Classifications

• • 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
  • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • 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

• This invention relates to variants of the lantibiotic mersacidin and their use.
• Mersacidin belongs to a group of bactericidal peptides that are called lantibiotics. The name signifies that these peptides contain the amino acids lanthionine and/or 3-methyllanthionine. Mersacidin has activity against methicillin-resistant Staphylococcus aureus (MRSA) and is therefore of considerable interest in medicine.
• MRSA methicillin-resistant Staphylococcus aureus
• HIL HIL Y-85,54728
• Mersacidin is produced by processing of a small protein of 68 amino acids.
• the N-terminal 48 amino acids of the protein form a leader sequence, and the C-terminal 20 amino acids are a propeptide sequence which is processed by modifying enzymes to produce mersacidin.
• the sequence of the mersacidin gene, mrsA is provided as SEQ ID NO:1 and its translation as SEQ ID NO:2.
• the sequence of the mature peptide, which includes post-translationally modified residues, is shown in Figure 1 (SEQ ID NO:3).
• the mrsA gene forms part of the mrs gene cluster of about 12.3 kb (Altena et al, 2000).
• the gene cluster includes regulatory genes which control the production of mersacidin by regulating the expression of the mrsA gene and/or its modifying enzymes.
• the mrsA gene is expressed in early stationary phase of the growth of the Bacillus HIL strain.
• Variants of naturally occurring antibiotics can be useful in medicine. Variants can be produced synthetically, semi-synthetically (e.g. by chemical modification of fermented products) or by genetic changes to the organisms which produce them. Potentially, mersacidin could be varied by all three routes, with the latter two being of particular interest. For example, modification of amino acids could be used to produce variants which have altered activity profiles, as well as properties such as bioavailability, biodistribution or the ability to overcome resistance mechanisms to mersacidin itself. Altered amino acids may also be useful to introduce reactive side-chains allowing modification of the peptide by chemical means.
• both the S 161 and E17A were essentially inactive (about 1 ,000-fold greater Minimum Inhibitory Concentration (MIC) measured against M. luteus) while the F3L peptide was weakly active (MIC of 12.5 mg/l against M. luteus, compared to wild-type of 0.195).
• MIC Minimum Inhibitory Concentration
• the variants of the present invention have anti-bacterial activity which in many cases is of a comparable or even better level than that of mersacidin itself.
• the invention thus provides novel antibiotic compounds, genes encoding such compounds, methods of making such compounds and their use in the treatment of human or animal subjects, particularly in conditions requiring anti-bacterial therapy.
• Figure 1 shows the structure of mersacidin.
• the compound is produced from 20 amino acids, 8 of which, including the C-terminal cysteine, are involved in lanthionine bridge formation.
• the compound includes the non-naturally occurring amino acids dehydroalanine (Dha) and [2- aminobutyric acid] (Abu), which are produced by post-translational modification of serine and threonine respectively.
• Dha dehydroalanine
• Abu [2- aminobutyric acid]
• the 2-aminobutyric acid moieties are further combined with cysteine moieties to form thioether crosslinks known as methyllanthionines.
• the invention provides a mersacidin variant wherein the variant comprises a modification to position 3, 5, 6, 7, 8, 9, 10, 11 , 14 or 16 of mersacidin as set out in Table 1 below: Table 1
• Dha is dehydroalanine and Dhb is dehydrobutyrine.
• Dhb is dehydrobutyrine.
• the variant comprises a modification to position 3, 6, 7, 8, 9, 10, 11, 14 or 16 of mersacidin as set out in Table 2 below: Table 2
• the variant comprises a modification to position 3, 6, 7, 8, 9, 10, 11 , 14 or 16 of mersacidin as set out in Table 3 below:
• the variant comprises a modification to position 3, 7, 8, 9, 10, 11 , 14 or 16 of mersacidin as set out in Table 4 below: Table 4
• Variants which comprise a modification selected from the group F3W, G8A, G9A, G9H, V11 I, V11 L, L14I, L14M, L14V, Dha16G and Dha16Dhb are particularly preferred.
