WO2016130024A1 - Endolysin expression platform - Google Patents

Endolysin expression platform Download PDF

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Publication number
WO2016130024A1
WO2016130024A1 PCT/NZ2016/050013 NZ2016050013W WO2016130024A1 WO 2016130024 A1 WO2016130024 A1 WO 2016130024A1 NZ 2016050013 W NZ2016050013 W NZ 2016050013W WO 2016130024 A1 WO2016130024 A1 WO 2016130024A1
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seq
variant
acid sequence
polypeptide
fragment
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PCT/NZ2016/050013
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French (fr)
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Saul PURTON
Henry Nicholas TAUNT
Laura STOFFELS
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Supreme Biotechnologies Limited
University College London
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61P17/10—Anti-acne 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01028—N-Acetylmuramoyl-L-alanine amidase (3.5.1.28)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10333—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10351—Methods of production or purification of viral material

Definitions

  • the invention relates to an expression platform for expressing an endolysin.
  • the invention relates to a cyanobacterial expression platform for expressing an endolysin with antibacterial activity, particularly against Propionibacterium acnes which is a causative bacterium for acne vulgaris.
  • Antibiotics are still the major weapons against severe infectious diseases and it is therefore very important to preserve the efficacy of the available antibiotics, but also to develop new antibiotics with different targets and new strategies to circumvent the resistance mechanisms of the microorganisms.
  • antibiotics developed as a defence mechanism of microorganisms against competitors. At the same time mechanisms to resist the antimicrobial substances also evolved in microorganisms to keep up in the battle of competing microorganisms. Different mechanisms are known that cause resistance against antimicrobial agents.
  • Today, 15 classes of antibiotics are known that target different physiological and metabolic functions, but resistance mechanisms have developed against every class. Beside the increasing occurrence of resistance against conventional antibiotics, there are further limitations of these drugs. Most antibiotics have a broad-spectrum against a range of bacterial classes and therefore each treatment affects not only the pathogen, but also the beneficial and protective human bacterial flora.
  • Bacteriophages are viruses that infect bacteria and are the most abundant entities on the planet. The majority are tailed bacteriophages, comprising a tail and a head that encapsulates the DNA or RNA genome.
  • the nucleic acids can be single or double stranded, and some bacteriophages have a circular genome, while others carry a linear genome.
  • the tail is involved in the attachment to the bacterial cell and the injection of the nucleic acids.
  • Endolysins are bacteriophage-encoded enzymes that are produced in bacterial cells after they have been infected by a bacteriophage. These proteins are also known as bacteriophage lysozymes, lysins or muralytic/mureolytic enzymes. To infect a bacterial cell, bacteriophages attach to the cell surface, often to receptors, and insert their genetic material into the cell. The bacterial DNA and protein synthesis machinery changes to produce bacteriophage particles and the host genome is degraded. At the same time, endolysins and another type of proteins, called holins, are produced and accumulate in the cytoplasm of the infected bacterium.
  • the holin proteins help to tightly control the moment of the endolysin-mediated cells lysis.
  • the hydrophobic proteins assemble into oligomers and insert into the cell membrane.
  • the insertion of the holin proteins results in the creation of pores, which allow the endolysin to pass across the cytoplasmic membrane and enter the peptidoglycan.
  • the endolysins cleave specific bonds of the peptidoglycan, which results in the disruption of the cell wall and the high internal pressure of 20 - 50 atmospheres causes the osmotic lysis and the death of the bacterial cell.
  • the cell lysis results in the release of the phage progenies, which can now infect new bacterial cells.
  • Bacteriophages are highly specific to a target bacterium.
  • endolysins are usually only effective against the species of bacteria in which they are naturally produced, or in some cases, against other species within the same genus.
  • Endolysins that are specific for Gram- positive bacteria usually have a modular organisation. Many of these endolysins have a two- domain structure with an N-terminal catalytic domain and a C-terminal cell binding domain connected by a linker. However, there are also endolysins with different domain architectures. Despite their modular structure, nearly all known endolysins specific to Gram-positive bacteria are single polypeptides encoded by one gene.
  • Endolysins attach the enzyme tightly to its substrate after cell lysis. Endolysins are also able to enter and cleave the peptidoglycan of Gram positive bacteria from outside of the cell (i.e. 'exolysins'). Therefore, the attachment prevents the endolysins from attacking surrounding cells of the target bacterium, which can still function as a host for the bacteriophage. In contrast, the outer membrane of Gram-negative bacteria protects uninfected cells against lysis by endolysins from outside. Endolysins that are specific to Gram negative bacteria are therefore usually smaller single-domain proteins without a cell binding domain.
  • the cell wall binding domain is responsible to a large degree for the specificity of the endolysin for certain species or genera.
  • the domain binds noncovalently to molecules in the cell envelope, which can be part of the peptidoglycan or other cell wall associated molecules. It has been shown that the LysM domain binds to the N-acetyl-glucosamine acid of the peptidoglycan.
  • the catalytic domain is responsible for the enzymatic cleavage of the peptidoglycan and can be an N-acetyl- -D-glucosaminidase or N-acetyl-p-D-muramidase (lysozymes), which both cleave one of the two glycosidic bonds of the carbohydrate strand.
  • Other catalytic domains have endopeptidase activity and cleave a specific bond of the peptide chains.
  • these domains can be N-acetylmuramoyl-Lalanine amidases, which hydrolyse the amide bond between the sugar strand and the peptide chain.
  • Catalytic domains can also have an influence on the specificity of an endolysin.
  • the peptidoglycan structures differ between both Gram-types, but also between different bacterial species. The absence or presence of the specific target bond contributes therefore to a limitation of the host range.
  • Endolysins have been studied by different research groups and it has been shown for several endolysins, that when applied externally to Gram-positive bacteria, these enzymes are able to lyse and kill cells of the target bacterium. It was believed that endolysins could not be used as a treatment against Gram-negative bacteria, since the outer membrane represents a barrier for the enzymes.
  • endolysins have a high specificity for the cell wall of bacterial species in which they are naturally produced. Some endolysins are even effective only against certain serotypes or strains, whereas others lyse bacteria within the same genus. In contrast to conventional antibiotics, which usually have a broad-spectrum, endolysins could be used to specifically eliminate the pathogen without harming the commensal bacterial flora.
  • the usage of narrow spectrum antibiotics for the treatment is highly desirable, but requires accurate and practicable diagnostic methods. With the development of better diagnostic methods, the demand for narrow-spectrum antibiotics will most likely greatly increase.
  • endolysins cleave the peptidoglycan from outside and do not need to get into the cell, several of the commonly known resistance mechanisms cannot affect the activity of endolysins. Intracellular resistance mechanisms such as antibiotic-efflux pumps, cytoplasmic enzymes that degrade or modify antimicrobials, and the reduction of membrane permeability, can be therefore excluded.
  • Propionibacterium acnes is a Gram-positive, anaerobic bacterium of the phylum Actinobacteria, and it is a prevalent human skin commensal that colonises mainly areas that are rich in fatty acids such as the face, scalp and upper back.
  • P. acnes is believed to play a role in the inflammatory stages of acne vulgaris. It has been shown that antibiotic treatments that reduced the number of P. acnes cells on the skin have a therapeutic effect on the severity of the acne condition. Furthermore, patients with acne vulgaris who did not respond to an antibiotic treatment were more likely to carry P. acnes strains which were resistant to this antibiotic.
  • Acne vulgaris is a skin condition that affects about 80% of the population at some point in life, mainly in adolescence. It can be very painful, cause psychological problems and can result in lifelong scarring in severe cases. Furthermore, P. acnes can cause eye lid infections and postoperative as well as device-related (e.g. joint prostheses) infections, especially in immunocompromised patients. Acne vulgaris is treated with topical or oral antibiotics, but shows high levels of resistance against the prescribed antibiotics. Alternatively, chemical treatments with substances such as benzyl peroxide are used, which can have strong side effects.
  • endolysins are to be used as antibacterial agents then a suitable expression platform must be found that can be used for efficient large-scale commercial production, but this platform also needs to fulfil all safety criteria for the production of therapeutic proteins. Complicating this search, however, is that the production cost for recombinant proteins is generally high. It would be therefore desirable to use a system with low costs for the production of recombinant endolysins in order to compete, cost-wise, with small molecule drugs.
  • microalgae which includes eukaryotic as well as prokaryotic, photosynthetic microorganisms and includes cyanobacteria (traditionally called blue-green algae), have started to get progressively more attention as production platforms for biological products.
  • photosynthetic organisms to utilise sunlight, inorganic nutrients and CO2 for the production of biological products makes these organisms appealing.
  • microalgae are believed to offer several advantages for the production of recombinant proteins, including the use of simple and inexpensive nutrients.
  • Several microalgae and plants are classified as GRAS organisms (Generally Regarded as Safe), which means these organisms are safe for human consumption.
  • these microalgae and plants are free of endotoxins and viral or prion contaminants, which is important especially for the production of therapeutic proteins.
  • microalgae can be grown in full containment under sterile conditions in photobioreactors (PBRs), which prevents the release of transgenes to the environment. Growth in PBRs reduces the risk of environmental contamination of the production system and therefore the contamination of the therapeutic protein.
  • PBRs photobioreactors
  • C. reinhardtii The production of recombinant therapeutic protein in the Chlamydomonas reinhardtii chloroplast has been known for over a decade and several studies since then have demonstrated the production of a variety of proteins such as antibodies, hormones and vaccines.
  • the use of C. reinhardtii is particularly well-suited for production of recombinant proteins in PBRs, and several investigations have already contributed to an improvement of the expression levels, including studies that investigated different promoters and UTRs as well as codon optimisation strategies.
  • Gp20 was even attempted with two differently codon-optimised versions of the gp20 gene and the expression of both proteins was attempted with two different promoters, and through translational fusions of these non-expressing endolysins to expressed proteins. In all efforts made, the endolysin did not accumulate to detectable levels in the C. reinhardtii chloroplast.
  • a particular polypeptide, or fragment, variant, derivative or fusion thereof which is believed to be effective in the treatment and/or prevention of infections of Propionibacterium acnes, may be formed using recombinant techniques.
  • the recombinant techniques make use of a cyanobacterial expression platform.
  • the cyanobacterial expression platform is particularly well suited for large-scale production of the polypeptide, or fragment, variant, derivative or fusion thereof. Such large-scale production may involve the use of a photobioreactor (PBR).
  • PBR photobioreactor
  • the invention provides a cyanobacterial expression platform capable of producing a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
  • the cyanobacterial expression platform comprises a strain of Synechocystis sp., such as Synechocystis PCC 6803.
  • the invention provides the use of a cyanobacterial expression platform to produce a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
  • the invention provides an isolated nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
  • the invention provides a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
  • the invention provides a host cell comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof or a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
  • the invention provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof comprising culturing a population of cyanobacterial host cells comprising:
  • nucleic acid molecule encoding the polypeptide comprising the amino acid
  • sequence of SEQ ID No: 1 or a fragment, variant, derivative or fusion thereof; or b) a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof under conditions in which the polypeptide is expressed.
  • the invention provides a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof produced by a process comprising the step of expressing the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof in a population of cyanobacterial host cells.
  • the invention provides a pharmaceutical composition comprising the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
  • the invention provides the use of the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof in the
  • the manufacture of a medicament for the treatment and/or prevention of a bacterial infection is manufacture of a medicament for the treatment and/or prevention of a bacterial infection.
  • the bacterial infection is caused by Propionibacterium acnes.
  • the invention provides a method of treating and/or preventing a bacterial infection comprising administering the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof to the subject in need thereof.
  • the bacterial infection is caused by Propionibacterium acnes.
  • the invention provides polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof for use in the treatment and/or prevention of a bacterial infection.
  • the bacterial infection is caused by Propionibacterium acnes.
  • the treatment and/or prevention may be to enhance the cosmetic appearance of the subject.
  • FIG. 1 Strains of Synechocystis sp. PCC 6803 expressing the endolysin genes under the control of the constitutive promoter of the psbAII gene and the nickel-inducible promoter nrsB as well as the corresponding wild type strain.
  • the diagram shows the Synechocystis sp. PCC 6803 strains that were created to study the expression of gp20 in the cyanobacterium.
  • FIG. 3 Schematic diagram showing the binding sites of the used sets of primers and the expected fragment sizes for the different transformants.
  • the primer psbAII.F binds to the 5 ' UTR of psbAII
  • psbAII.R binds just outside of the insertion site of the gene of interest.
  • these primers result in fragments sizes of 0.3 kb + the size of the gene of interest (kb) for Syn6803_AII_x transformants and 1 .1 kb kb + the size of the gene of interest (kb) for Syn6803_nrsB_x transformants.
  • FIG. 4 Western blot analysis of whole cell extracts of Syn6803_nrsB_gp20i and Syn6803_nrsB_gp202 transformants showing the presence or absence of HA-tagged Gp20 protein.
  • the gene expression in Syn6803_nrsB_gp20i and Syn6803_nrsB_gp202 was induced with 6.4 ⁇ of NiC .
  • PCC 6803 wild type (WT 6803) was used as a negative control.
  • the expected band size for Gp20 is 32.3 kDa. Protein sizes were determined using the PageRulerTM prestained protein ladder (Thermo Scientific). The black box indicates that all samples were analysed on the same gel and membrane and the white line indicates that lanes were cut out between the samples for the preparation of the figure.
  • the gene expression was induced with 6.4 ⁇ of NiCb for 5.5 hours, harvested and concentrated to 100x the culture volume.
  • AAATTATCAG CTGCTGACTT AAAAGCAGGT CGTCGTGGTG TTTGTGGTCA TGTTGATGTA 480 ACTGACGCTT GGCACCAAAG CGATCATGAT GATCCAGGAC CATGGTTTCC TTGGGACAAA 540 TTCATGGCTG TTGTAAACGG TGGTAGCGGT GACAGCGGAG AATTAACAGT TGCAGATGTA 600 AAAGCTTTAC ACGATCAAAT TAAACAATTA TCAGCACAAT TAACTGGTAG CGTGAACAAA 660
  • AAATTATCAG CTGCTGATTT AAAAGCTGGT CGTCGTGGTG TTTGTGGTCA CGTAGACGTA 480 ACTGATGCTT GGCACCAATC TGATCACGAC GACCCAGGTC CATGGTTCCC TTGGGATAAA 540
  • AAAGCTTTAC ACGACCAAAT TAAACAATTA TCAGCTCAAT TAACAGGTTC AGTAAACAAA 660
  • Cyanobacteria have minimal nutrient requirements and are capable of photoautotrophic, mixotrophic or heterotrophic growth, and their cultivation is inexpensive. Furthermore, several species of cyanobacteria have GRAS status. Species such as Arthrospira plantesis and Arthrospira maxima (better known as Spirulina) as well as Nostoc flagelliforme and Nostoc commune are even used as food supplements for vitamins, minerals and ⁇ -carotene.
  • Cyanobacteria are classified as Gram-negative bacteria, but their peptidoglycan is thicker and has a higher degree of cross-linking than that of most Gram-negative bacteria.
  • the peptide chains that crosslink the polysaccharide strands have the typical Gram-negative composition and differ therefore from the peptidoglycan of S. pneumoniae, S. aureus and other Gram-positive bacteria.
  • These cell walls lack for example the glycine bridge, which contains the bond that is cleaved by the endopeptidase domain of other endolysins.
  • the cytoplasmic membrane would present a barrier, in the absence of holins that prevents the endolysins from reaching the peptidoglycan.
  • Cyanobacteria have been seldom considered as production platforms for recombinant proteins despite the fact that genetic engineering tools for the expression of foreign genes have been developed for several species. In most cases the introduction of foreign genes is used for metabolic engineering and the creation of novel biosynthetic pathways. For example, Lan & Liao (Lan, E.I. & Liao, J.C., 201 1. Metabolic engineering of cyanobacteria for 1 -butanol production from carbon dioxide. Metabolic Engineering, 13(4), pp.353-363) expressed a transenoyl-CoA reductase gene for the production of butanol in Synechococcus elongatus PCC 7942, whereas Guerrero et al.
  • the group also investigated the effect of supplementing the feed of turbot (Scophthalmus maximus L) with the transgenic Synechocystis cells producing fGH and found that it can be used as an efficient growth promoter without any observed side effects (Liu, S. et al., 2007. Effect of growth hormone transgenic Synechocystis on growth, feed efficiency, muscle composition, haematology and histology of turbot (Scophthalmus maximus L); Aquaculture Research, 38(12), pp.1283-1292.). These results indicated therefore that Synechocystis sp. PCC 6803 does not contain major amounts of endotoxins that harm turbot following oral administration. It may be possible to use the crude extract of Synechocystis sp. PCC 6803 on humans.
  • PA6 The genome of PA6 consists of double-stranded DNA and has a length of 29.7 kb.
  • Farrar et al. (2007) showed that the bacteriophage could infect 32 different isolates of P. acnes, including clinical isolates with antibiotic resistances to erythromycin and clindamycin.
  • PA6 showed a high specificity for P. acnes and did not infect other bacteria that are common commensals in the human skin microflora such as Propionibacterium granulosum, Propionibacterium avidum, Staphylococcus epidermis or Corynebacterium bovis.
  • ORFs Open Reading Frames
  • Gp20 was found to have homology to the amidases of the mycobacteriophages PG1 and Che8 and a high similarity (67% identity for amino acids 2 to 145, N-terminal catalytic domain) to another N-acetyl-muramoyl-L-alanine amidase, which is an autolysin of P. acnes.
  • the downstream ORF21 is predicted to encode the holin of the lytic system. The gene arrangement is typical for endolysin and holin genes in bacteriophage genomes.
  • N-terminal domain and also the C-terminal domain, that is predicted to be responsible for cell binding, are highly conserved within different P. acnes bacteriophage endolysins and have sequence identities of 92 to 96%. Gp20 from phage PA6 can be therefore seen as a representative example for P. acnes bacteriophage endolysins in general.
  • amino acid refers to any natural or unnatural amino acid. Such amino acids may be in the 'L' form or 'D' form, and may further include omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (such as ⁇ , ⁇ -disubstituted amino acids and N-alkyl amino acids) and chemically derivatised amino acids. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, so long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
  • isolated refers to where the polypeptide or fragment, variant, derivative or fusion thereof of the invention, specifically the wildtype endolysin of bacteriophage PA6, is provided in a form other than that in which it may be found naturally.
  • amino acid sequence of SEQ ID No: 1 is capable of binding specifically to and/or lysing cells of Propionibacterium acnes.
  • the expression "capable of binding specifically to cells of Propionibacterium acnes” means that the entity is capable of binding preferentially to cells of Propionibacterium acnes.
