumoniae, obtenues par recombinaison et présentant une activité hydrolytique contre les capsules polysaccharidiques bactériennes. Les protéines formées des séquences d'acides aminées SEQ ID NO. 1-gp31 et SEQ ID NO. 2-gp32 empêchent le développement du biofilm formé par les diverses souches de Klebsiella pneumoniae et le dégradent et elles présentent un effet synergique dans le cadre de la lutte contre le biofilm formé par les souches de K. pneumoniae, E. cloacae et S. aureus.
PCT/PL2014/000126 2013-11-04 2014-11-04 Protéine possédant une activité hydrolytique contre les capsules polysaccharidiques bactériennes, polynucléotide codant pour ladite protéine, vecteur biologiquement actif, composition contenant la protéine et son utilisation WO2015065209A1 (fr)

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PL40591113A PL405911A1 (pl) 2013-11-04 2013-11-04 Białko o aktywności hydrolitycznej wobec polisacharydowych otoczek bakteryjnych, polinukleotyd kodujący to białko, biologicznie czynny wektor, kompozycja zawierająca białko i jego zastosowanie
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113755469A (zh) * 2021-11-09 2021-12-07 上海瑞宙生物科技有限公司 肺炎克雷伯菌荚膜多糖解聚酶及其应用

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EP0219009B1 (fr) 1985-10-07 1990-12-05 Microlife Technics, Inc. Bactérie pour l'expression d'une dépolymérase de polysaccharide contenant un plasmide recombinant
US20090191254A1 (en) 2004-12-06 2009-07-30 The Govt. Of The U.S.A., Centers For Disease Control And Prevention Inhibition of biofilm formation using bacteriophage
WO2010141135A2 (fr) 2009-03-05 2010-12-09 Trustees Of Boston University Bactériophages exprimant des peptides antimicrobiennes et utilisations afférentes
US8377431B2 (en) 2007-09-13 2013-02-19 Intron Biotechnology, Inc. Bacteriophage or lytic protein derived from the bacteriophage which effective for the treatment of Staphylococcus aureus biofilm
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EP0219009B1 (fr) 1985-10-07 1990-12-05 Microlife Technics, Inc. Bactérie pour l'expression d'une dépolymérase de polysaccharide contenant un plasmide recombinant
EP2570130A1 (fr) 2003-07-23 2013-03-20 Biocontrol Limited Agents thérapeutiques contenant des bactériophages contre P. aeruginosa
US20090191254A1 (en) 2004-12-06 2009-07-30 The Govt. Of The U.S.A., Centers For Disease Control And Prevention Inhibition of biofilm formation using bacteriophage
US8377431B2 (en) 2007-09-13 2013-02-19 Intron Biotechnology, Inc. Bacteriophage or lytic protein derived from the bacteriophage which effective for the treatment of Staphylococcus aureus biofilm
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