WO2005019250A1 - Peptides antibiotiques diriges contre staphylococcus presentant un large spectre d'action - Google Patents

Peptides antibiotiques diriges contre staphylococcus presentant un large spectre d'action Download PDF

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Publication number
WO2005019250A1
WO2005019250A1 PCT/DK2004/000553 DK2004000553W WO2005019250A1 WO 2005019250 A1 WO2005019250 A1 WO 2005019250A1 DK 2004000553 W DK2004000553 W DK 2004000553W WO 2005019250 A1 WO2005019250 A1 WO 2005019250A1
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Prior art keywords
polypeptide
seq
precursor
fragment
variant
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PCT/DK2004/000553
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English (en)
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Angus King
Martin Hansen
Vladimir P. Korobov
Thomas Kofoed
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Ace Biosciences A/S
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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)
    • 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
    • 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 relates to antibiotic (poly)peptides, in particular low-molecular- mass polypeptides with a broad spectrum of antimicrobial activity that are synthesised by bacteria of the genus Staphylococcus, in particular S. warneri.
  • Bacteria are known to secrete specific peptide substances into the environment, inhibiting the micro-flora of the adjacent surroundings as a means of controlling the sources of nutrition and territory.
  • a first type of such substances are the bacteriocins, which represent common simple or complex peptide molecules with a molecular weight above 10 kDa. As a rule, their effect is manifested within a genus. The release of typical bacteriocins into the environment resembles suicide in that both producing cells. and the remaining cells of bacterial populations are killed.
  • a second type of antibacterial peptide substances comprises specific peptide compounds of small size with a molecular mass below 10 kDa that are similar to bacteriocins in their bactericidal effects.
  • Low-molecular-weight bacteriocin-like peptides are detected in cells and supernatants from cultivation media of bacteria from different genera - Bacillaxeae, Enterobacteriaceae, Micrococcaceae, Streptococcaceae, Peptococcaceae, Lactobacillaceae, Actinoplanaceae, Streptomyceae, and also from the bacteria of genera Lactococcus lactis and Enterococcus faecalis.
  • Such peptides do not have any effect on cells from the parent or producer strain but result in the inhibition of growth or death of sensitive cells from other species.
  • Low-molecular-weight antibacterial peptides also exhibit inhibiting effects on the growth of viruses and fungi.
  • antimicrobial peptides Due to peculiarities of their chemical molecular structure, low-molecular-weight peptides usually exhibit marked thermal stability, resistance to pH alterations, and some tolerance to the effect of proteases. A specific feature of some antimicrobial peptides appears to be their hypoallergenic properties. There have only been very rare occurrences of bacterial resistance to these peptides observed over a 30-year period during which the most familiar peptide of this kind, nisin, has been used as a food preservative. These characteristics of antimicrobial peptides make these compounds also promising for practical use in control of infectious agents in animals and humans.
  • Staphylococci are active producers of both staphylocins (typical bacteriocins), and bacteriocin-like peptide compounds with low molecular mass.
  • staphylocins typically bacteriocins
  • bacteriocin-like peptide compounds with low molecular mass typically lysostaphin (S. simulans), epidermin, Pep 5 (S. epid ⁇ rmidis) and gallidermin (S. gallinarum) are best studied.
  • US patent 5,703,040 describes a method for the production of a partially purified protein-like compound with a molecular weight of about 6.4 kDa from the cultivation media of Staphylococcus aureus KSI 1829.
  • the compound exhibits antibacterial action to Streptococci, Corynebacteria, Bordetella, Moraxella, and the bacterium Haemophilus parasuis.
  • RU patent 2,200,195 describes detection of an antibacterial activity in the culture medium of Staphylococcus warneri strain IEGM KL1.
  • the antibacterial activity was resistant to boiling, to DNase and to RNase, but sensitive to trypsin. Ultrafiltration indicated that the activity was due to substances smaller than 3,000 Da. However, no further purification, identification or characterisation of the antibacterial substance(s) was described in RU 2,200,195.
  • the present invention relates to isolated antimicrobial polypeptides, in particular an isolated polypeptide synthesised by S. warneri (also termed “warnerin” herein) and variants, fragments and precursors of said polypeptide. More specifically the invention relates to an isolated antimicrobial polypeptide, wherein said polypeptide - comprises the sequence Dha-Val-Val-Xaa1-Dhb-Dhb-Xaa2-Xaa3-Ala (SEQ ID NO:1), wherein Xaa1 is Lys or Gin, Xaa2 is Leu or lie, Xaa3 is Lys or Gin, and comprises the sequence Lys-Gly-Ala-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8- Xaa9-Asn-Xaa10-Dhb-Gly-Lys-Xaa11 (SEQ ID NO:2), wherein Xaa4 is Thr or an Abu-part of a
  • the invention furthermore relates to pharmaceutical and other compositions comprising a polypeptide, fragment, variant or precursor of the invention and a pharmaceutically-acceptable or other carrier.
  • the invention furthermore relates to methods of producing a polypeptide, fragment, variant or precursor of the invention, and to polynucleotides, expression vectors, and heterologous host cells for use in such methods.
  • the present invention relates to uses of polypeptides, fragments, variants and precursors of the invention to combat microorganisms.
  • Figure 2. Dynamics of the antimicrobial factor's release into the producer's medium: Quantitative estimation of the content of antibacterial factor in a cultivation medium. Black spots evidence for the growth of S. epidermidis.
  • Figure 3. Detection of antibacterial activity in a cultivation medium. Antibacterial test on a solid LB agarose medium. Numerals - hours of cultivation.
  • Figure 4 Purification using a heparin-agarose column: Isolation of fractions possessing antibacterial activity from the supernatants of the S. warneri cultivation medium with different carriers. Gradient elution of fractions exhibiting antibacterial activity from the affinity sorbents (Heparin-agarose). Column dimensions: 16 x 200 mm.
  • Figure 5 Desalination of purified fractions exhibiting antibacterial activity on the columns PD-10. The volume of the column - 3 ml, the volume of the sample - 2 ml. Detection of the release of active fraction along the area of lysis of the indicator culture lawn from S. epidermidis in 12 hours after the application of 5 ⁇ fraction eluate and cultivation at 37 e C.
