Modified bacteriophage
Inventors
Fairhead, Heather • Wilkinson, Adam • Holme, Sarah • Pitts, Katy • Jackson, Alison
Assignees
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Publication Number
US-10106784-B2
Publication Date
2018-10-23
Expiration Date
Abstract
Provided is a modified bacteriophage capable of infecting a target bacterium, which bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacterium, wherein the SASP gene is under the control of a constitutive promoter which is foreign to the bacteriophage and the SASP gene.
Core Innovation
The disclosure describes a modified bacteriophage for inhibiting or preventing growth of a target bacterium in or on a subject by administering a therapeutically effective amount of the modified bacteriophage. The bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP that is toxic to the target bacterium. The SASP gene is placed under the control of a constitutive bacterial promoter that is foreign to the bacteriophage and functionally linked to the SASP gene.
The constitutive foreign promoter drives production of toxic levels of SASP when present in multiple copies in the target bacterium, while the bacteriophage contains a single copy of the SASP gene functionally linked to the constitutive bacterial promoter. The disclosure positions this design as enabling SASP production independent of the phage lifecycle, including contexts where bacterial DNA-degradation limits host range. The modified bacteriophage is further described as acting as a delivery vector for toxic SASP in such conditions.
The disclosure describes selecting constitutive bacterial promoters from S. aureus and related promoter variants, including pdhA, rpsB, pgi, and fbaA, including sequences with more than 90% identity. A specific example configuration replaces the φ11 holin gene with an fbaA-SASP-C cassette using B. megaterium SASP-C together with a cadmium resistance marker, producing SASPject vectors. The disclosure reports that killing depends on the SASP gene and that superinfection immunity does not prevent SASP toxicity.
Claims Coverage
The claims cover a method of treating a bacterial infection by administering a modified bacteriophage with a toxic α/β SASP gene expressed from a foreign constitutive bacterial promoter, configured so that the bacteriophage carries a single copy of the SASP gene while the promoter supports toxic SASP production when the gene is present in multiple copies in the target bacterium.
Treating a bacterial infection with a modified bacteriophage carrying a toxic α/β SASP gene
Administering a therapeutically effective amount of a modified bacteriophage that inhibits or prevents growth of a target bacterium, wherein the bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP toxic to the target bacterium.
Foreign constitutive bacterial promoter controlling toxic SASP production in multiple copies
Placing the SASP gene under the control of a constitutive bacterial promoter that is foreign to the bacteriophage and functionally linked to the SASP gene, wherein the promoter drives production of toxic levels of SASP when present in multiple copies in the target bacterium.
Single-copy SASP gene functionally linked to the constitutive promoter
Configuring the bacteriophage to contain a single copy of the SASP gene functionally linked to said constitutive bacterial promoter.
Constitutive promoter selection from pdhA, rpsB, pgi, fbaA or >90% identity variants
Using a constitutive promoter chosen from pdhA, rpsB, pgi, fbaA, or sequences having more than 90% identity thereto.
Non-lytic bacteriophage
Employing the modified bacteriophage as a non-lytic bacteriophage.
SASP gene inserted into a lysis gene
Inserting a SASP gene into a lysis gene of the bacteriophage.
φ11 holin gene modified to express Bacillus megaterium SASP-C under an fbaA constitutive promoter
Providing a bacteriophage that includes a φ11 bacteriophage with a holin gene in which is inserted a gene encoding Bacillus megaterium SASP-C under an fbaA constitutive promoter.
Topical administration
Carrying out the method using the modified bacteriophage applied topically.
Overall, the independent claim is centered on administering a modified bacteriophage whose single-copy toxic α/β SASP gene is controlled by a foreign constitutive bacterial promoter that supports toxic SASP levels when present in multiple copies in the target bacterium, with dependent claims refining promoter choice, phage phenotype (non-lytic), SASP insertion into lysis genes (including an example involving a φ11 holin modification and an fbaA-driven Bacillus megaterium SASP-C cassette), and administration as topical.
Stated Advantages
SASP production independent of the phage lifecycle.
Broader strain coverage enabled by SASP production independent of phage lifecycle.
Phage can act as a delivery vector even when bacterial DNA-degradation limits host range.
Documented Applications
Treating a bacterial infection in or on a subject by administering a therapeutically effective amount of a modified bacteriophage.
Topical infections, including dental caries, respiratory infections, eye infections, and carriage reduction via composition and use routes described as topical and other routes.
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