• the mersacidin variants may comprise a combination of two or more of the above modifications, for example from 1 to 4, such as 2 or 3 of the modifications (with the remaining residues being that of the wild-type mersacidin sequence).
• a variant comprising any one of the above-mentioned modifications may be a variant consisting of two, three or four changes in combination, or just consisting of a single positional change.
• the change F3W to provide a mersacidin variant ("mersacidin F3W") which has activity against a range of microoganisms which is more potent than mersacidin itself.
• the mersacidin variant may comprise F3W together with one, two or three other changes.
• Such mersacidins include mersacidin F3W G8A, mersacidin F3W G9A, mersacidin F3W G9H, mersacidin F3W V111 1 mersacidin F3W V11 L, mersacidin F3W L14I, mersacidin F3W L14M, mersacidin F3W L14V, mersacidin F3W Dha16G and mersacidin F3W Dha16Dhb.
• the mersacidins include mersacidin G8A G9A, mersacidin G8A G9H, mersacidin G8A V11 I, mersacidin G8A V11 L, mersacidin G8A L14I, mersacidin G8A L14M, mersacidin G8A L14V, mersacidin G8A Dha16G and mersacidin G8A Dha16Dhb.
• the mersacidins include mersacidin G9A V111, mersacidin G9H V111 1 mersacidin V111 L14I, mersacidin V111 L14M, mersacidin V111 L14V, mersacidin V111 Dha16G and mersacidin V111 Dha16Dhb.
• the mersacidins include mersacidin G9A L14I, mersacidin G9H L14I, mersacidin V11 L L14I, mersacidin L14I Dha16G and mersacidin L14I Dha16Dhb.
• the mersacidins include Dha16Dhb, mersacidin G9A Dha16Dhb, mersacidin G9H Dha16Dhb, mersacidin V11 L Dha16Dhb, mersacidin L14M Dha16Dhb, and mersacidin L14V Dha16Dhb.
• the mersacidin variants of the invention may be provided in substantially isolated form, e.g. free or substantially free of material with which they are associated with in a host cell used for their production.
• the mersacidin variant may be in the form of a salt, particularly a pharmaceutically acceptable salt.
• a salt particularly a pharmaceutically acceptable salt.
• These include basic salts, such as an alkali or alkaline earth metal salt, e.g. a sodium, potassium, calcium or magnesium salt.
• the salt may also be an acid addition salt such as those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toiuenesulfonic acid, salicylic acid and the like.
• a potassium salt is preferred. The preparation of
• the mersacidin variant may be prepared in the form of a pharmaceutical composition
• the composition may be in the form of a liquid, gel or solid.
• Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
• Oral, nasal and topical administration may include administration by way of aerosols.
• Topical formulations may also be present in the form of creams, ointments or gels, depending upon the site of intended use.
• Topical compositions of the invention may be in any pharmaceutical form normally used for topical application, in particular in the form of an aqueous, aqueous-alcoholic or oily solution, an oil-in-water or water-in-oil or multiple emulsion, an aqueous or oily gel, a liquid, pasty or solid anhydrous product.
• the composition may also contain the usual adjuvants in the cosmetics and dermatological fields, such as one or more of a hydrophilic or lipophilic gelling agent, hydrophilic or lipophilic active agent, preserving agent and antioxidant.
• the proportion of the fatty phase can range from 5 to 80% by weight, and preferably from 5 to 50% by weight, relative to the total weight of the composition.
• the oils, the emulsifiers and the co-emulsifiers used in the composition in emulsion form are chosen from those used conventionally in the field considered.
• the emulsifier and the co- emulsifier are present in the composition in a proportion ranging from 0.3 to 30% by weight, and preferably from 0.5 to 20% by weight, relative to the total weight of the composition.
• Oils which can be used include mineral oils (liquid petroleum jelly), oils of plant origin (avocado oil, soybean oil), oils of animal origin (lanolin), synthetic oils (perhydrosqualene), silicone oils (cyclomethicone) and fluoro oils (perfluoropolyethers).
