WO1995030751A1 - Lanthionine-containing antimicrobial compound - Google Patents
Lanthionine-containing antimicrobial compound Download PDFInfo
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- WO1995030751A1 WO1995030751A1 PCT/EP1995/001587 EP9501587W WO9530751A1 WO 1995030751 A1 WO1995030751 A1 WO 1995030751A1 EP 9501587 W EP9501587 W EP 9501587W WO 9530751 A1 WO9530751 A1 WO 9530751A1
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- peptide
- pharmaceutically acceptable
- peptide according
- acid addition
- acceptable acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention generally relates to a lanthionine-containing antimicrobial peptide obtainable from the culture medium of the bacterial strain Staphylococcus hyicus strain 664, to a pharmaceutically acceptable acid addition salt thereof, and to a method of producing said peptide.
- the invention further relates to a pharmaceutical composition comprising the lanthionine-containing antimicrobial peptide according to the invention, and to a method of producing said composition.
- the lanthionine-containing peptide according to the invention or its acid addition salts can be used as a pharmaceutical agent, but especially as a therapeutic agent in the treatment or prevention of bovine mastitis (anti-mastitis drug). Teat dipping drugs are included..
- Mastitis is an inflammatory disease of the mammalian mammary gland.
- Dairy cattle are highly specialised for milk production. They produce much more milk than is needed to nourish a calf. This super production, and the convention of milking dairy cows 2 or at most 3 times during 24 hours, renders their mammary glands susceptible to bacterial infections.
- they are milked by a mechanical apparatus that passes from cow to cow and so infection is transmitted from one animal to another.
- the mammary gland has a number of natural defense mechanisms against bacterial pathogens (Cullor et al., 1990). These can be overcome by high levels of bacterial challenge and also by compromise of the defense mechanisms. This compromise can be brought about by poor management or through physiological changes at certain times in the lactation cycle (Cullor et al., 1990). The period around drying off and calving are associated with a relatively high incidence of mastitis.
- Mastitis can be caused by many species of bacteria. Those most commonly implicated in bovine mastitis fall into 2 categories I and II: Category I embraces host pathogens such as Staphylococcus aureus and Streptococcus agalactiae. These live on the skin of the udder or in the udder and individual cows are the source of infection to others in the herd. Category II embraces environmental pathogens such as Streptococcus uberis and Escherichia coli. As their name suggests these category II bacteria are found in the immediate environment of the dairy cow and thus present a constant risk (Cullor et al., 1990).
- host pathogens such as Staphylococcus aureus and Streptococcus agalactiae. These live on the skin of the udder or in the udder and individual cows are the source of infection to others in the herd.
- Category II embraces environmental pathogens such as Streptococcus uberis and Escherichia coli.
- Mastitis caused by the bacteria characterized above can manifest as either clinical or subclinical disease (Cullor et al., 1990). Clinical disease can vary from mildly affected quarters with changes in the milk through severely infected quarters with eventual loss of that quarter, to a systemically ill cow that may die. Milder presentations are more usual.
- Subclinical mastitis as its name suggests is not obviously present. It is, however, very prevalent in many dairy herds. Subclinically affected quarters have bacteria present and the cell content of milk is greater than normal. This syndrome is accompanied by lower production. Indeed, it has been estimated that up to 70 % of the economic losses sustained by farmers because of mastitis can be attributed to lost production from subclinical disease (Philpot, W.N., 1984).
- mastitis is controlled through the exercise of scrupulous hygiene at milking, by detection of chronic subclinically infected cows and either milking them after the
- an object of the invention to provide an antimicrobial compound that may be advantageously used in the treatment or prevention of mastitis, especially in cows, and more especially in cows, which are not in the dry period.
- This object was surprisingly met within the scope of the present invention by providing a further lanthionine-containing antimicrobial peptide or a pharmaceutically acceptable acid addition salt thereof that may be used as a pharmaceutical agent, but proved especially useful in the treatment of bovine mastitis.
- the lanthionine-containing antimicriobial peptide according to the invention which is also designated hyicin M51 is obtainable from the culture medium of the bacterial strain Staphylococcus hyicus strain664 using for example consecutive steps of conventional column chromatography. It has a molecular weight of about 2119 ⁇ 2 Dalton and can be cleaved by trypsin to yield two fragments of a molecular weight of about 1256 ⁇ 2 Dalton and about 881 ⁇ 2 Dalton, respectively.
- the present invention is therefore drawn to a lanthionine-containing antimicrobial peptide obtainable from the culture medium of the bacterial strain Staphylococcus hyicus strain 664, or a pharmaceutically acceptable acid addition salt thereof; to an antimicrobial or pharmaceutical composition comprising said peptide in addition to a suitable carrier, and to methods for the preparation of said peptide and said compositions.
- the invention further comprises the use of the inventive peptide and compositions to treat or prevent mastitis in a mammal or to prepare a drug for treating or preventing mastitis in a mammal.
