Vectors encoding antirepressor proteins and uses thereof

Abstract

This disclosure relates to compositions and methods for inducing bacterial lysis using antirepressor proteins that are capable of inhibiting prophage-encoded repressor proteins. Provided herein are polynucleotides and vectors encoding the antirepressor proteins and methods of use thereof to lyse bacteria and to treat bacterial infections. In an embodiment, the antirepressor protein is derived from a phage-inducible chromosomal island (PICI), for example Sam protein from Staphylococcus aureus pathogenicity island (SaPI), and the bacterial infection is caused by S. aureus harbouring a prophage φNM1.

Description

[0001] VECTORS ENCODING ANTIREPRESSOR PROTEINS AND USES THEREOF

[0002] Technical field

[0003] The present invention relates, in general terms, to compositions and methods for lysing bacteria and more specifically to methods of lysing bacteria using polynucleotides encoding antirepressor proteins.

[0004] Background

[0005] Antimicrobial resistance is a global threat to public health that is diminishing the effectiveness of antibiotics against bacterial infections. Bacteriophage therapy has emerged as one of the most promising alternatives to traditional antibiotics. Phages have several advantages over antibiotics as therapeutics, namely, they are highly host-specific and do not target human cells, and arc also self-amplifying as they can replicate and spread among target hosts. However, a key obstacle to the efficacy of phage therapy is the polylysogenic nature of most bacterial strains, wherein prophages resident in bacterial chromosomes can shield their hosts from destruction by infecting phages. Master repressor proteins continuously expressed by the prophages to maintain dormancy can shut down the transcription of lytic genes in incoming viral genomes.

[0006] Prophages can be induced to re-enter the lytic cycle, whereupon they lyse and kill their host in the process of phage maturation. In many studied systems, prophage activation is driven by the bacterial SOS response to DNA damage, which leads to cleavage of the repressor protein. Genotoxic agents can be delivered to trigger prophage activation, but such agents are generally indiscriminate and will also damage host cells. They are thus not suitable to complement phage therapy.

[0007] It would be desirable to overcome or alleviate at least one of the above-described problems, or at least to provide a useful alternative.

[0008] Summary

[0009] Disclosed herein is a method of inducing lysis of a bacterium harbouring a prophage, the method comprising contacting the bacterium with an effective amount of a polynucleotide encoding at least one antirepressor protein capable of inhibiting a repressor protein encoded by the prophage, wherein expression of the at least one antirepressor protein induces bacterial lysis.

[0010] Disclosed herein is a polynucleotide encoding at least one antirepressor protein capable of inhibiting a repressor protein encoded by a prophage in a bacterium.

[0011] Disclosed herein is a vector comprising a polynucleotide as defined herein.

[0012] Disclosed herein is a pharmaceutical composition comprising a polynucleotide or a vector as defined herein.

[0013] Disclosed herein is a method of treating an infection in a subject caused by a bacterium harbouring a prophage, the method comprising administering to the subject an effective amount of a polynucleotide, vector or pharmaceutical composition as defined herein.

[0014] Disclosed herein is a method of selecting a subject with a bacterial infection for treatment, the method comprising: (a) detecting a prophage in a sample from the subject; and (b) treating a subject found to have a prophage.

[0015] Disclosed herein is a polynucleotide, vector or pharmaceutical composition as defined herein, for use in treating an infection in a subject caused by a bacterium harbouring a prophage.

[0016] Disclosed herein is the use of a polynucleotide, vector or pharmaceutical composition as defined herein in the manufacture of a medicament for treating an infection in a subject caused by a bacterium harbouring a prophage.

[0017] Brief description of the drawings

[0018] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which: Figure 1 is a linear schematic of: (A) the wild- type SaPIl island; and (B) an exemplary antimicrobial prophage activator (APA) derived from SaPIl in which the genes for antibiotic resistance and toxins have been deleted (indicated by white arrows). Gene annotations are as follow: 1 : ear (antibiotic resistance); 2: tsst-1 (toxin); 3: terS (SaPI DNA packaging); 4: ptiA (phage interference); 5: ptiM (phage interference); 6: cpmB (phage interference); 7: cpmA (phage interference); 8: ptiB (phage interference); 9: hyp; 10: ppi (phage interference); 11: hyp; 12: sam (antirepressor); 13: hyp; 14: xis (excisionase); 15: str (SaPI regulation); 16: stl (SaPI regulation); 17: seq (toxin); 18: sek (toxin); 19: t (integrase).

[0019] Figure 2 is an illustration of APA-induced prophage activation and dissemination. First, an APA particle injects DNA into a S. aureus cell harbouring prophage tbNMl. The APA expresses a Sam protein which activates the d>NM I prophage. Second, the APA element and <hNM 1 genome replicate. Finally, infectious NM1 phages and APA particles arc formed and the cell is lysed, thus releasing the particles to infect nearby cells.

[0020] Figure 3 shows that APA-induced prophage activation leads to host cell killing. (A) A S. aureus ArecA strain harbouring prophage 4>NM1 was infected with APA1 at a multiplicity of infection (MOI) of 0.001 and sampled over time. Cell lysates were filtered and assayed for plaque-forming units (PFU) on a non-lysogenic strain of S. aureus and represented as the PFU / ml. Buffer indicates buffer only, APA1 indicates wild-type APA1, APA1 Asam indicates deletion of the sam gene; and No cells indicates no S. aureus present. (B) Wildtype S. aureus strain Newman was infected with APA1 and the optical density (ODeoo) was measured over time at several MOI.

[0021] Detailed description

[0022] The inventor has developed an antimicrobial therapy that can induce prophages residing in bacterial chromosomes to enter the lytic cycle, without having to activate bacterial SOS systems. The therapeutic is based on the delivery of antirepressor proteins capable of inhibiting repressor proteins expressed by the prophages. De -repression initiates the phage lytic cycle to cause bacterial lysis. The inventors have further developed a delivery system that allows genes encoding the antirepressor proteins to replicate in target bacteria and be propagated to neighbouring bacteria, thus initiating a cascade of dc-rcprcssion and bacterial lysis.

