- Email: [email protected]
Programming Bacteriophages by Swapping Their Specificity Determinants
TIMI 1254 No. of Pages 2
Spotlight
Programming Bacteriophages by Swapping Their Specificity Determinants Moran[1_TD$IF] G. Goren,1,z Ido Yosef,1,z and Udi Qimron1,* Bacteriophages, bacteria's natural enemies, may serve as potent antibacterial agents. Their specificity for certain bacterial sub-species limits their effectiveness, but allows selective targeting of bacteria. Lu and colleagues present a platform for such targeting through alteration of bacteriophages’ host specificity by swapping specificity domains in their host-recognition ligand. Bacteriophages are viruses that propagate in bacteria and usually kill them. Ever since their discovery a century ago, bacteriophages have been considered potential tools in the fight against bacterial pathogens. Nevertheless, there are several major barriers to their use: poor accessibility to the infected tissue, sequestration of the phage by the spleen and liver, neutralization by antibodies, evolution of resistant bacteria, and narrow host range [1]. With respect to phages, narrow host range means infection of only a limited number of hosts. This specific drawback, however, is an advantage in the sense that phages can be programmed to kill specific bacteria with minimal disturbance to the natural microbial flora. Data collected from the recent Human Microbiome Project initiative, which studied the components of the bacterial flora during health and disease, suggest that manipulating bacterial communities of the microbiome may affect health, immunity, and
nutritional states of individuals [2]. Due to their specificity, bacteriophages may serve as an efficient tool in shifting the microbiome balance toward a desired composition by selectively killing specific bacterial species. Thus, control of bacteriophage specificity is expected to be highly valuable for extending their pathogen-killing efficiency and selectively shaping desired bacterial populations. Lu and colleagues used an elegant platform for engineering bacteriophages to change their host specificity [3]. Instead of engineering the phages in their natural host, they cloned the phage's DNA in yeast. In contrast to natural hosts, yeast can maintain the entire genome of the phage since they typically tolerate phage-derived gene products. In addition, since phage propagation is not a prerequisite for the process, manipulated phage DNA can replicate in yeast even if the yeast lack genes that are essential for propagation in its natural host. To clone the entire phage genome into yeast, phage DNA was either synthesized de novo from several PCR fragments that were consequently ligated in the correct order, or supplied as an extracted genome. The natural or synthetic phage genome was then ligated into a yeast artificial chromosome (YAC), allowing further manipulations. The manipulated genome, encoded by the YAC, was then extracted from the yeast and transformed into bacteria that support the production of phage components and their assembly into a functional phage. Since the assembled phages usually cannot propagate in these bacteria, they are thereafter propagated in their natural host. Using this technology, Lu and colleagues initially constructed eight different phages, infecting four different host species. They later swapped the tail and tail-fiber genes of the phages encoding the proteins that mediate the recognition of a specific host strain. Phages engineered with tails encoded by phages with different host specificity acquired the ability to recognize and infect new hosts (Figure 1). The authors also showed that even a partial domain of
the tail fiber alone can determine specificity changes upon swapping. Specifically, the authors showed that bacteriophage T7 gains the ability to infect Escherichia coli strains that are normally infected only by bacteriophages T3 or 13a upon swapping their tail-fiber-encoding genes, or even the partial gene encoding the C-terminal domain. Furthermore, the swapping enabled overcoming species barriers. For example, swapping the tail-fiber-encoding gene of T3 with that of Yersinia phage R expanded T3's ability to infect Yersinia pseudotuberculosis, in addition to its ability to infect its natural host E. coli. Finally, the authors showed that use of engineered bacteriophages with swapped receptors can specifically eliminate unwanted bacterial species within a mixed bacterial population with a minimal effect on adjacent species. A platform for phage and bacterial engineering had been demonstrated previously [4,5]. Extension of phage host range by isolating mutant phages with altered ligand-recognition properties, crossing species barriers, had also been shown previously [6]. However, combining the powerful engineering technologies to carry out specificity swapping between phages by complete swapping of entire sets of proteins is novel. Demonstrating the modularity of tail and tail-fiber proteins from various members of the T7 phage group and their ability to be assembled and function together despite significant sequence divergence is a significant achievement. These studies should make it easier to obtain regulatory approval for phages used in therapeutics or bacterial population editing. Typically, a cocktail of phages is required for efficient treatment (e.g[6_TD$IF]., [7]). Each phage of the cocktail must undergo strict regulatory approval that requires labor and associated costs. The new study should allow the use of a single phage with many tail variations, thus simplifying the approval process for various applications and allowing easy adaptation
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1254 No. of Pages 2
CRISPR-Cas system has also been recently used for eliminating and selecting desired populations in bacterial mixtures [8–10]. Combining the specificity of the CRISPR-Cas system with that of phages may prove to be synergistic in shaping bacterial populations. Department [5_TD$IF]of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv
1
69978, Israel z
Equal contribution.