• mineral oils liquid petroleum jelly
• oils of plant origin oils of plant origin
• lanolin oils of animal origin
• synthetic oils perhydrosqualene
• silicone oils cyclomethicone
• fluoro oils perfluoropolyethers
• Fatty alcohols cetyl alcohol
• waxes can also be used as fatty substances.
• Emulsifiers and co-emulsifiers which can be used include, for example, of fatty acid esters of polyethylene glycol, such as PEG 20 stearate, and fatty acid esters of glycerol, such as glyceryl stearate.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
• conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used.
• the active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, acetylated triglycerides and the like, as the carrier.
• Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
• a carrier such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like
• the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
• composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to alleviate the symptoms of the subject being treated.
• a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like.
• excipients such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like.
• Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like.
• Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously.
• Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
• Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
• the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc.
• Another approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., US Patent No. 3,710,795.
• Dosage forms or compositions containing active ingredient in the range of 0.1 to 95% with the balance made up from non-toxic carrier may be prepared.
• percentages of active ingredient of 0.1 % to 50% in solution are employable.
• compositions of a mersacidin variant of the invention may also comprise a second active agent, including a different mersacidin variant including those described herein, a different antibacterial agent, or another agent intended to treat a second symptom or cause of a condition to be treated.
• a second active agent including a different mersacidin variant including those described herein, a different antibacterial agent, or another agent intended to treat a second symptom or cause of a condition to be treated.
• an antibiotic of the above classes can be, for example, one of the following:
• ⁇ -Lactam Antibiotics imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefinenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flo
• Ketolides ABT-773, Telithromycin (HMR 3647), HMR3562, HMR3004, HMR3787, ABT-773, CP- 654,743, C2-fluoro ketolide, A1957730, and TE802.
• Quinolones amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, PD131628, PD138312, PD140248, Q-35, AM-1155, NM394, T-3761 , rufloxacin, OPC-17116, DU-6859a, and DV-7751a.
• Tetracyclines chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, and tetracycline.
• Glycopeptides vancomycin and derivatives thereof.
• Aminoglycosides amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, clindamycin, and lincomycin.
• Rifamycins rifamycin SV, rifamycin O, rifabutin, rifampicin, rifampin, and rifalizil.
• the composition may comprise a second agent intended to treat a further symptom or cause of a condition to be treated by the mersacidin variant.
• the composition may comprise an analgesic agent, e.g. a non-steroidal antiinflammatory compound.
• analgesic agent e.g. a non-steroidal antiinflammatory compound.
• the composition may comprise a dermatological agent such as a steroid, for treatment of inflammation of the skin.
• agents which may be useful in dermatological applications include retinoids, bactericidal agents such as benzoyl peroxide and anti-fungal agents.
• the mersacidin variant to be combined with a second active agent may be any one of the variants mentioned above, including mersacidin F3W, mersacidin G8A, and mersacidin F3W G8A.
• Mersacidin variants of the invention may be administered to a human or animal subject in methods of treatment, for example in the treatment of bacterial infection, particularly MRSA (methicillin resistant staphylococcus aureus) infection.
• Such treatment may comprise the step of administering to a subject in need of treatment an effective amount of said mersacidin variant or composition thereof.
• the invention also provides a mersacidin variant or composition thereof for use in a method of treatment or prophylaxis of the human or animal body.
• the invention also provides a mersacidin variant or composition thereof for use in a specific method of treatment or prophylaxis of the human or animal body, the specific method including those described herein below.
• the invention also provides the use of a mersacidin variant or composition thereof for the manufacture of a medicament for use in a specific method of treatment or prophylaxis of the human or animal body, the specific method including those described herein below.
• variants or compositions thereof of the invention may be used for the treatment of bacterial infections, including systemic bacterial infections, caused by bacteria including Clostridium difficile, Staphylococcus spp., Streptococcus spp, Enterococcus spp, Propionibacterium acnes, and Helicobacter pylori.
• the Staphylococcus spp. may be coagulase-negative staphylococci.