[0023] The deliver}' system is based on engineered genetic elements known as phage-inducible chromosomal islands (PICIs), which are mobile genetic elements that reside quiescently in the genome of bacterial hosts until induced to excise and replicate, typically by infection of the bacterial host with certain phages. The engineered PICIs are also referred to as Antimicrobial Prophage Activators (APAs) in this disclosure. APAs encoding antirepressor proteins can be packaged into phage particles by mutated phages that are unable to package their own genomes, resulting in infectious particles that resemble phages but comprise only APAs and no phage genomic material. When phage particles containing the APAs are injected into pathogenic bacteria, the antirepressor genes they cany are expressed, resulting in the inactivation of phage master repressors in the bacteria, which in turn induces resident prophages to enter the lytic cycle. In this manner, lysogenic protection is circumvented while bypassing the SOS response. The activated prophages then perform two functions: they enter the lytic cycle and lyse the host cell, and they package and disseminate APAs to nearby cells to initiate additional rounds of bacterial killing and dissemination.

[0024] The APA approach is superior to conventional phage therapy because it circumvents the major hurdle of lysogenic protection by activating resident prophages to kill the host and then uses the prophages to self-amplify and disseminate to neighbouring cells. Bacterial pathogens such as Staphylococcus aureus arc especially susceptible to APAs, since the majority of sequenced strains are shown to carry at least one and sometimes up to four prophage genomes integrated in their chromosomes.

[0025] Finding phage candidates for phage therapy is slow and costly because suitable phages have to be individually identified for each patient. Typically, target bacterial strains are isolated from patients and then sent to various phage labs for susceptibility testing. Currently, there is no way to determine phage susceptibility in a high-throughput manner based on the genome sequence of the target bacterial strain. Advantageously, with APA therapy, the genome sequence of a target bacterial strain can reveal the repressor genes encoded by resident prophages, and appropriate antirepressors can then be chosen based on known antirepressor-repressor interacting pairs. This allows the identification of a suitable APA using only the genome sequence of the target bacterial strain. In order for a phage to infect and lyse a strain, at least two steps are required: the phage must recognise a bacterial surface receptor, and the phage lysis proteins (holin and lysin) must be compatible with the membrane of the cell. APA therapy is not limited by host range because APAs adopt the host range of phage mutants used to provide structural proteins to encapsidate APAs into particles. APA therapy also does not need lysis proteins because the APAs induce the resident prophages to lyse the host cell.

[0026] Accordingly, disclosed herein are polynucleotides and vectors encoding one or more antirepressor proteins that are capable of inhibiting prophage-encoded repressors in bacteria and thus mobilising resident prophages and inducing bacterial lysis. Phage vectors may be used to deliver the polynucleotides into bacterial hosts. The polynucleotide may be configured for packaging into phage particles in the bacterial host such that they can be propagated to other bacteria following bacterial lysis. In some embodiments, the polynucleotides contain a mobile genetic clement, such as a phage-inducible chromosomal island (PICI), that is capable of being packaged into phage particles encoded by prophages in the bacteria. Also disclosed herein are methods of inducing bacterial lysis and treating bacterial infections using the polynucleotides and vectors.

[0027] General definitions

[0028] As used herein, a “repressor protein” refers to a protein produced by a bacteriophage, particularly a temperate phage, to inhibit the expression of genes required for the lytic cycle and thus maintain the prophage during lysogeny. The skilled person can identify repressor proteins from phage and prophage genomes using methods known in the art, for example, based on homology searching. Examples of repressor proteins include the phage Cl protein, Bacteriophage 434 Cl protein, P22 C2 protein, and Bacteriophage Mu RepC protein.

[0029] As used herein, an “antirepressor protein” refers to a protein capable of inhibiting the activity of a repressor protein and thus activating the phage lytic cycle. The antirepressor protein may, for example, bind to a repressor protein and prevent the repressor from interacting with other subunits or with a target nucleic acid, promote the degradation of the repressor protein, or modify the repressor protein in a way that inhibits its function. Antirepressor proteins may be specific for a repressor or may be capable of inhibiting different repressors.

[0030] The terms “polynucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to refer to polymers of nucleotides, and include but are not limited to single- and double-stranded DNA and RNA, DNA / RNA hybrids including polynucleotide chains of regularly and / or irregularly alternating deoxyribosyl moieties and ribosyl moieties, and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included.

[0031] By “vector” is meant any vehicle suitable for delivering a nucleic acid into a cell, and in particular into a bacterium. Vectors include but are not limited to plasmids, cosmids, fosmids, phagemids, bacteria artificial chromosomes, polymeric nanoparticles (e.g., Nanocin™ or chitosan particles), viruses, virus-like particles, bacteriophages and phage-like particles.

[0032] A “prophage”, as used herein, refers to a dormant form of a bacteriophage which has integrated its genome into the genome of a bacterial host cell. A prophage is part of the lysogenic cycle of a temperate phage. The prophage state is maintained by the expression of specific repressor proteins which inhibit the transcription of phage lytic genes. A prophage can be induced to enter the lytic cycle under certain conditions, such as DNA damage, specific environmental signals, or host infection with another phage. Induction typically involves the inactivation or degradation of prophage repressor proteins, leading to the excision of the prophage from the bacterial chromosome, followed by active replication, assembly, and release of new phage particles through host lysis. Many bacteria, including pathogenic bacteria such as Staphylococcus aureus. Salmonella spp., and Vibrio cholerae, harbour prophages which encode virulence factors contributing to disease or which encode antibiotic resistance elements.

[0033] The terms “phage vector”, “phage particle” and “transducing phage particle” are used interchangeably herein to refer to a phage or a phage-like structure that is capable of delivering a nucleic acid payload (such as DNA) to a bacterial host. The phage vector may comprise components (such as a capsid, tail or collar) from a single phage species or from different phage species. These components may be wild-type (phage-endogenous) or may contain modifications designed to, c.g., alter the host range, or to display additional moictics. Phage vectors herein may be assembled in a bacterial host from an extrachromosomal vector (such as a plasmid or phagemid) or using machinery encoded by a phage.

[0034] As used herein, the term “induce” or “inducing” when referring to a genetic element resident in a genome (such as a prophage), refers to the mobilisation of the genetic element from a latent or repressed state. Induction generally results in the expression of genes within the clement and the excision of the clement from the host genome, and may also involve replication of the element and / or packaging into a phage particle for transmission.