  • Lane 1 Molecular weight markers.
  • Lane 3 Duplicate gel seen in lanes 1 & 2, overlayed with S. epidermidis culture and grown at 37 e C overnight.
  • the clear area in lane 3 shows bacterial killing.
  • Figure 7 Chromatographic confirmation of purity of isolated antibacterial peptide fraction. Chromatographic isolation of antibacterial peptide homogenous fraction under the use of an acetonitrile gradient with "gentle slope”.
  • Figure 8. Activity of warnerin upon treatment with nucleases and proteases Rows 1 ,4,7 and 10; Warnerin + enzyme + buffer + LB + S. epidermidis.
  • RNAase 1 mg/ml was prepared by boiling for 5 minutes, centrifugation and removal of precipitate material in 100mM Tris.HCI pH7.2. 1 mg/ml Proteinase K and 1 mg/ml trypsin were prepared separately in 50mM Tris.HCI pH7.5. DNAase: Deoxyribonu- clease, RNAase: Ribonuclease, LB: Lauria Bertani broth.
  • Figure 9 MALDI spectrum of intact peptide, with internal calibration
  • Figure 16 Possible sequence of mercaptoethanol treated fragment 1312 Figure 17. Fragmentation spectrum of 1544. The sequences indicates are in reverse order, i.e. from C-terminus to N-terminus.
  • Figure 20 Genomic fragment encoding warnerin Figure 21. Warnerin without leader before and after post-translational modification
  • Peptide and polypeptide - are used interchangeably herein.
  • Isolated - is used to characterise polypeptides and polynucleotides disclosed herein that have been identified and separated and/or recovered from a component of their natural environment. Contaminant components of their natural environment are materials that would typically interfere with the various uses for the polypeptide, and may include proteinaceous and/or non-proteinaceous solutes.
  • about - When used in connection with the value for a molecular mass of a protein, "about” is meant to indicate a molecular mass close to ( ⁇ 10%) the indicated value.
  • E.g. "about 3000 Da” is meant to indicate between 2700 and 3300 Da, such as between 2900 and 3100 Da, for example between 2950 and 3050 Da, such as between 2990 and 3010 Da, for example between 2995 and 3005 Da, or exactly 3000 Da.
  • Fragment - refers to a non-full-length part of a polypeptide.
  • the length of fragments may vary from 2 amino acids to the full-length polypeptide minus one amino-acid residue.
  • fragments are less than 28 amino acids, e.g. less than 25 amino acids, such as less than 20 amino acids, e.g. less than 10 amino acids in length.
  • fragments are more than 5, such as more than 10, e.g. more than 15, such as more than 20, e.g. more than 25, such as more than 27 amino acids in length.
  • a fragment consists of a part of an amino-acid sequence which is less than 100% in length as compared to the full-length polypeptide.
  • the length of the fragment is between 10% and 99%, such as between 50% and 99%, for example between 75% and 99% of the length of the full-length polypeptide.
  • Further preferred fragments are fragments containing at least 5, such as at least 7, e.g. at least 9, such as at least 11 , e.g. at least 13, such as at least 15, e.g. at least 17, such as at least 19, e.g. at least 21 , such as at least 23, e.g. at least 25, such as at leas 27 consecutive amino acids of the full-length warnerin polypeptide.
  • Variant- Variants can be naturally-occurring variants, but also variants that have not been found in nature. Furthermore, the invention also encompasses variants of fragments of the invention and variants of precursors of the invention. Variants are determined on the basis of their degree of identity or their homology with a predetermined amino-acid sequence, said predetermined amino-acid sequence preferably being selected from the group of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27; or, when the variant is a fragment, a fragment of one of these specific sequences.
  • variants preferably have at least 50%, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 96% sequence identity, such as at least 98 % sequence identity with SEQ ID NO:27.
  • Identities between amino-acid sequences may be calculated using well known algorithms, such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90.
  • Variants are also determined based on a predetermined number of conservative amino-acid substitutions as defined herein below.
  • a conservative amino-acid substitution as used herein relates to the substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids exhibit similar or substantially similar characteristics.
  • one amino acid may be substituted for another within the groups of amino acids indicated herein below: i) Amino acids having aliphatic side chains (Gly, Ala, Val, Leu, lie) ii) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro) iii) Amino acids having acidic side chains (Asp, Glu) iv) Amino acids having basic side chains (Lys, Arg, His) v) Amino acids having amide side chains (Asn, Gin) vi) Amino acids having hydroxy side chains, and their dehydrated derivatives (Ser, Thr, Dha, Dhb) vii) Amino acids having sulphor-containing side chains (Cys, Met) viii) Lanthionine and methyllanthionine
  • the same variant or fragment thereof may comprise at least one conservative amino-acid substitution from one or more than one group of conservative amino- acids as defined herein above.
  • Conservative substitutions may be introduced in any position of SEQ ID NO:26 or SEQ ID NO:27, and it may also be desirable to intro-wit non-conservative substitutions in any one or more positions.
  • a non- conservative substitution leading to the formation of a functionally equivalent variant of warnerin would for example i) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or ii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iii) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and vice versa).
  • Variants obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydropho- bicity and hydrophilicity values and the relative similarity of the amino acid side- chain substituents, including charge, size, and the like.
  • Exemplary amino-acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Variants may also differ from the pre-determined sequence in that one or more amino acids have been added or deleted.
  • the addition or deletion of an amino acid is preferably an addition or deletion of from 1 to 20 amino acids, for example from 1 to 10 amino acids, such as from 1 to 5 amino acids, e.g. 1 ,2,3,4 or 5 amino acids.
  • sterically similar variants may be formulated to mimic the key portions of the polypeptide structure. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • the present invention relates to functionally equivalent variants comprising substituted amino acids having hydrophilic or hydropathic indices that are within +/-2.5, for example within +/- 2.3, such as within +/- 2.1 , for example within +/- 2.0, such as within +/- 1.8, for example within +/- 1.6, such as within +/- 1.5, for example within +/- 1.4, such as within +/- 1.3 for example within +/- 1.2, such as within +/- 1.1 , for example within +/- 1.0, such as within +/- 0.9, for example within +/- 0.8, such as within +/- 0.7, for example within +/- 0.6, such as within +/- 0.5, for example within +/- 0.4, such as within +/- 0.3, for example within +/- 0.25, such as within +/- 0.2 of the value of the amino acid it has substituted.
  • hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554,101 , each incorporated herein by reference).
  • amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala- nine (+1.8); glycine (-0.4 ); threonine (-0.7 ); serine (-0.8 ); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (- 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5) (Kyte & Doolittle, 1982).
  • amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+- .1 ); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine
  • Antimicrobial activity indicates the ability to kill microorganisms (a cidal activity) and/or prevent propagation or growth of microorganisms (a static activity). Antimicrobial activities include antibacterial, antifungal, antiparasitic as well as antiviral activity.
  • unusual amino acids refers to amino acids in antimicrobial peptides, such as lantibiotics, that are formed by post-translational modification, such as didehydroalanine (Dha), didehydroamino-2- butyric acid (Dhb), lanthionine, (beta)-methyllanthionine, and 2-aminobutyric acid (Abu).
  • didehydroalanine (Dha) can be formed by post-translational modification of serine residues
  • didehydroamino-2-butyric acid (Dhb) can be formed by post-translational modification of threonine residues.
  • Lanthionines can be formed from a dehydrated serine and a cysteine. The two parts of the resulting lanthionine are termed “Ala-part of lanthionine” herein. Methyllanthionines can be formed from a dehydrated threonine and a cysteine. The two parts of the resulting methyllan- thionine are termed "Abu-part of methyllanthionine” and "Ala-part of methyllanthionine” for the derivative of the dehydrated threonine and the derivative of the cysteine, respectively. See the figures for illustration.
  • Precursor - refers to a polypeptide from which an antimicrobial peptide of the invention can be formed, preferably by post-translational modification.
  • a precursor may or may not possess antimicrobial activity.
  • post-translational modifications can include dehydration of specific hydroxyl amino acids and/or formation of thioether amino acids via addition of neighbouring cysteines to didehydro amino acids.
  • a precursor can be a naturally-occurring precursor, or a precursor that has not been found in nature.
  • Purified - indicates that the polypeptide of the invention has been separated from other components such as cellular components, or other cell-derived or other components found in the culture medium.
  • purified indicates that the polypeptide makes up at least 50%, such as 60%, for example 70%, such as 80%, for example 90%, such as 95%, for example 98%, such as 99%, for example essentially 100% of the total macromolecular content of the composition on a weight or molecule basis.
  • Segment- refers to a part of a polypeptide, i.e. a part of the polypeptide chain.
  • polypeptides of the invention in a main aspect, relates to an isolated antimicrobial polypeptide, wherein said polypeptide comprises the sequence Dha-Val-Val-Xaa1 -Dhb-Dhb- Xaa2-Xaa3-Ala (SEQ ID NO:1), wherein Xaa1 is Lys or Gin, Xaa2 is Leu or lie, Xaa3 is Lys or Gin, and comprises the sequence Lys-Gly-Ala-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Asn-
  • Xaa10-Dhb-Gly-Lys-Xaa11 (SEQ ID NO:2), wherein Xaa4 is Thr or an Abu-part of a methyllanthionine, Xaa5 is Leu or lie, Xaa6 is Thr or an Abu-part of a methyllanthionine, Xaa7 is Gly or Cys or an Ala-part of a methyllanthionine, Xaa8 is Gly or Cys or an Ala-part of a methyllanthionine, Xaa9 is Cys or an Ala-part of a methyllanthionine, Xaa10 is Leu or lie, Xaa11 is Lys or Gin, and wherein SEQ ID NO:1 is located N-terminally of SEQ ID NO: 2, or a fragment, a variant, or a precursor of said polypeptide;
  • the invention relates to an isolated antimicrobial polypeptide which has a molecular mass of about 3000 Da, such as between 2900 and 3100 Da, for example between 2950 and 3050 Da, such as between 2990 and 3010 Da, for example between 2995 and 3005 Da, wherein said polypeptide comprises at least one amino acid, such as at least 2 amino acids, for example at least 3 amino acids, such as at least 4 amino acids each individually selected from the group of Dha and Dhb;
  • the invention relates to an isolated antimicrobial polypeptide which is derivable, by post-translational modification, from the precursor sequence of SEQ ID NO:25 and/or the sequence of SEQ ID NO:26.
  • polypeptide of the invention possess all three of the above defined characteristics, i.e. the polypeptide
  • - comprises the sequence Dha-Val-Val-Xaa1-Dhb-Dhb-Xaa2-Xaa3-Ala (SEQ ID NO:1), wherein Xaa1 is Lys or Gin, Xaa2 is Leu or He, and Xaa3 is Lys or Gin; and comprises the sequence Lys-Gly-Ala-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Asn- Xaa10-Dhb-Gly-Lys-Xaa11 (SEQ ID NO:2), wherein Xaa4 is Thr or an Abu-part of a methyllanthionine, Xaa5 is Leu or lie, Xaa6 is Thr or an Abu-part of a methyllanthionine, Xaa7 is Gly or Cys or an Ala-part of a methyllanthionine, Xaa8 is Gly or Cys or
  • NO:1 is located N-terminally of SEQ ID NO: 2, and
  • polypeptide has a molecular mass of about 3000 Da, such as between 2900 and 3100 Da, for example between 2950 and 3050 Da, such as between 2990 and 3010 Da, for example between 2995 and 3005 Da, wherein said polypeptide comprises at least one amino acid, such as at least 2 amino acids, for example at least 3 amino acids, such as at least 4 amino acids, each individually selected from the group of Dha and Dhb; and
  • - is derivable, by post-translational modification, from the precursor sequence of SEQ ID NO:25 and/or the sequence of SEQ ID NO:26.
  • the isolated antimicrobial polypeptide - has a molecular mass of about 3000 Da, such as between 2900 and 3100 Da, for example between 2950 and 3050 Da, such as between 2990 and 3010 Da, for example between 2995 and 3005 Da, wherein said polypeptide comprises at least one amino acid, such as at least 2 amino acids, for example at least 3 amino acids, such as at least 4 amino acids each individually selected from the group of Dha and Dhb; and - comprises the sequence of SEQ ID NO:1. More preferably, the above polypeptide also comprises the sequence of SEQ ID NO:2.
