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![• Phages are used not only to treat and prevent human bacterial infections but also
to control plant diseases, detect pathogens and assess food safety
• Like certain antibiotics, phages can cause rapid and massive bacterial lysis via
release of lipopolysaccharides [LPS], which induce adverse immune responses in
the human host
• Bacteria frequently live in biofilm communities surrounded by extracellular
polymeric substances (EPS), which act as a barrier to phage penetration.
• By genetically engineering phages, it may be possible to overcome many of
these limitations.](https://image.slidesharecdn.com/phageengineering-231215183943-8b0055bf/85/Phage-engineering-pptx-5-320.jpg)
Phage engineering.pptx
- 2. Phage Engineering for Antimicrobials Dr.J Sai Prasad Assistant Professor Agricultural Microbiology Professor Jayashankar Telangana State Agricultural University, Hyderabad. T.S.
- 3. Contents • Introduction • Techniquesfor Engineering Phages • Synthetic Phages for pathogen control • Phage derived antimicrobials • Drug delivery system by engineered Phages • Conclusion • Future perspectives
- 4. Introduction • Bacteriophages (phages)the most abundant biological particles on earth. Highly versatile and potential for various applications. • Phages are viruses that infect bacteria; their self-replication depends on access to a bacterial host having additional advantages for their use as antimicrobials. • Discovered independently by Frederick Twort in 1915 and by Félix d’Hérelle in 1917 and used early on as antimicrobial agents. • The rising tide of antibiotic resistance has revived interest in phages as antibacterial agents. • Unlike most antibiotics, phages are typically highly specific for a particular bacterial species or strains and thus expected to target effects on commensal microflora Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 5. • Phages areused not only to treat and prevent human bacterial infections but also to control plant diseases, detect pathogens and assess food safety • Like certain antibiotics, phages can cause rapid and massive bacterial lysis via release of lipopolysaccharides [LPS], which induce adverse immune responses in the human host • Bacteria frequently live in biofilm communities surrounded by extracellular polymeric substances (EPS), which act as a barrier to phage penetration. • By genetically engineering phages, it may be possible to overcome many of these limitations.
- 6.
- 7. • Homologous recombination isa natural phenomenon. • A reporter gene, usually encoding luciferase or a fluorescent protein, is commonly cloned along with the gene of interest to facilitate the identification of mutant phages by detecting the reporter. Homologous Recombination Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 8. Bacteriophage Recombineering ofElectroporated DNA • Co-electroporating the recombineering substrates, i.e., phage DNA and dsDNA, into electrocompetent bacterial cells carrying a plasmid that encodes proteins promoting high levels of homologous recombination, such as the RecE/RecT-like proteins. Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 9. In Vivo Recombineering •Bacterial cells carrying a defective prophage and the pL operon under the control of a temperature-sensitive repressor (A) are infected with the phage to be manipulated (B) and subsequently transformed with dsDNA or ssDNA (C). • Following phage infection, the recombination functions are induced by heating the mid- log-phase bacterial culture to 42°C. Recombination then occurs (D), after which recombinant phage particles are recovered (E). Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 10. Rebuilding/Refactoring Phage GenomesIn Vitro • Phage genomes can be manipulated and edited in vitro before they are introduced into their bacterial hosts. • Once the phage DNA has been purified (A), it is digested using native restriction sites (B), and independent pieces (C) can be subcloned and further manipulated (D). Once released from the vector, the recombinant section is ligated to the rest of the phage genome (E) and electroporated into the phage host for recovery of engineered phage particles (F). Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 11. Yeast-Based Assembly ofPhage Genomes • Propagating phage genomes in a bacterial host can be toxic for the host, thus limiting the efficiency of phage genome engineering. It can overcome by using Saccharomyces cerevisiae • Purified phage DNA (A) is electroporated into S. cerevisiae together with linear YAC molecules (B). Recombination in the yeast cell enables genomic subcloning (YAC backbone in green) (C), which upon YAC purification and electroporation (D) allows the recovery of functional phage particles in bacteria(E). Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 12. Cell-Free Transcription-Translation Systems •Advantages of using in vitro or yeast-based genome modification is that phage genomes can be engineered without causing toxicity to the host. • Purified phage genome DNA is combined with cell-free expression systems (A) that enable gene transcription (B), translation (C), DNA replication (D), and assembly of whole phage particles (E). Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 13. SYNTHETIC PHAGES FORPATHOGEN CONTROL Natural Phage-Based Antimicrobials Sl No Name Mechanism Antimicrobial / control org. 1 PhagoBurn, a clinical trial by the Rose. T et al., (2014) Normal phages have shown no adverse effects related to its application Evaluate phage cocktail for the treatment of E. coli and P. aeruginosa infections in burn wound patients 2 Bruttin and Brüssow (2005) Treated E. coli T4 phage orally to 5 human volunteers. Controls the E. coli diarrheal infections. No adverse effects observed in a safety study. Rose. T et al, (2014) & Bruttin and Brüssow (2005)
- 14. Engineered Phages forEnhanced Antibacterial Activity for individual pathogen Sl no Name Mechanism Antimicrobial / control org. 1 Edgar et al. (2012) Engineered temperate phages to deliver genes encoding rpsL and gyrA, which confer sensitivity to streptomycin and nalidixic acid antibiotics into bacteria respectively E. coli K-12 resistant to these antibiotics were then lysogenized with the engineered phages (carrying rpsL or gyrA), and restores antibiotic efficiency by reversing pathogen resistance. Edgar et al. (2012), Appl Environ Microbiol 78:744–751.