*Correspondence: [email protected] (U. Qimron). http://dx.doi.org/10.1016/j.tim.2015.10.006 References 1. Lu, T.K. and Koeris, M.S. (2011) The next generation of bacteriophage therapy. Curr. Opin. Microbiol. 14, 524–531
Figure 1. Swapping Tail-fiber Domains of Phages Results in Altered Phage Specificity. DNA encoding a specificity domain in the tail fiber enables phages to recognize and infect a new host. DNA encoding the ligand as well as matching receptor-ligand pairs are colored green, red, brown, and blue.
2. Round, J.L. and Mazmanian, S.K. (2009) The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323 3. Ando, H. et al. (2015) Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems 1, 187–196
of an already approved phage for alterna- However, most bacteriophages do not encode their own RNA polymerase, and tive applications. depend on their host's genes. Thus, the This study proved to be efficient on vari- proof of concept presented here is limited ous phages of the T7 group. Following to the repertoire of hosts infected by the transformation of the YAC encoding the T7 group and, for example, exclude the desired phage DNA, phage capsids were entire group of Gram-positive bacteria [6]. produced in the E. coli host, even for Further work is required to extend the phages whose natural host is not E. coli. study to a variety of bacteria that are This ability to produce infective particles in infected by a single phage scaffold with a non-host strain is due mainly to the fact alternative specificity domains. that members of the T7 group are almost independent of their host's genes. Most of The timeliness of the study is reflected in the required components for infective par- the recent information gained from microticles, including their own RNA polymer- biome studies suggesting ways to shape ase, are encoded in their genome. the microbiome, and the fact that the
2
Trends in Microbiology, Month Year, Vol. xx, No. yy
4. Gibson, D.G. et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 5. Jaschke, P.R. et al. (2012) A fully decompressed synthetic bacteriophage oX174 genome assembled and archived in yeast. Virology 434, 278–284 6. Molineux, I.J. (2005) The T7 Group. In The Bacteriophages (Abedon, S.T. and Calendar, R.L., eds), pp. 275–299, Oxford University Press 7. Abuladze, T. et al. (2008) Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl. Environ. Microbiol. 74, 6230–6238 8. Bikard, D. et al. (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 9. Yosef, I. et al. (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc. Natl. Acad. Sci. U.S.A. 112, 7267–7272 10. Citorik, R.J. et al. (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145
Spotlight
Programming Bacteriophages by Swapping Their Specificity Determinants Moran[1_TD$IF] G. Goren,1,z Ido Yosef,1,z and Udi Qimron1,* Bacteriophages, bacteria's natural enemies, may serve as potent antibacterial agents. Their specificity for certain bacterial sub-species limits their effectiveness, but allows selective targeting of bacteria. Lu and colleagues present a platform for such targeting through alteration of bacteriophages’ host specificity by swapping specificity domains in their host-recognition ligand. Bacteriophages are viruses that propagate in bacteria and usually kill them. Ever since their discovery a century ago, bacteriophages have been considered potential tools in the fight against bacterial pathogens. Nevertheless, there are several major barriers to their use: poor accessibility to the infected tissue, sequestration of the phage by the spleen and liver, neutralization by antibodies, evolution of resistant bacteria, and narrow host range [1]. With respect to phages, narrow host range means infection of only a limited number of hosts. This specific drawback, however, is an advantage in the sense that phages can be programmed to kill specific bacteria with minimal disturbance to the natural microbial flora. Data collected from the recent Human Microbiome Project initiative, which studied the components of the bacterial flora during health and disease, suggest that manipulating bacterial communities of the microbiome may affect health, immunity, and
nutritional states of individuals [2]. Due to their specificity, bacteriophages may serve as an efficient tool in shifting the microbiome balance toward a desired composition by selectively killing specific bacterial species. Thus, control of bacteriophage specificity is expected to be highly valuable for extending their pathogen-killing efficiency and selectively shaping desired bacterial populations. Lu and colleagues used an elegant platform for engineering bacteriophages to change their host specificity [3]. Instead of engineering the phages in their natural host, they cloned the phage's DNA in yeast. In contrast to natural hosts, yeast can maintain the entire genome of the phage since they typically tolerate phage-derived gene products. In addition, since phage propagation is not a prerequisite for the process, manipulated phage DNA can replicate in yeast even if the yeast lack genes that are essential for propagation in its natural host. To clone the entire phage genome into yeast, phage DNA was either synthesized de novo from several PCR fragments that were consequently ligated in the correct order, or supplied as an extracted genome. The natural or synthetic phage genome was then ligated into a yeast artificial chromosome (YAC), allowing further manipulations. The manipulated genome, encoded by the YAC, was then extracted from the yeast and transformed into bacteria that support the production of phage components and their assembly into a functional phage. Since the assembled phages usually cannot propagate in these bacteria, they are thereafter propagated in their natural host. Using this technology, Lu and colleagues initially constructed eight different phages, infecting four different host species. They later swapped the tail and tail-fiber genes of the phages encoding the proteins that mediate the recognition of a specific host strain. Phages engineered with tails encoded by phages with different host specificity acquired the ability to recognize and infect new hosts (Figure 1). The authors also showed that even a partial domain of