  • Xaa1 is a lysine. In other embodiments, Xaa1 is a glutamine.
  • Xaa2 is in some embodiments an isoleucine and in other embodiments a leucine.
  • Xaa3 is in some embodiments a lysine and in other embodiments a glutamine.
  • Xaa4 is in some embodiments a threonine and in other embodiments an Abu-part of a methyllanthionine.
  • Xaa5 is in some embodiments an isoleucine and in other embodiments a leucine.
  • Xaa6 is in some embodiments a threonine and in other embodiments an Abu-part of a methyllanthionine.
  • Xaa7 is in some embodiments a glycine, in other embodiments a cysteine and in yet other embodiments an Ala-part of a methyllanthionine.
  • Xaa8 is in some embodiments a glycine, in other embodiments a cysteine and in yet other embodiments an Ala-part of a methyllanthionine.
  • Xaa9 is in some embodiments a cysteine and in other embodiments an Ala-part of a methyllanthionine.
  • Xaa10 is in some embodiments a leucine and in other embodiments an isoleucine.
  • Xaa11 is in some embodiments a lysine and in other embodiments a glutamine.
  • Xaa4 and Xaa6 are both an Abu-part of a methyllanthionine and Xaa7 and Xaa9 are both an Ala-part of methyllanthionine and these four residues form two methyllanthionines.
  • Xaa4 forms a methyllanthionine with Xaa7 and Xaa6 forms a methyllanthionine with Xaa9.
  • Xaa4 forms a methyllanthionine with Xaa9 and Xaa6 forms a methyllanthionine with Xaa7.
  • a polypeptide segment of about 541 Da separates the segments defined in SEQ ID NO:1 and SEQ ID NO:2.
  • the segment defined in SEQ ID NO:1 is preceded by a polypeptide segment of about 143 Da.
  • the sequence of SEQ ID NO:2 comprises the C-terminus of the polypeptide.
  • the polypeptide of the invention preferably contains no aspartic acid and no glutamic acid residues.
  • polypeptide of the invention comprises the sequence of SEQ ID NO:27, even more preferably comprising thioether linkages located as shown in figure 21.
  • the invention relates in one aspect to an isolated antimicrobial polypeptide which is derivable, by post-translational modification, from the precursor sequence set forth in SEQ ID NO:25 and/or the sequence of SEQ ID NO:26.
  • Said post-translational modification is preferably a type of modification which can be performed by Staphylococcus warneri.
  • the post- translational modification comprises removal of the leader peptide and/or dehydration of at least one serine and/or threonine. In a more preferred embodiment, it comprises dehydration of all serines and threonines.
  • the post-translational modification further comprises formation of at least one thioether linkage. More preferably, 3 thioether linkages are formed.
  • the polypeptide of the invention has antibacterial activity against Staphylococcus epidermis.
  • the polypeptide preferably preserves its antibacterial action after boiling for 5 min of an aqueous solution of the polypeptide at a concentration of 1 micrograms/ml and/or after freezing and storage at -18 Q C for 36 months.
  • the polypeptide of the invention contains two preferred cleavage sites for trypsin.
  • polypeptide of the invention is isolated and/or purified. Fragments, variants and precursors
  • the invention also relates to fragments, variants and precursors of the above defined polypeptide of the invention. Preferred are fragment variants and precursors of SEQ ID NO:27. Fragments, variants and precursors have been defined above.
  • the invention also relates to variants of fragments of the polypeptide of the invention, to variants of precursors of the polypeptide of the invention, and to fragments of precursors of polypeptides of the invention.
  • Preferred fragments are fragments with a length of at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90% of the length of the polypeptide of the invention.
  • a variant or precursor of a polypeptide of the invention has at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90% sequence identity to said polypeptide.
  • variants and variants have retained at least some or all of the antimicrobial activity of the polypeptide of the invention. Without being bound by a specific theory, it is expected that the methyllanthionine and lanthionine residues are important for the activity of the peptide.
  • variants or fragments of the polypeptide of the invention that comprise at least one, such as two, more preferably three, meth- ylanthionines or lanthionines are preferred.
  • variants or fragments of SEQ ID NO:27 that comprise at least one, such as two, more preferably three, methylanthionines or lanthionines.
  • fragments and variants of the invention have a pi of more than 9, such as more than 10.
  • fragments and variants of the polypeptide of the invention preferably have more than 5 positively charged residues, such as more than 6, more than 7, more than 8 or more than 9 positively charged residues, preferably lysines or arginines, most preferably lysines.
  • the invention relates to a precursor comprising the sequence set forth in SEQ ID NO:3.
  • the invention relates to a precursor comprising the sequence set forth in SEQ ID NO:26, such as the precursor set forth in SEQ ID NO:25.
  • Precursors of the polypeptides of the invention can e.g. be used to synthesise polypeptide of the invention in vitro.
  • the antimicrobial activity of the polypeptide, fragment, variant or precursor of the invention is an antibacterial activity.
  • the minimal inhibitory concentration against S. epidermis 33 under the test-conditions described herein in the examples is 5 micrograms/ml or less.
  • the minimal inhibitory concentration against S. epidermis 33 under the test-conditions described herein in the examples is preferably a molar concentration which is equivalent to 5 micrograms/ml of the peptide of SEQ ID NO:27 or less.
  • the minimal inhibitory concentration is, or is equivalent to, 2 micrograms SEQ ID NO:27-peptide/ml or less, for example 1 microgram/ml or less, such as 0.5 micrograms/ml or less, for example 0.25 micrograms/ml or less, such as 0.1 micrograms/ml or less, for example 0.05 micrograms/ml or less such as 0.02 micrograms/ml or less, for example 0.01 micrograms/ml or less against S. epidermis 33 under the test-conditions described herein in the examples.
  • the invention also relates to isolated polynucleotides comprising a sequence encoding a polypeptide of the invention or a variant, fragment or precursor of the invention.