- the invention contemplates a method of treating or preventing mastitis in a mammal comprising administering to said mammal a pharamaceutically effective amount of the lanthionine- containing antimicrobial peptide according to the invention or a pharmaceutically acceptable acid addition salt thereof.
- the bacteria are heat-inactivated in a first step by exposing the culture to temperatures between 50° C and 80° C, but preferably between 60° C to 70° C, for a time sufficient to heat-inactivate the bacteria. In the case of, for example, 65° C a period of 30 to 40 minutes of exposure will be sufficient.
- the bacteria are removed from the culture medium using filtration or centhfugation and the cleared supernatant is applied to a first chromatographic column.
- the column material can be added directly to the bacterial culture without prior removal of the bacteria.
- the resin is filtered off, washed and packed into a column.
- Amberlite XAD-7 (Sigma) is used, in which case stirring of the culture in the presence of the resin for 1 to 3 hours ensures efficient adsorption of the peptide to the resin, which is washed after filtration with water and a solution of Na-citrate containing 20% to 30% isopropanol.
- the peptide is eluted from the resin using for example a solution of Na-citrate containing 40% to 50% isopropanol in the case of ar Amberlite XAD-7 resin.
- Active fractions are pooled and further purified in a similiar fashion using chromatographic materials such as for example SP Sepharose Fast Flow (Pharmacia). For storage purposes it is preferred to pool and lyophylise the fractions containing the antimicrobial peptide.
- the peptide is to be used for analytical purposes it can be further purified using for example HPLC.
- HPLC RP-HPLC is done on a Vydac C18 column monitoring the protein by UV detection.
- the peptide according to the invention can be eluted from the colu» — ' ⁇ applying a linear gradient of acetonitrile in 0.1% TFA, elution being followed by drying O ⁇ the active fractions applying vaccuum centrifugation.
- Purified hyicin M51 shows a strong antimicrobial activity against bacteria, especially against bacteria commonly implicated in mammalian and preferably bovine mastitis.
- bacteria include but are not limited to Staphylococcus aureus V557, Staphylococcus aureus Newbould 305, Streptococcus uberis, Streptococcus faecalis, Pasteurella haemolytica, Listeria innocua, Bacillus cereus, Micrococcus luteus, and Leuconostoc mesenteroides, and the Minimal Inhibitory Concentrations (MICs) for these bacteria are in the range from 0.01 g/ml to 100 g/ml, but preferably from 0.05 g/ml to 50 g/ml, and most preferably from 0.1 g/ml to 10 g/ml.
- MICs Minimal Inhibitory Concentrations
- amino acid sequence analysis of hyicin M51 is complicated by the fact that the second amino acid after the N-terminal He is an unusual amino acid blocking further sequence analysis from the N-terminal end. Therefore, the amino acid sequence has to be determined by the combination of experimental data obtained from mass spectroscopy, NMR spectroscopy, and DNA sequencing. The sequence as determined is highly homologous to the lanthionine-containing peptide gallidermin (EP-342486-A1), which has a molecular weight of about 2164 ⁇ 2 Dalton. The following peptide structure of hyicin M51 is revealed by amino acid sequence analysis
- Xaa 1 , Xaa 2 , and Xaa 3 can be any amino acid with the proviso, that the molecular wheight of the complete peptide is 2119 ⁇ 2 Dalton.
- Xaa 1 and Xaa 3 are 2,3-Didehydrobutyrin (Dhb), and Xaa 2 is 2,3-Didehydroalanine (Dha).
- Xaa 1 , Xaa 2 , and Xaa 3 can be any amino acid with the exception that Xaa 1 , Xaa 2 , and Xaa 3 must not be Ala, Lys, and ⁇ , ⁇ -Didehydrobutyric acid, respectively, within the same peptide.
- the present invention also relates to an antimicrobial composition
- an antimicrobial composition comprising in addition to a suitable carrier the lant. ionine-containing antimicrobial peptide according to the invention in an antimicrobially effective amount.
- the present invention relates to a pharmaceutical composition comprising in addition to a suitable carrier a pharmaceutically effective amount of the lanthionine-containing antimicrobial peptide according to the invention or of a pharmaceutically acceptable acid addition salt thereof.
- a pharmaceutical composition is provided for the treatment and prevention of mastitis in a mammal, which preferably is a cow, comprising in additon to a suitable carrier a pharmaceutically effective amount of the antimicrobial compound according to the invention or one of the pharmaceutically acceptable acid salts thereof.
- the prepeptide form of the lanthionine-containing antimicrobial peptide according to the invention is encoded by nucleic acid molecules.
- the structural gene of the prepeptide is encoded by bacterial DNA, whereas the template for translation of the prepeptide consists of RNA. Therefore, nucleic acid molecules encoding the lanthionine-containing antimicrobial peptide or the corresponding prepeptide constitute another embodiment of the invention.