  • the expression "capable of lysing cells of Propionibacterium acnes” means that the entity retains (at least in part) the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells, such as cells of Propionibacterium acnes.
  • polypeptide, or fragment, variant, derivative or fusion thereof need not retain all of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells. Rather, it is simply necessary for said polypeptide, fragment, variant, derivative or fusion to retain at least 10% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells. Preferably, however, the polypeptide, fragment, variant, derivative or fusion exhibits at least 30%, for example at least 50%, 70%, 90%, 100%, 150%, 200% or more, of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells.
  • the fragment may comprise or consist of at least 50 contiguous amino acids of SEQ ID NO: 1 , for example at least 100, 150, 200, 250 or 265 contiguous amino acids of SEQ ID NO: 1.
  • catalytic domain that is responsible for cell wall degradation
  • cell wall binding domain that recognises a cell surface motif and permits attachment of the endolysin to that target cell.
  • the catalytic (enzymatic) domain can be identified by its amino acid homology to other similar regions of lytic enzymes that share the same type of lytic activity.
  • the catalytic domain may correspond to a N-acetylmuramoyl-L-alanine amidase.
  • the polypeptide, or fragment, variant, derivative or fusion thereof has homology to the amidases of the mycobacteriophages PG1 and Che8 and a high similarity (such as, for example, 67% identity for amino acids 2 to 145, N-terminal catalytic domain) to a N-acetyl-muramoyl-L-alanine amidase which is an autolysin of P. acnes as taught in Farrar et al (2007).
  • the polypeptide, or fragment, variant, derivative or fusion thereof will contain a catalytic domain corresponding to a N-acetylmuramoyl-L-alanine amidase.
  • the polypeptide, or fragment, variant, derivative or fusion thereof will contain a catalytic domain corresponding to the catalytic domain of the polypeptide of SEQ ID No. 1.
  • polypeptide, or fragment, variant, derivative or fusion thereof comprises the cell wall binding domain of the polypeptide of SEQ ID No. 1 . In some embodiments the polypeptide or fragment, variant, derivative or fusion thereof comprises the cell wall binding domain of SEQ ID No: 1 and a catalytic domain different to that of SEQ ID No: 1 . In some embodiments the polypeptide or fragment, variant, derivative or fusion thereof comprises the catalytic domain of SEQ ID No: 1 and a cell wall binding domain different to that of SEQ ID No: 1.
  • the term 'variant' includes reference to polypeptide insertions, deletions and/or substitutions, either conservative or non-conservative, relative to the amino acid sequence of SEQ ID NO: 1 .
  • the polypeptide may comprise an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1 , more preferably at least 70% or 80% or 90% identity to said sequence, and most preferably at least 95% or 97% identity to said amino acid sequence.
  • the above sequence identity may be over the full length of the amino acid sequence of SEQ ID NO: 1 or over a portion thereof.
  • the sequence identity is over at least 50 amino acids of the amino acid sequence of SEQ ID NO: 1 , such as over at least 100, 150, 200, 250, 265 or more amino acids therein.
  • percent identity which is typically calculated in relation to polypeptides whose sequence has been aligned optimally.
  • polypeptide of the invention may comprise one or more amino acids that are modified or derivatised.
  • polypeptide may comprise or consist of a derivative of the amino acid sequence of SEQ ID NO: 1 , or of a fragment, variant or fusion thereof.
  • polypeptide may conveniently be blocked at its N- or C- terminus so as to help reduce susceptibility to exoproteolytic digestion, e.g., by amidation.
  • the polypeptide, or fragment, variant, fusion or derivative thereof is cyclic.
  • the polypeptide, or fragment, variant, fusion or derivative thereof is linear.
  • the polypeptide may comprise or consist of a fusion of the amino acid sequence of SEQ ID NO: 1 , or of a fragment, variant or derivative thereof.
  • the term 'fusion' refers to a polypeptide which is fused to any other polypeptide.
  • the polypeptide may comprise one or more additional amino acids, inserted internally and/or at the N- and/or C-termini of the amino acid sequence of SEQ ID NO: 1 , or of a fragment, variant or derivative thereof.
  • fusion proteins of Gp20 are disclosed in US 2012/0195872 A1 , the entire contents of which are incorporated in entirety.
  • the fusion comprises one or more additional amino acids conjugated at the N- and/or C-terminus of the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or of a fragment, variant, or derivative thereof.
  • the polypeptide of the first aspect of the invention comprises a fragment of SEQ ID NO: 1 consisting of the cell wall binding domain (or a variant of such a domain sequence which retains the cell wall binding activity thereof), to which is fused an enzymatic domain from a different source.
  • the invention also provides the polypeptide of the first aspect of the invention comprising a fragment of SEQ ID NO: 1 consisting of the enzymatic domain (or a variant of such a domain sequence which retains the enzymatic activity thereof), to which is fused a cell wall binding domain from a different source.
  • polypeptide or fragment, variant, derivative or fusion thereof comprises one or more functional groups that may be protonated or deprotonated (for example at physiological pH)
  • the polypeptide or fragment, variant, derivative or fusion thereof may be prepared and/or isolated as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to the salt of a given compound, wherein the salt is suitable for administration as a pharmaceutical.
  • such salts may be formed by the reaction of an acid or a base with an amine or a carboxylic acid group respectively.
  • Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids.
  • inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.
  • organic acids include 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-toluenesulfonic acid, salicylic acid and the like.
  • Pharmaceutically acceptable base addition salts may be prepared from inorganic and organic bases.
  • Corresponding counterions derived from inorganic bases include the sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • Organic bases include primary, secondary and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N- alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine.
  • Acid/base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid/base forms.
  • the compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention.
  • solvate is a complex of variable stoichiometry formed by a solute (in this invention, a polypeptide or fragment, variant, derivative or fusion thereof of the invention) and a solvent. Such solvents should not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol or acetic acid. Methods of solvation are generally known within the art.
  • the compounds of the invention may be in the form of a "pro-drug".
  • pro-drug is used in its broadest sense and encompasses those derivatives that are converted in vivo to the peptides of the invention. Such derivatives would readily occur to those skilled in the art and include, for example, compounds where a free hydroxy group is converted into an ester derivative or a ring nitrogen atom is converted to an N-oxide.
  • ester derivatives include alkyl esters (for example acetates, lactates and glutamines), phosphate esters and those formed from amino acids (for example valine).
  • Any compoimd that is a prodrug of a polypeptide or fragment, variant, derivative or fusion thereof of the invention is within the scope and spirit of the invention.
  • Conventional procedures for the preparation of suitable prodrugs according to the invention are described in text books, such as "Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985 - the entire contents of which is incorporated herein by reference.
  • polypeptides of the invention lyse cells of Propionibacterium acnes.
  • the polypeptide is capable of lysing cells of multiple strains of Propionibacterium acnes.
  • the polypeptide may be capable of lysing one or more of the strains of Propionibacterium acnes lysed by the PA6 lysin of SEQ ID NO: 1 .
  • the polypeptides of the invention are substantially incapable of lysing bacteria which are commensal members of the microbiota of healthy skin (and not known to cause adverse effects on the host).
  • the polypeptide does not lyse cells of Propionibacterium granulosum, Propionibacterium avidum, Staphylococcus epidermis or Corynebacterium bovis.
  • the polypeptide of the invention is capable of lysing cells of Propionibacterium acnes.
  • the polypeptide may exhibit at least 10% of the lysis activity of the polypeptide of SEQ ID NO: 1 on cells of Propionibacterium acnes, for example at least 40%, 60%, 80%, 100% or more, for example such that the polypeptide may exhibit a greater lysis activity than the polypeptide of SEQ ID NO: 1 on cells of Propionibacterium acnes.
  • the polypeptide is capable of lysing cells of pathogenic bacteria selectively, i.e. to a greater extent than cells of non-pathogenic bacteria.
  • polypeptides or a fragment, variant, fusion or derivative thereof, for use in the first aspect of the invention are known and are described in more detail in the Examples section below.
  • the polypeptide, or fragment, variant, fusion or derivative thereof is or comprises a recombinant polypeptide.
  • the term "genetic construct” refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule.
  • a genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • the insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA.
  • the genetic construct may be linked to a vector.
  • vector refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell.
  • the vector may be capable of replication in at least one additional host system.
  • expression construct refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • An expression construct typically comprises in a 5' to 3' direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and c) a terminator functional in the host cell into which the construct will be transformed.
  • coding region or "open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences.
  • the coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon.
  • a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences.
  • “Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
  • noncoding region refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency. Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
  • promoter refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
  • transgene is a polynucleotide that is taken from one organism and introduced into a different organism by transformation.
  • the transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
  • An "inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,
  • Read-through transcription will produce a transcript that undergoes complementary base- pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
  • nucleic acid molecule (or polynucleotide) encoding the polypeptide, or fragment, variant, fusion or derivative thereof, may be expressed in a suitable host and the polypeptide obtained therefrom.
  • An expression vector may be constructed comprising a nucleic acid molecule which is capable, in an appropriate host, of expressing the polypeptide encoded by the nucleic acid molecule.
  • nucleic acid refers to DNA (e.g., cDNA) or RNA.
  • the nucleic acid molecule according to the invention comprises the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3, with or without a HA-Tag.
  • the invention also relates to nucleic acids that differ in sequence from SEQ ID NOs: 2 and 3, referred to herein as variants. For example, nucleic acid sequences having at least 70% sequence identity (e.g., at least 80%, 90%, 95%, 99% or 100% sequence identity) to any of the nucleic acid sequences shown in SEQ ID NOs: 2 and 3 are provided.
  • the first and second sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined.
  • the number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value.
  • the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence.
  • a single sequence can align differently with other sequences and hence, can have different percent sequence identity values over each aligned region.
  • Two sequences can be aligned to determine percent sequence identity using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389-3402), which is incorporated into BLAST (basic local alignment search tool) programs available at ncbi.nlm.nih.gov.
  • the vector comprises the nucleic acid or a variant thereof according to the invention.
  • the vector is typically an expression vector, such as pLAH.AII or pLAH.nrsB.
  • a host cell may be used as an expression platform to express the polypeptide, or fragment, variant, fusion or derivative thereof according to the invention.
  • the host cell will typically comprise the nucleic acid according to the invention.
  • the host cell is typically a cyanobacterial cell, and the expression will typically use a population of such host cells to generate sufficient polypeptide, or fragment, variant, fusion or derivative thereof to provide a therapeutic or prophylactic effect.
  • the host cell will typically be a species of cyanobacteria that can be genetically transformed using recombinant techniques. Examples of such cyanobacterial host cells include strains of Synechocystis sp.
  • Synechocystis sp. PCC 6803 Synechococcus sp. PCC7942, Synechococcus elongatus PCC7942, as well as Anabaena sp. PCC7120, Leptolyngbya sp. BL0902, Nostoc punctiforme ATCC29133, Synechococcus strains CC931 1 , Synechococcus strains CC9605, Anacystis nidulans R2, Spirulina platensis strain C1 (Arthrospira sp.
  • the cyanobacterial host cell be a strain of Synechocystis sp., such as Synechocystis PCC 6803.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide, or fragment, variant, fusion or derivative thereof as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
  • the polypeptide, or fragment, variant, fusion or derivative thereof may be used following isolation from the host cell or may be used without isolation from the host cell.
  • the host cell may undergo a treatment, such as disruption of the cell wall (for example by vortexing, the use of a cell disrupter, a French Pressure cell press or sonication), and the crude product used in the pharmaceutical composition.
  • the crude product may be used with or without subsequent purification.
  • composition is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.
  • polypeptide, or fragment, variant, fusion or derivative thereof as hereinbefore described, or pharmaceutically acceptable salt thereof may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the compound is within the scope of the invention.
  • the compound could be administered with one or more therapeutic agents in combination.
  • the combination may allow for separate, sequential or simultaneous administration of the polypeptide or fragment, variant, derivative or fusion thereof as hereinbefore described with the other active ingredient(s).
  • the combination may be provided in the form of a pharmaceutical composition.
  • the host cell used to express the polypeptide, or fragment, variant, fusion or derivative thereof may also produce one or more additional antibacterial agents.
  • the polypeptide, or fragment, variant, fusion or derivative thereof may be administered in combination with the one or more additional antibacterial agents.
  • the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the animal (preferably mammal) to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art.
  • any formulation containing the polypeptide active care should be taken to ensure that the activity of the polypeptide is not destroyed in the process and that the polypeptide is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the polypeptide by means known in the art, such as, for example, micro encapsulation.
  • the route of administration chosen should be such that the polypeptide reaches its site of action.
  • Those skilled in the art may readily determine appropriate formulations for the polypeptides of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including carotenoids such as astaxanthin, phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.
  • carotenoids such as astaxanthin
  • phenolic compounds such as BHT or vitamin E
  • reducing agents such as methionine or sulphite
  • metal chelators such as EDTA.
  • the polypeptide of the invention will be applied topically such as in a cream, lotion or gel.
  • polypeptide may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery.
  • Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.
  • compositions include oral and enteral formulations of the present invention, in which the active polypeptide may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • Liquid formulations may also be administered enterally via a stomach or oesophageal tube.
  • the present invention also extends to any other forms suitable for administration, for example compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions.
  • Pharmaceutically acceptable vehicles and/or diluents include 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 conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • therapeutically effective amount refers to that amount which is sufficient to effect treatment, as defined below, when administered to an animal, preferably a mammal, more preferably a human in need of such treatment.
  • the therapeutically effective amount will vary depending on the subject and nature of bacterial infection being treated, the severity of the infection and the manner of administration, and may be determined routinely by one of ordinary skill in the art.
  • the pharmaceutical composition may comprise an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to inhibit at least in part the growth of cells of Propionibacterium acnes in a subject who is infected or susceptible to infection with such cells.
  • the pharmaceutical formulation comprises an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to kill cells of Propionibacterium acnes in the subject.
  • polypeptide, or fragment, variant, fusion or derivative thereof is intended primarily for use in the treatment or prevention of Propionibacterium acnes infection in humans, the polypeptide, or fragment, variant, fusion or derivative thereof may also be used on other animals.
  • treatment covers any treatment of a condition or disease in an animal, preferably a mammal, more preferably a human, and includes: (i) inhibiting the bacterial infection, eg arresting its proliferation; (ii) relieving the infection, eg causing a reduction in the severity of the infection; or (iii) relieving the conditions caused by the infection, eg symptoms of the infection.
  • prevention covers the prevention or prophylaxis of a condition or disease in an animal, preferably a mammal, more preferably a human and includes preventing the bacterial infection from occurring in a subject which may be predisposed to infection but has not yet been diagnosed as being infected.
  • Synechocystis sp. PCC 6803 is a Gram-negative, fresh-water cyanobacterium that was originally isolated from a lake in California in 1968 by R. Kunisawa. It possesses coccoid, non- filamentous cells with a size of 1.5 to 2 pm. Synechocystis can grow photoautotrophically as well as mixotrophically or heterotrophically using glucose or organic acids as additional carbon sources. The cyanobacterium is also able to grow under anaerobic conditions in the presence of carbon sources. The organism is mesophilic and basophilic and achieves optimal growth at temperatures of 30°C to 34°C and at pH values between 8 and 10. The cell division of Synechocystis cells occurs by binary fission.
  • cyanobacteria are classified as Gram- negative bacteria, they have a relatively thick peptidoglycan layer.
  • Synechocystis cells are surrounded by a four layer cell envelope, which is composed of the cytoplasmic membrane, followed by the peptidoglycan cell wall and the outer membrane. The last layer is mostly formed by a S-layer or other carbohydrate structures.
  • the cyanobacterium is able to take up exogenous DNA.
  • Synechocystis carries a genome that is comprised of one circular chromosome and seven additional plasmids.
  • the main chromosome has a size of 3.5 Mb, whereas the sizes of the plasmids vary between 2.3 and 120 kb.
  • Synechocystis cell Multiple copies (approximately ten) of the genome are present in each Synechocystis cell.
  • the genome of Synechocystis has been fully sequenced and has a GC content of 47.7%.
  • the annotated genome and information about gene functions and mutants are available in the databases CyanoBase and CyanoMutants.
  • Synechocystis may be manipulated using several different promoter systems, selectable markers and transformation vectors.
  • the majority of promoters and expression elements that are used in Synechocystis are derived from strongly expressed endogenous genes encoding for proteins involved in photosynthesis and carbon fixation such as psbAII, psaA, psaD and rbcL. Nevertheless, a few foreign promoters have been shown to result in efficient expression as well, such as the inducible E. coli promoters trc10, trc20 and A1 lacO-1 .
  • antibiotic resistance genes are mainly used as selectable markers.
  • the most frequently used antibiotic resistance marker genes are nptl and nptll encoding for neomycin phosphotransferases conferring resistance against neomycin and kanamycin and the aadA gene, which confers resistances against streptomycin and spectinomycin.
  • nrsBRS promoter system kanamycin resistant
  • nrsBRS promoter system kanamycin resistant
  • nrsBRS promoter system kanamycin resistant
  • the target bacterium used for antibacterial activity studies Propionibacterium acnes ATCC 6919 DSM number: 1897, type strain, other collection numbers: NCTC 737 DSMZ - German Collection of Microorganisms and Cell Cultures
  • the Synechocystis sp. PCC 6803 strains are listed in Table 1. The strains were maintained on BG1 1 agar plates. Cultures on solid medium were grown in an illuminated incubator with a light intensity of 30 - 50 pmol/m 2 /s at 30 °C and subsequently stored under dim light (5 - 10 ⁇ / ⁇ 2 /5) at 20 °C. The Synechocystis strains were transferred to fresh plates every three to four weeks.
  • Liquid BG1 1 cultures were grown in Erienmeyer flasks (liquid medium to air ratio 1 :5 or 1 :4) in an illuminated incubator with a light intensity of 100 - 200 pmol/m 2 /s, at 120 rpm shaking and at a temperature of 25 °C. Small flasks and pre-cultures were inoculated with a loop full of cells from agar plates, whereas larger cultures of more than 50 ml were inoculated from a pre-culture with a 1 % inoculum.
  • P. acnes was grown in liquid brain heart infusion medium at 30 °C for five to seven days under anaerobic conditions.
  • P. acnes was cultured on Columbia blood agar plates or brain heart infusion medium agar plates at 30 °C under anaerobic conditions.