the tail fiber alone can determine specificity changes upon swapping. Specifically, the authors showed that bacteriophage T7 gains the ability to infect Escherichia coli strains that are normally infected only by bacteriophages T3 or 13a upon swapping their tail-fiber-encoding genes, or even the partial gene encoding the C-terminal domain. Furthermore, the swapping enabled overcoming species barriers. For example, swapping the tail-fiber-encoding gene of T3 with that of Yersinia phage R expanded T3's ability to infect Yersinia pseudotuberculosis, in addition to its ability to infect its natural host E. coli. Finally, the authors showed that use of engineered bacteriophages with swapped receptors can specifically eliminate unwanted bacterial species within a mixed bacterial population with a minimal effect on adjacent species. A platform for phage and bacterial engineering had been demonstrated previously [4,5]. Extension of phage host range by isolating mutant phages with altered ligand-recognition properties, crossing species barriers, had also been shown previously [6]. However, combining the powerful engineering technologies to carry out specificity swapping between phages by complete swapping of entire sets of proteins is novel. Demonstrating the modularity of tail and tail-fiber proteins from various members of the T7 phage group and their ability to be assembled and function together despite significant sequence divergence is a significant achievement. These studies should make it easier to obtain regulatory approval for phages used in therapeutics or bacterial population editing. Typically, a cocktail of phages is required for efficient treatment (e.g[6_TD$IF]., [7]). Each phage of the cocktail must undergo strict regulatory approval that requires labor and associated costs. The new study should allow the use of a single phage with many tail variations, thus simplifying the approval process for various applications and allowing easy adaptation
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1254 No. of Pages 2
CRISPR-Cas system has also been recently used for eliminating and selecting desired populations in bacterial mixtures [8–10]. Combining the specificity of the CRISPR-Cas system with that of phages may prove to be synergistic in shaping bacterial populations. Department [5_TD$IF]of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv
1
69978, Israel z
Equal contribution.
*Correspondence: [email protected] (U. Qimron). http://dx.doi.org/10.1016/j.tim.2015.10.006 References 1. Lu, T.K. and Koeris, M.S. (2011) The next generation of bacteriophage therapy. Curr. Opin. Microbiol. 14, 524–531
Figure 1. Swapping Tail-fiber Domains of Phages Results in Altered Phage Specificity. DNA encoding a specificity domain in the tail fiber enables phages to recognize and infect a new host. DNA encoding the ligand as well as matching receptor-ligand pairs are colored green, red, brown, and blue.
2. Round, J.L. and Mazmanian, S.K. (2009) The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323 3. Ando, H. et al. (2015) Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems 1, 187–196
of an already approved phage for alterna- However, most bacteriophages do not encode their own RNA polymerase, and tive applications. depend on their host's genes. Thus, the This study proved to be efficient on vari- proof of concept presented here is limited ous phages of the T7 group. Following to the repertoire of hosts infected by the transformation of the YAC encoding the T7 group and, for example, exclude the desired phage DNA, phage capsids were entire group of Gram-positive bacteria [6]. produced in the E. coli host, even for Further work is required to extend the phages whose natural host is not E. coli. study to a variety of bacteria that are This ability to produce infective particles in infected by a single phage scaffold with a non-host strain is due mainly to the fact alternative specificity domains. that members of the T7 group are almost independent of their host's genes. Most of The timeliness of the study is reflected in the required components for infective par- the recent information gained from microticles, including their own RNA polymer- biome studies suggesting ways to shape ase, are encoded in their genome. the microbiome, and the fact that the
2
Trends in Microbiology, Month Year, Vol. xx, No. yy
4. Gibson, D.G. et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 5. Jaschke, P.R. et al. (2012) A fully decompressed synthetic bacteriophage oX174 genome assembled and archived in yeast. Virology 434, 278–284 6. Molineux, I.J. (2005) The T7 Group. In The Bacteriophages (Abedon, S.T. and Calendar, R.L., eds), pp. 275–299, Oxford University Press 7. Abuladze, T. et al. (2008) Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl. Environ. Microbiol. 74, 6230–6238 8. Bikard, D. et al. (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 9. Yosef, I. et al. (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc. Natl. Acad. Sci. U.S.A. 112, 7267–7272 10. Citorik, R.J. et al. (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145