- the nucleic acid molecule comprises the nucleotide sequence
- the nucleic acid molecules according to the invention can be obtained by a method comprising (a) construction of a genomic library of Staphylococcus hyicus strain 664 in a microorganism;
- chromosomal DNA of Staphylococcus hyicus strain 664 is either partially or completely digested with a restriction enzyme.
- a suitable restriction enzyme preferably does not cut within the coding region of the hyicin M51 gene.
- the restriction fragments obtained are ligated to a plasmid vector either immediately after restriction digestion or after an optional size fractionation step.
- the resultant collection of various DNA fragments cloned within plasmid vectors is subsequently transformed into a microorganism.
- the microorganism used is E.coli or a bacteriophage infecting E.coli.
- the genomic library obtained can be used to screen for sequences homologous to the structural gene of gallidermin. Screening procedures using hybridization with a suitable nucleic acid probe are described in Maniatis et al. Suitable probes can be obtained by restriction digestion of the gallidermin gene or by amplification of defined regions of the gallidermin gene using the polymerase chain reaction. Instead of radioactively labeled nucleotides, non-radioactive labels such as digoxigenin (DIG) labeled nucleotides can be used for labelling of the probe.
- DIG digoxigenin
- clones of the genomic library are screened with the gallidermin probe.
- Positive clones i.e. clones showing hybridization with the probe used, are further characterized by purifying and sequencing of the cloned DNA comprised by said clone, to identify the structural gene of the hyicin M51 peptide.
- the structural gene of hyicin M51 can be used biotechnologically to produce hyicin M51 in a microbial host.
- said host has to be equipped with the enzymatic machinery which performs the posttranslational modifications found in hyicin M51.
- a comprehensive overview on the subject including the gene organisation of the epidermin operon can be found in "Nisin and Novel Lantibiotics" (Jung, G. and Sahl, H-G., 1991).
- the DNA sequence of the structural gene for hyicin M51 constitutes a preferred embodiment of the present invention. Flanking sequences can easily be obtained by complete sequence analysis of the 6.5kb DNA fragment isolated from the genomic library of Staphylococcus hyicus which fragment comprises the structrual gene for hyicin M51. Additional clones comprising Staphylococcus hyicus genomic DNA adjacent to said 6.5kb fragment and clones of the whole operon can be obtained easily by procedures well known in the state of the art.
- a DNA molecule comprising the isolated operon can then be cloned in a plasmid which is able to be replicated and expressed in bacterial strains such as other strains of gram positive bacteria and staphylococci.
- Augustin et al (1991) for example describe the cloning and transfer of the epidermin genes from S. epidermidis to S. carnosus. Transfer of the genes necessary for the production of lantibiotics can also be achieved without detailed knowledge of all the genes involved.
- McKay et al (1984) and EP-137869 for example describe the transfer of nisin production by conjugative transfer. Hansen et al (1991 ) achieved transfer of subtilin production between two strains ob B. subtilis by integrative transformation.
- microbial hosts devoid of the enzymatic machinery to produce hyicin M51 might be genetically modified in such a way as to be able to perform the posttranslational modifications needed. This could be achieved by expressing the genes encoding the enzymes responsible for the posttranslational modifications in said microbial host. The relevant genes might be introduced into the microbial host by way of transformation or conjugative transfer.
- an antibiotic peptide such as the peptide according to the invention which on the one hand fully controls bovine mastitis and is non-toxic to warm-blooded animals, and which on the other hand exerts only minor effects on bacteria in milk that play a role in cheese and yoghurt production.
- Another embodiment of the present invention is directed to a method of treating or preventing mastitis in a mammal comprising administering to said mammal, which preferably is a cow, goat, or ewe, a pharmaceutically effective amount of the lanthinone-containing antimicrobial peptide according to the invention or one of the pharmaceutically acceptable acid addition salts thereof.
- the administration can be done either before or after the infection by the mastitis causing pathogens has occurred, and it can be done prepartum or postpartum.
- the present invention is concerned with treating or preventing mastitis.
- treating is meant curing or ameliorating an animal that has contracted mastitis.
- Preventing mastitis means preventing the occurrence of the infection, or tempering the severity of the infection if it is later contracted.
- the lanthionine-containing antimicrobial peptide according to the invention and pharmaceutically acceptable acid addition salts thereof are highly active against pathogens causing bovine mastitis, and what is even more surprising, they show almost no inhibition in their antipathogenic action in the presence of milk. What is most important, this lack of inhibition in the presence of milk allows reduction of the therapeutic dose and thus the amount of residual lanthionine-containing peptide in the milk such that the utilization of the milk from treated cows in cheese and yoghurt production is significantly improved.
- one important aspect of the present invention is the treatment or prevention of mastitis in mammals by the administration of a lanthionine-containing antimicrobial peptide according to the invention or a pharmaceutically acceptable acid addition salt thereof to the subject mammal.