  • the polynucleotide of the invention is the sequence of SEQ ID NO:24 or a variant of fragment thereof.
  • Such polynucleotides can be obtained or constructed using standard techniques known in the art and be used in production of the polypeptide. For example, such polynucleotides can be introduced into the original production strain by transformation in order to obtain a strain with multiple copies of a polynucleotide encoding the polypeptide of the invention. This would normally result in the production of larger amounts of the polypeptide.
  • polynucleotides can be introduced into a heterologous production organism.
  • the term 'heterologous' refers to any other strain or species than the one from which the polypeptide originates (i.e. any other strain than S. warneri IEGM KL1), preferably another Staphylococcus species.
  • Such warnerin-overproducing homologous strains and wamerin-producing heterologous strains are a further aspect of this invention.
  • Methods for protein engineering and heterologous production of lantibiotics have been described in e.g. Kuipers et al.(1996) Ant. v. Leeuwenhoek 69:161-169 and Heidrich et al. (1998) Appl. Environ. Microbiol.
  • the invention furthermore relates to an expression vector comprising a polynucleotide of the invention and to a heterologous host cell comprising such an expression vector and/or a polynucleotide of the invention. Furthermore, the invention relates to Staphylococcus warneri ACE Neo-mut-DSM 16081 strain which has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the registration nr.: Staphylococcus warneri ACE Neo-mut-DSM 16081.
  • the polypeptide of the invention is preferably purified.
  • the invention relates to a method for producing a polypeptide, fragment, variant or precursor as defined herein, comprising the steps of a. culturing a strain capable of producing said polypeptide, fragment, variant or precursor under conditions wherein the polypeptide is produced, b. harvesting the polypeptide from the culture, and c. purifying the polypeptide.
  • the purification in step c. is continued until the polypeptide of the invention comprises at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 98%, such as at least 99%, for example essentially 100% of the total protein content, e.g. using the procedures described herein in the Examples. Methods for quantifying the degree of purification are well-known to those skilled in the art.
  • the invention relates to compositions comprising a carrier and a polypeptide, fragment, variant or precursor as defined herein.
  • Such warnerin- containing compositions are preferably stabilised using methods known in the art, such as the methods described in US 5,763,395.
  • Suitable carriers are defined below.
  • the polypeptide, fragment, variant or precursor makes up at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 98%, such as at least 99%, for example essentially 100% of the total protein content of the composition.
  • the invention relates to a composition
  • a composition comprising an antimicrobial polypeptide, a carrier and optionally other substances, wherein the polypeptide has a molecular mass of about 3000 Da, such as between 2900 and 3100 Da, for example between 2950 and 3050 Da, such as between 2990 and 3010 Da, for example between 2995 and 3005 Da, wherein said polypeptide comprises at least one amino acid, such as at least 2 amino acids, for example at least 3 amino acids, such as at least 4 amino acids each individually selected from the group of Dha and Dhb. and wherein said polypeptide makes up at least 1 %, such as at least 5%, for exam- pie at least 10%, such as at least 20%, for example at least 30%, such as at least
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide, fragment, variant or precursor of the invention and a pharma- ceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conven- tional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the pharmaceutical composition is suitable for topical admini- stration.
  • the polypeptide may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavouring agents, foaming agents, and humectants.
  • the active compounds may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavouring agent, such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
  • the active compounds may also be administered parenterally, e.g., formulated for intravenous, intramuscular, or subcutaneous injection.
  • Solutions of the active com- pounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and ster- ile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dis- persion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the various steril- ised active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previ- ously sterile-filtered solution thereof.
  • Polypeptides, fragments, variants, precursors, compositions and pharmaceutical compositions of the invention can be used to combat microorganisms in any envi- ronment capable of sustaining microbial growth.
  • This refers to any fluid, substance or organism where microbial growth can occur or where microbes can exist.
  • Such environments can be, for example, animal tissue or bodily fluids, water and other liquids, food, food products or food extracts, crops and certain inanimate objects. It is not necessary that the environment promotes the growth of the microbe, only that it permits its subsistence. Any use for which nisin, subtilin or other known lantibiotics are used are also envisioned for the polypeptides of this invention and fragments, variants or precursors thereof.
  • a preferred use is use as a food additive.
  • the inven ⁇ tion thus also relates to an edible product comprising a polypeptide, fragment, variant or precursor as defined herein in an amount sufficient to prevent undesirable microbial growth and/or kill undesirable microorganisms.
  • said edible product is a meat or diary product.
  • the invention also relates to use of a polypeptide, fragment, variant or precursor as defined herein for the preparation of such an edible product.
  • the present invention relates to methods for treating and preventing infections by administering to an individual in need thereof a therapeutically-effective amount of a polypeptide, fragment, variant or precursor of the invention as defined herein.
  • the polypeptide, fragment, variant, precursor or pharmaceutical composition of the invention as defined herein is used for the prevention (i.e. prophylaxis) or treatment of microbial infection in a mammal, preferably a human being.
  • the invention also relates to the polypeptide, fragment, variant, precursor or phar- maceutical composition of the invention for use as a medicament and to use of the polypeptide, fragment, variant, precursor or pharmaceutical composition of the invention for the manufacture of a medicament for the prevention or treatment of microbial infection in a mammal.
  • said mammal is a human being.
  • said infection is a bacterial infection, more preferably caused by Gram-positive bacteria.
  • the invention furthermore relates to a kit of parts comprising a polypeptide, fragment, variant, precursor, composition or pharmaceutical composition of the invention as defined herein, and a scheme instructing a user of the dosage to be used in function of the weight and/or age and/or clinical diagnosis of an individual that is to be treated for an antimicrobial infection.
  • the polypeptide of the invention may be used alone or in combination with other active compounds.
  • Non-limiting examples of such other active compounds are compounds that have antimicrobial activity or compounds that stimulate the immune system of the host.
  • the polypeptide of the invention may be useful as an enhancer of the activity of another compound. Effects of combinations of the polypeptide of the invention with other compounds may be additive or synergistic.
  • Infections that may be treated with polypeptides of the invention include those caused by or due to microorganisms.