  • RYpsa.R B) GGATTTCTCCTTATAATAAC or pSRSapI (B) rbcL.R CAAACTTCACATGCAGCA Sequencing of atpA.F (A) C AAGT G ATCTT ACC AC TC AC ASapI (A) and psa.F (B) GTTC ACGC GT A AG C T T T C TT AATTC AA C ATTT pSRSapI (B) inserts
  • psbAILF A) CTCAGAACTATGGTAAAGGCG PCR screening and psbAII.R GGTCTGCCTC GTGAAGAAGGTGTT sequencing for the insertion of the GOI ursBseq.F (B) CCGTCTCATCTTCCACCAGC into pLAH.AII (A) and pLAH.nrsB (B)
  • DNA fragments were separated on 1 % (w/v) agarose gels made with 1x TAE buffer (40 mM Tris, 1 mM sodium EDTA, 17.5 mM glacial acetic) and 1 ⁇ g/ml ethidium bromide.
  • the samples were mixed with 6x DNA Loading Dye (Fermentas) before loading onto the gel that had been submerged in 1x TAE buffer in an electrophoresis tank. The electrophoresis was performed at 85 V for 30 to 120 min. Subsequently, the DNA fragments were visualized using a UV transilluminator (UVP Gel Documentation System).
  • UV transilluminator UV transilluminator
  • the O'GeneRulerTM 1 kb Plus DNA Ladder was used to estimate the size of the DNA fragments.
  • Plasmid isolation was performed with the GENEJet Plasmid Miniprep Kit from Fermentas or the QIAprep Spin Midiprep Kit from Qiagen according to the manufacturer's instructions for lower quantities of a plasmid (up to 20 ⁇ g).
  • the commercial QIAfilter Plasmid Midi Kit (Qiagen) was used according to the manufacturer's instructions.
  • PCR products were purified using the QIAquick PCR purification Kit from Qiagen (Hilden, Germany) and Gel extraction of DNA bands was performed using the QIAquick Gel Extraction Kit from Qiagen.
  • Restriction enzyme digests were performed using restriction endonuclease enzymes according to the manufacturer's instructions from New England Biolabs (NEB) and Fermentas (Thermo Scientific), respectively. Ligations were performed using T4 DNA ligase from NEB with 3 to 5 times more insert than vector according to the manufacturer's instructions. To avoid relegation of digested vectors in some cases the 5' phosphate was removed after the digest using the Antarctic phosphatase (NEB) according to the manufacturer's instructions. DNA sequencing
  • DNA sequencing of plasmids and PCR products was performed using the Scientific Support Services of The Wolfson Institute for Biomedical Research, University College London. The sequencing results were aligned and analysed using MacVector 12.6.0.
  • Liquid BG1 1 medium (50 ml) was inoculated with a loopful of Synechocystis cells from a freshly restreaked plate and allowed to grow in an illuminated shaking incubator at 25 °C and a light intensity of 50 - 100 ⁇ / ⁇ 2 /5 for four days.
  • the cultures were grown to an OD 7 50nm of 0.4 - 0.8 and the concentration (cells/ml) was calculated by multiplying the ODzsonm value obtained by 1.15 x 10 8 .
  • the culture was harvested by centrifugation at 3,000 x g for 10 minutes and washed in 2 ml of fresh BG1 1 medium and spun down a second time.
  • the cell pellet was resuspended in BG1 1 medium to a final concentration of 4 x 10 8 cells/ml.
  • 1 -5 ⁇ g of the vector DNA was added to 200 ⁇ of the cell suspension and incubated at 30 °C for 4-6 hours.
  • the cells were spread onto BG1 1 plates, allowed to dry, and then incubated for 2-3 days to allow expression of the introduced resistance marker. Subsequently, the plates were overlaid with 3 ml of 0.6% (w/v) agar containing kanamycin to give a final concentration of 200 ⁇ g/ml in the plates.
  • the plates were incubated for approximately two weeks in a light intensity of ⁇ 40 pmol/m 2 /s at 30 °C and were then examined for the presence of putative transformant colonies. The colonies were restreaked three times to reached homoplasmicity before the insertion of the GOI was analysed by PCR.
  • a loop full of cells from an agar plate was resuspended in 20 ⁇ of sterile ddH20, followed by the addition of 20 ⁇ ethanol (100%) and 200 ⁇ of chelex (5%, w/v).
  • the mixture was incubated at 98 °C for five minutes and centrifuged at 21 ,000 x g for two minutes at room temperature. Subsequently, 2 ⁇ of the supernatants were used in 50 ⁇ PCR reactions.
  • NCBI littp • > ww Jicbi.iiliii.iiih.gov
  • SDS-PAGE was carried out using a Protein gel tank system from Biorad holding 80 x 83 x mm gels with 10 wells.
  • the gels were prepared based on the Laemmli gel recipe stated Table 5 with a lower 15% resolving gel and an upper 3.75% stacking gel.
  • Samples were prepared in Solution A or by the addition of 4x protein gel sample loading buffer (Table 6), boiled at 99 °C for three minutes and centrifuged at 21 ,000 x g for 20 minutes before they were loaded onto the gel. The gels were run in reservoir buffer at 120V for 120 minutes.
  • the gel was stained with Coomassie staining solution (Table 7) for one hour at room temperature and destained in destaining solution for at least two hours. Subsequently, the gel was scanned using the Odyssey® imaging system from LI-COR.
  • the gel was soaked in Towbin buffer together with 8 pieces of 3MM Whatman paper and an AmershamTM HybondTM-ECL nitrocellulose membrane (GE Healthcare), all of the same size as the gel, for about 20 minutes.
  • a sandwich was assembled in the following order from the anode side: 4 pieces of 3MM Whatman paper, the nitrocellulose membrane, the gel, and another 4 pieces of Whatman paper on top. The transfer was performed at a voltage of 20 V for approximately one hour using a Trans-Blot SD semi-dry electrophoretic transfer cell from Biorad.
  • the membrane was subsequently blocked in blocking buffer under gently shaking at room temperature for one hour or at 4 °C overnight. After a quick rinse with TBS-T, the membrane was incubated with the primary antibody in blocking buffer at room temperature for 1 - 3 hours under gently shaking. Afterwards the membrane was quickly rinsed and then washed for 5 - 10 minutes under vigorous shaking with TBS-T. The washes were repeated three times, before the secondary antibody in blocking buffer was applied for one hour, followed by three more washes. In experiments where horseradish peroxidase-linked secondary (ECL) antibodies were used, the membrane was first incubated with SuperSignal® West Pico Chemiluminescence Substrate (Thermo Scientific) for five minutes at room temperature.
  • ECL horseradish peroxidase-linked secondary
  • the membrane was exposed to a sheet of Hyperfilm ECL (GE Healthcare) in the dark room.
  • the film was developed using a Xograph automatic film developer. The exposure time varied between 10 seconds and 20 minutes depending on the strength of the signal.
  • IRDye® secondary antibodies DylightTM 800
  • IR signal infrared fluorescence signal
  • the infrared fluorescent signals of the bands in the western blot analysis measured using the Odyssey® Infrared Imaging system were analysed using the Image Studio Software 3.1 .4 from LI-COR Biosciences for most analysis.
  • the Odyssey® Infrared Imaging system application software 3.0 was used. Both programs express the signal in different value dimensions.
  • the newer software expresses the signal in values of several millions, whereas the older software states values between 0.01 and 200.
  • LI-COR states that its system can be used to directly detect the amount of antigen in western blot analysis over a wide range of quantities (www.licor.com).
  • the Odyssey® Infrared Imaging system and the Image Studio Software can be therefore used to compare the amount of the protein of interest between samples of one performance. Quantifications were always performed with samples on the same gel and membrane, since gel to gel (and membrane to membrane) differences in the IR fluorescence signal can occur. When more than nine samples were compared, which exceeded the capacity of one gel, the gels were run in parallel and were blotted at the same time onto nitrocellulose membranes. All further steps were performed under the same conditions and the membranes were scanned in the Odyssey® system at the same time, which greatly minimised variations between samples on different gels. In figures that show comparisons between samples from different gels and membranes, the borders of the membranes are indicated by black boxes.
  • Protein concentrations were determined with the Bio-Rad protein assay, based on the method of Bradford, using the microassay procedure for 96-well microtitre plates.
  • the standards 25, 50, 100, 200, 300, 400, 500 pg/ml
  • samples were measured in triplicates.
  • Synechocystis cultures are grown to an OD750nm of 1 and harvested by centrifugation at 8,000 x g for 10 minutes or using a cream separator (Motor Sich, Ukraine) (for 30 litre cultures).
  • the cell pellet is resuspended in 20 mM NaPi-buffer (NahbPC , the pH is adjusted to 6.9 with NaOH) to a concentration of 100 times the culture volume.
  • protease inhibitor (Roche cOmplete, EDTA-free) is added to the cell suspension. Subsequently, the cells are broken using a French pressure cell press or a cell disrupter.
  • the broken cell suspension is centrifuged at 21 ,000 x g for five minutes (or 3,000 x g for 20 minutes) and the supernatant is recovered and is referred to as crude extract in this study.
  • crude extract is centrifuged at 100,000 x g for one hour in an ultracentrifuge.
  • the samples (soluble protein extract or combined DEAE cellulose elution fractions) are stirred on ice and grounded ammonium sulphate is slowly added to it until the desired ammonium sulphate concentration is reached. The stirring is continued for 30 minutes before the solution is centrifuged at 3,000 x g for 30 minutes. The pellet is resuspended in a smaller volume of 20 mM NaPi-buffer and used for further analyses. The supernatant is treated stepwise with higher concentrations of ammonium sulphate in the same way as described above. Purification with anti-HA (influenza haemagglutinin peptide) resin
  • HA resin 100 - 200 ⁇ is incubated with 500 - 800 ⁇ of the sample for 4 to 18 h at 4 °C on a rotating incubator. The tube is gently centrifuged 3 times to allow the resin to settle down before the flow through of the sample was removed.
  • the resin is washed five times with 1 ml NaPi-buffer.
  • the HA tagged protein is eluted from the resin by incubation with 300 - 500 ⁇ 0.1 M Glycine-HCI buffer (pH 2.5) for two minutes and immediately neutralized with 1 M Tris-HCI buffer (pH 8.0).
  • the resin is eluted by incubation with 100 pg/ml HA peptide for 10 minutes.
  • a liquid culture of the target bacterium was grown overnight and 200 ⁇ of the culture were spread onto Columbia blood agar plates or other appropriate nutrient agar plates. Subsequently, the plates were dried for five to ten minutes before 20 - 50 ⁇ of each sample were spotted onto the plates. The plates were incubated overnight regarding to the growth conditions of the target bacterium and inspected for inhibition zones on the next morning.
  • a culture of the target bacterium of interest was grown under the growth conditions described above. Subsequently, the cultures were harvested and the cells were resuspended in 20 mM NaPi-buffer.
  • the TRAs were first performed in 1 ml cuvettes and the OD was measured at 600 nm manually over a times course. Subsequently, the TRAs were performed in 96-well microtitre plates with a final assay volume of 200 ⁇ .
  • An ELx 808 microplate reader (BIO-TEK INSTRUMENTS INC.) was used to measure the OD at 595 nm automatically every two minutes over times courses of 60 to 500 minutes at 37°C.
  • the start OD595 or 600 nm values of the bacterial suspensions were 0.1 to 1 .0 depending on the assay.
  • the assays were started by the addition of the endolysin containing samples and corresponding control samples.
  • Most TRAs were performed by adding 20 ⁇ of the endolysin or control preparation to 180 ⁇ of suspension of the target bacterium.
  • Synechocystis -produced endolysin samples were prepared by breaking Synechocystis cells by vortexing in the presence of glass beads (212 - 300 pm) for two minutes followed by two minutes on ice for five cycles. Subsequently, the supernatant was separated from the cell debris by centrifugation (21 ,000 x g for five minutes) and used in the TRAs.
  • the vector pLAH.AII (Al-Haj 2014) carries an expression cassette, which is flanked with upstream and downstream flanking regions of the psbAII gene that include the 5 ' UTR and 3 ' UTR of the psbAII gene and the psbAII promoter. These elements allow the expression of the gene of interest and the insertion into the psbAII locus by homologous recombination.
  • the expression cassette of pLAH.nrsB is also flanked with upstream and downstream regions of the psbAII gene, but these elements exclude the psbAII promoter.
  • the vector pLAH.nrsB (Al-Haj 2014) lacks the psbAII promoter and carries instead the nickel inducible endogenous promoter from nrsB that is part of the nrsBACD operon.
  • the four proteins encoded by nrsBACD are involved in the resistance of Synechocystis to nickel.
  • the nrsB promoter is controlled by a two-component transduction system that is involved in nickel sensing and induces the promoter in the presence of nickel.
  • the transduction system is encoded by the genes nrsR and nrsS, which are located upstream of nrsBACD operon in the opposite orientation.
  • the expression vector pLAH.nrsB carries beside the promoter nrsB also nrsR and nrsS.
  • Gp20 is comprised of 286 amino acids and has a predicted mass of 31 ,237 Da.
  • the gp20i gene was codon optimised for the C. reinhardtii chloroplast to a Codon Adaption Index (CAI) of 0.8 with respect to the codon usage table from Nakamura et al. (Nakamura, Y., Gojobori, T. & Ikemura, T., 2000. Codon usage tabulated from international DNA sequence databases: status for the year 2000.
  • CAI Codon Adaption Index
  • gp20 genes were synthesised by GENEART (Regensburg, Germany) with a C-terminal human influenza haemagglutinin (HA) epitope tag for detection.
  • the created gp20 genes have a length of 858 bp and, including the HA-tag coding sequence and two stop codons (TAA), a length of 891 bp.
  • the HA-tagged Gp20 protein has a molecular weight of 32.3 kDa.
  • the vector pLAH.AII (Al-Haj 2014) was used for the creation of Syn6803_AII_gp20i-HA.
  • the expression cassette of pLAH.AII carries the restriction sites Ndel and BamHI for the insertion of the gene of interest.
  • the gp20i-HA gene was amplified in a PCR with the primers Syn6803gp20.F and Syn6803gp201.R, which bind at the ends of the gene and contain the restriction sites of Asel and BamHI respectively, since the gp20 gene also contains an Ndel site.
  • the PCR products were digested with Asel and BamHI followed by ligation with pLAH.AII, which was cut by Ndel and BamHI.
  • the vector pLAH.nrsB (Al-Haj 2014) was used for the creation of Syn6803_nrsB_gp20i-HA and Syn6803_nrsB_gp202-HA.
  • the expression cassette of pLAH.nrsB carries also the restriction sites Ndel and BamHI for the insertion of the gene of interest.
  • the creation of the lines was performed as described above, except that the primers Syn6803gp20.F and Syn6803gp202.R were used for the amplification of ⁇ 20 ⁇ - ⁇ .
  • the successful transformation was confirmed as described above.
  • the PCR screening confirmed once more that all analysed colonies had been successfully transformed.
  • the concentration of 6.4 ⁇ NiC was chosen in this study, because an earlier study that used the nrsB promoter for the expression of the reporter gene luxAB found that induction with 6.4 ⁇ Ni 2+ results in the highest level of bioluminescence (Peca, L. et al., 2008. Construction of bioluminescent cyanobacterial reporter strains for detection of nickel, cobalt and zinc. FEMS Microbiology Letters, 289(2), pp.258-264). The study created a whole-cell bioluminescent reporter that quantitatively responds to Ni 2+ and other metal ions and showed that increasing concentrations of Ni 2+ result in a dose dependent bioluminescence signal, but concentrations above 6.4 ⁇ resulted in a reduced bioluminescence.
  • the transgenic lines carrying the gp20 genes were analysed for HA-tagged protein in western blot analyses.
  • the strains were cultured as described above.
  • the induction of Syn6803_nrsB_gp20i-HA and Syn6803_nrsB_gp20 2 -HA was performed at an OD 75 onm of 0.8 and the cultures were harvested 5.5 hours after the start of the induction. After harvest the samples were concentrated to 100 times the culture volume.
  • Western blot analysis with anti-HA antibodies and the Odyssey® Infrared (IR) Imaging System confirmed the successful production of the Gp20 protein in Syn6803_nrsB_gp20i-HA ( Figures 6 and 7), an endolysin that was not produced to detectable levels in the C. reinhardtii chloroplast.
  • the western blot analysis suggested that the codon optimised version gp20i results in higher expression levels than the gp20 ⁇ gene.
  • the genes have a shared sequence identity of 85.7%.
  • the gp20i contains the UCA codon six times, which is less frequently used for serine in Synechocystis, whereas gp20 ⁇ contains it 13 times. This could be therefore an explanation for the lower protein levels.
  • the western blot analysis needs to be performed with a higher number of transformants and different induction times before it can be concluded that the gene sequence of gp20i results in higher protein yields compared to gp20 ⁇ .
  • the three transgenic lines were broken by vortexing with glass beads (212 - 300 pm) for 2 min followed by 2 min on ice for five cycles. Subsequently the supernatant was separated from the cell debris by centrifugation (21 ,000 x g for 5 min). The whole cell extract before centrifugation and the supernatants (crude extract) were analysed by western blot analysis for the HA-tagged endolysins. All samples were additionally treated with SDS and boiling to break leftover unbroken cells.
  • Gp20 A demonstration of the lytic activity of Synec/iocysi/ ' s-produced Gp20 would not only demonstrate that it can be produced as an active enzyme in the cyanobacterium, but would also deliver the experimental proof that Gp20 on its own has antibacterial activity against P. acnes.
  • Gp20 could be purified using either a FPLC or by developing an ion exchange column protocol.
  • sequence for a His-tag or Strep-tag could be attached to the gene and commercial available columns could be used for the purification. In this way it would be possible to separate Gp20 from the phycobilliproteins that strongly interfere with the TRA and to concentrate the protein to levels that result in detectable activity.
  • crude extract containing Gp20 or the purified enzyme could be mixed with P. acnes cells and a decrease in cfu after incubation on agar plates could be analysed.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

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Abstract

The invention relates to an expression platform for expressing an endolysin. In particular the invention relates to a cyanobacterial expression platform for expressing an endolysin with antibacterial activity, particularly against Propionibacterium acnes which is a causative bacterium for acne vulgaris. The cyanobacterial expression platform is particularly well suited for large-scale production of the endolysin, or fragment, variant, derivative or fusion thereof. Such large-scale production may involve the use of a photobioreactor (PBR). In some embodiments, the cyanobacterial expression platform comprises a strain of Synechocystis sp., such as Synechocystis PCC 6803. The invention provides polypeptide comprising the amino acid sequence of SEQ ID No: 1, or a fragment, variant, derivative or fusion thereof for use in the treatment and/or prevention of a bacterial infection, such as bacterial infection which is caused by Propionibacterium acnes.