• the Staphylococcus spp may be in particular Staphylococcus, epidermidis.
• the Staphylococcus spp may be Staphylococcus aureus including drug-resistant species, such as MRSA, VISA (Vancomycin Intermediate Staph, aureus), VRSA (Vancomycin Resistant Staph, aureus), GISA (glycopeptide- intermediate Staph, aureus), LRSA (linezolid-resistant Staph, aureus), or mupirocin-resistant Staph, aureus.
• MRSA microsomal growth factor
• VISA VISA
• VRSA VRSA
• VRSA Vancomycin Resistant Staph, aureus
• GISA glycopeptide- intermediate Staph, aureus
• LRSA linezolid-resistant Staph, aureus
• Streptococcus pyogenes may be Streptococcus pyogenes, Streptococcus agalactiae, or Streptococcus pneumoniae.
• Enterococcus spp. include Enterococcus faecium, Enterococcus. faecalis.
• the variants and composition may be used for systemic treatment of bacteraemia (including catheter related bacteraemia), pneumonia, skin and skin structure infections (including surgical site infections), endocarditis and osteomyelitis.
• the variants or compositions may also be used for topical treatment of skin infections including acne ie. Propionibacterium acnes.
• the variants and compositions thereof may also be used in the treatment of eye infections, such as conjunctivitis, and for oral treatment for gut super-infection, such as that caused by Clostridium difficile including multiply-resistant C. difficile (pseudomembranous colitis), or gut infections associated with Helicobacter pylori.
• the variants may also be used in the treatment or prevention of infection of the skin in wounds or burns.
• the variants and compositions thereof may be used in prophylactic methods, such as for the clearance of the nares to prevent transmission of MRSA. This may be practiced on subjects at risk of infection (e.g. patients entering a hospital) or on health professionals or other carers at risk of being carriers of such infections. Prophylactic clearance of gut flora ahead of abdominal surgery is also contemplated.
• the effective amount of the mersacidin variant to be administered will ultimately be at the discretion of the physician, taking into account the severity of the disease in a particular subject (e.g. a human patient or animal model) and the overall condition of the subject. Suitable dose ranges will typically be in the range of from 1 to 50 mg/kg, e.g. from 5 to 25 mg/kg, with doses typically being administered in twice daily, daily or every other day as the physician finds appropriate.
• nucleic Acids in another aspect, provides a nucleic acid, generally a DNA, coding for a peptide precursor of a mersacidin variant of the invention.
• precursor it is meant coding for the naturally occurring amino acids which are post-translationally modified by other elements of the mrsA gene cluster to produce mersacidin.
• mersacidin GIODha may be encoded by a sequence at which codon 10 is for serine.
• the nucleic acid may be fused in-frame to nucleic acid encoding the N-terminal 48 amino acids of the mrsA protein leader sequence.
• the nucleic acid, or its fusion may be present in a replicable vector.
• the vector e.g. a plasmid vector, may contain an origin of replication (e.g. a replication origin functional in a bacterial host cell, particularly a Bacillus host cell), together with other elements such as an antibiotic marker gene.
• One or more other genes of the mrsA gene cluster may be present in the vector.
• the mrsR1 gene may be present in the vector.
• the nucleic acid sequence may also form part of the mersacidin biosynthesis gene cluster, in which it has replaced the mrsA wild-type gene. Such a replacement may be achieved by homologous recombination.
• Nucleic acids of the invention may be made by any standard methodology known as such in the art. Typically, the nucleic acids are made by oligonucleotide mutagenesis of the mrsA gene, as described by Szekat et a/, though any other suitable method may be used.
• the nucleic acids of the invention may be present in a host cell, particularly a bacterial host cell such as a Bacillus host cell (e.g. Bacillus sp. HIL Y-85, 54728 or a derivative thereof).
• a Bacillus host cell e.g. Bacillus sp. HIL Y-85, 54728 or a derivative thereof.