  • microorganisms include bacteria (e.g. Gram-positive, Gram-negative), fungi, (e.g. yeast and moulds), parasites (e.g. protozoans, nema- todes, cestodes and trematodes), and viruses. Specific organisms in these classes are well known (see for example, Davis et al., Microbiology, 3rd edition, Harper & Row, 1980).
  • clinical indications include, but are not limited to: 1/infections following insertion of intravascular devices or peritoneal dialysis catheters; 2/infection associated with medical devices or prostheses; 3/infection during hemodialysis; 4/S. aureus nasal and extra-nasal carriage; 5/burn wound infections; 6/surgical wounds, 7/acne, including severe acne vulgaris; 8/nosocomial pneumonia; 9/meningitis; 10/cystic fibrosis; 11 /infective endocarditis; 12/osteomyelitis; and 13/sepsis in an immunocompromised host.
  • contaminated intravascular devices such as central venous catheters, or peritoneal dialysis catheters. These catheters are cuffed or non-cuffed, although the infection rate is higher for non-cuffed catheters. Both local and systemic infection may result from contaminated intravascular devices. More than 25,000 patients develop device-related bacteremia in the United States each year. The main organisms responsible are coagulase-negative staphylococci (CoNS), Staphylococcus aureus, Enterococcus spp, E. coli and Candida spp.
  • CoNS coagulase-negative staphylococci
  • Staphylococcus aureus Staphylococcus aureus
  • Enterococcus spp E. coli
  • Candida spp Candida spp.
  • the polypeptide of the invention and/or antibiotic preferably as an ointment or cream, can be applied to the catheter site prior to insertion of the catheter and then again at each dressing change.
  • the polypeptide may be incorporated into the ointment or cream at a concentration preferably of about 0.5 to about 2% (w/v)
  • the polypeptide of the invention and/or other antibiotic can be coated, either covalently bonded or by any other means, onto the medical device either at manufacture of the device or after manufacture but prior to insertion of the device.
  • the polypeptide is preferably applied as a 0.5 to 2% solution.
  • topical antimicrobial substances such as Bacitracin, Tetracycline, or Chlorhexidine
  • the polypeptide of the in- vention alone or in combination with an antibiotic are preferably applied intra- nasally, formulated for nasal application, as a 0.5 to 2% ointment, cream or solution. Application may occur once or multiple times until the colonisation of staphylococci is reduced or eliminated. 5/Burn wound infections. Although the occurrence of invasive burn wound infections has been significantly reduced, infection remains the most common cause of morbidity and mortality in extensively burned patients. Infection is the predominant determinant of wound healing, incidence of complications, and outcome of burn pa- tients.
  • the main organisms responsible are Pseudomonas aeruginosa, S. aureus, Streptococcus pyogenes, and various Gram-negative organisms. Frequent de- bridements and establishment of an epidermis, or a surrogate such as a graft or a skin substitute, is essential for prevention of infection.
  • the polypeptide alone or in combination with antibiotics can be applied to burn wounds as an ointment or cream and/or administered systemically. Topical application may prevent systemic infection following superficial colonisation or eradicate a superficial infection.
  • the polypeptide is preferably administered as a 0.5 to 2% cream or ointment. Application to the skin could be done once a day or as often as dressings are changed.
  • the systemic administration could be by intravenous, intramuscular or subcutaneous injections or infusions. Other routes of administration can also be used.
  • the antimicrobial polypeptide alone or with an antibiotic can be incorporated into soap or applied topically as a cream, lotion or gel to the affected areas either once a day or multiple times during the day.
  • the length of treatment may be for as long as the lesions are present or used to prevent recurrent lesions.
  • the peptide antibiotic could also be administered orally or systemically to treat or prevent acne lesions.
  • Nosocomial pneumonias account for nearly 20% of all nosocomial infections. Patients most at risk for developing nosocomial pneumonia are those in intensive care units, patients with altered levels of consciousness, elderly patients, patients with chronic lung disease, ventilated patients, smokers and post-operative patients. In a severely-compromised patient, multiantibiotic-resistant nosocomial pathogens are likely to be the cause of the pneumonia.
  • the main organisms responsible are P. aeruginosa, S. aureus, Klebsiella pneumoniae and En- terobacter spp.
  • the polypeptide alone or in combination with other antibiotics can be administered systemically to treat pneumonia. Administration could be once a day or multiple administrations per day.
  • the antimicrobial polypeptide can also be admin- istered directly into the lung via inhalation or via installation of an endotracheal tube.
  • Cystic fibrosis is the most common genetic disorder of the Caucasian population. Pulmonary disease is the most common cause of premature death in cystic fibrosis patients. Optimum antimicrobial therapy for CF is not known, and it is generally believed that the introduction of better anti-pseudomonal antibiotics has been the major factor contributing to the increase in life expectancy for CF patients.
  • the most common organisms associated with lung disease in CF are S. aureus , P. aeruginosa and H. influenzae.
  • the polypeptide alone or in combination with other antibiotics could be administrated orally or systemically or via aerosol to treat cystic fibrosis.
  • treatment is effected for up to 3 weeks during acute pulmonary disease and/or for up to 2 weeks every 2-6 months to prevent acute exacerbations.
  • Infective endocarditis results from infection of the heart valve cusps, although any part of the endocardium or any prosthetic material inserted into the heart may be involved. It is usually fatal if untreated. Most infections are nosocomial in origin, caused by pathogens increasingly resistant to available drugs. The main organisms responsible are Viridans streptococci, Enterococcus spp, S. aureus and CoNS. The antimicrobial polypeptide alone or in combination with other antibiotics could be administered orally or systemically to treat endocarditis, although systemic administration would be preferred. Treatment is preferably for 2-6 weeks in duration and may be given as a continuous infusion or multiple admini- stration during the day.
  • 12/Osteomyelitis In early acute disease the vascular supply to the bone is compromised by infection extending into surrounding tissue. Within this necrotic and ische- mic tissue, the bacteria may be difficult to eradicate even after an intense host re- sponse, surgery, and/or antibiotic therapy.
  • the main organisms responsible are S. aureus, E. coli, and P. aeruginosa.