- a preferred embodiment of the invention is the treatment or prevention of mastitis in cattle.
- the present invention contemplates employing any form of the inventive peptides or pharmaceutically acceptable acid addition salts thereof alone or in combination.
- the present invention encompasses using native forms of the active agents. However, since the production of recombinant peptides has substantial advantages relative to the purification of native peptides, recombinant peptides are a preferred embodiment. It is also contemplated that synthetic forms of the antimicrobial peptides according to the invention and muteins showing minor structural differences to the native product exhibiting the same biological activity, are also within the scope of the invention.
- the active agents of the present invention are usually prepared and stored as ready-to-use liquid formulations.
- the aqueous solution is generally applicable, but the formulation can also be adapted to the specific type of administration.
- the formulation can also contain non-ionic surfactants that carry no discrete charge when dissolved in aqueous media and are selected from ethoxylated esters of fatty acids and triglycerides.
- the hyicin M51 formulation may also contain EDTA to improve the antimicrobial spectrum and stabilizing agents such as methionines, ascorbic acid, and preservatives such as propylene glycol.
- the active agents of the present invention are administered by intramammary injection; however, effective dosages may be administered parenterally, percutaneously, by implant and also by dipping. In a preferred embodiment of the present invention the administration is carried out via intramuscular, subcutaneous, or intravenous injection.
- the active agents according to the present invention are generally administered using a pharmaceutically acceptable vehicle or excipient. Suitable vehicles are, for example, water, saline, mannitol, dextran, amino acids, glycerol, or the like, in various combinations, in addition, if desired, the vehicle may contain auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents.
- the active ingredient will typically range from about 0.001% to about 95% (w/w) of the composition administered, or even higher or lower if appropriate.
- Parenteral administration may be conventionally accomplished by subcutaneous, intradermal, intramuscular, and even intravenous injection. Needle-less air-blast injection devices may be equally useful. Parenteral administration is well known in the art and may be carried out in ways usual in the animal veterinary or human medical art.
- Sustained action of the active agent to achieve prolonged release can be obtained by formulating the protein in a matrix that will physically inhibit rapid dissolution.
- the formulated matrix is injected into the animal's body where it remains as a depot from which the protein is slowly released.
- Useful adjuvants are polymers and copolymers of lactides and glycosides.
- gelling agents like aluminum, calcium or magnesium monostearate, or carbohydrates (cellulose, pectin, dextran derivatives), polysiloxanes or proteins (gelatin, collagen) could extend the releasing time of the active agents of the present invention after parenteral application.
- Percutaneous administration is also meant to include implantation of controlled release devices, e.g.
- implantable matrices from polymeric materials can be used subcutaneously to deliver the compound over the required period of time. This can also be achieved by implantation of minipumps containing aqueous solutions of the protein. Such implantation techniques are also well known in the art and often used in medical treatment.
- Polysiloxane carriers are described in the art for a variety of hormonal delivery forms and may be adapted to the release of the active agents of the present invention.
- a collagen delivery system for the release of antibiotics is described in the German Offenlegungsschrift DE-3,429,038. This system can also be adapted for the lanthionine-containing anitmicrobial peptides according to the invention and pharmaceutically acceptable acid addition salts thereof.
- Slow release formulations and other pharmaceutical or veterinary formulations of the inventive peptide and its pharmaceutically acceptable acid addition salts can be prepared by adapting, for example, the formulation of lanthionine-containing peptides or other protein formulations already described in the art.
- a “therapeutically effective amount" of an active agent of the present invention is a dose sufficient to either prevent or treat mastitis in a subject to which the active agent is administered.
- the dosages of the active agents of the present invention which can treat or prevent mastitis can be easily determined in view of this disclosure by one of ordinary skill in the art by running routine trials with appropriate controls. Comparison of the appropriate treatment groups to the controls will indicate whether a particular dosage is effective in preventing or treating a disease used in a controlled challenge. In general, effective dosage will vary depending on the mode of administration. It has been found that in the case of an intramammary injection using hyicin M51 administration of 0.1 mg to 10mg per quarter is sufficient to control mastitis due to Staphylococcus aureus.
- effective dosages will depend on the weight of the animal and will typically run in the range of from about 2 g/kg to about 800 g/kg, preferably 20 g/kg to about 200 g/kg. More typically, the dosage will be at least about 50 g/kg, but less than 150 g/kg.
- an effective administration of an active agent according to the present invention will in part depend on the number and timing of the dosages. For example, multiple administrations of a dosage may be given to an animal, typically at least about 12hours apart. In most circumstances it may be desirable to administer the active agent at least three times. It may even be desirable to administer even more dosages to the animal, such as six, seven, eight, or even nine or more over an equal number of days or longer. It is believed that the precise combination of dosage and timing will be subject to a wide range of variation and that numerous combinations effective in treating or preventing a disease can be readily established by those of ordinary skill in the art in view of the present disclosure.