Description

Endolysin expression platform
FIELD OF THE INVENTION
The invention relates to an expression platform for expressing an endolysin. In particular the invention relates to a cyanobacterial expression platform for expressing an endolysin with antibacterial activity, particularly against Propionibacterium acnes which is a causative bacterium for acne vulgaris.
BACKGROUND
The increase of antibiotic resistance in the last decades has severely reduced the reliability of antibiotic treatments. In particular, the prevalence and range of multi-drug resistant strains has increased alarmingly.
At the same time the development of new antibiotics has severely declined in the last decades, mainly owing to the lower profitability of antibiotics compared to other drugs. The risk of resistance development makes it important to limit the widespread use of newly-developed antibiotics to maintain their efficacy, which negatively affects the profits from that drug.
Antibiotics are still the major weapons against severe infectious diseases and it is therefore very important to preserve the efficacy of the available antibiotics, but also to develop new antibiotics with different targets and new strategies to circumvent the resistance mechanisms of the microorganisms.
The discovery and development of new antibiotics has declined severely in the last decades, and the last truly new class was discovered in the 1980s.
Over 2 billion years ago, antibiotics developed as a defence mechanism of microorganisms against competitors. At the same time mechanisms to resist the antimicrobial substances also evolved in microorganisms to keep up in the battle of competing microorganisms. Different mechanisms are known that cause resistance against antimicrobial agents. Today, 15 classes of antibiotics are known that target different physiological and metabolic functions, but resistance mechanisms have developed against every class. Beside the increasing occurrence of resistance against conventional antibiotics, there are further limitations of these drugs. Most antibiotics have a broad-spectrum against a range of bacterial classes and therefore each treatment affects not only the pathogen, but also the beneficial and protective human bacterial flora. Reoccurring exposure to antibiotics results in the spread of antimicrobial resistance genes within the commensal bacterial flora, which functions as a reservoir for resistance genes and can pass the genes on to pathogenic bacteria. It would be therefore highly desirable to develop narrow-spectrum antibiotics that specifically kill only the targeted pathogen, to reduce the collateral effects on the commensal bacterial flora and to decrease the spread of antimicrobial resistance genes.
Bacteriophages are viruses that infect bacteria and are the most abundant entities on the planet. The majority are tailed bacteriophages, comprising a tail and a head that encapsulates the DNA or RNA genome. The nucleic acids can be single or double stranded, and some bacteriophages have a circular genome, while others carry a linear genome. The tail is involved in the attachment to the bacterial cell and the injection of the nucleic acids.
Bacteriophages were first described by Felix d'Herelle in 1917. Research into the use of bacteriophages in therapy has taken place, however, the demand for such therapies strongly declined after the break-up of the Soviet Union which until that point had been a major consumer. In the 1930's, several companies in the US, France and Germany produced phage preparations as antimicrobials e.g. Eli Lilly (US). However, after the discovery and spread of antibiotics and only mixed success, phage therapy disappeared in Western countries in the 1940s.
The increase in antibiotic resistant strains and the expansion in our understanding of bacteriophage biology has now caused a rediscovery of phage therapy in western countries. With the rediscovery of phage therapy, attention was also drawn to bacteriophage-encoded endolysins. These enzymes are used by most bacteriophages to digest the peptidoglycan of the bacterial cell wall for bacteriophage progeny release at the end of the lytic cycle, and have the potential to be used on their own as antibacterial agents.
Endolysins are bacteriophage-encoded enzymes that are produced in bacterial cells after they have been infected by a bacteriophage. These proteins are also known as bacteriophage lysozymes, lysins or muralytic/mureolytic enzymes. To infect a bacterial cell, bacteriophages attach to the cell surface, often to receptors, and insert their genetic material into the cell. The bacterial DNA and protein synthesis machinery changes to produce bacteriophage particles and the host genome is degraded. At the same time, endolysins and another type of proteins, called holins, are produced and accumulate in the cytoplasm of the infected bacterium. The holin proteins help to tightly control the moment of the endolysin-mediated cells lysis. At the end of the lytic cycle and once a critical holin concentration has been reached, the hydrophobic proteins assemble into oligomers and insert into the cell membrane. The insertion of the holin proteins results in the creation of pores, which allow the endolysin to pass across the cytoplasmic membrane and enter the peptidoglycan. Subsequently, the endolysins cleave specific bonds of the peptidoglycan, which results in the disruption of the cell wall and the high internal pressure of 20 - 50 atmospheres causes the osmotic lysis and the death of the bacterial cell. The cell lysis results in the release of the phage progenies, which can now infect new bacterial cells.
Bacteriophages are highly specific to a target bacterium. In addition their endolysins are usually only effective against the species of bacteria in which they are naturally produced, or in some cases, against other species within the same genus. Endolysins that are specific for Gram- positive bacteria usually have a modular organisation. Many of these endolysins have a two- domain structure with an N-terminal catalytic domain and a C-terminal cell binding domain connected by a linker. However, there are also endolysins with different domain architectures. Despite their modular structure, nearly all known endolysins specific to Gram-positive bacteria are single polypeptides encoded by one gene.
The cell binding domains of endolysins attach the enzyme tightly to its substrate after cell lysis. Endolysins are also able to enter and cleave the peptidoglycan of Gram positive bacteria from outside of the cell (i.e. 'exolysins'). Therefore, the attachment prevents the endolysins from attacking surrounding cells of the target bacterium, which can still function as a host for the bacteriophage. In contrast, the outer membrane of Gram-negative bacteria protects uninfected cells against lysis by endolysins from outside. Endolysins that are specific to Gram negative bacteria are therefore usually smaller single-domain proteins without a cell binding domain.
Furthermore, the cell wall binding domain is responsible to a large degree for the specificity of the endolysin for certain species or genera. The domain binds noncovalently to molecules in the cell envelope, which can be part of the peptidoglycan or other cell wall associated molecules. It has been shown that the LysM domain binds to the N-acetyl-glucosamine acid of the peptidoglycan. The catalytic domain is responsible for the enzymatic cleavage of the peptidoglycan and can be an N-acetyl- -D-glucosaminidase or N-acetyl-p-D-muramidase (lysozymes), which both cleave one of the two glycosidic bonds of the carbohydrate strand. Other catalytic domains have endopeptidase activity and cleave a specific bond of the peptide chains. Furthermore, these domains can be N-acetylmuramoyl-Lalanine amidases, which hydrolyse the amide bond between the sugar strand and the peptide chain.
Catalytic domains can also have an influence on the specificity of an endolysin. The peptidoglycan structures differ between both Gram-types, but also between different bacterial species. The absence or presence of the specific target bond contributes therefore to a limitation of the host range.
Endolysins have been studied by different research groups and it has been shown for several endolysins, that when applied externally to Gram-positive bacteria, these enzymes are able to lyse and kill cells of the target bacterium. It was believed that endolysins could not be used as a treatment against Gram-negative bacteria, since the outer membrane represents a barrier for the enzymes.
Most endolysins have a high specificity for the cell wall of bacterial species in which they are naturally produced. Some endolysins are even effective only against certain serotypes or strains, whereas others lyse bacteria within the same genus. In contrast to conventional antibiotics, which usually have a broad-spectrum, endolysins could be used to specifically eliminate the pathogen without harming the commensal bacterial flora. The usage of narrow spectrum antibiotics for the treatment is highly desirable, but requires accurate and practicable diagnostic methods. With the development of better diagnostic methods, the demand for narrow-spectrum antibiotics will most likely greatly increase.
Since endolysins cleave the peptidoglycan from outside and do not need to get into the cell, several of the commonly known resistance mechanisms cannot affect the activity of endolysins. Intracellular resistance mechanisms such as antibiotic-efflux pumps, cytoplasmic enzymes that degrade or modify antimicrobials, and the reduction of membrane permeability, can be therefore excluded.
Propionibacterium acnes is a Gram-positive, anaerobic bacterium of the phylum Actinobacteria, and it is a prevalent human skin commensal that colonises mainly areas that are rich in fatty acids such as the face, scalp and upper back. P. acnes is believed to play a role in the inflammatory stages of acne vulgaris. It has been shown that antibiotic treatments that reduced the number of P. acnes cells on the skin have a therapeutic effect on the severity of the acne condition. Furthermore, patients with acne vulgaris who did not respond to an antibiotic treatment were more likely to carry P. acnes strains which were resistant to this antibiotic.
Acne vulgaris is a skin condition that affects about 80% of the population at some point in life, mainly in adolescence. It can be very painful, cause psychological problems and can result in lifelong scarring in severe cases. Furthermore, P. acnes can cause eye lid infections and postoperative as well as device-related (e.g. joint prostheses) infections, especially in immunocompromised patients. Acne vulgaris is treated with topical or oral antibiotics, but shows high levels of resistance against the prescribed antibiotics. Alternatively, chemical treatments with substances such as benzyl peroxide are used, which can have strong side effects.
Especially, for the treatment of skin infections in the face and upper body the use of a narrow- spectrum antibiotic would be desirable to reduce the risk of opportunistic infections by fungi or bacteria.
SUMMARY OF THE INVENTION
If endolysins are to be used as antibacterial agents then a suitable expression platform must be found that can be used for efficient large-scale commercial production, but this platform also needs to fulfil all safety criteria for the production of therapeutic proteins. Complicating this search, however, is that the production cost for recombinant proteins is generally high. It would be therefore desirable to use a system with low costs for the production of recombinant endolysins in order to compete, cost-wise, with small molecule drugs.
In recent decades microalgae, which includes eukaryotic as well as prokaryotic, photosynthetic microorganisms and includes cyanobacteria (traditionally called blue-green algae), have started to get progressively more attention as production platforms for biological products. Primarily, the ability of photosynthetic organisms to utilise sunlight, inorganic nutrients and CO2 for the production of biological products makes these organisms appealing. In this respect, microalgae are believed to offer several advantages for the production of recombinant proteins, including the use of simple and inexpensive nutrients. Several microalgae and plants are classified as GRAS organisms (Generally Regarded as Safe), which means these organisms are safe for human consumption. Furthermore, these microalgae and plants are free of endotoxins and viral or prion contaminants, which is important especially for the production of therapeutic proteins.
There are other advantages provided by microalgae. First, if suitable conditions are found, then microalgae can be grown in full containment under sterile conditions in photobioreactors (PBRs), which prevents the release of transgenes to the environment. Growth in PBRs reduces the risk of environmental contamination of the production system and therefore the contamination of the therapeutic protein.
The production of recombinant therapeutic protein in the Chlamydomonas reinhardtii chloroplast has been known for over a decade and several studies since then have demonstrated the production of a variety of proteins such as antibodies, hormones and vaccines. The use of C. reinhardtii is particularly well-suited for production of recombinant proteins in PBRs, and several investigations have already contributed to an improvement of the expression levels, including studies that investigated different promoters and UTRs as well as codon optimisation strategies.
Despite this knowledge, the successful expression of a number of endolysins (full-length Pal, Cpl-1 and φ1 1 ) in other expression platforms (C. reinhardtii chloroplast and E .coli, variously), it has been previously discovered (Taunt, H.N., 2013. The Synthesis of Novel Antibacterial Proteins in the C. reinhardtii Chloroplast; PhD thesis; London: University College London) that the Propionibacterium acnes specific endolysin Gp20 was not able to be expressed in C. reinhardtii chloroplast to detectable levels.
The expression of Gp20 was even attempted with two differently codon-optimised versions of the gp20 gene and the expression of both proteins was attempted with two different promoters, and through translational fusions of these non-expressing endolysins to expressed proteins. In all efforts made, the endolysin did not accumulate to detectable levels in the C. reinhardtii chloroplast.
It has now been found that a particular polypeptide, or fragment, variant, derivative or fusion thereof, which is believed to be effective in the treatment and/or prevention of infections of Propionibacterium acnes, may be formed using recombinant techniques. In particular the recombinant techniques make use of a cyanobacterial expression platform. The cyanobacterial expression platform is particularly well suited for large-scale production of the polypeptide, or fragment, variant, derivative or fusion thereof. Such large-scale production may involve the use of a photobioreactor (PBR).
In one aspect the invention provides a cyanobacterial expression platform capable of producing a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof. In some embodiments, the cyanobacterial expression platform comprises a strain of Synechocystis sp., such as Synechocystis PCC 6803.
In one aspect the invention provides the use of a cyanobacterial expression platform to produce a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
In one aspect the invention provides an isolated nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
In one aspect the invention provides a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
In one aspect the invention provides a host cell comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof or a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
In one aspect the invention provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof comprising culturing a population of cyanobacterial host cells comprising:
a) a nucleic acid molecule encoding the polypeptide comprising the amino acid
sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof; or b) a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof under conditions in which the polypeptide is expressed.
In one aspect the invention provides a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof produced by a process comprising the step of expressing the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof in a population of cyanobacterial host cells.
In one aspect the invention provides a pharmaceutical composition comprising the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
In one aspect the invention provides the use of the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof in the
manufacture of a medicament for the treatment and/or prevention of a bacterial infection. In some embodiments the bacterial infection is caused by Propionibacterium acnes.
In one aspect the invention provides a method of treating and/or preventing a bacterial infection comprising administering the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof to the subject in need thereof. In some embodiments the bacterial infection is caused by Propionibacterium acnes.
In one aspect the invention provides polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof for use in the treatment and/or prevention of a bacterial infection. In some embodiments the bacterial infection is caused by Propionibacterium acnes.
In some embodiments the treatment and/or prevention may be to enhance the cosmetic appearance of the subject.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 Strains of Synechocystis sp. PCC 6803 expressing the endolysin genes under the control of the constitutive promoter of the psbAII gene and the nickel-inducible promoter nrsB as well as the corresponding wild type strain. The diagram shows the Synechocystis sp. PCC 6803 strains that were created to study the expression of gp20 in the cyanobacterium. The wild type strain was used as the recipient strain and as a negative control. Expression elements are indicated in dark grey and genes in light grey and the Tn903 kanamycin resistance cassette (KmR) in white, UE = upstream element, DE = downstream element;
Figure 2 Codon usage tables from Nakamura et al. (2000) for Synechocystis sp PCC 6803. The tables show in the first row the codon triplet, in the second the frequency of the codon per thousand codons and in brackets the number of times the codon was found in the analysed coding sequences (CDS). In Synechocystis sp. PCC 6803 3623 CDS with 1 ,161 ,949 codons were analysed. Codons with a frequency of 5.0 and below stop codons are highlighted in grey. The tables were taken from httpi //www .kazusa.or.jp/codon;
Figure 3 Schematic diagram showing the binding sites of the used sets of primers and the expected fragment sizes for the different transformants. The primer psbAII.F binds to the 5'UTR of psbAII, whereas psbAII.R binds just outside of the insertion site of the gene of interest. Together these primers result in fragments sizes of 0.3 kb + the size of the gene of interest (kb) for Syn6803_AII_x transformants and 1 .1 kb kb + the size of the gene of interest (kb) for Syn6803_nrsB_x transformants. Primers are shown as grey arrows, expression elements are indicated in dark grey and genes in white, Tn903 kanamycin resistance cassette (KmR) in white, UE = upstream element, DE = downstream element;
Figure 4 Western blot analysis of whole cell extracts of Syn6803_nrsB_gp20i and Syn6803_nrsB_gp202 transformants showing the presence or absence of HA-tagged Gp20 protein. The gene expression in Syn6803_nrsB_gp20i and Syn6803_nrsB_gp202 was induced with 6.4 μΜ of NiC . The western blot was performed with whole cell extract (= suspension of whole cells that were broken by the addition of SDS (2% w/v) and boiling) and anti-HA antibodies, IRDye® secondary antibodies and the Odyssey® Infrared Imaging System for detection (A) and quantification (B). Synechocystis sp. PCC 6803 wild type (WT 6803) was used as a negative control. The expected band size for Gp20 is 32.3 kDa. Protein sizes were determined using the PageRulerTM prestained protein ladder (Thermo Scientific). The black box indicates that all samples were analysed on the same gel and membrane and the white line indicates that lanes were cut out between the samples for the preparation of the figure.
Figure 5 Western blot analysis of whole cell extracts of Syn6803_nrsB_pal-HA,
Syn6803_nrsB_<|)1 1 -HA and Syn6803_nrsB_gp20i showing the relative amounts of Pal, φ1 1 and Gp20 protein. The gene expression was induced with 6.4 μΜ of NiCb for 5.5 hours, harvested and concentrated to 100x the culture volume. The western blot analysis was performed with whole cell extract (= suspension of whole cells that were broken by the addition of SDS (2% w/v) and boiling) and anti-HA antibodies, IRDye® secondary antibodies and the Odyssey® Infrared Imaging System for detection (A) and quantification (B).