• the host cell may comprise a mrsA gene cluster in which the mrsA gene is inactive, e.g. due to mutation of the gene sequence such that no transcription occurs, or due to the presence of a mutation which results in an inactive gene product (e.g. mersacidin E17A).
• a restriction map of the mrs gene cluster is shown in Altena et al, 2000.
• the sequence of this cluster is available as GenBank accession number: AJ250862.
• the overlapping restriction fragments illustrated in Altena et al may be obtained by, for example, PCR amplification based on primers derived from AJ250862. These fragments are assembled using standard cloning procedures and the mrs gene cluster cloned into a suitable cloning vector.
• a vector may be pTRKH2 (O'Sullivan and Klaenhammer 1993).
• the vector may be transformed into a laboratory strain of B. subtilis such as B. subtilis 168 in order to replicate, and plasmid DNA isolated from this host.
• the plasmid may be integrated into this host, or recovered and introduced into other host cells, particularly low-GC Gram positive host cells.
• B. subtilis such as B. subtilis 168
• plasmid DNA isolated from this host may be integrated into this host, or recovered and introduced into other host cells, particularly low-GC Gram positive host cells.
• the present invention provides a bacterial host cell which carries a vector comprising the mrs gene cluster and one of a vector of the present invention or wherein the mrs gene cluster has been modified to produce a mersacidin variant of the present invention.
• the invention also provides a bacterial host cell in which the mrs gene cluster has been integrated into the genome, wherein said cell produces a mersacidin variant of the present invention.
• the host cell is Bacillus sp. HIL Y-85,54728.
• the invention may be a SIgH deficient Bacillus sp.
• HIL Y-85,54728 (“ASigH HIL Y-85,54728"), or a Bacillus species carrying the mrsA gene cluster in which the mrsA gene codes for a variant mersacidin of the present invention.
• ⁇ SigH HIL Y-85,54728 can provide certain advantages for improved production of mersacidin and its variants, as discussed herein below.
• Sigma H is the product of the sigH (or spoOH) gene. It is essential for transcription of genes that function in the transition from exponential to stationary phase and in the induction of sporulation. Mutants deficient in SigH do not sporulate. SigmaH activates transcription of a number of other regulatory proteins e.g. spoOA, spoOF, kinA, spoOM, spoVG, spoVS and the spollA family as well as the phr family of secreted peptide pheromones. For further details see Britton et al. J Bacteriol. 184, 4881 -90; 2002.
• ⁇ SigH HIL strains of the invention may be made utilising the HIL strain deposited as NCIMB Accession Number NCIMB 41211 , deposited 19th March 2004.
• the SigH gene in the HIL strain may be inactivated in accordance with standard techniques available in the art, including for example homologous recombination. Such techniques are described further in WO2005/093069, the contents of which are incorporated herein by reference.
• a construct such as a plasmid which contains part of a Bacillus SigH coding sequence is introduced into the HIL strain, e.g. by protoplast transformation.
• the vector contains a selectable marker such as a chloramphenicol acetyl transferase gene, and the transformed cells are selected for integration of the marker into the chromosome.
• the SigH coding sequence is widely available in the art, and is also available in databases, such as GenBank, accession no. NC_000964.
• the ⁇ SigH HIL may also be an HIL derivative in which the mrsA gene product is inactive, either because the mrsA gene is transcriptionally inactive, or because the gene product is a mutant which does not show antibacterial activity against bacteria which are normally killed by mersacidin.
• bacteria include Micrococcus luteus, such as M. luteus ATCC 4498.
• a ⁇ MrsA HIL in which the mrsA gene is inactivated by insertion into the mrsA gene of an erythromycin resistance gene is disclosed in Altena et al, 2000.
• Another ⁇ MrsA HIL is the E17A HIL disclosed by Szekat et al, 2003.
• a further ⁇ MrsA HIL is one in which the mrsA gene is altered to include a stop codon resulting in a truncated and inactive gene product. All these and other ⁇ MrsA HIL strains may be used to produce ⁇ MrsA ⁇ SigH HIL strains for use in the invention.