[0035] As used herein “sequence identity” refers to the number (or fraction expressed as a percentage %) of identical or similar' amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global, but for purposes herein alignment is generally a global alignment where the full-length of each sequence is compared. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. Sequence identity can be determined by taking into account gaps as the number of identical rcsiducs / lcngth of the shortest scqucnccxlOO. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalised). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions / length of the total aligned sequencexl 00.

[0036] Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that axe known to those skilled in the art, for instance, using standard alignment algorithms such as BLAST, ClustalW, MAFFT, EMBOSS, T-Coffee, with default parameters. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

[0037] Amino acid sub-classification

[0038] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in the table below under the heading of exemplar}' substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

[0039] Exemplary amino acid substitutions

[0040] The term “disrupt” refers to any genetic modification that decreases or eliminates expression and / or functional activity of a nucleic acid or an expression product thereof. For example, disruption of a gene includes within its scope any genetic modification that decreases or eliminates expression of the gene and / or the functional activity of a corresponding gene product (e.g., mRNA and / or protein). Genetic modifications include complete or partial inactivation, suppression, deletion, interruption, blockage, or down-regulation of a nucleic acid (e.g., a gene). Illustrative genetic modifications include, but are not limited to, gene knockout, inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or use of inhibitory nucleic acids (e.g., inhibitory RNAs such as sense or antisense RNAs, molecules that mediate RNA interference such as siRNA, shRNA, miRNA; etc.), inhibitory polypeptides (e.g., antibodies, polypeptide-binding partners, dominant negative polypeptides, enzymes etc.) or any other molecule that inhibits the activity of the gene or level or functional activity of an expression product of the gene.

[0041] By “pharmaceutically acceptable carrier” is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.c., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0042] The terns “treating” and “preventing” include: delaying or preventing the onset of symptoms of the disease, disorder or condition; reducing the severity of (i.c., alleviating) the symptoms of the disease, disorder or condition; reversing the symptoms of (i.e., ameliorating) the disease, disorder or condition; reducing morbidity of subjects having the disease, disorder or condition; reducing mortality of subjects having the disease, disorder or condition; delaying or preventing progression of the disease, disorder or condition (e.g., to a later stage); and / or otherwise inhibiting the symptoms or effects of the disease, disorder or condition for at least a period of time. It is to be understood that the terms “treating” and “preventing” do not imply that the disease, disorder or condition, or a symptom or effect thereof, is permanently delayed, reduced, alleviated, ameliorated or otherwise inhibited and therefore also encompasses the temporary delay, reduction, alleviation, amelioration or otherwise inhibition of the disease, disorder or condition, or a symptom or effect thereof.

[0043] As used herein a “therapeutically effective amount” or “effective amount” is an amount that is non-toxic to the subject and sufficient to effect desired outcomes in a subject (i.e., achieve therapeutic efficacy). For purposes of this disclosure, an effective amount of a polynucleotide, vector or composition is an amount that is sufficient to induce cell lysis or to palliate, ameliorate, stabilise, reverse, prevent, slow or delay the progression of a disease state. An effective amount can be administered in one or more administrations. The terms “subject” or “patient”, used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. A preferred subject is a human in need of treatment for an infection. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0044] A “sample” as used herein includes any biological specimen (e.g., fluids, cells or tissues) that may be extracted, untreated, treated, diluted or concentrated from a subject. Any suitable methods for obtaining a biological sample can be employed. Exemplary methods include, e.g., phlebotomy, fine needle aspiration, swab (e.g., buccal swab) and surgical biopsy. The sample may be pooled from multiple aliquots.

[0045] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0046] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0047] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0048] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase “consisting essentially of”, and variations such as “consists essentially of’ will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0049] Antirepressor proteins

[0050] Antirepressor proteins herein may be derived from phage-inducible chromosomal islands (PICIs), such as bacterial pathogenicity islands. The inventor has found that PICIs may encode proteins that can inhibit phage master repressor proteins and thus activate prophages latent in a bacterial genome. In some embodiments, the antirepressor protein is derived from a PICT that is native to a target bacterium containing the prophage. Tn some embodiments, the antirepressor protein is derived from a PICI that is native to a strain or species related to the target bacterium. In some embodiments, the antirepressor protein is derived from a Staphylococcus aureus pathogenicity island (SaPI).

[0051] In some embodiments, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 1-10. Sequence variations herein (such as the about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, or about 30% sequence variation) may be conservative amino acid substitutions or other natural or engineered sequence variations which do not substantially alter the ability of the polypeptide to function as an antirepressor. A skilled person can identify appropriate sequence variants using, for example, sequence alignment to identify conservative amino acid subsitutions, structural modelling using in silico tools (e.g., AlphaFold, SWISS-MODEL, Rosetta, PyMOL, ChimcraX, I-TASSER, etc.), and functional assays.

[0052] In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 1. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 2. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 3. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 4. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 5. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 6. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 7. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 8. In one embodiment, the antirepressor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 9. In one embodiment, the antircprcssor protein comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 10.

[0053] Other suitable antirepressor proteins may be identified by searching for proteins homologous to SEQ ID NO: 1-10 on known sequence databases (e.g., NCBI protein database, NCBI RcfScq, UniProt Knowledgebase (UniProtKB)).

[0054] In some embodiments, the antirepressor protein is capable of inhibiting a repressor protein encoded by a Staphylococcus phage. In some embodiments, the antirepressor protein is capable of inhibiting a repressor protein encoded by a phage selected from <1>NM1, 0NM4, 029, 052A, 085, 096, 0WHB1 131O, 0WHB 11325, 0WHB11359, 0WHB1 136O and 0WHB 11473.

[0055] In some embodiments, the antirepressor protein is capable of inhibiting a prophage repressor protein comprising an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%), about 93%, about 94%>, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 11-21.