  • the antimicrobial polypeptide could be administered systemically alone or in combination with other antibiotics. Treatment would be 2-6 weeks in duration.
  • the peptide antibiotic could be given as a continuous infusion or multiple administration during the day.
  • the polypeptide of the invention could be used as an antibiotic-impregnated cement or as antibiotic coated beads for joint replacement procedures.
  • Effective treatment of infection may be examined in several different ways.
  • the patient may exhibit reduced fever, a reduced number of microorganisms, a lower level of inflammatory molecules (e.g., IFN-.gamma., IL-12, IL-1 , TNF), and the like.
  • the in vivo therapeutic efficacy from administering an antimicrobial polypeptide and antibiotic agent in combination is based on a successful clinical outcome and does not require 100% elimination of the organisms involved in the infection. Achieving a level of antimicrobial activity at the site of infection that allows the host to survive or eradicate the microorganism is sufficient. When host defences are maximally effective, such as in an otherwise healthy individual, only a minimal antimicrobial effect may suffice.
  • Another preferred use according to the invention is use of the polypeptide, fragment, variant or precursor of the invention for coating of a medical device.
  • the invention also relates to a medical device, such as a catheter, coated with a polypeptide, fragment, variant or precursor of the invention.
  • the strain producing the low-molecular-weight antibacterial peptide was isolated from air in laboratory compartments in 1996 at the Laboratory of the Biochemistry of Microorganisms at the Institute of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences, Perm, Russia.
  • the bacterial strain was identified as being a strain of Staphylococcus warneri and deposited at the Ta- rasevitch Institute as Staphylococcus warneri IEGM KL1 , No. 260.
  • Strains of Micro- coccus luteus, Staphylococcus epidermidis 33, Staphylococcus aureus 209P; Streptococcus pyogenes were received from the Tarasevitch Institute.
  • the strain E E.
  • coli M17 was received from the Scientific-Production Association "Biomed", Perm, Russia. Strains Corynebacterium ammoniagenes, , Rhodococcus ruber, Bacillus subtilis were obtained from the Regional Collection of Alkanotrophic Microorganisms IEGM UB RAS, Perm, Russia.
  • Cultivation of the bacterial strain IEGM KL1 was carried out on a rich nutrient Lauria Broth (LB) medium containing 1 % Tryptone (Difco), 0.5% yeast extract, and 0.64% KCI under aeration at 37 S C.
  • Cultivation of other bacterial strains was carried out in the specified standard media of the suppliers. Typical curves for the warnerin producer's growth and for the level of antibacterial factors in the medium are represented in Fig.1.
  • Example 2 Secretion of an antibacterial factor from a producer strain into the growth medium
  • Determination of the level of antibacterial activity in preparations was carried out in immunological plates based on the principle of two-fold dilution of aliquots from cultivation media in LB medium (Fig. 2).
  • One hundred microliters of LB medium were introduced into each well of the plate.
  • the first well was supplemented with 100 mi- croliters of cultivation media that had been cleared by centrifugation (10000 g, 10 min) under sterile conditions followed by boiling for 5 min.
  • a serial dilution series was made ranging from 1:2 to 1 :256..
  • Ten microliters of S. epidermidis test-culture was subsequently added a to each well.
  • This test culture was prepared as follows: Cells from the loga- rithmic growth phase were washed twice with fresh cultivation medium and centrifugation at 10000 g for 10 minutes. Cells were then resuspended in fresh culture medium at a density of 1.8-2.0 x 10 5 CFU/ml and used as test culture. The efficacy of the inhibiting action of the preparations was evaluated after 12-16 hours of cultivation at 37 S C. The quantity of antibacterial factor in the preparations under investiga- tion was expressed in arbitrary units (AU) that represented reciprocal of the amounts from maximum dilutions at which a complete lack of growth of the S. epidermidis indicator strain was observed. To test the antibacterial peptide activity on a solid medium, the indicator culture S.
  • AU arbitrary units
  • epidermidis was added to a 0.7% LB-agar medium at a concentration of 1.8-2.0 x 10 4 CFU/ml.
  • Five microliters of culture supernatants was added to aga- rose in Petri dishes.
  • the antibacterial action was detected by the formation of areas of bacterial cell lysis at the sites of application after 12-16 hours of incubation at 37 e C.
  • triphenyltetrazolium chloride was introduced to a final con- centration of 0.1% at the end of the incubation period.
  • the same solution was layered onto the LB-agar surface during the tests on dishes.
  • a typical example of such tests for the level of the antibacterial factor production in the cultivation medium is represented in Fig.3.
  • aqueous solutions of warnerin at a concentration of 1 ⁇ g/ml were prepared and held in a boiling water bath for up to 60 minutes. Samples were then frozen at -18 Q C and stored for 60 min, 24 hours, or several months. Results of the heat treatment are shown in Table 2. Freezing and storing for long periods at -18 e C did not affect the antibacterial activity. Similar solutions of peptides isolated were examined for the resistance to enzymatic action. The addition of DNAse (1 mg/ml) and RNAse (1 mg/ml) to warnerin solutions did not result in any alteration in antibacterial activity after incubation at 37 9 C for 2 hours. However, treatment with proteinase K (1 mg/ml) under similar conditions lead to loss of antibacterial activity. Similar observations occurred after treatment with trypsin (1 mg/ml) after a 12 hour-incubation (shown in Fig.8).
  • Mass spectrometric analysis was done using MALDI-TOF (internal calibration) and ESI-MS.
  • the exact mass of the intact peptide as determined by ESI-MS was (monoisotopic): 2997.88 Da.
  • the exact mass of the intact peptide by MALDI-MS was: 2998.75 Da (M+H) monoisotopic: 2997.74 Da. (Shown in Fig. 9 and 10)
  • the observation of loss of 128 Da during treatment with trypsin from both the intact protein 2998 Da (peak at 2870 Da) and from the 1312 Da fragment (1184 Da) indicates that either the N-terminal is a Lys or the 2nd amino acid from the C-terminal is an Arg or Lys and the C-terminal end a Lys or a Gin (See Fig.11 ).