- the bacteriocin producer Staphylococcus hyicus 664 was obtained from the Czechoslovak National Collection of Type Cultures (CNCTC) and a sample of Staphylococcus hyicus strain 664 has been deposited with the DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig on November 17, 1994, under the accession number DSM 9547.
- An ISF 200 fermenter (Infors AG, Bottmingen, CH) containing 4 liters of BHI medium (Difco) with 0.05% antifoam Polywax Polyol PPG 2025 (BP Chemicals) was inoculated at 10% with an overnight culture of S. hyicus grown in BHI medium. The culture was grown at 37° C and O. ⁇ bar backpressure. p0 2 was maintained at >60% of saturation by varying the aeration rate between 5-7lt/min and stirrer speed between 200-400rpm. (Antibacterial activity was detected by spotting aliquots of the samples on agar plates seeded with an indicator bacteria as described in example F). After 6 hours the culture was heated to 65° C for 35 minutes to inactivate the bacteria. After cooling to 37° C the pH was adjusted to 4,9 with 3M HCL
- the resin was then transferred to a chromatographic column (Pharmacia XK 50) and washed with another 1000ml of 20mM Na-citrate pH4 containing 30% of isopropanol. Activity was eluted with 20mM Na-citrate pH4 containing 30% of isopropanol, active fractions were pooled (750ml), and 11ml of SP Sepharose Fast Flow (Pharmacia) that had been prewashed with water were added to the pool. After stirring for 30min at RT the resin was filtered off, resuspended in 0.01 M HCI and packed in a Pharmacia XK2.6 column. The column was washed with 100ml of 0.01 M HCI containing 30% methanol.
- the activity was eluted with the same buffer containing 0.3M NaCI. Active fractions were pooled (120ml) and 11 g of Amberlite XAD-7 (prewashed with H20) were added to the pool. After stirring for 45min the resin was filtered off and packed in a Pharmacia XK2.6 column. The column was washed with 100ml of 0.01 M HCI and eluted with 0.01 M HCI containing 50% isopropanol. The fractions containing activity were pooled and lyophilysed.
- the material was further purified by RP-HPLC on a Vydac C18 column (4.6mmx250mm). Activity was eluted using a linear gradient running from 75% solvent A, 25% solvent B to 60%A, 40%B. Solvent A was 90% H2O, 10% acetonitrile, 0.1% TFA, solvent B was 10% H2O, 90% acetonitrile, 0.1% TFA. Protein was monitored by UV detection at 213nm. Typically, fractions of 1ml were collected and analysed for activity. Active fractions were dried by vacuum centrifugation.
- Amino acid sequence information is obtained with a gas phase sequencer (Applied Biosystems, Foster City, California) using protocols supplied by the manufacturer. The results show that after the N-teminal lie the peptide is no longer available for sequencing indicating the presence of an unusual amino acid at position 2 of the peptide chain.
- Mass spectra are recorded on a Bio Ion 20252CF plasma desorption time of flight mass spectrometer (BIO ION KB, Uppsala, Sweden). The instrument is operated at an accelerating voltage of 15kV. A 1 ns time resolution is employed.
- Hyicin M51 and the two fragments TF1 and TF2 obtained by trypsin digestion are subjected to mass spectroscopy as described in example III.
- NMR measurements are done on a Varian Unity 500 spectrometer with a 1 H-frequency of 500MHz.
- CD 3 OH is used as solvent.
- 1 H-NMR spectra are recorded at temperatures of between 17.5° C and 40° C with presaturation to suppress water.
- Two-dimensional spectra are recorded at 25° C, at which temperature the signal overlap in the amide region is minimal.
- the CD 3 -multiplett signal of the solvent CD 3 OH is set to 3.30ppm.
- one-dimensional 13 C-NMR as well as two-dimensional DQF-H.H-COSY; TOCSY; NOESY; ROESY; H.C-COSY (HSQC); and H,C-COSY-long-range (HMBC)
- DQF H.H-COSY experiments provide proton signals, which are due to coupling over a distance of 2 or 3 chemical bonds; TOCSY experiments additionally provide proton signals, due to coupling over a distance of more than 3 chemical bonds; ROESY spectra provide proton signals resulting from dipolar coupling and provide additional information for the identification of individual spin systems; H.C-COSY experiments, finally, correlate protons, which are joined to a carbon atom directly.
- hyicin M51 He, Leu, 2x Gly, Ala, Pro, and Lys
- the sequential assignment of the individual amino acid residues is based on the interpretation of short-range ROESY cross signals between NH-, alpha- and beta protons of the i-th amino acid and the NH protons of the neighbouring amino acid i+1.