[0056] A single antirepressor protein may be capable of inhibiting two or more repressor proteins. For example, the inventor has found that the antirepressor from SaPIl can inhibit repressors encoded by the 0NM1, 029, 052A, 085, 096 phages (see Tables 1 and 2). The antirepressor from SaPI_J 11 can inhibit repressors encoded by the 0WHB 11310 and 0NM4 phages. The antirepressor from SaPl_PT1028 can inhibit repressors encoded by the <bWHB 11325 and 4>NM4 phages. The antirepressor from SaPI_JS395 and SaPI_CCPO53636 can inhibit repressors encoded by the WHB 11360, 4>WHB 11473 and <t>NM4 phages. The antirepressor from SaPI_EF900 can inhibit the repressor encoded by the <t>NM4 phage. The antirepressor from SaPI_OP007 can inhibit the repressor encoded by the <t>WHB 11359 phage.

[0057] Many pathogenic strains harbour multiple prophages, thus in some embodiments, polynucleotides herein may encode two or more antirepressor proteins, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more antirepressor proteins so as to induce multiple prophages that may be present in a bacterium. The two or more antirepressor proteins may target different prophage-encoded repressors, or there may be overlap in their repressor targets.

[0058] In some embodiments, the polynucleotide encodes two or more antircprcssor proteins comprising an amino acid sequence selected from an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 1-10.

[0059] In some embodiments, the polynucleotide encodes two or more antircprcssor proteins comprising an amino acid sequence selected from an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 10. For example, the polynucleotide may encode 2, 3, 4, 5 or 6 antirepressor proteins comprising an amino acid sequence selected from an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 10. These antirepressor proteins are fairly diverse in their amino acid sequence and in combination can provide broad spectrum activity against many Staphylococcal prophages. The gene for the antirepressor protein may be operably linked to a promoter for expression in the bacterial host. As used herein “operably linked” is the association of two or more nucleic acid fragments in a construct such that the function of one is controlled by the other, for example DNA encoding a protein associated with DNA encoding a promoter. The promoter may be, for example, an inducible promoter, constitutive promoter or an endogenous promoter for the antirepressor. In preferred embodiments, the gene for the antirepressor protein is operably linked to its endogenous promoter. Where two or more antirepressors arc used, their expression may be placed under the control of a single promoter or under separate promoters (e.g., under their endogenous promoters).

[0060] Table 1. Exemplary antirepressor proteins

[0061] Table 2. Exemplary phage-encoded repressor proteins

[0062] Polynucleotides

[0063] In some embodiments, polynucleotides herein are capable of being replicated and packaged into phage particles within the bacterium. The phage particle may comprise proteins encoded by the prophage in the bacterium, such as capsid, tail, tail fibre, tail spike, base plate, collar or neck proteins encoded by the prophage. Advantageously, this allows the antirepressors to be disseminated to other bacteria in the vicinity following bacterial lysis and phage dispersal.

[0064] The polynucleotide may encode its own replicon, such as its own replication origin (oh), primase (pri) and / or replicase (rep). The polynucleotide may additionally contain packaging sequences (c.g., pac or cos sites) for packaging into a phage capsid. In some embodiments, the polynucleotide comprises packaging sequences that are recognised by a terminase small subunit (TcrS) encoded by the prophage in the bacterium. In some embodiments, the polynucleotide encodes its own small terminase (TerS) to redirect the phage DNA packaging machinery to package the polynucleotide into the phage capsid.

[0065] In some embodiments, the polynucleotide is not capable of integrating into the bacterial genome. For example, the polynucleotide may contain a deleted or non-functional integrase or recombinase gene, or it may lack an insertion sequence (such as an attP or inverted repeat sequence). This ensures the polynucleotide remains mobile and can be packaged into phage particles and delivered to other bacteria.

[0066] In some embodiments, polynucleotides herein comprise or consist essentially of a mobile genetic element that can be packaged into a phage particle in the bacterium. The gene for the antirepressor protein may be contained in the mobile genetic element.

[0067] In one embodiment, polynucleotides herein comprise or consist essentially of a phageinducible chromosomal island (PICT). PICIs are mobile genetic elements found in bacterial genomes which can be induced by bacteriophages. They typically remain dormant in the bacterial chromosome until an infecting phage triggers the excision and autonomous replication of the PICT. Copies of the PICT are then packaged into particles of the infecting phage, cither by competing with the phage genome or by using specialised packaging signals. The packaged PICIs are then transferred to new bacterial hosts during subsequent phage infections. PICIs encode their own replicon and excisionase (Xis) and integrase (Int) proteins, and most also encode their own small subunit (TerS) of the phage terminase complex, which directs specific packaging of PICI genomes. PICIs typically also carry genes that confer selective advantages to the bacterial host, such as antibiotic resistance genes and genes for virulence factors. PICIs are typically flanked by direct repeat sequences for insertion at specific bacterial chromosomal sites. Examples of PICIs include but arc not limited to bacterial pathogenicity islands (e.g., Staphylococcal aureus Pathogenicity Islands, or SaPIs, and Vibrio cholera Pathogenicity Islands), Enterococcus faecalis V583 (EfCIV583), Streptococcus pyogenes Ml (SpyCIMl), and Streptococcal SpyCIMl-like elements.

[0068] In one embodiment, the PICI is a bacterial pathogenicity island.

[0069] In one embodiment, the PICI is a Staphylococcus pathogenicity island, such as a pathogenicity island from Staphylococcus aureus, Staphylococcus epidermidis. Staphylococcus hemolyticus or Staphylococcus saprophyticus.

[0070] In one embodiment, the PICI is a Staphylococcus aureus pathogenicity island (SaPI). Nonlimiting examples of SaPIs include SaPIl, SaPI2, SaPI3, SaPI4, SaPI5, SaPIbovl, SaPIbov2, SaPIbov3, SaPIbov4, SaPIbov5, SaPIml, SaPIm4, SaPInl, SaPI_Jll, SaPI_PT1028, SaPI_JS395, SaPI_CCP053636, SaPI_EF900.„ 16565 and SaPI„ HDX8245599.1. hr some embodiments, the PICI is SaPIl, SaPI2, SaPIbovl or SaPIbov2. In one embodiment, the PICI is SaPIl .

[0071] In some embodiments, this disclosure also provides for genetic modification of the mobile genetic element or PICI, for example, to insert one or more antirepressor genes and / or to remove virulence determinants or non-essential or accessory genes that makeup the PICI’s natural cargo. Advantageously, deleting these genes can help to detoxify the PICI and increase the PICI’s packaging capacity.

