Modified bacteriocins and methods for their use
View Patent ↗Modified forms of naturally occurring bacteriocins, such as the R-type pyocins of Pseudomonas aeruginosa , are disclosed as are methods for producing them in GRAS organisms. The bacteriocins are modified at the ends of their tail fibers in a region responsible for binding specificity and affinity to their cognate binding partners, or receptors, such as those on the surface of bacteria. Methods for the use of the modified bacteriocins, such as to bind receptors, including virulence or fitness factors, on the surfaces of bacteria, are also described.
1. A tail protein comprising a base plate attachment region (BPAR) from an R-type high molecular weight (hmw) bacteriocin tail fiber protein, wherein the BPAR comprises amino acids 1-161 of the N-terminal portion of the R-type bacteriocin tail fiber protein; and a heterologous receptor binding domain (RBD) from a bacteriophage tail protein.
2. The protein of claim 1 wherein the R-type hmw bacteriocin is an R-type pyocin and the bacteriophage is a podoviridae.
3. An R-type high molecular weight (hmw) bacteriocin having bactericidal activity comprising a tail protein comprising a base plate attachment region (BPAR) from an R-type hmw bacteriocin tail fiber protein, wherein the BPAR comprises amino acids 1-161 of the N-terminal portion of the R-type bacteriocin tail fiber protein; and a heterologous receptor binding domain (RBD) from a bacteriophage tail protein.
4. The R-type hmw bacteriocin of claim 3 wherein the BPAR comprises all or part of the amino acid sequence of SEQ ID NO:3 and the RBD comprises all or part of the amino acid sequence of SEQ ID NOS:60, 61, 62, 63, 64, 65, or 70.
5. A method of compromising the integrity of a cytoplasmic membrane of a pathogenic bacterium, said method comprising contacting said pathogenic bacterium with the bacteriocin of claim 3 to bind a virulence factor of the bacterium compromising the integrity of the cytoplasmic membrane.
6. The method of claim 5 wherein the pathogenic bacterium is E. coli O157.
7. The method of claim 6 wherein compromising the membrane results in sterilizing the E. coli O157.
8. The method of claim 6 wherein bacteriocin is detectably-labeled and E. coli O157 is detected.
9. The bacteriocin of claim 3 wherein the expressed RBD binds a corresponding receptor on a surface of a bacterial cell which leads to compromising the integrity of a cytoplasmic membrane of said cell.
10. The bacteriocin of claim 3 wherein the expressed RBD binds a virulence or fitness factor on a surface of a bacterial cell.
11. The bacteriocin of claim 3 wherein said BPAR comprises amino acids 1-161, 1-164, 1-169, 1-172, or 1-240 from SEQ ID NO:3.
12. The bacteriocin of claim 3 wherein said RBD is from the C-terminus of a P2, L-413c, PS 17, BPP-1, CTX, VHML, CUS3, epsilon 15 HK620, sf6, ST64T, or phiV10 phage.
13. The bacteriocin of claim 12 wherein the C-terminus of the RBD is from about 347 to about 755 amino acids in length.
14. The bacteriocin of claim 3 wherein the R-type bacteriocin is an R2-type pyocin and the RBD comprises the C-terminal portion of a phiV10 bacteriophage tail spike protein.
15. The bacteriocin of claim 3 wherein the RBD comprises amino acids 204-875, 211-875, or 217-875 from SEQ ID NO:60.
16. The bacteriocin of claim 9 wherein the bacterial cell is E. coli O157.
17. The bacteriocin of claim 12 wherein the RBD comprises amino acids 158-669 or amino acids 322-669 from SEQ ID NO:25; or amino acids 158-913 from SEQ ID NO:28.
18. The protein of claim 1 wherein said BPAR comprises amino acids 1-161, 1-164, 1-169, 1-172, or 1-240 from SEQ ID NO:3.
19. The protein of claim 1 wherein said RBD is from the C-terminus of a tail fiber protein of P2, L-413c, PS 17, BPP-1, CTX, VHML, CUS3, epsilon15, HK620, sf6, ST64T, or phiV10 phage.
20. The protein of claim 19 wherein the RBD comprises amino acids 204-875, 211-875 or 217-875 from SEQ ID NO:60.
21. The protein of claim 1 wherein the R-type hmw bacteriocin is an R2-type pyocin and the RBD comprises the C-terminal portion of a phiV10 bacteriophage tail spike protein.
22. The protein of claim 21 wherein the BPAR comprises ammo acids 1-161, 1-164, 1-169, 1-172, or 1-240 from SEQ ID NO: 3, and the RBD comprises amino acids 204-875, 211-874 or 217-875 from SEQ ID NO:60.
23. The protein of claim 1 wherein the BPAR comprises 95% sequence identity to amino acids 1-161, 1-164, 1-169, 1-172, or 1-240 from SEQ ID NO:3.