- the characterization of the thio-ether bridges constitutes the last step in the elucidation of the structure of hyicin M51. Assignment of the various Ala structures to the different thio-ether bridges is achieved by interpretation of the long-range ROESY signals. The following thio-ether bridges are identified:
- Chromosomal DNA is isolated from a culture of S. hyicus 664 grown overnight at 37° C in M17-G medium (Terzaghi et al, 1975). Cells are harvested by centrifugation and lysed by incubation for 30 minutes at 37° C in 25mM Tris-HCI pH8, 10mM EDTA, 50mM glucose containing 4mg/ml lysozyme and 40mg/l mutanolysin.
- Table 2 1 H-NMR values in ppm of Hyicin M51 (CD 3 OH, 10mmol, 25 C, TOCSY)
- DNA fragments of between 5kbp-10kbp are collected and ligated to BamHI linearized plasmid pUC18 which has additionally been treated with calf intestine phosphatase (CIP).
- CIP calf intestine phosphatase
- the ligation mixture containing the ligation products is used to transform E.coli strain TG1 to ampicillin resistance.
- the oligonucleotides are used in a polymerase chain reaction (PCR) to amplify a 151 bp DNA fragment of the gallidermin gene.
- PCR polymerase chain reaction
- thermostable Taq polymerase from Perkin Elmer Cetus (GeneAmp TM ) is used according to the manufacturer's instructions.
- Genomic DNA of a strain of Staphylococcus saprophyticus, which is known to produce gallidermin is used as the template DNA for amplification.
- the polymerase chain reaction is carried out in the presence of DIG-11-UTP purchased from Boehringer Mannheim.
- the DNA sequence reveals an open reading frame of 144bp (SEQ ID NO 1 ) with considerable homology to the gallidermin structural gene gdmA.
- the deduced amino acid sequence (SEQ ID NO 4) establishes the results obtained from NMR analysis of the hyicin M51 peptide. After posttranslational modification mature hyicin M51 differs from gallidermin in three amino acid residues. The leader peptides of the two peptides show much less homology.
- S. aureus Newbould 305 is a causative agent of Mastitis
- S. diacetylactis is used in the manufacture of yoghurt and cheese.
- nisin and hyicin M51 300, 100, 30, 10, 3, 1 , 0.3, 0.1 g/ml were prepared in milk and in M17 broth (Merck) and allowed to stand at room temperature for 30 min.
- nisin and hyicin M51 have comparable activities against Streptococcus diacetylactis, both in milk and broth. There is however a clear inhibition of nisin activity against S. aureus in milk, while hyicin M51 retains full activity under these conditions.
- Table 3 MIC plate assay (MICs for Nisin and Hyicin M51 in g/ml)
- Staphylococcus aureus Newbould 305 was grown in M17 broth (Merck) at 37° C to an optical density at 600nm of 1.0. Cells were collected by centrifugation and washed in 20 mM Tris-HCI pH 8.0. The bacterial pellet was resuspended in milk at a density of about 10 7 cells/ml.
- nisin is inhibited in the presence of milk (Jung et al., 1992).
- hyicin M51 does not show a similar inhibition in the presence of milk. It is this particular property that suits it so well for the use as a prophylactic and therapeutic agent for use in bovine mastitis since the therapeutic dose can be lowered accordingly.
- the minimal inhibitory concentrations (MIC) of hyicin M51 against a panel of test organisms were determined using an agar well diffusion method. Agar plates were overlaid with soft agar seeded with the test organism. When the agar had solified wells of 4mm diameter were plugged into the agar. 50 I of a series of two fold dilutions of the peptide in citrate buffer pH4 were applied to the wells and the plates were kept at 4° C for two hours prior to incubation at 37° C overnight. The MIC was determined as the lowest concentration of peptide giving a clear zone of lysis around the well. Indicator strains were grown in Tryptose broth (Difco).
- M17 medium (Merck) was used for plates and soft agar except for Pasteurella haemolytica (Tryptose supplemented with 5% of fetal calf's serum), Leuconostoc mesenteroides (Plate count agar - Merck -, + 15% saccharose), and Bacillus cereus (Plate count agar + 0.2% potato starch-Sigma 2004-). Results are shown in Table 5.
- a dairy herd is maintained to supply candidate cows for enrollment in trials.
- Cows are bought from local cattle dealers. These cows should have a minimal milk production of 15 l/day.
- Two milk samples are taken one day apart from all quarters for bacteriological examination. No cows from which mastitis pathogens can be recovered are entered into the tests, i >n-infected cows or goats are selected based on the lack of recovery of Staphylococcus aureus from two milk samples taken one day apart from all quarters.
- 3 quarters are inoculated via the intramammary route using a suspension of Staphylococcus aureus. The 4th quarter serves as a control.
- In goats 1 of 2 udder halves are inoculated.
- milk samples are taken from all quarters at least 3 times to see whether Staphylococcus aureus can be recovered, and therefore whether the quarter has become infected.
- a quarter is defined as being infected, if Staphylococcus aureus is recovered from at least two of the milk samples. Treatments are assigned randomly to infected quarters and injected via the intramammary route. Milk samples are taken daily until a minimum of 14 days after the last treatment.