[0072] The genetic modification may be a substitution, deletion or insertion of one or more nucleotides in the PICI, or a rearrangement of one or more segments of the PICI. The modification may be in a coding or non-coding region of the PICI, such as in a gene, gene regulatory region, or packaging site. The modification may delete or disrupt one or more genes, insert one or more genes, rearrange the sequence of genes, or modify gene expression.

[0073] In some embodiments, the PICI is engineered to delete or disrupt one of more genes. In some embodiments, the gene that is deleted or disrupted is a virulence gene (i.e., a gene coding for a toxin or other virulence factor), a resistance gene (i.e., a gene coding for antibiotic or antimicrobial resistance), an integrase gene (e.g., a tyrosine or serine integrase), an excisionase gene, a repressor gene, a capsid morphogenesis gene, a terminase gene, or a phage interference (i.e., a gene that interferes with or disrupts the phage life cycle, such as, for example, a gene that disrupts phage replication, gene expression, capsid assembly or genome packaging).

[0074] In one embodiment, the PICI is engineered to delete or isrupt an endogenous virulence or resistance gene in the PICI. Virulence genes include but arc not limited to genes for toxic shock toxin, enterotoxins (e.g., enterotoxin B, C, K, L or Q), exfoliative toxin ETA, biofilm- associated protein Bap, complement inhibitor SCIN, or von Willebrand factor-binding protein. Resistance genes include but are not limited to genes encoding resistance to penicillins, cephalosporins, carbapenems, sulphonamides, quinolones, aminoglycosides, macrolides, monobactams, lincosamides, glycopeptides, tetracyclines, rifamycins, triazoles, trimethoprims, doxycyclines, and multidrug transporters.

[0075] In one embodiment, the PICI is engineered to delete or disrupt an integrase gene. The PICI may also be engineered to delete or disrupt an insertion sequence (such as an attP site) so that it is incapable of integrating into the bacterial genome.

[0076] In one embodiment, the PICI is engineered to delete or disrupt a gene which interferes with or disrupts the phage life cycle (i.e., a phage interference gene). Such genes may include, but are not limited to, genes involved in capsid morphogenesis (e.g., cpmA and cpmB), genes for proteins that inhibit the phage small terminase (e.g., ppi), and genes for proteins that block transcription of late phage genes (e.g., ptiA,ptiB or ptiM).

[0077] In one embodiment, the PICI is engineered to insert one or more antirepressor genes, such as antirepressor genes that are not endogenous to the PICI. Polynucleotides herein may be of any length suitable for packaging into a phage capsid. Thus, for example, the polynucleotide may range in length from about 10 kb (at the smaller end of the size of a PICT) to about 46 kb (at the higher end of the size of a phage genome).

[0078] Vectors

[0079] Polynucleotides herein may be comprised in a vector for delivery into bacteria. Non-limiting examples of suitable vectors include plasmids, phagemids, cosmids, phage vectors, and phage-like particles.

[0080] In some embodiments, the vector is a phage or a phage-like particle (PLP). The phage may be a wild-type or engineered phage, e.g., a phage engineered to recognise a specific host.

[0081] The phage vector or PLP may comprise structural components from a single phage or from multiple phages. In some embodiments, the phage vector or PLP comprises one or more structural proteins from a bacteriophage that infects the target bacterium, preferably structural proteins such as tail proteins that allow the vector to attach to specific bacterial hosts and / or to insert its nucleic acid payload into the bacterial host.

[0082] In some embodiments, the phage vector or PLP is derived from a tailed phage. In some embodiments, the phagc-likc particle is derived from a double- stranded phage that uses a pac or / w-lype sequence for DNA packaging.

[0083] In some embodiments, the phage vector or PLP comprises one or more structural proteins from an 80a phage.

[0084] Recombinant vectors may be generated using methods known in the art. For example, phage vectors containing recombinant nucleic acids may be produced using host bacteria strains that carry a helper phage lysogen or a helper plasmid encoding genes for phage replication and assembly (e.g., structural or packaging proteins). The polynucleotide payload is transformed into the host bacteria, and phage production is induced (e.g., by chemical induction or using UV radiation or mitomycin C treatment) to produce phage particles containing the polynucleotide. The phage particles are released upon bacteria lysis, and may be collected and purified from the lysate using, for example, density gradient centrifugation, dialysis, ultrafiltration and / or chromatographic separation.

[0085] To produce phage vectors containing only the polynucleotide uncontaminated by phage or plasmid DNA, the gene for TerS may be deleted from the helper phage or plasmid, so that phage or plasmid genetic material cannot be packaged into phage capsids that are formed upon induction. Instead, the polynucleotide is packaged into the capsid by means or a TerS endogenous to or inserted into the polynucleotide.

[0086] A cocktail of different phages targeting the same bacterial host may be used to improve the efficacy of polynucleotide delivery.

[0087] Target bacteria and prophages

[0088] The polynucleotides, vectors and compositions of this disclosure may be used to target any bacteria harbouring a prophage that can be induced by the encoded antirepressor proteins. The bacteria may be Gram-positive or Gram-negative. Exemplary bacteria include but are not limited to Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophila, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. inlracellulare, M. kansaii, M. gordonae), Staphylococcus sp., Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus sp. (e.g.. Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (yiridans group), Streptococcus faecalis. Streptococcus bovis. Streptococcus (anaerobic sps.), or Streptococcus pneumonia), Serratia marcescens, Acinetobacter baumannii, Acinetobacter junii, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas sp., Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, pathogenic strains of Escherichia coli, Salmonella spp., Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Vibrio cholera, Leptospira, and Actinomyces israelii. The antirepressor proteins may be selected to target pathogenic bacteria harbouring prophages, and / or to exclude probiotic strains, such as bacteria of the Lactobacillus and Bifidobacterium genus. In some embodiments, the target bacterium is an antibiotic-resistant or multidrug-rcsistant bacterium. The bacterium may be resistant to one or more antibiotics, such as to P-lactams, extended spectrum P-lactams, aminoglycosides, ansamycins, carbacephem, carbapenems, any generation of cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptides, quinolones, fluoroquinolones, sulphonamides, tetracyclines, mycobacterial antibiotics, chloramphenicol, or mupirocin.