  • the exact masses of the two fragments found by MS were:
  • the intact peptide showed up to 7 additions of mercaptoetanol/ethanthiol (Fig. 14). Since each lanthionine, Dha and Dhb will give one addition, we expect the intact peptide to have 7 of these unusual amino acids.
  • the different products were used in MS/MS fragmentation studies. Especially the data from a 1544 Da fragment were very good and these were used in the sequence identification.
  • the peptide with a mass of 1544 Da corresponds to the 1312 Da peptide plus addition of three mercaptoethanol-groups and loss of 2 Da during disulfide- bridge formation. Investigation of the fragmentation spectrum gave the possible se- quence shown in figure 16. This structure will lead to the possible original structures shown in figures 17 and 18.
  • the placement of the thio-ether bonds is not verified and the C-terminal Lys may be a Gin. Furthermore, the method does not discriminate between the lie and Leu amino acids.
  • the sequence can be prepared from an unmodified precursor peptide comprising the following sequence: Gly-Ala-Thr-lle/Leu-Thr-Gly/Cys-Cys/Gly-Cys-Asn-Leu/lle- Thr-Gly-Lys-Lys/Gln (SEQ ID NO:3) (figure 22 shows SEQ ID NO:1-3).
  • N-terminal part of the polypeptide was analysed.
  • the N-terminal part was isolated by RP-HPLC (molecular weight of 1705 Da) and allowed to react with beta-mercaptoethanol to give between 2 and 4 substitutions. Subsequently, fragmentation studies were carried out wherein MALDI-TOF/TOF and ESI-MS/MS were used.
  • Figure 19 shows a possible sequence in accordance with the information obtained.
  • Test plates for immunological reactions with round bottom wells were used. 100 microliters of LB medium were pipetted into each well. 100 microliters of warnerin were introduced into the first well of a row, mixed, and 100 microliters of the peptide solution obtained were transferred to the next well of a row, etc., thus creating a sequential serial dilution series. A bacterial test-culture inoculum (10 microliters, prepared as above) was added to each well creating an initial cell concentration of 2 x 10 4 CFU/ml. Results are shown in Table 3.
  • the warnerin gene was obtained via sequencing from genomic DNA of Staphylococcus warneri ACE Neo-mut-DSM 16081 strain (a mutation which was introduced via UV incubation and led to higher production of the peptide). The mutation was not genetically analysed or specified.
  • the Staphylococcus warneri ACE Neo-mut-DSM 16081 strain has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the registration nr.: Staphylococcus warneri ACE Neo-mut-DSM 16081.
  • Genomic DNA was prepared using a modified protocol originally provided by Qiagen.
  • the modification included mainly the addition of high amounts of lysostaphin (1 OOU per 2 x 10 8 bacteria), which was required to break the Staphylococcus warneri mutated strain.
  • Sequencing was done according to standard protocols at the ABI 3100 Sequencer. Cloning was performed into the cloning vectors pBad and pET101 , both obtained from Invitrogen via standard PCR-cloning proto- cols.
  • Neo-forward 5'- CAG TTG TTA AAA CAA C - 3' (SEQ ID NO:6)
  • Neo-Seq-1 5'- GTT CGG ATT GTA GTC TGC -3' SEQ ID NO:7
  • neointernallfw 5'- GGA GCA ACA TTA ACT TGT GG SEQ ID NO:8
  • neointerna rw 5'- CCA CAA GTT AAT GTT GCT CC SEQ ID NO:9
  • Neo(rwint)RW2 5'- CTT TAT TCA TAT CGA CTC ACC SEQ ID NO:17
  • SEQ ID NO: 24 shows the warnerin ORF and 5' and 3' flanking regions (figure 20)
  • SEQ ID NO: 25 shows the warnerin precursor sequence (figure 20)
  • SEQ ID NO: 26 shows the warnerin peptide sequence without leader(figure 21)
  • SEQ ID NO: 27 shows the warnerin peptide including post-translational modifications (figure 21 ).
  • the 73 Da group at the N-terminus is a blocking group. Without being bound by one theory, this may well be a 2-hydroxypropionyl, having the following structure: CH3-CH(OH)-CO-.

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Abstract

L'invention concerne des peptides dirigés contre Staphylococcus warneri de faible masse moléculaire présentant un large spectre d'activité antimicrobienne. L'invention concerne également des variants, des fragments et des précurseurs de ces peptides. Elle se rapporte en outre à des compositions pharmaceutiques et analogues comprenant lesdits peptides, fragments, variants ou précurseurs. Par ailleurs, la présente invention porte sur des méthodes destinées à produire un polypeptide, un fragment, un variant ou un précurseur susmentionné, ainsi que sur des polynucléotides, des vecteurs d'expression et des cellules hôtes hétérologues destinés être utilisés dans ces méthodes. La présente invention concerne enfin des utilisations desdits polypeptides, fragments, variants et précurseurs en vue de combattre des micro-organismes.
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WO2008091416A2 (fr) 2006-09-28 2008-07-31 The Ohio State University Research Foundation Agents antimicrobiens antibiotiques et leurs procédés d'utilisation
US9017692B2 (en) 2011-12-05 2015-04-28 Ohio State Innovation Foundation Antimicrobial agent, bacterial strain, biosynthesis, and methods of use

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008091416A2 (fr) 2006-09-28 2008-07-31 The Ohio State University Research Foundation Agents antimicrobiens antibiotiques et leurs procédés d'utilisation
EP2069473A2 (fr) * 2006-09-28 2009-06-17 The Ohio State University Research Foundation Agents antimicrobiens antibiotiques et leurs procedes d'utilisation
EP2069473A4 (fr) * 2006-09-28 2009-11-11 Univ Ohio State Res Found Agents antimicrobiens antibiotiques et leurs procedes d'utilisation
US20110245152A1 (en) * 2006-09-28 2011-10-06 The Ohio State University Research Foundation a not-for-profit corporation Antibiotic antimicrobial agents and methods of their use
US8299020B2 (en) * 2006-09-28 2012-10-30 The Ohio State University Research Foundation Antimicrobial peptides and methods of their use
US9017692B2 (en) 2011-12-05 2015-04-28 Ohio State Innovation Foundation Antimicrobial agent, bacterial strain, biosynthesis, and methods of use

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