SEQ ID No. 1 :
MVRYIPAAHH SAGSNNPVNR VVIHATCPDV GFPSASRKGR AVSTANYFAS PSSGGSAHYV 60
CDIGETVQCL SESTIGWHAP PNPHSLGIEI CADGGSHASF RVPGHAYTRE QWLDPQVWPA 120
VERAAVLCRR LCDKYNVPKR KLSAADLKAG RRGVCGHVDV TDAWHQSDHD DPGPWFPWDK 180
FMAVVNGGSG DSGELTVADV KALHDQIKQL SAQLTGSVNK LHHDVGVVQV QNGDLGKRVD 240
ALSWVKNPVT GKLWRTKDAL WSVWYYVLEC RSRLDRLESA VNDLKK 286
Figure imgf000011_0001
ATGGTTCGTT ATATTCCAGC TGCTCATCAC TCAGCAGGTT CTAACAATCC TGTTAATCGT 60
GTAGTAATTC ATGCAACATG TCCAGATGTA GGATTTCCTA GTGCTAGCCG TAAAGGTCGT 120
GCTGTTTCAA CTGCTAATTA CTTCGCATCA CCATCTAGTG GTGGTAGCGC TCACTATGTG 180
TGTGACATTG GAGAAACAGT ACAATGTTTA TCTGAAAGTA CTATTGGATG GCACGCACCA 240
CCAAACCCTC ATTCATTAGG TATTGAAATT TGTGCTGATG GTGGTTCTCA TGCTAGCTTT 300 CGTGTGCCTG GTCACGCATA TACACGTGAA CAATGGTTAG ATCCACAAGT TTGGCCAGCT 360
GTTGAACGTG CTGCAGTATT ATGTCGTCGT TTATGTGACA AATACAATGT ACCTAAACGT 420
AAATTATCAG CTGCTGACTT AAAAGCAGGT CGTCGTGGTG TTTGTGGTCA TGTTGATGTA 480 ACTGACGCTT GGCACCAAAG CGATCATGAT GATCCAGGAC CATGGTTTCC TTGGGACAAA 540 TTCATGGCTG TTGTAAACGG TGGTAGCGGT GACAGCGGAG AATTAACAGT TGCAGATGTA 600 AAAGCTTTAC ACGATCAAAT TAAACAATTA TCAGCACAAT TAACTGGTAG CGTGAACAAA 660
TTACATCACG ATGTTGGTGT TGTACAAGTG CAAAACGGTG ACTTAGGAAA ACGTGTAGAC 720
GCTTTAAGCT GGGTGAAAAA TCCAGTGACT GGAAAATTAT GGCGTACAAA AGATGCTTTA 780
TGGTCTGTTT GGTATTATGT ATTAGAATGT CGTAGCCGTT TAGATCGTTT AGAAAGCGCT 840
GTAAATGACT TAAAAAAA 858
SEQ ID No. 3: qp202
ATGGTTCGTT ATATTCCAGC TGCTCACCAC TCTGCTGGTT CAAACAACCC AGTTAACCGT 60
GTAGTTATTC ACGCTACTTG TCCAGATGTT GGTTTCCCTT CTGCTTCACG TAAAGGTCGT 120
GCTGTTTCTA CTGCTAACTA CTTTGCTTCT CCATCTTCTG GTGGTTCAGC TCACTACGTA 180
TGTGACATCG GTGAAACAGT ACAATGTTTA TCAGAATCAA CTATTGGTTG GCACGCTCCA 240
CCAAACCCAC ACTCTTTAGG TATTGAAATT TGTGCTGATG GTGGTTCACA CGCTTCTTTC 300
CGTGTTCCAG GTCACGCTTA CACACGTGAA CAATGGTTAG ATCCACAAGT TTGGCCTGCT 360
GTTGAACGTG CTGCTGTTTT ATGTCGTCGT TTATGTGACA AATACAACGT TCCAAAACGT 420
AAATTATCAG CTGCTGATTT AAAAGCTGGT CGTCGTGGTG TTTGTGGTCA CGTAGACGTA 480 ACTGATGCTT GGCACCAATC TGATCACGAC GACCCAGGTC CATGGTTCCC TTGGGATAAA 540
TTCATGGCTG TTGTAAACGG TGGTTCAGGT GATTCTGGTG AATTAACAGT AGCTGATGTT 600
AAAGCTTTAC ACGACCAAAT TAAACAATTA TCAGCTCAAT TAACAGGTTC AGTAAACAAA 660
TTACACCACG ACGTAGGTGT TGTACAAGTA CAAAACGGTG ATTTAGGTAA ACGTGTAGAT 720 GCTTTATCTT GGGTTAAAAA CCCAGTTACA GGTAAATTAT GGCGTACTAA AGATGCTTTA 780
TGGTCAGTAT GGTACTACGT TTTAGAATGT CGTTCACGTT TAGACCGTTT AGAATCAGCT 840 GTAAACGATT TAAAAAAA 858
DETAILED DESCRIPTION OF THE INVENTION
Cyanobacteria as a production platform for endolysins
Cyanobacteria have minimal nutrient requirements and are capable of photoautotrophic, mixotrophic or heterotrophic growth, and their cultivation is inexpensive. Furthermore, several species of cyanobacteria have GRAS status. Species such as Arthrospira plantesis and Arthrospira maxima (better known as Spirulina) as well as Nostoc flagelliforme and Nostoc commune are even used as food supplements for vitamins, minerals and β-carotene.
Furthermore, several studies identified antimicrobial compounds in cyanobacteria. For example, Kawaguchipeptin B, an antibacterial cyclic undecapeptide has been isolated from the cyanobacterium Microcystis aeruginosa. In a separate study, two highly halogenated aromatic compounds, Ambigol A and B, have been isolated from Fischerella ambigua. It is therefore conceivable that the use of crude cyanobacterial extracts that contain endolysins may also provide potentially additive antibacterial properties.
Cyanobacteria are classified as Gram-negative bacteria, but their peptidoglycan is thicker and has a higher degree of cross-linking than that of most Gram-negative bacteria. However, the peptide chains that crosslink the polysaccharide strands have the typical Gram-negative composition and differ therefore from the peptidoglycan of S. pneumoniae, S. aureus and other Gram-positive bacteria. These cell walls lack for example the glycine bridge, which contains the bond that is cleaved by the endopeptidase domain of other endolysins. Furthermore, the cytoplasmic membrane would present a barrier, in the absence of holins that prevents the endolysins from reaching the peptidoglycan.
Cyanobacteria have been seldom considered as production platforms for recombinant proteins despite the fact that genetic engineering tools for the expression of foreign genes have been developed for several species. In most cases the introduction of foreign genes is used for metabolic engineering and the creation of novel biosynthetic pathways. For example, Lan & Liao (Lan, E.I. & Liao, J.C., 201 1. Metabolic engineering of cyanobacteria for 1 -butanol production from carbon dioxide. Metabolic Engineering, 13(4), pp.353-363) expressed a transenoyl-CoA reductase gene for the production of butanol in Synechococcus elongatus PCC 7942, whereas Guerrero et al. (Guerrero, F. et al., 2012. Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803. PLoS ONE, 7(1 1 ), p.e50470.) produced an ethylene-forming enzyme (EFE) in Synechocystis sp. PCC 6803 for the production of ethylene. Furthermore, applications in which the cyanobacterium can be used as the production and delivering system have been investigated. A study by (Zang et al. 2007) produced Paralichthys olivaceus (flounder) growth hormone (fGH) in Synechocystis sp. PCC 6803. The group also investigated the effect of supplementing the feed of turbot (Scophthalmus maximus L) with the transgenic Synechocystis cells producing fGH and found that it can be used as an efficient growth promoter without any observed side effects (Liu, S. et al., 2007. Effect of growth hormone transgenic Synechocystis on growth, feed efficiency, muscle composition, haematology and histology of turbot (Scophthalmus maximus L); Aquaculture Research, 38(12), pp.1283-1292.). These results indicated therefore that Synechocystis sp. PCC 6803 does not contain major amounts of endotoxins that harm turbot following oral administration. It may be possible to use the crude extract of Synechocystis sp. PCC 6803 on humans.
It has been shown that Synechocystis species have antibacterial activity against Gram-positive bacteria (Martins, R.F. et al., 2008. Antimicrobial and Cytotoxic Assessment of Marine Cyanobacteria - Synechocystis and Synechococcus. Marine Drugs, 6(1 ), pp.1-1 1 .)
The Propionibacterium acnes-specific endolysin Gp20 from bacteriophage PA6
Farrar et al. (Farrar, M.D. et al., 2007. Genome Sequence and Analysis of a Propionibacterium acnes Bacteriophage. Journal of Bacteriology, 189(1 1 ), pp.4161-4167.) were the first to sequence the genome of a bacteriophage that infects Propionibacterium acnes, a bacterium that is associated with inflammatory acnes vulgaris. The bacteriophage PA6 has a high genetically and morphologically similarity to several mycobacteriophages. Like Dp-1 and φ1 1 , PA6 belongs to the family of the Siphoviridae. The genome of PA6 consists of double-stranded DNA and has a length of 29.7 kb. Farrar et al. (2007) showed that the bacteriophage could infect 32 different isolates of P. acnes, including clinical isolates with antibiotic resistances to erythromycin and clindamycin. Advantageously, PA6 showed a high specificity for P. acnes and did not infect other bacteria that are common commensals in the human skin microflora such as Propionibacterium granulosum, Propionibacterium avidum, Staphylococcus epidermis or Corynebacterium bovis.
Farrar et al. (2007) identified 48 Open Reading Frames (ORFs) in the genome of PA6 and predicted that ORF20 encodes a putative N-acetylmuramoyl-L-alanine amidase and defined the encoded protein as Gp20. Gp20 was found to have homology to the amidases of the mycobacteriophages PG1 and Che8 and a high similarity (67% identity for amino acids 2 to 145, N-terminal catalytic domain) to another N-acetyl-muramoyl-L-alanine amidase, which is an autolysin of P. acnes. Furthermore, the downstream ORF21 is predicted to encode the holin of the lytic system. The gene arrangement is typical for endolysin and holin genes in bacteriophage genomes.
The study by Taunt (2013) confirmed using sequence alignments and database analysis, the prediction that ORF20 encodes an amidase at the N-terminus and therefore the endolysin of PA6. At the time Taunt started the investigation of the production of Gp20 in Chlamydomonas reinhardtii, PA6 was the only P. acnes bacteriophage that had been sequenced so far. Since then, several further P. acnes specific bacteriophages have been sequenced and described and further putative endolysins have been identified, but none of the studies has experimentally confirmed that the encoded proteins are able to cleave peptidoglycan. Marinelli et al. (Marinelli, L.J. et al., 2012, Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates; mBio, 3(5), pp.e00279-12) found that the genome sequence of all 1 1 bacteriophages they analysed exhibit a remarkably high nucleotide identity of more than 85%. Furthermore, the study found that ORF20 encodes the endolysin in all analysed P. acnes bacteriophages and that the N-terminal domains of these endolysins are predicted to be N-acetyl-muramoyl-L-alanine amidases. The N-terminal domain and also the C-terminal domain, that is predicted to be responsible for cell binding, are highly conserved within different P. acnes bacteriophage endolysins and have sequence identities of 92 to 96%. Gp20 from phage PA6 can be therefore seen as a representative example for P. acnes bacteriophage endolysins in general.
As used herein the term "amino acid" refers to any natural or unnatural amino acid. Such amino acids may be in the 'L' form or 'D' form, and may further include omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (such as α,α-disubstituted amino acids and N-alkyl amino acids) and chemically derivatised amino acids. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, so long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
As used herein the term "isolated" refers to where the polypeptide or fragment, variant, derivative or fusion thereof of the invention, specifically the wildtype endolysin of bacteriophage PA6, is provided in a form other than that in which it may be found naturally.
In some embodiments, the amino acid sequence of SEQ ID No: 1 , or fragment, variant, derivative or fusion thereof is capable of binding specifically to and/or lysing cells of Propionibacterium acnes.
As used herein the expression "capable of binding specifically to cells of Propionibacterium acnes" means that the entity is capable of binding preferentially to cells of Propionibacterium acnes.
As used herein the expression "capable of lysing cells of Propionibacterium acnes" means that the entity retains (at least in part) the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells, such as cells of Propionibacterium acnes.
The polypeptide, or fragment, variant, derivative or fusion thereof, need not retain all of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells. Rather, it is simply necessary for said polypeptide, fragment, variant, derivative or fusion to retain at least 10% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells. Preferably, however, the polypeptide, fragment, variant, derivative or fusion exhibits at least 30%, for example at least 50%, 70%, 90%, 100%, 150%, 200% or more, of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells.
The fragment may comprise or consist of at least 50 contiguous amino acids of SEQ ID NO: 1 , for example at least 100, 150, 200, 250 or 265 contiguous amino acids of SEQ ID NO: 1.
Many bacteriophage endolysins consist of two distinct domains - a catalytic (enzymatic) domain that is responsible for cell wall degradation, and a cell wall binding domain that recognises a cell surface motif and permits attachment of the endolysin to that target cell. The catalytic (enzymatic) domain can be identified by its amino acid homology to other similar regions of lytic enzymes that share the same type of lytic activity. In the present invention, the catalytic domain may correspond to a N-acetylmuramoyl-L-alanine amidase. Preferably, the polypeptide, or fragment, variant, derivative or fusion thereof has homology to the amidases of the mycobacteriophages PG1 and Che8 and a high similarity (such as, for example, 67% identity for amino acids 2 to 145, N-terminal catalytic domain) to a N-acetyl-muramoyl-L-alanine amidase which is an autolysin of P. acnes as taught in Farrar et al (2007). Preferably the polypeptide, or fragment, variant, derivative or fusion thereof will contain a catalytic domain corresponding to a N-acetylmuramoyl-L-alanine amidase. Preferably the polypeptide, or fragment, variant, derivative or fusion thereof will contain a catalytic domain corresponding to the catalytic domain of the polypeptide of SEQ ID No. 1.
In some embodiments the polypeptide, or fragment, variant, derivative or fusion thereof comprises the cell wall binding domain of the polypeptide of SEQ ID No. 1 . In some embodiments the polypeptide or fragment, variant, derivative or fusion thereof comprises the cell wall binding domain of SEQ ID No: 1 and a catalytic domain different to that of SEQ ID No: 1 . In some embodiments the polypeptide or fragment, variant, derivative or fusion thereof comprises the catalytic domain of SEQ ID No: 1 and a cell wall binding domain different to that of SEQ ID No: 1.
As used herein the term 'variant' includes reference to polypeptide insertions, deletions and/or substitutions, either conservative or non-conservative, relative to the amino acid sequence of SEQ ID NO: 1 .
For example, the polypeptide may comprise an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1 , more preferably at least 70% or 80% or 90% identity to said sequence, and most preferably at least 95% or 97% identity to said amino acid sequence. The above sequence identity may be over the full length of the amino acid sequence of SEQ ID NO: 1 or over a portion thereof. Preferably the sequence identity is over at least 50 amino acids of the amino acid sequence of SEQ ID NO: 1 , such as over at least 100, 150, 200, 250, 265 or more amino acids therein. There are a number of techniques available to the person skilled in the art to determine percent identity, which is typically calculated in relation to polypeptides whose sequence has been aligned optimally.
It will be appreciated by skilled persons that the polypeptide of the invention, or fragment, variant or fusion thereof, may comprise one or more amino acids that are modified or derivatised. Thus, the polypeptide may comprise or consist of a derivative of the amino acid sequence of SEQ ID NO: 1 , or of a fragment, variant or fusion thereof.
It will be appreciated that the polypeptide may conveniently be blocked at its N- or C- terminus so as to help reduce susceptibility to exoproteolytic digestion, e.g., by amidation. Thus, in one embodiment the polypeptide, or fragment, variant, fusion or derivative thereof, is cyclic. However, in a preferred embodiment, the polypeptide, or fragment, variant, fusion or derivative thereof, is linear.
The polypeptide may comprise or consist of a fusion of the amino acid sequence of SEQ ID NO: 1 , or of a fragment, variant or derivative thereof.
As used herein the term 'fusion', with respect to a polypeptide, refers to a polypeptide which is fused to any other polypeptide. For example, the polypeptide may comprise one or more additional amino acids, inserted internally and/or at the N- and/or C-termini of the amino acid sequence of SEQ ID NO: 1 , or of a fragment, variant or derivative thereof. Examples of fusion proteins of Gp20 are disclosed in US 2012/0195872 A1 , the entire contents of which are incorporated in entirety. In some embodiments the fusion comprises one or more additional amino acids conjugated at the N- and/or C-terminus of the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or of a fragment, variant, or derivative thereof.
Thus, as described above, in one embodiment the polypeptide of the first aspect of the invention comprises a fragment of SEQ ID NO: 1 consisting of the cell wall binding domain (or a variant of such a domain sequence which retains the cell wall binding activity thereof), to which is fused an enzymatic domain from a different source. The invention also provides the polypeptide of the first aspect of the invention comprising a fragment of SEQ ID NO: 1 consisting of the enzymatic domain (or a variant of such a domain sequence which retains the enzymatic activity thereof), to which is fused a cell wall binding domain from a different source.
Where the polypeptide or fragment, variant, derivative or fusion thereof comprises one or more functional groups that may be protonated or deprotonated (for example at physiological pH) the polypeptide or fragment, variant, derivative or fusion thereof may be prepared and/or isolated as a pharmaceutically acceptable salt. It will be appreciated that the polypeptide or fragment, variant, derivative or fusion thereof may be zwitterionic at a given pH. As used herein the expression "pharmaceutically acceptable salt" refers to the salt of a given compound, wherein the salt is suitable for administration as a pharmaceutical. For example, such salts may be formed by the reaction of an acid or a base with an amine or a carboxylic acid group respectively.
Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of organic acids include 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-toluenesulfonic acid, salicylic acid and the like.
Pharmaceutically acceptable base addition salts may be prepared from inorganic and organic bases. Corresponding counterions derived from inorganic bases include the sodium, potassium, lithium, ammonium, calcium and magnesium salts. Organic bases include primary, secondary and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N- alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine.
Acid/base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid/base forms.
The compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. The term "solvate" is a complex of variable stoichiometry formed by a solute (in this invention, a polypeptide or fragment, variant, derivative or fusion thereof of the invention) and a solvent. Such solvents should not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol or acetic acid. Methods of solvation are generally known within the art.
The compounds of the invention may be in the form of a "pro-drug". The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the peptides of the invention. Such derivatives would readily occur to those skilled in the art and include, for example, compounds where a free hydroxy group is converted into an ester derivative or a ring nitrogen atom is converted to an N-oxide. Examples of ester derivatives include alkyl esters (for example acetates, lactates and glutamines), phosphate esters and those formed from amino acids (for example valine). Any compoimd that is a prodrug of a polypeptide or fragment, variant, derivative or fusion thereof of the invention is within the scope and spirit of the invention. Conventional procedures for the preparation of suitable prodrugs according to the invention are described in text books, such as "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985 - the entire contents of which is incorporated herein by reference.
In one aspect the polypeptides of the invention lyse cells of Propionibacterium acnes. Preferably, the polypeptide is capable of lysing cells of multiple strains of Propionibacterium acnes. For example, the polypeptide may be capable of lysing one or more of the strains of Propionibacterium acnes lysed by the PA6 lysin of SEQ ID NO: 1 .
In one embodiment, the polypeptides of the invention are substantially incapable of lysing bacteria which are commensal members of the microbiota of healthy skin (and not known to cause adverse effects on the host). For example, it is advantageous if the polypeptide does not lyse cells of Propionibacterium granulosum, Propionibacterium avidum, Staphylococcus epidermis or Corynebacterium bovis. Most preferably, the polypeptide of the invention is capable of lysing cells of Propionibacterium acnes. For example, the polypeptide may exhibit at least 10% of the lysis activity of the polypeptide of SEQ ID NO: 1 on cells of Propionibacterium acnes, for example at least 40%, 60%, 80%, 100% or more, for example such that the polypeptide may exhibit a greater lysis activity than the polypeptide of SEQ ID NO: 1 on cells of Propionibacterium acnes.
Advantageously, the polypeptide is capable of lysing cells of pathogenic bacteria selectively, i.e. to a greater extent than cells of non-pathogenic bacteria.
Methods for the production of polypeptides, or a fragment, variant, fusion or derivative thereof, for use in the first aspect of the invention are known and are described in more detail in the Examples section below. Conveniently, the polypeptide, or fragment, variant, fusion or derivative thereof, is or comprises a recombinant polypeptide.
