Therapeutic phages and methods thereof
The present invention relates, in part, to engineered viruses (e.g., engineered phages), phage cocktails, and methods of producing and/or identifying viruses for targeting pathogenic bacteria.
1. A method of producing an engineered phage for killing a bacterial colony, the method comprising:
identifying islands within a bacterial genome of a first bacterium;
identifying a genomic island comprising a first prophage sequence within the bacterial genome from the first bacterium,
identifying close relatives of the first prophage sequence by sorting the first prophage sequence into a group with other prophage sequences based on genomic distances computed amongst the first and other prophages;
wherein the first prophage sequence comprises a genomic island comprising an integrase gene;
identifying relatives of a target bacterium based on genomic distance measurements;
producing an engineered phage genome comprising the first prophage sequence that lacks the integrase gene, wherein the first prophage sequence is effective to kill the target bacterium and the first bacterium is a relative of the target bacterium, wherein the engineered phage genome encodes the engineered phage; and
targeting a bacterial colony, which comprises the target bacterium, with the engineered phage genome, wherein the bacterial colony comprises a strain of a second bacterium that is different than a strain of the first bacterium.
2. The method of claim 1 , wherein the first prophage sequence encodes a temperate phage and wherein the engineered phage genome encodes a virulent phage.
3. The method of claim 1 , wherein the identifying step comprises identifying a plurality of prophage sequences within the bacterial genome from the first bacterium, wherein each of the plurality of prophage sequences comprises a genomic island comprising an integrase gene; and wherein the producing step comprises producing the engineered phage genome comprising at least one of the plurality of prophage sequences that lacks the integrase gene.
4. The method of claim 3 , wherein the identifying step comprises identifying a first prophage sequence and a second prophage sequence, and wherein the producing step comprises producing a first engineered phage genome comprising the first prophage sequence and a second engineered phage genome comprising the second prophage sequence, and wherein each of the first and second prophage sequences comprises a genomic island.
5. The method of claim 4 , wherein the targeting step comprises targeting the bacterial colony with the first engineered phage genome and with the second engineered phage genome.
6. The method of claim 1 , wherein the engineered phage genome comprises the first phage genome that lacks a repressor gene, a repressor-binding site, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, or a structural phage protein gene.
7. The method of claim 1 , further comprising, after producing an engineered phage genome:
incubating the engineered phage genome with a first bacterial lawn comprising a strain of the first bacterium, thereby producing a plaque disposed on the first bacterial lawn; and
identifying a genomic sequence from the plaque by using one or more primers to bind to the first prophage sequence or a portion thereof.
8. The method of claim 1 , further comprising, after producing an engineered phage genome:
incubating the engineered phage genome with a first bacterial lawn comprising a strain of the second bacterium, thereby producing a plaque disposed on the first bacterial lawn, wherein the first bacterium and the second bacterium are different; and
identifying a genomic sequence from the plaque by using one or more primers to bind to the first prophage sequence or a portion thereof.
9. The method of claim 7 , wherein at least one of the one or more primers comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:8-19, or a complement of any of these or a fragment thereof.
10. The method of claim 1 , wherein the first prophage sequence comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-7.
11. The method of claim 1 , wherein the first bacterium comprises Pseudomonas, Burkholderia , or Klebsiella.
12. The method of claim 1 , wherein the integrase gene encodes a tyrosine integrase, a tyrosine recombinase, or a serine recombinase.
13. The method of claim 1 , wherein the engineered phage genome encodes a virulent phage.
14. The method of claim 1 , further comprising, after producing an engineered phage genome:
incubating the engineered phage genome with a bacterial lawn comprising a strain of the second bacterium, wherein the first bacterium and the second bacterium are different,
thereby producing a plaque disposed on the bacterial lawn; and
identifying a genomic sequence from the plaque by using one or more primers to bind to the first prophage sequence or a portion thereof.
15. The method of claim 14 , wherein the engineered phage genome comprises the first phage genome that lacks a repressor gene, a repressor-binding site, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, or a structural phage protein gene.
16. The method of claim 1 , further comprising:
designing one or more primers to bind to the first prophage sequence or a portion thereof;
incubating the engineered phage with a bacterial lawn comprising a strain of the second bacterium, wherein the first bacterium and the second bacterium are different, thereby producing a plaque disposed on the bacterial lawn; and
identifying a genomic sequence from the plaque by using the one or more primers to bind to the first prophage sequence or a portion thereof.
17. The method of claim 16 , wherein at least one of the one or more primers comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8-19, or a complement of any of these or a fragment thereof.
18. The method of claim 1 further comprising: deleting an integrase gene from the genomic island of the first prophage sequence.
19. The method of claim 1 , wherein the first bacterium and the target bacterium are different and have a MASH distance of from about 0 to about 0.15.
20. The method of claim 5 , wherein the first engineered phage genome and the second engineered phage genome are from a different group based on genomic distances.