- 15. Engineered Phages withShifted or Broadened Host Ranges Sl n Name Mechanism Antimicrobial / control org. 1 Lin T-Y et al., A hybrid T3 and T7 phage (T3/7) was devised, in which part of the tail fiber gene of T3 (gp17) was replaced with that of phage T7. The T3/7 recombinant phage exhibited a broader host range and a better adsorption efficiency than the wildtype phages, i.e., T3 and T7 individually 2 Marzari et al. Filamentous coliphage-fd by adding a receptor-binding domain from the filamentous phage IKe. Coliphage fd, which normally infects E. coli, was also engineered to recognize Vibrio cholerae. Lin T-Y et al., (2012) & Marzari et al. (1997)
- 16. PHAGE-DERIVED ANTIMICROBIALS Lytic enzyme Model Targetpathogens Result summary Phage- derived lysins ABgp46 In vitro MDR A. baumannii, P. aeruginosa, and S.typhimurium Cross-inoculation significantly reduced bacterial density PlyCD In vitro Clostridium difficile Reduced C. difficile colonization PlyG In vitro Bacillus anthracis Eliminated B. anthracis spores and vegetative cells PlyF307 Murine MDR A. baumannii i.p. treatment rescued mice from lethal bacteremia PlySs2 Murine Streptococcus pyogenes and MRSA i.p. treatment reduced mortality from lethal bacteremia Cpl-1 Murine Streptococcus pneumoniae i.p. treatment rescued mice from lethal pneumonia Cocktail 6 In vitro, murine MRSA Effective against biofilms in vitro and protected mice from lethal sepsis Lin DM et al . World J Gastrointest Pharmacol Ther 2017; 8(3): 162-173
- 17. Lytic enzyme Model Target pathogens Result summary Bioeng. chimlysins CHAPKIn vitro MRSA Eliminated MRSA and dispersed biofilms ClyH Murine MRSA Treatment rescued mice from bacteremia Cpl-711 Murine S. pneumoniae Treatment rescued mice from bacteremia Ply187 Murine Staphylococcu s aureus Prevented bacterial endophthalmitis Lysin & antibiotic therapy CF-301 Murine MRSA Lysin treatment was most effective when combined with vancomycin or daptomycin MR-10 Murine Burn wound infection Lysin treatment was most effective when combined with minocycline Lin DM et al . World J Gastrointest Pharmacol Ther 2017; 8(3): 162-173
- 18. Sn Engineered phagesDrug activi Antimicrobial / control org. 1 Yacoby I. et al., (2006) used filamentous phages (fd and M13) to target S. aureus by target-specific peptides on the major coat protein Enhancing Antibiotic Activity Chemically conjugated the phages with chloramphenicol, bound to the target cells and chloramphenicol was released, retarding bacterial growth 2 Bar H et al., (2008) used genetically modified fUSE5-ZZ phages to display a ligand that leads to the target cancer cells and then loaded with cytotoxic drugs. Delivery of Anticancer Drugs The drug-carrying phages targeted ErbB2-overexpressing human breast adenocarcinoma. Once endocytosed, the phages releasing the drug inside the cancer cells and resulting in about 50% inhibition of target cell, a 1,000-fold improvement of hygromycin. Drug Delivery Systems by Engineered Phages Yacoby I. et al. (2006) & Bar H etal., (2008)
- 19. Published findings onphage therapy in humans and in animal models Causative agent Model Condition Oral Result summary Shigella dysenteriae Human Dysentery Oral All four treated individuals recovered after 24 h Vibrio cholerae Human Cholera Oral 68 of 73 survived in treatment group and only 44 of 118 in control group MDR S. aureus Human Diabetic foot ulcer Topical All 6 treated patients recovered P. aeruginosa Murine Sepsis Oral 66.7% reduced mortality S. aureus Rabbit Wound infection S.C. Co-administration with S. aureus prevented infection Lin DM et al . World J Gastrointest Pharmacol Ther 2017; 8(3): 162-173
- 20. Phage products Sl no.Phage products Control organisms 1 List shield, Ecoshield and SalmoFresh from Intralytix Listeria monocytogenes, E. coli O157:H7, and S. enterica in foods or food-processing environments. 2 Salmonelex and Listex P100 Salmonella and L. monocytogenes, reduce contamination during food processing 3 AgriPhage from OmniLytics Xanthomonas campestris and Pseudomonas syringae on tomato and pepper plants Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543
- 21. Conclusion of theSeminar • The prodigious diversity of phages has led to powerful applications for therapeutics, diagnostics, materials science, drug delivery systems and vaccines • The new genetic engineering technologies has led to a more precise and accelerated modification of phage genomes. • The use of phages and derived proteins for combating bacterial infections, specifically for multidrug-resistant bacteria, shows phage therapy as either an alternative or a supplement to antibiotics. • Furthermore, techniques to contain the use of genetically modified phages for materials science applications and to inactivate the phages after use may also help to mitigate these issues • Phage engineering is an area of research that is attracting intense interest and has great potential utility, but it has yet to be fully exploited.