[0089] In some embodiments, the target bacterium is a Staphylococcus bacterium, such as Staphylococcus aureus. In some embodiments, the target bacterium is an antibiotic-resistant or multidrug-resistant Staphylococcus bacterium. In one embodiment, the target bacterium is Staphylococcus aureus.

[0090] In some embodiments, the bacterium is one that contains a prophage selected from 0NM1, 0NM4, 029, 052A, 085, 096, 0WHBH31O, 0WHB 11325, WHB 11359, 0WHB 11360. and 0WHB1 1473.

[0091] In some embodiments, the bacterium is one that expresses a prophage repressor protein comprising an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) to an amino acid sequence in SEQ ID NO: 11-21.

[0092] Pharmaceutical compositions

[0093] This disclosure also provides for pharmaceutical compositions comprising polynucleotides or vectors as defined herein. The composition may comprise compounds or agents to slow polynucleotide or vector degradation and / or improve polynucleotide or vector delivery of nucleic acid to the bacterium. In some embodiments, the composition comprises Nanocin1Mor chitosan particles to slow vector degradation.

[0094] The form of the pharmaceutical composition is not particularly limited, but generally comprises a polynucleotide or vector as defined herein and at least one inactive ingredient, such as a pharmaceutically acceptable carrier, diluent or excipient. Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilisers, gels, binders, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the polynucleotide or vector, its use in the pharmaceutical composition is contemplated.

[0095] Suitable diluents and excipients also include, for example, water, saline, dextrose, glycerol, and the like, and combinations thereof. In addition, if desired, substances such as wetting, solubilising or emulsifying agents, stabilising or pH buffering agents, viscosity controlling agents, preservatives, antioxidants, emollients, odour controlling or fragrance compounds may also be present. Pharmaceutically acceptable salts can also be present, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

[0096] The pharmaceutical composition may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and liposomes. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions may be administered intravenously (by injection or infusion), orally, intranasally (by nebuliser or nasal spray or drop), topically, rectally, vaginally, through bronchoscopy or endoscopy, or through a local implant or dressing.

[0097] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. A polynucleotide, vector or pharmaceutical composition of the present disclosure can be administered on multiple occasions. Intervals between single dosages can be, for example, hourly, daily, weekly, monthly or longer. Intervals can also be irregular as indicated by clinical progress or by changes in measured biomarkers (such in biomarkers of infection. Alternatively, the polynucleotide, vector or pharmaceutical composition can be administered as a sustained release formulation, in which case less frequent administration is required.

[0098] It may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specification for the dosage unit forms may be dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding.

[0099] Bacterial lysis and methods of treatment

[0100] Disclosed herein is a method of inducing lysis of a bacterium harbouring a prophage, the method comprising contacting the bacterium with an effective amount of a polynucleotide encoding at least one antirepressor protein capable of inhibiting a repressor protein encoded by the prophage, wherein expression of the at least one antirepressor protein encoded by the polynucleotide or vector induces bacterial lysis.

[0101] In one embodiment, the method comprises contacting the bacterium with an effective amount of a vector comprising the polynucleotide. The vector may be a phage vector.

[0102] In one embodiment, the bacterium is contacted with the polynucleotide or vector in vitro. In one embodiment, the bacterium is contacted with the polynucleotide or vector in vivo.

[0103] The polynucleotide or vector may be introduced into the bacterial host by any method known to the skilled person, such as electroporation, transduction, conjugation, thermal shock, lipid- assisted or liposome-assisted transformation, or chemically mediated transformation. In one embodiment, a phage vector containing the polynucleotide is used to deliver the antirepressor gene into the bacterial host.

[0104] Disclosed herein is a method of treating an infection in a subject caused by a bacterium harbouring a prophage, the method comprising administering an effective amount of a polynucleotide, vector or pharmaceutical composition as defined herein to the subject. The polynucleotide, vector or pharmaceutical composition may be administered in combination with a phage therapy. For example, phage-mediated antirepressor therapy may be combined with phage therapy using lytic phages to improve bacterial clearance.

[0105] The effective amount may vary depending on the type of vector used, the mode of administration and the nature of the infection (e.g., the bacteria burden, local or disseminated infection, etc.).

[0106] Where phages are used as the vector, the amount of phage to be used may be defined by the multiplicity of infection (MOI), which refers to the ratio of infectious phage particles to bacterial cells. Suitable MOIs may range from less than 0.01 to greater than 100, depending, for example, on the sensitivity of the bacterial strain and the kinetics of phage replication. A suitable phage dose may be determined by exposing a known concentration of bacteria to serial dilutions of the phage preparation and assessing bacterial lysis through methods such as optical density reduction, CFU counts, or viability assays. For in vivo administration, an estimate of the bacterial load may be obtained, e.g., by CFU counting, turbidity or metabolic assays, or flow cytometry, and used to determine a suitable phage dose. Suitable starting doses may be in the range of 107-IO10plaque forming units per ml (PFU / ml). A skilled person may also take into account factors such as the delivery route of the phage, tissue penetration, phage stability, clearance rate, and the nature and extent of host immune responses to determine the effective dose.

[0107] Disclosed herein is a method of selecting a subject with a bacterial infection for treatment, the method comprising: (a) detecting a prophage in a sample from the subject; and (b) treating a subject found to have a prophage.

[0108] The sample may be a biological sample, including but not limited to body fluids (e.g., whole blood, plasma, serum, lymph, pus, exudates, saliva, sputum, urine, cerebrospinal fluid, synovial fluid, GI tract contents, bile, semen, mucus, aspirates, lavage fluid) and tissue samples obtained via biopsy or surgical excision. Samples also include stool samples and swabs and scrapings collected from the skin, mucosal surfaces, wounds and sites of infection. The sample may be lysed to release nucleic acid for testing. In some embodiments, bacterial isolates may be cultured from the sample (e.g., using microbial media or nutrient agar) to increase the available bacteria for testing. The presence of inducible prophages may be assessed by treating the sample or bacterial isolate with an inducing agent (e.g., mitomycin C or UV light) to trigger the bacterial SOS response and phage lytic cycle, followed by isolation of released phages for visualisation (e.g., by electron microscopy) and / or sequencing.