- a quarter is defined as being cured if milk samples from that quarter become negative (i.e., no Staphylococcus aureus is recovered) within 7 days after the last treatment and stay negative throughout the sampling period.
- This experimental model is used to have a fixed framework on which to evaluate treatments.
- Staphylococcus aureus has the advantage that it is the most difficult Gram positive bacterium to treat and one of the most important bacteria causing bovine mastitis. Therefore, the cure rates are expected to be lower than for other Gram positive bacteria, such as Streptococci and coagulase-negative Staphylococci.
- a data entry/management system created using the well known statistical analysis computer program SAS, is employed.
- quarters are treated as independent statistical units and cure rates are based on results from individual quarters. This is not correct, since in the same cow quarters do not behave independently.
- the correlation between quarters in a cow is estimated to be 0.25. From our data, we estimate that the correlation in this disease model is between 0.15 and 0.35.
- One consequence of using such a method is that more weight is given to a cow for which three out of three quarters cured compared to three cows for which one out of one quarter cured. This result coincides with the observation that the more quarters are infected, the less they will cure. This problem does not arise in goats, because we infect one half only of each goat's udder.
- cowfactors in the analysis: breed, age, lactation number, lactation stage, daily milk production prior to treatment and number of times used for trials. Appropriate adjustments are also made for goats.
- Quarter factors that must be considered are the following: number of bacteria present before treatment, number of bacteria present at the time of treatment, SCC at treatment, position (front or rear), infection history, and treatment history.
- the odds ratio between treatments A and B is 3 it means that cows treated with A are three times more likely to cure than those treated with B.
- a 95 % confidence interval for the odds ratio is computed. Treatments are statistically different when the 95 % confidence interval does not include 1.
- Teat dipping is one of the most frequently used and most effective measures taken to prevent mastitis in dairy cows. This widely practiced procedure involves dipping the 4 teat of every cow, after every milking, in an antibacterial solution. This procedure leaves a coating of antibacterial substances on the teat surface and a droplet of this substance collects at the dependent part of the teat over the teat orifice. After milking a droplet of milk often covers the teat orifice and is a good medium for bacterial growth. From this position bacteria can readily penetrate the streak canal. Teat dipping provides protection against these possibilities.
- herds.teats are dipped before milking to kill off any pathogens resident on the teat surface before the milking machine is applied.
- agents need to be non-toxic and non-disruptive to cheese and yoghurt manufacture.
- Products currently used for teat dipping contain a variety of different active ingredients such as iodophors, chlorhexidiene and the lanthionine containing peptide, nisin.
- hyicin M51 and its pharmaceutically acceptable acid additional salts are very suitable for use in teat dips because of their rapid killing effect and preferential activity against major mastitis pathogens. Rapid killing gives a special application as an agent for pre-dipping.
- mice 50 cattle are recruited according to the protocol already described for trials of hyicin M51 in therapy of subclinical staphylococcal mastitis. This trial then proceeds according to protocol recognised by the US National Mastitis Council (NMC) (Hogan et al. 1990) for testing efficacy of teat germicides during experimental exposure.
- NMC National Mastitis Council
- Delvotest P is a commercially available, widely used, kit for detection of antimicrobial agents in milk. It relies on inhibition of the growth of Bacillus stearothermophilus.
- the indicator bacteria are suspended in an agar plug in a plastic test tube together with a pH indicator. Growth of bacteria results in acid production and colour change. In the presence of inhibiting substances the bacteria do not grow and there is no colour change.
- Bacillus stearothermophilus is a good representative of bacteria used in cheese and yoghurt manufacture and hence it is widely used as the indicator strain in antibiotic residue detection.
- Acidification is the first and most important step in the manufacturing processes when milk is fermented. If acidification proceeds normally then it is very likely that all other steps will be normal. Therefore one can investigate the ability of different concentrations of hyicin M51 or its pharmaceutically acceptable acid addition salts on the kinetics of acidification of milk by different bacterial strains used in these manufacturing processes.
- Lactobacillus lactis - diacetylactis Preparation of stock cultures: Lyophylised cultures as used in commercial conditions are obtained from Rudolf Wittwer, CH-5002 Rombach, Aarau, Switzerland. These cultures are reconstituted according to the manufacturers instructions and reactivated by passage three times in sterile skimmed milk. Each passage is incubated for 16 - 18 hours at the optimum temperature for each bacterial strain.
- the culture obtained after the third passage is diluted to 2% with whole sterile (UHT) milk, split into aliquots in 10ml tubes and frozen at -20° C for use in experiments.
- UHT whole sterile
- test samples The antimicrobial agent under test (hyicin M51 or one of its pharmaceutically acceptable acid addition salts) is diluted in sterile (UHT) whole milk over the required dilution range. These samples are then inoculated with 2% by volume of the experimental culture of the strain of bacteria being tested. The samples are then mixed and incubated at optimal temperature for the bacterial strain under evaluation. The pH is measured each two hours for a period of eight hours and then at 18 hours after inoculation. The rate of acidification of milk containing different concentrations of active ingredients can then be compared to the rate without any active ingredient.