- 22. Future Perspectives • Engineeredphage for single homogenous biofilm control technology could be expanded to target the heterogeneous extracellular composition of biofilms • Many strategies for engineering phages require the ability to genetically modify the bacterial hosts, which is still a challenge for many bacterial species. • Despite the potential benefits, the acceptance of genetically modified phages for real-world applications may vary across different regions of the world. • In the case of human use, the choice of compelling areas of tremendous medical need and explicit demonstrations of safety will both be important.
Editor's Notes
- #8 Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA
- #9 double-stranded-break repair initiated by RecE/RecT and Redα/Redβ. First, RecE or Redα degrades the DNA in a 5′–3′ direction, starting from the DSB, thereby creating a 3′ ssDNA overhang. Then, RecT or Redβ binds to the ssDNA, forming a recombinogenic proteonucleic filament which is used in recombination, either by single strand annealing or by strand invasion.
- #10 The bacterial RNA polymerase (Pol) binds and transcribes from the early promoters pL and pR. RNA Pol transcribes as far as the transcription terminators tL1 and tR1 beyond pL and pR, respectively
- #13 Cell-free transcription-translation systems offer a potential solution to this problem. For example, such systems have been used to replicate, synthesize, and assemble the T7 phage genome (Fig. 8) (87). In this case, as little as 1 nM phage genomic DNA, combined with a TX-TL cell-free system prepared from E. coli BL21 Rosetta2, resulted in the assembly of approximately 0.1 to 1 billion infectious T7 phage particles/ml of reaction mixture within a few hours of incubation
- #14 Phage therapy pharmacology phage cocktails. Phagoburn is a current European Union financed clinical study focused on testing the medical uses of bacteriophage for treating wounds. The main objective of Phagoburn is to assess the safety, effectiveness and pharmacodynamics of two therapeutic phage cocktails to treat E. coli and P. aeruginosa burn wound infections.
- #15 mutations of rpsL and gyrA in drug-resistant
- #16 gp 17 - Gp 17 - Enterobacteria phage T7M - gp 17 gene & protein extending filamentous phage host range by the grafting of a heterologous receptor binding domain. ... fd and IKe are two similar filamentous phage which infect their hosts by means of pili found on the host membrane: fd infects bacteria bearing F pili, whereas IKe infects bacteria bearing N or I pili.
- #17 MDR- multi drug resistant, Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes infections in different parts of the body. It's tougher to treat than most strains of staphylococcus aureus -- or staph -- because it's resistant to some commonly used antibiotics, Endolysin PlyF307 derived from an A. baumannii prophage is effective against exponentially growing A. baumannii cells Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. ... Streptococcus pneumoniae Streptococcus suis-PlySs2 PlyG by screening a library of proteins from the gammaphage, a member of a family of double-stranded DNA phage associated with B anthracis
- #20 Infectious ileocecitis--appendicitis mimicking syndrome. Subcutaneous-SC
- #21 Intralytix is a biotechnology company based in Baltimore, Maryland. Intralytixspecializes in bacteriophage-based products used to control bacterial pathogens in environmental, food processing, and medical settings. List shield, Ecoshield and SalmoFresh- 100% natural, safe, and effective products for reducing contamination of various foods Baltimore, Maryland, U.S.A. - have received GRAS Very positive' following a European Food Safety Authority (EFSA) opinion on its technology for Listeria monocytogenes in Ready to Eat (RTE) foods. Safety and efficacy of Listex P100 used during processing of meat and poultry, fish and shellfish and dairy products was assessed. AgriPhage from OmniLytics- U.S.A.