• the invention also provides a method of making a mersacidin variant which method comprises culturing a host cell of the invention in a culture medium and recovering the mersacidin variant from the medium.
• the mersacidin variant may be recovered from the medium by standard techniques in the art, such as separation from other components of the culture medium by chromatographic means. Such means include the use of hydrophobic resins, reversed phase chromatography, ion exchange chromatography and HPLC. The recovery of mersacidin is illustrated in US-A- 5,112,806.
• One process which may be used is to bind the mersacidin variant from the culture supernatant onto a hydrophobic resin such as HP20, then elute with acetonitrile-water or methanol-water. This is followed by dilution with water so as to allow binding onto a hydrophobic column such as a C18 reversed phase resin.
• the mersacidin is then eluted with acetonitrile or methanol and the eluate evaporated to reduce volume.
• the pH is then adjusted to about pH 2.5 with phosphate buffer and the solution bound onto a strong cation exchanger such Varian SCX, followed by elution with 50% methanol, 25OmM phosphate buffer pH7.
• the eluate is desalted on another C18 column, eluted with methanol, then lyophilised.
• variants have a different charge from mersacidin
• alterations to the process may be introduced.
• the ion exchange step may be altered or omitted if the charge is different and hplc might be utilised.
• the product may be released by treatment with methanol, acetonitrile or similar solvents,
• Reference herein to "recovery” or “recovering” includes the purification of the mersacidin or variant thereof to a degree such that it will be suitable for pharmaceutical use. Thus generally recovery will include the steps of removal of the microorganism (e.g. by centrifugation or filtration), separating the lantibiotic from other bacterial components present in the culture medium, and optionally if desired components of the culture medium. Thus the mersacidin variant will be in substantially isolated form.
• the mersacidin variant may be recovered in a solution, such as a buffer required to elute the mersacidin variant from a chromatography column, or it may be recovered in the form of a lyophilized fraction.
• Bacillus sp. HIL Y-85,54728 was deposited on 19 th March 2004 with NCIMB, Accession No. NCIMB 41211 in the name of Novacta Biosystems Limited.
• Example 1 Production of Mersacidin Variants.
• mersacidin may be performed using methods known per se in the art, e.g. as disclosed by Szekat et al, ibid.
• Mersacidin variants having antibacterial activity as determined by a bioassay using agar plates containing Micrococcus luteus ATCC 4698 as indicator strain were made. These variants are set out in Table 1 : Table 1
• Fermentation samples were spun down at 4000 rpm for 10 min in 15 ml centrifuge tubes.
• the supematants were transferred to 50 ml centrifuge tubes containinglOO mg of conditioned resin Diaion HP-20 (Supelco). After incubation at room temperature for 6 hours with shaking, the supematants were discarded and the resin containing the mersacidin variant was washed with 2 x 10 ml of water. A second washing step was carried out with 2 x 10 ml of methanohwater (1:1).
• Mersacidin variants were eluted from the resins with 1 ml of 100% methanol. The eluates were evaporated to dryness and resuspended in 0.250 ml of methanohwater (1 :1) and analysed by LC-MS, HPLC and bioassay.
• Fermentation broth samples and/or the concentrated resin eluates were transferred to HPLC vials and 20 ⁇ l of each sample was analysed by LC-MS using the HPLC gradient conditions listed in Table 5 and the mass spectrometry conditions listed in Table 6.
• samples Prior to bioassay of the components in broth samples and fermentation concentrate, samples were fractionated using an analytical HPLC coupled to a 96 well microtitre plate fraction collector. In general, 0.2 ml of broth sample or fermentation concentrate was loaded onto the column and the components were resolved and collected as described in Table 7. The fractions in the 96 well microtitre plates were evaporated to dryness and the resulting residues were dissolved in 50 ⁇ l of methanohwater (1 :1). For each variant the resuspended residues from fractions 36 to 43 were loaded onto bioassay agar plates containing Micrococcus luteus ATCC 4698 as indicator strain. The bioassay plates containing the mersacidin variant samples were left at room temperature for 1 hour to allow diffusion of the sample into the agar prior to incubation at 30 0 C overnight.