- UHT sterile
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE NO
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE NO
- MOLECULE TYPE peptide
- HYPOTHETICAL NO
- ANTI-SENSE NO
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP95917968A EP0759077A1 (en) | 1994-05-07 | 1995-04-26 | Lanthionine-containing antimicrobial compound |
JP7528644A JPH09512711A (en) | 1994-05-07 | 1995-04-26 | Lanthionine-containing antibacterial compound |
AU24086/95A AU2408695A (en) | 1994-05-07 | 1995-04-26 | Lanthionine-containing antimicrobial compound |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9409200A GB9409200D0 (en) | 1994-05-07 | 1994-05-07 | Antimicrobial compound |
GB9425886.0 | 1994-12-21 | ||
GBGB9425886.0A GB9425886D0 (en) | 1994-12-21 | 1994-12-21 | Antimicrobial compound |
GB9409200.4 | 1994-12-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995030751A1 true WO1995030751A1 (en) | 1995-11-16 |
Family
ID=26304850
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1995/001587 WO1995030751A1 (en) | 1994-05-07 | 1995-04-26 | Lanthionine-containing antimicrobial compound |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0759077A1 (en) |
JP (1) | JPH09512711A (en) |
AU (1) | AU2408695A (en) |
CA (1) | CA2186621A1 (en) |
WO (1) | WO1995030751A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006104403A1 (en) * | 2005-03-29 | 2006-10-05 | Blis Technologies Limited | Skin treatment compositions |
WO2009111838A1 (en) * | 2008-03-13 | 2009-09-17 | Agriculture Victoria Services Pty Limited | Method of treatment using antimicrobial composition |
US8383125B2 (en) | 2006-03-23 | 2013-02-26 | Agriculture Victoria Services Pty, Limited | Antimicrobial protein |
US9173972B2 (en) | 2000-09-20 | 2015-11-03 | The University Of Georgia Research Foundation, Inc. | Methods and compositions for promoting wound healing |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0181578A2 (en) * | 1984-11-06 | 1986-05-21 | Dr. Karl Thomae GmbH | Antibiotic polypeptide, process for its preparation and its use |
EP0342486A1 (en) * | 1988-05-18 | 1989-11-23 | Dr. Karl Thomae GmbH | Antibiotic |
-
1995
- 1995-04-26 WO PCT/EP1995/001587 patent/WO1995030751A1/en not_active Application Discontinuation
- 1995-04-26 EP EP95917968A patent/EP0759077A1/en not_active Withdrawn
- 1995-04-26 CA CA002186621A patent/CA2186621A1/en not_active Abandoned
- 1995-04-26 AU AU24086/95A patent/AU2408695A/en not_active Abandoned
- 1995-04-26 JP JP7528644A patent/JPH09512711A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0181578A2 (en) * | 1984-11-06 | 1986-05-21 | Dr. Karl Thomae GmbH | Antibiotic polypeptide, process for its preparation and its use |
EP0342486A1 (en) * | 1988-05-18 | 1989-11-23 | Dr. Karl Thomae GmbH | Antibiotic |
Non-Patent Citations (2)
Title |
---|
J R BROADBENT ET AL.: "Nisin inhibits several Gram-positive, mastitis causing pathogens", JOURNAL OF DAIRY SCIENCE, vol. 72, no. 12, December 1989 (1989-12-01), CHAMPAIGN, ILLINOIS US, pages 3342 - 3345 * |
N SCHNELL ET AL.: "Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-ringd", NATURE., vol. 333, 19 May 1988 (1988-05-19), LONDON GB, pages 276 - 278 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9173972B2 (en) | 2000-09-20 | 2015-11-03 | The University Of Georgia Research Foundation, Inc. | Methods and compositions for promoting wound healing |
US9314482B2 (en) | 2000-09-20 | 2016-04-19 | The University Of Georgia Research Foundation, Inc. | Methods and compositions for promoting wound healing |
WO2006104403A1 (en) * | 2005-03-29 | 2006-10-05 | Blis Technologies Limited | Skin treatment compositions |
US8383125B2 (en) | 2006-03-23 | 2013-02-26 | Agriculture Victoria Services Pty, Limited | Antimicrobial protein |
WO2009111838A1 (en) * | 2008-03-13 | 2009-09-17 | Agriculture Victoria Services Pty Limited | Method of treatment using antimicrobial composition |
Also Published As
Publication number | Publication date |
---|---|
CA2186621A1 (en) | 1995-11-16 |
EP0759077A1 (en) | 1997-02-26 |
JPH09512711A (en) | 1997-12-22 |
AU2408695A (en) | 1995-11-29 |
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