The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system.
The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5' to 3' direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and c) a terminator functional in the host cell into which the construct will be transformed.
The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences.
"Operably-linked" means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency. Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
The term "promoter" refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
A "transgene" is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
An "inverted repeat" is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,
(5')GATCTA TAGATC(3')
(3')CTAGAT ATCTAG(5')
Read-through transcription will produce a transcript that undergoes complementary base- pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
Thus, a nucleic acid molecule (or polynucleotide) encoding the polypeptide, or fragment, variant, fusion or derivative thereof, may be expressed in a suitable host and the polypeptide obtained therefrom. An expression vector may be constructed comprising a nucleic acid molecule which is capable, in an appropriate host, of expressing the polypeptide encoded by the nucleic acid molecule.
As used herein, unless the context requires otherwise, the term "nucleic acid" refers to DNA (e.g., cDNA) or RNA. In some embodiments the nucleic acid molecule according to the invention comprises the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3, with or without a HA-Tag. The invention also relates to nucleic acids that differ in sequence from SEQ ID NOs: 2 and 3, referred to herein as variants. For example, nucleic acid sequences having at least 70% sequence identity (e.g., at least 80%, 90%, 95%, 99% or 100% sequence identity) to any of the nucleic acid sequences shown in SEQ ID NOs: 2 and 3 are provided.
To calculate the percent sequence identity of two sequences, the first and second sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align differently with other sequences and hence, can have different percent sequence identity values over each aligned region. Two sequences can be aligned to determine percent sequence identity using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389-3402), which is incorporated into BLAST (basic local alignment search tool) programs available at ncbi.nlm.nih.gov.
In some embodiments the vector comprises the nucleic acid or a variant thereof according to the invention. The vector is typically an expression vector, such as pLAH.AII or pLAH.nrsB.
It will be understood that the invention is predicated, in part, on the discovery that a host cell may be used as an expression platform to express the polypeptide, or fragment, variant, fusion or derivative thereof according to the invention. In this respect the host cell will typically comprise the nucleic acid according to the invention. The host cell is typically a cyanobacterial cell, and the expression will typically use a population of such host cells to generate sufficient polypeptide, or fragment, variant, fusion or derivative thereof to provide a therapeutic or prophylactic effect. For example, the host cell will typically be a species of cyanobacteria that can be genetically transformed using recombinant techniques. Examples of such cyanobacterial host cells include strains of Synechocystis sp. such as Synechocystis sp. PCC 6803, Synechococcus sp. PCC7942, Synechococcus elongatus PCC7942, as well as Anabaena sp. PCC7120, Leptolyngbya sp. BL0902, Nostoc punctiforme ATCC29133, Synechococcus strains CC931 1 , Synechococcus strains CC9605, Anacystis nidulans R2, Spirulina platensis strain C1 (Arthrospira sp. PCC9438), Fischerella muscicola PCC 7414, Chlorogloeopsis fritschii PCC 6912 and Thermosynechococcus elongatus BP-1 , as well as Nostoc flagelliforme and Nostoc commune. Preferably the cyanobacterial host cell be a strain of Synechocystis sp., such as Synechocystis PCC 6803.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide, or fragment, variant, fusion or derivative thereof as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent. The polypeptide, or fragment, variant, fusion or derivative thereof may be used following isolation from the host cell or may be used without isolation from the host cell. The host cell may undergo a treatment, such as disruption of the cell wall (for example by vortexing, the use of a cell disrupter, a French Pressure cell press or sonication), and the crude product used in the pharmaceutical composition. The crude product may be used with or without subsequent purification.
The term "composition" is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.
While the polypeptide, or fragment, variant, fusion or derivative thereof as hereinbefore described, or pharmaceutically acceptable salt thereof, may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the compound is within the scope of the invention. For example, the compound could be administered with one or more therapeutic agents in combination. The combination may allow for separate, sequential or simultaneous administration of the polypeptide or fragment, variant, derivative or fusion thereof as hereinbefore described with the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition. In some embodiments, the host cell used to express the polypeptide, or fragment, variant, fusion or derivative thereof may also produce one or more additional antibacterial agents. In such embodiments, the polypeptide, or fragment, variant, fusion or derivative thereof may be administered in combination with the one or more additional antibacterial agents.
As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the animal (preferably mammal) to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the polypeptide active care should be taken to ensure that the activity of the polypeptide is not destroyed in the process and that the polypeptide is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the polypeptide by means known in the art, such as, for example, micro encapsulation. Similarly the route of administration chosen should be such that the polypeptide reaches its site of action.
Those skilled in the art may readily determine appropriate formulations for the polypeptides of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including carotenoids such as astaxanthin, phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.
Preferably, the polypeptide of the invention will be applied topically such as in a cream, lotion or gel.
Other dosage forms are envisaged. For example the polypeptide may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery. Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.
Other pharmaceutical forms include oral and enteral formulations of the present invention, in which the active polypeptide may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. Liquid formulations may also be administered enterally via a stomach or oesophageal tube.
The present invention also extends to any other forms suitable for administration, for example compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions.
Pharmaceutically acceptable vehicles and/or diluents include 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 conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The term "therapeutically effective amount" refers to that amount which is sufficient to effect treatment, as defined below, when administered to an animal, preferably a mammal, more preferably a human in need of such treatment. The therapeutically effective amount will vary depending on the subject and nature of bacterial infection being treated, the severity of the infection and the manner of administration, and may be determined routinely by one of ordinary skill in the art.
The pharmaceutical composition may comprise an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to inhibit at least in part the growth of cells of Propionibacterium acnes in a subject who is infected or susceptible to infection with such cells. Preferably, the pharmaceutical formulation comprises an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to kill cells of Propionibacterium acnes in the subject.
While the polypeptide, or fragment, variant, fusion or derivative thereof is intended primarily for use in the treatment or prevention of Propionibacterium acnes infection in humans, the polypeptide, or fragment, variant, fusion or derivative thereof may also be used on other animals.
The terms "treatment" and "treating" as used herein cover any treatment of a condition or disease in an animal, preferably a mammal, more preferably a human, and includes: (i) inhibiting the bacterial infection, eg arresting its proliferation; (ii) relieving the infection, eg causing a reduction in the severity of the infection; or (iii) relieving the conditions caused by the infection, eg symptoms of the infection. The terms "prevention" and preventing" as used herein cover the prevention or prophylaxis of a condition or disease in an animal, preferably a mammal, more preferably a human and includes preventing the bacterial infection from occurring in a subject which may be predisposed to infection but has not yet been diagnosed as being infected.
It should be noted that various changes and modifications to the presently preferred
embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.
Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof.
Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.
EXAMPLES
Synechocystis sp. PCC 6803 is a Gram-negative, fresh-water cyanobacterium that was originally isolated from a lake in California in 1968 by R. Kunisawa. It possesses coccoid, non- filamentous cells with a size of 1.5 to 2 pm. Synechocystis can grow photoautotrophically as well as mixotrophically or heterotrophically using glucose or organic acids as additional carbon sources. The cyanobacterium is also able to grow under anaerobic conditions in the presence of carbon sources. The organism is mesophilic and basophilic and achieves optimal growth at temperatures of 30°C to 34°C and at pH values between 8 and 10. The cell division of Synechocystis cells occurs by binary fission. Although cyanobacteria are classified as Gram- negative bacteria, they have a relatively thick peptidoglycan layer. Synechocystis cells are surrounded by a four layer cell envelope, which is composed of the cytoplasmic membrane, followed by the peptidoglycan cell wall and the outer membrane. The last layer is mostly formed by a S-layer or other carbohydrate structures. The cyanobacterium is able to take up exogenous DNA. Synechocystis carries a genome that is comprised of one circular chromosome and seven additional plasmids. The main chromosome has a size of 3.5 Mb, whereas the sizes of the plasmids vary between 2.3 and 120 kb. Multiple copies (approximately ten) of the genome are present in each Synechocystis cell. The genome of Synechocystis has been fully sequenced and has a GC content of 47.7%. The annotated genome and information about gene functions and mutants are available in the databases CyanoBase and CyanoMutants.
Synechocystis may be manipulated using several different promoter systems, selectable markers and transformation vectors. The majority of promoters and expression elements that are used in Synechocystis are derived from strongly expressed endogenous genes encoding for proteins involved in photosynthesis and carbon fixation such as psbAII, psaA, psaD and rbcL. Nevertheless, a few foreign promoters have been shown to result in efficient expression as well, such as the inducible E. coli promoters trc10, trc20 and A1 lacO-1 .
For the selection of Synechocystis transformants, antibiotic resistance genes are mainly used as selectable markers. The most frequently used antibiotic resistance marker genes are nptl and nptll encoding for neomycin phosphotransferases conferring resistance against neomycin and kanamycin and the aadA gene, which confers resistances against streptomycin and spectinomycin.
Table 1 Synechocystis sp. PCC 6803 strains used or created
Strain Genotype/phenotype Reference
Synechocystis sp. PCC 6803 Wild type, large, glucose-tolerant Williams (1988)
Syn6803_nrs5 _pal-HA Carrying pal with HA-tag sequence under nrsBRS This study
promoter system, kanamycin resistant
Syn6803_nrsB ll-HA Carrying φΐΐ with HA-tag sequence under psbAII This study
promoter, kanamycin resistant
Syn6S03_AII ll-HA Carrying φΐΐ with HA-tag sequence under This study
nrsBRS promoter system, kanamycin resistant
Syn6803_nrsB_gp20, Carrying gp20/ with HA-tag sequence under This study
nrsBRS promoter system, kanamycin resistant
Syn6803 _AII_gp201-HA Carrying gp20j with HA-tag sequence under This study
psbAII promoter, kanamycin resistant
Syn6803_nrsB_gp202-HA Carrying gp202 with HA-tag sequence under This study
nrsBRS promoter system, kanamycin resistant
[Williams, J., 1988. Construction of Specific Mutations in Photosystem II Photosynthetic Reaction Center by Genetic Engineering Methods in Synechocystis 6803. In Methods in Enzymology. Molecular Genetics. Academic Press.]
The target bacterium used for antibacterial activity studies: Propionibacterium acnes ATCC 6919 DSM number: 1897, type strain, other collection numbers: NCTC 737 DSMZ - German Collection of Microorganisms and Cell Cultures The medium for the cultivation of Synechocystis sp. PCC 6803: BG1 1 (Blue Green Medium) (Castenholz 1988) - to make 1 L of 1x BG1 1 : Combine: 100x BG1 1 10 ml, trace elements 1 ml and iron stock 1 ml; autoclave and allow to cool; then add: phosphate stock 1 ml, Na2CC>3 stock 1 ml, NaHCOs stock 10 ml and TES buffer 10 ml.
Culture conditions for Synechocystis sp. PCC 6803
The Synechocystis sp. PCC 6803 strains are listed in Table 1. The strains were maintained on BG1 1 agar plates. Cultures on solid medium were grown in an illuminated incubator with a light intensity of 30 - 50 pmol/m2/s at 30 °C and subsequently stored under dim light (5 - 10 μιτιοΙ/ιτι2/5) at 20 °C. The Synechocystis strains were transferred to fresh plates every three to four weeks. Liquid BG1 1 cultures were grown in Erienmeyer flasks (liquid medium to air ratio 1 :5 or 1 :4) in an illuminated incubator with a light intensity of 100 - 200 pmol/m2/s, at 120 rpm shaking and at a temperature of 25 °C. Small flasks and pre-cultures were inoculated with a loop full of cells from agar plates, whereas larger cultures of more than 50 ml were inoculated from a pre-culture with a 1 % inoculum.
Cutlure conditions for Propionibacterium acnes
P. acnes was grown in liquid brain heart infusion medium at 30 °C for five to seven days under anaerobic conditions. Alternatively, P. acnes was cultured on Columbia blood agar plates or brain heart infusion medium agar plates at 30 °C under anaerobic conditions.
Growth measurements for Synechocystis sp. PCC 6803
Growth of Synechocystis was measured by recording the optical density at 750 nm with a Unicam UV/Vis Spectrometer (Thermo Electron Corporation, USA). Samples with an OD750nm over 0.8 were diluted with the adequate medium.
Polymerase chain reaction (PCR)
In vitro gene amplification was carried out by the polymerase chain reaction using the Phusion polymerase (Fermentas or Thermo Scientific). The components and protocol of a 50 μΙ PCR reaction are shown in Table 2: Table 2 Components and protocol used for PCR reactions
Components Concentration
HF buffer 1 X
Template DNA 50 - 100 llg
Primers 10 pmol each
dNTPs 1 inM (250 μΜ each)
Pliusioii polymerase 0.6 ϋ
ddH20 Acid to a final volume of 50 ΐ
Step Teinpei atui e Time
Initial deiiaturatioii 98°C 2 mill
Dena turation 98°C 10 sec
25 - 30 x Aimealiiig Tm + 3°C of the lower primer Tm 30 sec
Elongation ~ 1 liiin'kb
Final elongation 72°C 10 inin
Oligonucleotide primers
The primers used for polymerase chain reactions in this study are shown in Table 3. All primers were synthesised by Eurofins MWG (Ebersberg, Germany).
Table 3 Oligonucleotide primers used in this study
Primer name Sequence (5' to 3') Fimcfioii
FLA K1 GTC ATTGCG AAA AT AC TG PC R screening for l bcL.Fn C GG AT GT AAC TC A ATCGG TAG successful
Transformation of C, rbcL.R CAAACTTCACATGCAGCA
reinhardtii with the atpA.R (A) ACGTCCACAGGCGTCGT
vectors pASapI (A)
RYpsa.R (B) GGATTTCTCCTTATAATAAC or pSRSapI (B) rbcL.R CAAACTTCACATGCAGCA Sequencing of atpA.F (A) C AAGT G ATCTT ACC AC TC AC ASapI (A) and psa.F (B) GTTC ACGC GT A AG C T T T C TT AATTC AA C ATTT pSRSapI (B) inserts
Pal.F ATC TGCTCT T C T AT GGGTGTT GATATTG
ψπ.ι ACGTGCTCTTCTATGCAAGCTAAATTAAC Attachment of a His-
T GTTGC ATGC TT ATT AATGATGATGAT GA TG AT tag to pa} and 411
Hista .R
G AGC GTAATC TGGAAC
Removal of the HA-
ATGCATGCTTAAACTTTAGATGTAATTAAACC
HAreinovaLR tag together with
ATCAGGTTC
Pai.Fw
psbAILF (A) CTCAGAACTATGGTAAAGGCG PCR screening and psbAII.R GGTCTGCCTC GTGAAGAAGGTGTT sequencing for the insertion of the GOI ursBseq.F (B) CCGTCTCATCTTCCACCAGC into pLAH.AII (A) and pLAH.nrsB (B)
AGCG ATT AATGC AAG C T AAAT T AAC AAAAAAC Insertion of ΦΠ into
Syn6803 >l l .F
GAATTTATTG the vectors
TGATGGATCCTTATTAAGCGTAGTCTGGAACA pLAH.AII and
SyiH,.SIl3<:> l l .R
TCG pLAHiii'sB
Syii6803pal,F ATGCCATATGGGTGTTGATATTGAAAAAGGTG Insertion of pa! into
The vectors
TGATGGATCCTTATTAAGCGTAATCTGGAACA
Syn6803paI.R pLAH.AII and
TCG
pLAH.nrsB
Syii6803gp20.F GCATATTAATGGTTCGTTATATTCCAGCTGC
Inseitiou of gf 20 into GCATGGATCCTTATTAAGCATAGTCAGGTACA
Syii6803gp201.R The vectors
TCATA
pLAH.AII and
GCATGGATCCTTATTAAGCGTAATCTGGAACA
Syn6803«p20;.R pLAH rirsB
TCG Agarose gel electrophoresis
DNA fragments were separated on 1 % (w/v) agarose gels made with 1x TAE buffer (40 mM Tris, 1 mM sodium EDTA, 17.5 mM glacial acetic) and 1 μg/ml ethidium bromide. The samples were mixed with 6x DNA Loading Dye (Fermentas) before loading onto the gel that had been submerged in 1x TAE buffer in an electrophoresis tank. The electrophoresis was performed at 85 V for 30 to 120 min. Subsequently, the DNA fragments were visualized using a UV transilluminator (UVP Gel Documentation System). The O'GeneRuler™ 1 kb Plus DNA Ladder (Fermentas) was used to estimate the size of the DNA fragments.
Plasmid isolation
Plasmid isolation was performed with the GENEJet Plasmid Miniprep Kit from Fermentas or the QIAprep Spin Midiprep Kit from Qiagen according to the manufacturer's instructions for lower quantities of a plasmid (up to 20 μg). For the isolation of larger quantities (up to 200 pg) of plasmids the commercial QIAfilter Plasmid Midi Kit (Qiagen) was used according to the manufacturer's instructions.
DNA purification and gel extraction
PCR products were purified using the QIAquick PCR purification Kit from Qiagen (Hilden, Germany) and Gel extraction of DNA bands was performed using the QIAquick Gel Extraction Kit from Qiagen.
Restriction enzyme digests, dephosphorylation and ligations
Restriction enzyme digests were performed using restriction endonuclease enzymes according to the manufacturer's instructions from New England Biolabs (NEB) and Fermentas (Thermo Scientific), respectively. Ligations were performed using T4 DNA ligase from NEB with 3 to 5 times more insert than vector according to the manufacturer's instructions. To avoid relegation of digested vectors in some cases the 5' phosphate was removed after the digest using the Antarctic phosphatase (NEB) according to the manufacturer's instructions. DNA sequencing
DNA sequencing of plasmids and PCR products was performed using the Scientific Support Services of The Wolfson Institute for Biomedical Research, University College London. The sequencing results were aligned and analysed using MacVector 12.6.0.