• Table 5 HPLC conditions used in the analysis of broth samples and fermentation concentrate samples by LC-MS
• Table 7 Analytical HPLC conditions used to fractionate broth samples and fermentation concentrate:
• MICs Minimum inhibitory concentrations for all organisms with the exception of Streptococcus pneumoniae were determined by two-fold serial antibiotic dilutions in Mueller- Hinton broth (MHB) supplemented with 50 ⁇ g/ml calcium as calcium chloride dihydrate.
• Antimicrobial agent stock solutions were prepared and stored according to NCCLS standard M7-A6.
• Actively growing broth cultures were diluted to contain 10 5 to 10 6 CFU/ml by adjusting to an absorbance of 0.2 - 0.3 at 600nm, equivalent to the McFarland 0.5 standard. They were then diluted a further 1 :100 in broth.
• the assays were performed in duplicate in sterile 96-well microtitre plates in a total volume of 200 ⁇ l (160 ⁇ l broth, 20 ⁇ l antibiotic, 20 ⁇ l inoculum) in a concentration range from 64 ⁇ g/ml to 0.06 ⁇ g/ml.
• the 12 th well of the microtitre plate contained no antimicrobial agent. Vancomycin was used as a reference antibiotic for quality control.
• Example 3 Further MIC Data.
• the G8H variant was tested as described in Example 2.
• the MIC (in ⁇ g/ml) against eight of the Example 2 strains was as set out in Table 11 : Table 11 :
• Example 5 Activity against fusidic acid resistant Staphylococcus aureus.
• Example 6 Activity against mupirocin resistant Staphylococcus aureus.
• MlCs were determined, as described in Example 2, for five variants against strains of Streptococcus pyogenes. Results are shown in Table 14 in ⁇ g/ml: Table 14:
• Example 8 Activity against viridans Streptococcus.
• Example 9 Activity against Propionibacterium acnes.
• Test organisms were selected from 3-7 day growth on Wilkens-Chalgren agar (WCA) supplemented with furazolidone (1-2 ⁇ g/ml).
• WAA Wilkens-Chalgren agar
• furazolidone 1-2 ⁇ g/ml
• Fresh Wilkens-Chalgren broth (WCB) was inoculated by direct colony suspension with single colonies of P. acnes and adjusted to a density equivalent to the McFarland 0.5 standard (1 x 10 8 CFU/ml), then further diluted in sterile WCB, supplemented with 50 ⁇ g/ml Ca 2+ (as calcium chloride dihydrate), for a final inoculum in sterile 96- well microtitre plates of approximately 10 5 CFU/ml.
• MICs Minimum inhibitory concentrations for C. difficile were determined and antimicrobial agent stock solutions were prepared and stored according to the NCCLS reference agar dilution method for anaerobic bacteria (M11-A5, 2001). Two-fold serial antibiotic dilutions were prepared in Wilkens-Chalgren agar (WCA). Test organisms were selected from 48 hour growth on Braziers (C.C.E.Y.) agar, subcultured in Schaedler broth to a density equivalent to a McFarland 0.5 standard (1 x 10 8 CFU/ml), with a final inoculum onto WCA plates supplemented with 50 ⁇ g/ml Ca 2+ (as calcium chloride dehydrate) of approximately 10 5 CFU/spot.
• Ca 2+ calcium chloride dehydrate
• Bacteroides fragilis ATCC 25285 was included as a reference control strain and Metronidazole was used as a reference antibiotic for quality control. All manipulations were performed in duplicate in ambient atmosphere in pre-reduced media with only brief exposure to air. Plates were incubated anaerobically for 48 hours at 37 0 C with the MIC defined as the concentration of drug where a marked reduction occurred in the appearance of growth on the test plate compared to growth on the control plate.
• SEQ ID NO:1 MrsA gene sequence of the MrsA encoding sequence including the leader sequence and the propeptide region.
• the propeptide encoding region is shown underlined:

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