[0109] The prophage and any prophage-encoded repressor protein may be detected using molecular and bioinformatic approaches known in the art. For example, nucleic acid extracted from the sample may be subjected to PCR amplification using primers specific for known prophage sequences, such as repressor genes, integrase genes or conserved att sites. For broader detection, metagenomic or whole genome sequencing may be performed on the sample and used to identify prophage sequences. Bioinformatic tools and algorithms, such as PHASTER, PHASTEST, Prophage Hunter, Phigaro and PhiSpy can be used to analyse sequence datasets or assemblies for prophage regions or prophage-encoded repressors.

[0110] In some embodiments, the method further comprises administering an effective amount of a polynucleotide, vector or pharmaceutical composition as defined herein to the subject. The polynucleotide, vector or pharmaceutical composition may be administered in combination with a phage therapy for the target bacteria.

[0111] By way of non-limiting example, where an infection or sample is found to contain the prophages, the subject may be treated with compositions encoding an antirepressor comprising an amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1). Where an infection or sample is found to contain the <FWHB 1 1310 prophage, the subject may be treated with compositions encoding an antirepressor comprising an amino acid sequence of SEQ ID NO: 4 (or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4). Where an infection or sample is found to contain the <bWHB l 1325 prophage, the subject may be treated with compositions encoding an antirepressor comprising an amino acid sequence of SEQ ID NO: 5 (or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 5). Where an infection or sample is found to contain the 11360 and / or 4>WHB 11473 prophages, the subject may be treated with compositions encoding an antirepressor comprising an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 (or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 7). Where an infection or sample is found to contain the 4>NM4 prophage, the subject may be treated with compositions encoding an antirepressor comprising an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 (or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Where an infection or sample is found to contain the «T»WHB 1 1359 prophage, the subject may be treated with compositions encoding an antirepressor comprising an amino acid sequence of SEQ ID NO: 10 (or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 10).

[0112] Disclosed herein is a polynucleotide, vector or pharmaceutical composition as defined herein, for use in treating an infection in a subject caused by a bacterium harbouring a prophage, optionally in combination with a phage therapy.

[0113] Disclosed herein is the use of a polynucleotide, vector or pharmaceutical composition as defined herein, in the manufacture of a medicament for treating an infection in a subject caused by a bacterium harbouring a prophage, optionally in combination with a phage therapy.

[0114] The polynucleotides, vectors or compositions of this disclosure can be administered to humans, and are also suitable for use in a veterinary context and accordingly can be given to non-human animals where targeting bacteria would be desirable.

[0115] The chosen route of administration may depend on the site of infection. In non-limiting examples, the polynucleotides, vectors and compositions may be administered intravenously (by injection or infusion), orally, intranasally (by nebuliser or nasal spray or drop), topically, rectally, vaginally, through bronchoscopy or endoscopy, or through a local implant or dressing.

[0116] Compositions herein and any partners in combination therapy may be administered in a single dose, in multiple doses, in a continuous or intermittent manner (e.g., at regular intervals), depending, for example, upon the infection and on the subject’s clinical condition. In non-limiting examples, the compositions may be administered continuously for a preselected period of time or in a series of spaced doses. Component therapies of a combination therapy may be administered concurrently or sequentially. For instance, phage therapy using lytic phages may be administered together with or following antirepressor therapy.

[0117] It is to be noted that dosage values may vary with the type and severity of the infection or condition to be treated. It is to be further understood that for any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0118] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0119] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0121] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described. EXAMPLES

[0122] Example 1: Production of phage-derived particles containing an engineered SaPI encoding an antirepressor Sam protein

[0123] It was found that Staphylococcus aureus Pathogenicity Islands (SaPIs) encode a small protein that bypasses the bacterial SOS response to directly induce prophages to enter the lytic cycle. The SaPIs arc a family of mobile genetic elements (MGEs) that arc extremely diverse and commonly found in most S. aureus genomes. Normally, SaPIs reside quiescently in the chromosomes of their S. aureus hosts until they are induced to excise and replicate by phage-encoded antirepressor proteins. Though they are highly mobile, the SaPIs lack the machinery for horizontal exchange and exploit phages for mobility by encapsidating their genomes into infective phage-derived particles that are transferred at extremely high frequencies. It was found that upon injection into a new host cell, SaPIs express Sam (SaPI activator of MGEs), a small protein encoded by nearly all SaPIs that binds to phage master repressors to disable their function. This enables SaPIs to trigger resident prophages to enter the lytic cycle, resulting in a SaPI pseudo-lytic cycle in which they infect, replicate, lyse from lysogenic host cells, and spread to new host cells.

[0124] To exploit Sam protein activation of prophages, SaPIs were genetically engineered to target the bacterial polylysogeny shield by exploiting the resident prophages of pathogenic bacteria as a vulnerability for therapy. The engineered SAPI is also referred to as an Antimicrobial Prophage Activator (APA) herein. Figure 1 shows an exemplary APA element (APA1) derived from SaPIl (GenBank accession number U93688) in which genes encoding antibiotic resistance and toxins have been deleted.

[0125] The SaPI may be engineered to encode multiple Sam proteins, including non-native Sam proteins, so as to induce different resident prophages in a bacterial host. An example of a SaPIl-derived APA encoding five Sam proteins (from SaPIl, SaPI_Jll, SaPI_PT1028, SaPI _JS395 and SaPI_CCPO53636) in which the toxin genes have been deleted is provided in SEQ ID NO: 22.

[0126] To produce APA particles, the APA1 element is maintained in an S. aureus strain that has an 80a phage with its small terminase gene deleted (80a terS). Phages that lack their small tcrminasc gene produce all their structural proteins and can lyse their way out of the cell, but are unable to package their own genomes into capsids. A strain with 80a AterS and APA1 will produce only APA particles and no mature 80a phages. Thus, APA particles resemble phages because they are packaged into phage structural proteins and can inject the APA1 DNA element into S. aureus strains.

[0127] Example 2: Phage-derived particles encoding Sam protein can induce lysis of lysogenic bacteria

[0128] APA particles can be used to infect 5. aureus strains that are l sogenic for targeted prophages such as NM1. Figure 2 depicts how APAs can infect and disseminate in lysogenic strains. First, an APA particle injects its DNA into a S. aureus cell. The Sam protein induces the resident prophage to enter the lytic cycle. Next, the phage genome and APA DNA replicate and phage structural proteins are made. Lastly, mature phages and new APA particles are formed and lysis of the host cell releases the APA particles for dissemination to start the process again in nearby cells. The infectious phage particles released will be inhibited by lysogenic protection when they infect sibling cells.