Transformation of Synechocystis sp. PCC 6803
The transformation method used was adapted from Williams (Williams, J., 1988. Construction of Specific Mutations in Photosystem II Photosynthetic Reaction Center by Genetic Engineering Methods in Synechocystis 6803. In Methods in Enzymology; Molecular Genetics; Academic Press) by Al-Haj (Al-Haj, L.A., 2014. Development of genetic engineering tools for the cyanobacterium Synechocystis PCC 6803 for advanced biofuel production; PhD thesis; London: University College London). Liquid BG1 1 medium (50 ml) was inoculated with a loopful of Synechocystis cells from a freshly restreaked plate and allowed to grow in an illuminated shaking incubator at 25 °C and a light intensity of 50 - 100 μιτιοΙ/ιτι2/5 for four days. The cultures were grown to an OD750nm of 0.4 - 0.8 and the concentration (cells/ml) was calculated by multiplying the ODzsonm value obtained by 1.15 x 108. Subsequently, the culture was harvested by centrifugation at 3,000 x g for 10 minutes and washed in 2 ml of fresh BG1 1 medium and spun down a second time. The cell pellet was resuspended in BG1 1 medium to a final concentration of 4 x 108 cells/ml. In duplicates, 1 -5 μg of the vector DNA was added to 200 μΙ of the cell suspension and incubated at 30 °C for 4-6 hours. The cells were spread onto BG1 1 plates, allowed to dry, and then incubated for 2-3 days to allow expression of the introduced resistance marker. Subsequently, the plates were overlaid with 3 ml of 0.6% (w/v) agar containing kanamycin to give a final concentration of 200 μg/ml in the plates. The plates were incubated for approximately two weeks in a light intensity of ~40 pmol/m2/s at 30 °C and were then examined for the presence of putative transformant colonies. The colonies were restreaked three times to reached homoplasmicity before the insertion of the GOI was analysed by PCR.
Isolation of DNA from Synechocystis sp. PCC 6803
To confirm that the gene of interest had been inserted into Synechocystis sp. PCC 6803 or to sequence the transgenic lines, DNA was extracted using the chelex-based method adapted from (Werner & Mergenhagen 1998). A loop full of cells from an agar plate was resuspended in 20 μΙ of sterile ddH20, followed by the addition of 20 μΙ ethanol (100%) and 200 μΙ of chelex (5%, w/v). The mixture was incubated at 98 °C for five minutes and centrifuged at 21 ,000 x g for two minutes at room temperature. Subsequently, 2 μΙ of the supernatants were used in 50 μΙ PCR reactions.
Bioinformatics
For the alignments of DNA and protein sequences, virtual restriction enzyme digests, reverse complementation of DNA sequences and translation of DNA sequences the software MacVector 12.6.0 was used. Furthermore, the free online molecular analysis tools and databases that were used in this study is shown in Table 4.
Table 4: The free online molecular analysis tools and databases used in this study
Protein and D A sequence databases
UiiiProt B./Swiss-Prat littp: ' www.imiproT.org
NCBI littp: •> ww Jicbi.iiliii.iiih.gov
C. reinhardrii chloroplast genome hop: '/www .chlainy.org/elil oro/def a u 11. html
Bioiufoi maticv tools
Reverse complementation littp: ywmv.bioMoraMtics.org/siiis/revjcoim>.ht
Primer design littp: 'Vmv\v.bioinfonnatics.org/siiis2
Restriction digest littp ://tools.iieb . com<'NEBciitter2/
Conversion tool littp: "mw.attotroo.com/cybeiTory/aiialysisitrans.litiii
Sequence alignment littp: 'www.ebi . ac ,ak/Tools/msa/c is†a]w2/
Protein and nucleotide BLAST littp: '/blast Jicbi.nlm.iiih. gov/Bla st. cgi
Endolysiu database
pliiBIOTICS littp: ' www.phibiotics , org
Codon usage database
Nakamura et al. 2000 littp: •'/wwwJcazusa. or.jp/codon/
Culture conditions of microorganisms
DSMZ littp: 7mvw.dsmz.de
Protein analysis
Sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE)
SDS-PAGE was carried out using a Protein gel tank system from Biorad holding 80 x 83 x mm gels with 10 wells. The gels were prepared based on the Laemmli gel recipe stated Table 5 with a lower 15% resolving gel and an upper 3.75% stacking gel. Samples were prepared in Solution A or by the addition of 4x protein gel sample loading buffer (Table 6), boiled at 99 °C for three minutes and centrifuged at 21 ,000 x g for 20 minutes before they were loaded onto the gel. The gels were run in reservoir buffer at 120V for 120 minutes. To visualize all proteins in the gel or to check if the western blotting was successful the gel was stained with Coomassie staining solution (Table 7) for one hour at room temperature and destained in destaining solution for at least two hours. Subsequently, the gel was scanned using the Odyssey® imaging system from LI-COR.
Table 5: Laemmli gel recipe
15% Resolving gel (40 ml) Volume in 40 ml
A ylamide bisa ylaiuide (Sigma)
40% stock, acrylamide:bisacrylaiiiide = 37: 1 15 nil
Resolving buffer (8x stock)
0.25M Tris, 1.92M glycine. 1 % SDS. pH8.3 5 ml
I 0¾ SDS 0.4 ml
Distilled H:0 18 ml
10% ammonium persulphate (AMPS) 1.5 ml
Tetraiiiethylethyienediaiiiiiie (TEMED) 15 μΐ
3.75% Stacking gel (20 ml) Volume in 20 ml
Aciylaniide/bisaeiylaiiiide (Sigma)
1.88 nil
40% stock. aciylaiuide:bisaciylaiiiide = 37: 1
Stacking buffer (4x stock)
5 ml 0.5M Tiis-HCl (pH 6.8)
10% SDS 0.2 ml
Distilled H2C) 12 ml
10% aimiioniiini persulphate (AMPS) 1.5 ml
Tetranietliylethylenediaiiiiue (TEMED) 15 μΐ
Reservoir buffer (10 x)
Tris 0.25 M
Glycine 1.92 M
SDS I %
The pH was adjusted to 8.3. Table 6: protein gel sample loading buffer
Protein gel sample loading buffer (4x)
Tns-HCl pH 6.8 50 niM
SDS %
Glycerol 10 % β- ercaptoethanol i %
EDTA (Ethylenediaininetetraacetic acid) 12, 5 niM
Bronioplieiiol blue 0.02 %
Solution A
Tris-HCi pH 8.3 0.8 M
Sorbitol 0.2 M β-Mercaptoetlianol 1
SDS 1
Table 7: Coomassie staining solution
Coomassie Blue R staining solution
Coomassie Brilliant Blue R niM
Methanol 50 % (v/ )
Acetic acid 10 % (v/v)
Destainiug solution
Methanol 40 % (v v)
Acetic acid 10 % (V 'V) Western blot analysis (semi-dry)
After electrophoresis, the gel was soaked in Towbin buffer together with 8 pieces of 3MM Whatman paper and an AmershamTM HybondTM-ECL nitrocellulose membrane (GE Healthcare), all of the same size as the gel, for about 20 minutes. For the transfer a sandwich was assembled in the following order from the anode side: 4 pieces of 3MM Whatman paper, the nitrocellulose membrane, the gel, and another 4 pieces of Whatman paper on top. The transfer was performed at a voltage of 20 V for approximately one hour using a Trans-Blot SD semi-dry electrophoretic transfer cell from Biorad.
Table 8: Buffers and solutions used in western blot analysis
Towbin buffer
Tri.8 25 niM
Glycine 192 niM
Methanol 20 % (v/v)
TBS-T
TBS buffer
Tris base 20 niM
NaCl 137 mM
HCl H 7.4 1 M
Tween-20 0.1 %
Blocking buffer
TBS-T
Skimnied milk 0.5 - 5 Immuno-detection
The membrane was subsequently blocked in blocking buffer under gently shaking at room temperature for one hour or at 4 °C overnight. After a quick rinse with TBS-T, the membrane was incubated with the primary antibody in blocking buffer at room temperature for 1 - 3 hours under gently shaking. Afterwards the membrane was quickly rinsed and then washed for 5 - 10 minutes under vigorous shaking with TBS-T. The washes were repeated three times, before the secondary antibody in blocking buffer was applied for one hour, followed by three more washes. In experiments where horseradish peroxidase-linked secondary (ECL) antibodies were used, the membrane was first incubated with SuperSignal® West Pico Chemiluminescence Substrate (Thermo Scientific) for five minutes at room temperature. Subsequently, the membrane was exposed to a sheet of Hyperfilm ECL (GE Healthcare) in the dark room. The film was developed using a Xograph automatic film developer. The exposure time varied between 10 seconds and 20 minutes depending on the strength of the signal. For quantitative western blot analysis IRDye® secondary antibodies (DylightTM 800) were used and the infrared fluorescence signal (IR signal) was excited at 785 nm and detected using the Odyssey® Infrared Imaging system (Li-COR Biosciences).
Quantification of western blot analysis
The infrared fluorescent signals of the bands in the western blot analysis measured using the Odyssey® Infrared Imaging system were analysed using the Image Studio Software 3.1 .4 from LI-COR Biosciences for most analysis. For a few earlier analysis shown in this study the Odyssey® Infrared Imaging system application software 3.0 was used. Both programs express the signal in different value dimensions. The newer software expresses the signal in values of several millions, whereas the older software states values between 0.01 and 200.
To quantify the signal using the Image Studio Software, rectangles were drawn around each band to define the area for quantification. The program automatically subtracts a background value from the signal, which is determined as the median of the signal measured in an area of 2 pixels above and below the rectangle. LI-COR states that its system can be used to directly detect the amount of antigen in western blot analysis over a wide range of quantities (www.licor.com).
The Odyssey® Infrared Imaging system and the Image Studio Software can be therefore used to compare the amount of the protein of interest between samples of one performance. Quantifications were always performed with samples on the same gel and membrane, since gel to gel (and membrane to membrane) differences in the IR fluorescence signal can occur. When more than nine samples were compared, which exceeded the capacity of one gel, the gels were run in parallel and were blotted at the same time onto nitrocellulose membranes. All further steps were performed under the same conditions and the membranes were scanned in the Odyssey® system at the same time, which greatly minimised variations between samples on different gels. In figures that show comparisons between samples from different gels and membranes, the borders of the membranes are indicated by black boxes.
Antibodies
Table 9: Antibodies used in this study
Antibodies Source Dilution
Primary antibodies
Anti-HA (polyclonal, produced in rabbit) Sigma-Aldricli 1 2.000 (Western blot)
1 : 750 (ELISA)
Anti-Pal (polyclonal, produced in rabbit) Enrogentec 1 :2,000 (Western blot)
1 : 100 to 1 : 12,500 (ELISA)
Anti-D 1 (polyclonal, produced in rabbit) Gift from P. Nixon. 1 2,000 (Western blot)
Imperial College
London, UK
Anti-rbcL (polyclonal, produced in rabbit) Gift from J. Gray. 1 : 20,000 (Western blot)
University of
Cambridge. UK
Secondary antibodies
I t 1 anti-rabbit IgG, horseradish GE Healthcare 1 : 10.000 (Western) peroxidase-linked species- specific whole 1 : 1.000 (Anti-HA ELISA) antibody (from donkey) 1 :5.000 (Anti-Pal ELISA)
Anti-rabbit IgG (H&L). Dylight™ 800 Thermo Scientific 1 :20.000 conjugated (from goat) Protein concentration assay
Protein concentrations were determined with the Bio-Rad protein assay, based on the method of Bradford, using the microassay procedure for 96-well microtitre plates. The standards (25, 50, 100, 200, 300, 400, 500 pg/ml) and samples were measured in triplicates. The assay was performed by mixing 160 μΙ of each standard or sample with 40 μΙ of Bio-Rad protein assay dye reagent concentrate, followed by incubation at room temperature for 10 minutes. Subsequently, the absorbance was measured at 595 nm. Linear regression analysis of the results provided a function of: y = 0.0005573x with R2 = 0.9918414.
Protein purification methods
Preparation of crude extract and soluble protein extract
Synechocystis cultures are grown to an OD750nm of 1 and harvested by centrifugation at 8,000 x g for 10 minutes or using a cream separator (Motor Sich, Ukraine) (for 30 litre cultures). The cell pellet is resuspended in 20 mM NaPi-buffer (NahbPC , the pH is adjusted to 6.9 with NaOH) to a concentration of 100 times the culture volume. In some cases protease inhibitor (Roche cOmplete, EDTA-free) is added to the cell suspension. Subsequently, the cells are broken using a French pressure cell press or a cell disrupter. The broken cell suspension is centrifuged at 21 ,000 x g for five minutes (or 3,000 x g for 20 minutes) and the supernatant is recovered and is referred to as crude extract in this study. To remove further cell debris, membranes and non-soluble proteins and gain a soluble protein extract the crude extract is centrifuged at 100,000 x g for one hour in an ultracentrifuge.
Ammonium sulphate precipitation
The samples (soluble protein extract or combined DEAE cellulose elution fractions) are stirred on ice and grounded ammonium sulphate is slowly added to it until the desired ammonium sulphate concentration is reached. The stirring is continued for 30 minutes before the solution is centrifuged at 3,000 x g for 30 minutes. The pellet is resuspended in a smaller volume of 20 mM NaPi-buffer and used for further analyses. The supernatant is treated stepwise with higher concentrations of ammonium sulphate in the same way as described above. Purification with anti-HA (influenza haemagglutinin peptide) resin
HA resin (100 - 200 μΙ) is incubated with 500 - 800 μΙ of the sample for 4 to 18 h at 4 °C on a rotating incubator. The tube is gently centrifuged 3 times to allow the resin to settle down before the flow through of the sample was removed.
Subsequently, the resin is washed five times with 1 ml NaPi-buffer. The HA tagged protein is eluted from the resin by incubation with 300 - 500 μΙ 0.1 M Glycine-HCI buffer (pH 2.5) for two minutes and immediately neutralized with 1 M Tris-HCI buffer (pH 8.0). Alternatively, the resin is eluted by incubation with 100 pg/ml HA peptide for 10 minutes.
Antibacterial activity assays
Spot tests
A liquid culture of the target bacterium was grown overnight and 200 μΙ of the culture were spread onto Columbia blood agar plates or other appropriate nutrient agar plates. Subsequently, the plates were dried for five to ten minutes before 20 - 50 μΙ of each sample were spotted onto the plates. The plates were incubated overnight regarding to the growth conditions of the target bacterium and inspected for inhibition zones on the next morning.
Counting colony forming units (cfu)
Cells from a liquid culture of the target bacterium were mixed with endolysin containing samples and control samples (crude extracts or purified endolysins) and incubated for 10 to 60 minutes. Subsequently, the mixture was diluted in serial 10 fold dilution steps before 20 μΙ of the dilutions were spotted on appropriate nutrient agar plates and incubated overnight regarding to the growth conditions of the target bacterium. On the next morning the colonies in each spot were counted and the colony forming units per ml of cell suspension were calculated. Furthermore, the differences in cfu between the endolysin treated and the control samples were calculated.
Alternatively, samples that had been first analysed in a TRA to follow the lysis of the target bacterium were used for the cfu assay. As soon as the decrease in OD750nm in the presence of the endolysin had plateaued in the TRA, the mixtures of bacterial suspension and endolysin containing sample as well as the control assays were diluted and spotted on the agar plates as described above. Turbidity reduction assays (TRAs)
A culture of the target bacterium of interest was grown under the growth conditions described above. Subsequently, the cultures were harvested and the cells were resuspended in 20 mM NaPi-buffer. The TRAs were first performed in 1 ml cuvettes and the OD was measured at 600 nm manually over a times course. Subsequently, the TRAs were performed in 96-well microtitre plates with a final assay volume of 200 μΙ. An ELx 808 microplate reader (BIO-TEK INSTRUMENTS INC.) was used to measure the OD at 595 nm automatically every two minutes over times courses of 60 to 500 minutes at 37°C. The start OD595 or 600 nm values of the bacterial suspensions were 0.1 to 1 .0 depending on the assay. The assays were started by the addition of the endolysin containing samples and corresponding control samples. Most TRAs were performed by adding 20 μΙ of the endolysin or control preparation to 180 μΙ of suspension of the target bacterium.
Endolysin preparations used in the TRAs
Synechocystis -produced endolysin samples were prepared by breaking Synechocystis cells by vortexing in the presence of glass beads (212 - 300 pm) for two minutes followed by two minutes on ice for five cycles. Subsequently, the supernatant was separated from the cell debris by centrifugation (21 ,000 x g for five minutes) and used in the TRAs.
Synthesis of bacteriophage endolysins in the cyanobacterium Synechocystis sp. PCC 6803
Creation of transgenic lines of Synechocystis sp. PCC 6803 for the synthesis of bacteriophage endolysins
Six different transgenic lines of Synechocystis sp. PCC 6803 carrying a synthetic endolysin gene in their genome were created to study the synthesis of endolysins in the cyanobacterium. The lines differ in regard to the promoters used for the expression of the genes and the endolysin genes they are carrying (Figures 1 shows the lines for the expression of gp20).
All lines were created using one of the two Synechocystis transformation vectors pLAH.AII and the pLAH.nrsB (Al-Haj 2014) and the Synechocystis sp. PCC 6803 wild type strain as recipient strain. The vectors carrying a Tn903 kanamycin resistance cassette and successful transformants can be therefore selected for kanamycin resistance. Both vectors insert the genes of interest into the locus of the psbAII gene. The psbAII encodes together with psbAI and psbAIII the D1 subunit of the photosystem II. It has been shown that a knockout of psbAII does not have an adverse effect on the growth of Synechocystis.
Promoters and 573' untranslated regions (UTR)
The vector pLAH.AII (Al-Haj 2014) carries an expression cassette, which is flanked with upstream and downstream flanking regions of the psbAII gene that include the 5'UTR and 3'UTR of the psbAII gene and the psbAII promoter. These elements allow the expression of the gene of interest and the insertion into the psbAII locus by homologous recombination. The expression cassette of pLAH.nrsB is also flanked with upstream and downstream regions of the psbAII gene, but these elements exclude the psbAII promoter. The vector pLAH.nrsB (Al-Haj 2014) lacks the psbAII promoter and carries instead the nickel inducible endogenous promoter from nrsB that is part of the nrsBACD operon. The four proteins encoded by nrsBACD are involved in the resistance of Synechocystis to nickel.
The nrsB promoter is controlled by a two-component transduction system that is involved in nickel sensing and induces the promoter in the presence of nickel. The transduction system is encoded by the genes nrsR and nrsS, which are located upstream of nrsBACD operon in the opposite orientation. The expression vector pLAH.nrsB carries beside the promoter nrsB also nrsR and nrsS.
Design and codon-optimisation of the synthetic endolysin genes
All codons are used in Synechocystis and the cyanobacterium shows less bias for particular codons for each amino acid.
The sequence of the Gp20 protein is available in the database UniProtKB/Swiss-Prot (Entry: A4K487; Entry name: A4K487_9CAUD; Protein name: Gp20, EC=3.5.1 .28; Organism: Propionibacterium phage PA6 (Taxonomic identifier: 376758)) and is replicated in SEQ ID No. 1 herein.
Gp20 is comprised of 286 amino acids and has a predicted mass of 31 ,237 Da. The gp20i gene was codon optimised for the C. reinhardtii chloroplast to a Codon Adaption Index (CAI) of 0.8 with respect to the codon usage table from Nakamura et al. (Nakamura, Y., Gojobori, T. & Ikemura, T., 2000. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research, 28(1 ), pp.292-292.) (http://www.kazusa.or.jp/codon), whereas the gp20∑ gene was designed using the CUO Moptomiser subroutine, which was developed by Henry Taunt and Khai Kong Jien. This program codon-optimises genes for the C. reinhardtii chloroplast not only in respect to the codon usage, but also takes into account codon-pairing and the usage of codons found in a subset of highly expressing C. reinhardtii chloroplast genes. The DNA sequences of the gp20i and gp20∑ gene are shown in SEQ ID No. 1 and SEQ ID No. 2 respectively. The UCA codon appears six times in gp20i and 13 times in gp20∑. The codon usage table for Synechocystis sp PCC 6803 is shown in Figure 3.
Both gp20 genes were synthesised by GENEART (Regensburg, Germany) with a C-terminal human influenza haemagglutinin (HA) epitope tag for detection. The created gp20 genes have a length of 858 bp and, including the HA-tag coding sequence and two stop codons (TAA), a length of 891 bp. The HA-tagged Gp20 protein has a molecular weight of 32.3 kDa.
Creation of the transgenic lines
Figure imgf000046_0001
The vector pLAH.AII (Al-Haj 2014) was used for the creation of Syn6803_AII_gp20i-HA. The expression cassette of pLAH.AII carries the restriction sites Ndel and BamHI for the insertion of the gene of interest. The gp20i-HA gene was amplified in a PCR with the primers Syn6803gp20.F and Syn6803gp201.R, which bind at the ends of the gene and contain the restriction sites of Asel and BamHI respectively, since the gp20 gene also contains an Ndel site. The PCR products were digested with Asel and BamHI followed by ligation with pLAH.AII, which was cut by Ndel and BamHI. Subsequently, the construct was transformed into Synechocystis sp. PCC 6803. The colonies obtained were restreaked two times to achieve homoplasmicity. The correct insertion of the genes into the genome of Synechocystis was confirmed by PCR analysis with the primers psbAII.F and psbAII.R (Figure 4) and by sequencing. The PCR screening showed that all analysed colonies had been successfully transformed.
Syn6803_nrsB_gp20i-HA and Syn6803_nrsB_gp202-HA
The vector pLAH.nrsB (Al-Haj 2014) was used for the creation of Syn6803_nrsB_gp20i-HA and Syn6803_nrsB_gp202-HA. The expression cassette of pLAH.nrsB carries also the restriction sites Ndel and BamHI for the insertion of the gene of interest. The creation of the lines was performed as described above, except that the primers Syn6803gp20.F and Syn6803gp202.R were used for the amplification of ςρ20∑-ΗΑ. The successful transformation was confirmed as described above. The PCR screening confirmed once more that all analysed colonies had been successfully transformed.
Confirmation of the production of the endolysins in Synechocystis sp. PCC 6803
After the successful creation of the transgenic Synechocystis sp. PCC 6803 lines carrying the gp20 genes under the control of two different promoters, the next stage was to analyse whether the endolysins were accumulating to detectable levels in the cyanobacterium, particularly since the gene product did not accumulate to detectable levels in the C. reinhardtii chloroplast (Taunt 2013).
During early trials into the viability of Synechocystis as an expression platform, it was observed that the addition of nickel had a negative impact on the growth of Synechocystis including the wild type strain. The addition of 6.4 μΜ Ni2+ resulted, independent of the OD750nm the culture had reached at the time of the addition, in a slowing down of the growth and a plateauing of the OD750nm (Figure 5) These results indicate that the used nickel concentration used has a negative effect on the growth and health of Synechocystis during longer induction periods, which then results in a decrease of the recombinant protein yield after induction for 9.5 hours or longer.
The concentration of 6.4 μΜ NiC was chosen in this study, because an earlier study that used the nrsB promoter for the expression of the reporter gene luxAB found that induction with 6.4 μΜ Ni2+ results in the highest level of bioluminescence (Peca, L. et al., 2008. Construction of bioluminescent cyanobacterial reporter strains for detection of nickel, cobalt and zinc. FEMS Microbiology Letters, 289(2), pp.258-264). The study created a whole-cell bioluminescent reporter that quantitatively responds to Ni2+ and other metal ions and showed that increasing concentrations of Ni2+ result in a dose dependent bioluminescence signal, but concentrations above 6.4 μΜ resulted in a reduced bioluminescence. These results suggest that higher concentrations of Ni2+ have a negative impact on either the activity or production of the luminescent protein or the health of the cells. However, the study performed the induction for only three hours. It is therefore conceivable that extending the induction time to 9.5 hours or more has a negative impact on Synechocystis cells already at a concentration of 6.4 μΜ. Another study showed that nrs mRNA can be already detected after induction by 0.45 μΜ Ni2+ for one hour, but the mRNA levels were higher after induction with 17 μΜ Ni2+. Higher concentrations of Ni2+ did not increase the mRNA levels any further (Garcia-Dominguez, M. et al., 2000. A Gene Cluster Involved in Metal Homeostasis in the Cyanobacterium Synechocystis sp. Strain PCC 6803. Journal of Bacteriology, 182(6), pp.1507-1514). In contrast, the study by Al-Haj (Al-Haj, L.A., 2014. Development of genetic engineering tools for the cyanobacterium Synechocystis PCC 6803 for advanced biofuel production. PhD thesis. London: University College London) and a second study (Y. Lui, unpublished) did not observe a difference in recombinant protein yields after induction with Ni2+ concentrations between 0.8 and 6.4 μΜ.
It is therefore conceivable that an induction with lower Ni2+ concentrations results in the same rate of protein accumulation, but might have less impact on the health of the Synechocystis cells and enable an induction over a longer period, which might result in the accumulation of higher final yields of the endolysins. This might be therefore a strategy to maximise the yield of endolysin protein under the control of the nrsBRS system in Synechocystis.
Expression of gp20 in Syn6803_nrsB_gp20i-HA, Syn6803_nrsB_gp202-HA and Syn6803_AII_
The transgenic lines carrying the gp20 genes were analysed for HA-tagged protein in western blot analyses. The strains were cultured as described above. The induction of Syn6803_nrsB_gp20i-HA and Syn6803_nrsB_gp202-HA was performed at an OD75onm of 0.8 and the cultures were harvested 5.5 hours after the start of the induction. After harvest the samples were concentrated to 100 times the culture volume. Western blot analysis with anti-HA antibodies and the Odyssey® Infrared (IR) Imaging System, confirmed the successful production of the Gp20 protein in Syn6803_nrsB_gp20i-HA (Figures 6 and 7), an endolysin that was not produced to detectable levels in the C. reinhardtii chloroplast.
The western blot analysis suggested that the codon optimised version gp20i results in higher expression levels than the gp20∑ gene. The genes have a shared sequence identity of 85.7%. The gp20i contains the UCA codon six times, which is less frequently used for serine in Synechocystis, whereas gp20∑ contains it 13 times. This could be therefore an explanation for the lower protein levels. Though, the western blot analysis needs to be performed with a higher number of transformants and different induction times before it can be concluded that the gene sequence of gp20i results in higher protein yields compared to gp20∑. It is important to note that the levels of Gp20 produced in Synechocystis are a significantly lower than the amounts of two other endolysins produced in the same system - Pal and φ1 1 and that approximately ten-fold more concentrated samples were used compared to the western blot analysis showing the accumulation of Pal and φ1 1 in the previous sections. Figure 7 shows a comparison of samples from Syn6803_nrsB_gp20i-HA with samples from Syn6803_nrsB_pal-HA and Syn6803_nrsB_φ1 1 -HA, which were cultured, induced and prepared in the same way. This western blot analysis showed that under these conditions approximately 50 times more full-length φ1 1 and 60 times more Pal accumulated in Synechocystis using the same expression system. These results suggest, together with the fact that Gp20 could not be produced in the C. reinhardtii chloroplast, that either the synthesis of the protein is less efficient or that it is less stable than the endolysins Pal, Cpl-1 and φ1 1 .
Cell breakage of Synechocystis sp. PCC 6803 and production of endolysin protein preparations
In order to perform assays analysing the antibacterial activity of the endolysin against the target bacteria, it was necessary to break the Synechocystis cells and to recover the endolysins without denaturing the proteins. Previous protocols have used vortexing in the presence of glass beads to break Synechocystis cells. This method was therefore used for cell breakage and it was evaluated whether the endolysins can be recovered and separated from the cell debris afterwards. Other methods that could be used include using a cell disrupter, a French Pressure cell press or sonication. Alternatively, the cells could be broken enzymatically or in a combination of mechanical and enzymatic breakage.
The three transgenic lines were broken by vortexing with glass beads (212 - 300 pm) for 2 min followed by 2 min on ice for five cycles. Subsequently the supernatant was separated from the cell debris by centrifugation (21 ,000 x g for 5 min). The whole cell extract before centrifugation and the supernatants (crude extract) were analysed by western blot analysis for the HA-tagged endolysins. All samples were additionally treated with SDS and boiling to break leftover unbroken cells.
These analyses showed that the endolysin could be recovered into the supernatant after cell breakage using glass beads. Vortexing in the presence of glass beads is an easy and quick method and it was possible to recover all three endolysins into the crude extract after cell breakage. Furthermore, the procedure can be easily performed under sterile conditions. This method was therefore used to break the Synechocystis cells for the performance of activity assays. Investigation of the antibacterial activity of Synechocystis-produced endolysin Gp20
A study by Farrar et al. (2007) sequenced the genome of the bacteriophage PA6 that infects Propionibacterium acnes and predicted that ORF20 encodes an endolysin. However, it has never been shown that Gp20 could be prepared as a recombinant protein in a cyanobacterium such as Synechocystis (which can be grown in a photobioreactor) nor has it been confirmed experimentally that the protein cleaves the peptidoglycan of P. acnes in a human subject. A demonstration of the lytic activity of Synec/iocysi/'s-produced Gp20 would not only demonstrate that it can be produced as an active enzyme in the cyanobacterium, but would also deliver the experimental proof that Gp20 on its own has antibacterial activity against P. acnes.
Gp20 could be purified using either a FPLC or by developing an ion exchange column protocol. Alternatively, the sequence for a His-tag or Strep-tag could be attached to the gene and commercial available columns could be used for the purification. In this way it would be possible to separate Gp20 from the phycobilliproteins that strongly interfere with the TRA and to concentrate the protein to levels that result in detectable activity. Alternatively, crude extract containing Gp20 or the purified enzyme could be mixed with P. acnes cells and a decrease in cfu after incubation on agar plates could be analysed.
The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.
Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.
All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to".

Claims

Claims:
1 . A cyanobacterial expression platform configured to produce a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
2. The cyanobacterial expression platform according to claim 1 wherein the cyanobacterial expression platform comprises a strain of Synechocystis sp..
3. The cyanobacterial expression platform according to claim 2 wherein the strain of Synechocystis sp. is Synechocystis PCC 6803.
4. The cyanobacterial expression platform according to claim 1 wherein the polypeptide, fragment, variant, derivative or fusion exhibits at least 30% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells.
5. The cyanobacterial expression platform according to claim 1 wherein the polypeptide, fragment, variant, derivative or fusion exhibits at least 30% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells of Propionibacterium acnes.
6. The cyanobacterial expression platform according to claim 1 wherein the fragment comprises or consists of at least 50 contiguous amino acids of SEQ ID NO: 1 .
7. The cyanobacterial expression platform according to claim 1 wherein the variant comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1 .
8. The cyanobacterial expression platform according to claim 1 wherein the derivative has been formed by modification or derivatisation of one or more of the amino acid residues to block the N- or C- terminus and/or cyclise the polypeptide.
9. The cyanobacterial expression platform according to claim 1 wherein the fusion comprises one or more additional amino acids, inserted internally and/or at the N- and/or C- termini of the amino acid sequence of SEQ ID NO: 1.
10. The cyanobacterial expression platform according to claim 1 wherein the cyanobacterial expression platform comprises a genome comprising the nucleotide acid sequence of SEQ ID No: 2 or a variant thereof.
1 1. The cyanobacterial expression platform according to claim 10 wherein the variant of the nucleotide acid sequence of SEQ ID No: 2 has at least 70% sequence identity to the nucleotide acid sequence of SEQ ID No: 2.
12. The cyanobacterial expression platform according to claim 1 wherein the cyanobacterial expression platform comprises a genome comprising the nucleotide acid sequence of SEQ ID No: 3 or a variant thereof.
13. The cyanobacterial expression platform according to claim 12 wherein the variant of the nucleotide acid sequence of SEQ ID No: 3 has at least 70% sequence identity to the nucleotide acid sequence of SEQ ID No: 3.
14. Use of a cyanobacterial expression platform to produce a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
15. Use according to claim 14 wherein the cyanobacterial expression platform comprises a strain of Synechocystis sp..
16. Use according to claim 15 wherein the strain of Synechocystis sp. is Synechocystis PCC 6803.
17. Use according to claim 14 wherein the polypeptide, fragment, variant, derivative or fusion exhibits at least 30% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells.
18. Use according to claim 14 wherein the polypeptide, fragment, variant, derivative or fusion exhibits at least 30% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells of Propionibacterium acnes.
19. Use according to claim 14 wherein the fragment comprises or consists of at least 50 contiguous amino acids of SEQ ID NO: 1.
20. Use according to claim 14 wherein the variant comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1 .
21. Use according to claim 14 wherein the derivative has been formed by modification or derivatisation of one or more of the amino acid residues to block the N- or C- terminus and/or cyclise the polypeptide.
22. Use according to claim 14 wherein the fusion comprises one or more additional amino acids, inserted internally and/or at the N- and/or C-termini of the amino acid sequence of SEQ ID NO: 1 .
23. Use according to claim 14 wherein the cyanobacterial expression platform comprises a genome comprising the nucleotide acid sequence of SEQ ID No: 2 or a variant thereof.
24. Use according to claim 23 wherein the variant of the nucleotide acid sequence of SEQ ID No: 2 has at least 70% sequence identity to the nucleotide acid sequence of SEQ ID No: 2.
25. Use according to claim 14 wherein the cyanobacterial expression platform comprises a genome comprising the nucleotide acid sequence of SEQ ID No: 3 or a variant thereof.
26. Use according to claim 25 wherein the variant of the nucleotide acid sequence of SEQ ID No: 3 has at least 70% sequence identity to the nucleotide acid sequence of SEQ ID No: 3.
27. An isolated nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
28. The isolated nucleic acid molecule according to claim 27 wherein the polypeptide, fragment, variant, derivative or fusion exhibits at least 30% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells.
29. The isolated nucleic acid molecule according to claim 27 wherein the polypeptide, fragment, variant, derivative or fusion exhibits at least 30% of the ability of the wildtype endolysin of bacteriophage PA6 to lyse bacterial cells of Propionibacterium acnes.
30. The isolated nucleic acid molecule according to claim 27 wherein the fragment comprises or consists of at least 50 contiguous amino acids of SEQ ID NO: 1 .
31. The isolated nucleic acid molecule according to claim 27 wherein the variant comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1 .
32. The isolated nucleic acid molecule according to claim 27 comprising the nucleotide acid sequence of SEQ ID No: 2 or a variant thereof.
33. The isolated nucleic acid molecule according to claim 32 wherein the variant of the nucleotide acid sequence of SEQ ID No: 2 has at least 70% sequence identity to the nucleotide acid sequence of SEQ ID No: 2.
34. The isolated nucleic acid molecule according to claim 27 comprising a genome comprising the nucleotide acid sequence of SEQ ID No: 3 or a variant thereof.
35. The isolated nucleic acid molecule according to claim 34 wherein the variant of the nucleotide acid sequence of SEQ ID No: 3 has at least 70% sequence identity to the nucleotide acid sequence of SEQ ID No: 3.
36. A vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
37. The vector according to claim 36 wherein the vector is selected from the pLAH.AII expression vector or pLAH.nrsB expression vector.
38. A cyanobacterial host cell comprising: a) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof; b) a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof; and/or c) a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
39. The cyanobacterial host cell according to claim 38 wherein the cyanobacterial host cell is a strain of Synechocystis sp..
40. The cyanobacterial host cell according to claim 38 wherein the host cell is Synechocystis PCC 6803.
41. A pharmaceutical composition comprising: a) a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof; and/or b) a cyanobacterial host cell according to claim 38.
42. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof, the method comprising culturing a population of cyanobacterial host cells comprising:
a) a nucleic acid molecule encoding the polypeptide comprising the amino acid
sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof; or b) a vector comprising the nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof
under conditions in which the polypeptide is expressed.
43. A polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof produced by a process comprising the step of expressing the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof in a population of cyanobacterial host cells.
44. Use of the polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof in the manufacture of a medicament for the treatment and/or prevention of a bacterial infection.
45. Use according to claim 44 wherein the bacterial infection is caused by Propionibacterium acnes.
46. Use according to claim 44 wherein the treatment and/or prevention is to enhance the cosmetic appearance of the subject.
47. A method of treating and/or preventing a bacterial infection comprising administering the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof to the subject in need thereof.
48. Method according to claims 47 wherein the bacterial infection is caused by
Propionibacterium acnes.
49. Method according claim 47 wherein the treatment and/or prevention is to enhance the cosmetic appearance of the subject.
50. Polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof for use in the treatment and/or prevention of a bacterial infection.
51. Polypeptide according to claim 50 wherein the bacterial infection is caused by
Propionibacterium acnes.
52. Polypeptide according to claim 50 wherein the treatment and/or prevention is to enhance the cosmetic appearance of the subject.
53. A genetic construct which comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID No: 1 , or a fragment, variant, derivative or fusion thereof.
54. The genetic construct of claim 53, wherein the genetic construct is an expression construct.
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WO2012085259A2 (en) * 2010-12-23 2012-06-28 Lysando Holding Ag Antimicrobial agents

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US11903984B1 (en) 2017-04-21 2024-02-20 Phi Therapeutics, Inc. Compositions comprising Propionibacterium acnes bacteriophages for treating acne
WO2020104216A1 (en) * 2018-11-19 2020-05-28 Unilever N.V. An antimicrobial composition for selectively inhibiting growth of p. acnes bacteria
CN115461034A (en) * 2020-03-02 2022-12-09 联合利华知识产权控股有限公司 An effective anti-acne personal care composition
WO2023168418A1 (en) * 2022-03-03 2023-09-07 Massachusetts Institute Of Technology Cell-wall binding protein specifically targeting cutibacterium acnes

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