[0129] To demonstrate that APA1 can bypass the SOS response to activate prophages, a S. aureus ArecA strain harbouring prophage <bNMl was infected with APA1 and sampled over time. The lysates were filtered and assayed for plaque forming units (PFU) on a non-lysogcnic strain of S. aureus and represented as the PFU / ml. Figure 3 A shows that APA1 induced strong activation of the <bNMl prophage. Activation was SOS-independent because the RecA protein is required for the bacterial SOS response. As proof of concept for APA therapy, a clinical S. aureus isolate (Newman) with four resident prophages was infected with APA1 and assayed for bacteria cell killing as measured by optical density of the culture. Figure 3B shows that the uninfected Newman cultures grew rapidly and increased in cell density while the infected cultures were lysed and exhibited much lower optical density. Since the Newman strain was lysed even at low multiplicities of infection (MOls), this result shows that APA1 was able to self-amplify and disseminate to nearby cells after the initial rounds of infection.

[0130] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of inducing lysis of a bacterium harbouring a prophage, the method comprising contacting the bacterium with an effective amount of a polynucleotide encoding at least one antirepressor protein capable of inhibiting a repressor protein encoded by the prophage, wherein expression of the at least one antirepressor protein induces bacterial lysis.

2. The method of claim 1, wherein the at least one antirepressor protein is derived from a phage-inducible chromosomal island (PICI).

3. The method of claim 1 or 2, wherein the at least one antirepressor protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 1-10.

4. The method of any one of claims 1 to 3, wherein the polynucleotide encodes two or more antirepressor proteins.

5. The method of claim 4, wherein the two or more antirepressor proteins comprise an amino acid sequence selected from an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 10.

6. The method of any one of claims 1 to 5, wherein the polynucleotide is capable of being packaged into a phage particle in the bacterium.

7. The method of claim 6, wherein the phage particle is encoded by the prophage in the bacterium.

8. The method of any one of claims 1 to 7, wherein the polynucleotide is not capable of integrating into the bacterial genome.

9. The method of any one of claims 1 to 8, wherein the polynucleotide comprises anengineered phage-inducible chromosomal island (PICT) comprising a deletion or disruption of one or more genes selected from the group consisting of a virulence gene, resistance gene, integrase gene, excisionase gene, repressor gene, phage interference gene, capsid morphogenesis gene and terminase gene.

10. The method of claim 9, wherein the PICT is a Staphylococcus aureus pathogenicity island (SaPI).

11. The method of claim 10, wherein the PICT is SaPIl.

12. The method of any one of claims 1 to 11, wherein the polynucleotide is comprised in a phage vector.

13. The method of any one of claims 1 to 12, wherein the prophage is selected from <t>NMl,, , , , , , ,14. The method of any one of claims 1 to 13, wherein the bacterium is an antibiotic-resistant bacterium.

15. The method of any one of claims 1 to 14, wherein the bacterium is a Staphylococcus bacterium.

16. The method of claim 15, wherein the bacterium is Staphylococcus aureus.

17. A polynucleotide encoding at least one antirepressor protein capable of inhibiting a repressor protein encoded by a prophage in a bacterium.

18. The polynucleotide of claim 17, wherein the polynucleotide comprises an engineered phage-inducible chromosomal island (PICT) comprising a deletion or disruption of one or more genes selected from the group consisting of a virulence gene, resistance gene, integrase gene, excisionase gene, repressor gene, phage interference gene, capsid morphogenesis gene and terminase gene.

19. The polynucleotide of claim 17 or 18, wherein the at least one antirepressor protein is derived from a phage-inducible chromosomal island (PICT).

20. The polynucleotide of any one of claims 17 to 19, wherein the at least one antirepressor protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 1-10.

21. The polynucleotide of any one of claims 17 to 20, wherein the polynucleotide encodes two or more antirepressor proteins.

22. The polynucleotide of claim 21, wherein the two or more antirepressor proteins comprise an amino acid sequence selected from an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 10.

23. The polynucleotide of any one of claims 17 to 22, wherein the polynucleotide is capable of being packaged into a phage particle in the bacterium.

24. The polynucleotide of claim 23, wherein the phage particle is encoded by the prophage in the bacterium.

25. The polynucleotide of any one of claims 17 to 24, wherein the polynucleotide is not capable of integrating into the bacterial genome.

26. The polynucleotide of claim 25, wherein the PICT is a Staphylococcus aureus pathogenicity island (SaPI).

27. The polynucleotide of claim 26, wherein the PICI is SaPIl.

28. The polynucleotide of any one of claims 17 to 27, wherein the prophage is selected from29. A vector comprising a polynucleotide of any one of claims 17 to 28.

30. The vector of claim 29, wherein the vector is a phage vector.31 . A pharmaceutical composition comprising a polynucleotide of any one of claims 17 to 28, or a vector of claim 29 or 30.

32. A method of treating an infection in a subject caused by a bacterium harbouring a prophage, the method comprising administering to the subject an effective amount of a polynucleotide of any one of claims 17 to 28, a vector of claim 29 or 30, or a pharmaceutical composition of claim 31.

33. A method of selecting a subject with a bacterial infection for treatment, the method comprising: (a) detecting a prophage in a sample from the subject; and (b) treating a subject found to have a prophage.

34. The method of claim 33, wherein step (b) comprises administering to the subject an effective amount of a polynucleotide of any one of claims 17 to 28, a vector of claim 29 or 30, or a pharmaceutical composition of claim 31 .

35. A polynucleotide of any one of claims 17 to 28, a vector of claim 29 or 30, or a pharmaceutical composition of claim 31, for use in treating an infection in a subject caused by a bacterium harbouring a prophage.

36. Use of a polynucleotide of any one of claims 17 to 28, a vector of claim 29 or 30, or a pharmaceutical composition of claim 31 in the manufacture of a medicament for treating an infection in a subject caused by a bacterium harbouring a prophage.

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