Bacteriophage and phage therapy

1. Bacteriophage and phage therapy

2. Bacteriophage • Phage therapy

3. Bacteriophage: basic definition • Viruses that infect bacteria and require bacterial host for replication • Obligate intracellular parasites that consist of a protein coat surrounding a central nucleic acid core • Considerable diversity exists among bacterial viruses in both physical structure and nucleic acid content

4. 肌尾病毒科 丝状病毒科 短杆状噬菌体属 长尾病毒科 Bacteriophages are diverse, But, most phages have DNA genomes 短尾病毒科 滴状病毒科 脂毛噬菌体科 丝状病毒科 被盖病毒科 芽生病毒科 福塞尔噬菌体科 覆盖层病毒科 古噬菌体科 轻小病毒科 囊状病毒科

5. Basic anatomy(解剖) of a phage Binary symmetry Tailed phage Tadpole like Icosahedral symmetry Helical symmetry 基片 T2 bacteriophage

6. Bacteriophage: the basics • Tend to have large genomes and complex structure • Most phage are dsDNA viruses but some are ssDNA, ssRNA, or dsRNA • Phage head (protein) contains nucleic acid • Viral ligand配体 that interacts with bacterial receptor located on tail fibers • Sheath (hollow) contracts收缩 in some phage during binding to bacterial cell • Most infect by drilling a hole through peptidoglycan and injecting DNA directly into bacterial cell while the rest of the virus stays extracellular

7. Steps in phage replication • Infection • Adsorption of the phage to the bacterial cells • Sheath contraction • Nucleic acid injection • Phage multiplication: • Lytic (replication culminates最终 in lysing and thus killing bacterium) • Lysogenic/temperate (quiescent state in the bacterium such that host is unharmed)

8. Lysogenic Pathway Lytic Pathway Replication of phage DNA and Protein Integration (prophage) Assembly Lysis & Release

10. （1）The T4 infectious cycle

11. T4 DNA characteristics • 169 kb double stranded, linear molecule (genome 166 kb pairs) • Encodes 144 genes • Terminally redundant - 3 kb pairs (2% at left end repeated at the right end) • Circularly permuted置换(circular DNA cut at different points yields linear DNA, different permutations排列) • Replication forms concatemers多联体 • Tandemly串联 linked genome copies

12. Adsorption • DNA injection Energy independent These steps require energy

13. Absorption/Attachment • Tail fibers attach to specific receptors on the bacterial cell (receptor may be protein, LPS, lipoprotein)

14. Sheath contraction and nucleic acid injection • Rest of phage remains extracellular, only nucleic acid enters bacterium

15. Adsorption • DNA injection • Synthesis of early mRNA Transcribed using host RNA polymerase, new proteins include modifiers of transcriptional specificity, especially, a new sigma factor (s), that recognizes only phage promoters Other early genes encode proteins required to take over host cell and synthesize viral nucleic acids

16. Adsorption • DNA injection • Synthesis of early mRNA • Degradation of host DNA Phage DNA is protected from degradation by incorporation of hydroxymethyl cytosine (HMC) 5-羟甲基胞嘧啶, and glucosylation糖基化. Phage DNA is protected from degradation by viral nucleases and host restriction enzymes.

17. Assembly

19. Lysis and release phase Eclipse phase Intracellular accumulation phase Host cell lysis and release of ~300 virions Involves holin穿孔素and endolysin内溶素

21. Overview of Gene Regulation during Phage Infection • Early genes are expressed that encode regulatory proteins and proteins involved in DNA metabolism. • Later gene expression involves synthesis of proteins that play a structural or packaging role. • Synthesis of proteins that help to release the phage from the cell.

22. Lysogeny • Temperate phages can convert host to lysogen • Establishment likely with high level of infection of bacterial culture, starvation • Stable association of phage DNA with bacterial cell (prophage) • Normal growth and division of lysogen • Environmental cues （诱导）may induce lytic cycle

24. Phage can transfer new functions to a bacterial cell • Confer antibiotic-resistance • Allow bacteria to use certain nutrients • Allow bacteria to colonize new environment • Increase pathogenic potential of bacteria

25. Transduction • The transfer of viral or bacterial, or both, DNA from one cell to another via bacteriophage

26. Mechanisms of transduction • Bacterial gene transfer via a virus particle: • During assembly stage of lytic replication, DNA fragments from host genome are packaged instead of phage DNA GENERALIZED • During excision切除 of prophage, fragment of chromosomal DNA can be excised with phage DNA SPECIALIZED

27. Lysogenic conversion: Prophage genes (inserted into chromosome) can increase the survival fitness of the host cell in particular situations: • Phage repressor and super-infection exclusion provide immunity to the host against lytic infection • Can convert non-pathogenic bacterial strains into pathogenic ones (encode toxins, for example)

28. Bacterial gene transfer During lytic replication (accidental packaging of chromosomal DNA): During switch from lysogenic state to lytic replication (improper excision from chromosome) :

29. leu+ leu+ leu+ leu- leu+ Conversion of a leu(-) strain to leu(+) by transduction Gain of nutrient utilization gene leusine

30. Mechanisms of transduction • Bacterial gene transfer: • During assembly stage of lytic replication, DNA fragments from host genome are packaged instead of phage DNA • During excision of prophage, chromosomal DNA can be excised with phage DNA

31. Ⅱ. Phage therapy: alternative to antibiotics 替代抗生素吗？

32. Phage therapy • The therapeutic use of bacteriophages to treat pathogenic bacterial infections. Although extensively used and developed mainly in former Soviet Union countries for about 90 years, this method of therapy is still being tested elsewhere for treatment of a variety of bacterial and poly-microbial biofilm infections, and has not yet been approved in countries other than Georgia. Phage therapy has many potential applications in human medicine as well as dentistry牙科, veterinary science兽医, and agriculture.

33. Phages have been investigated as a potential means to eliminate pathogens like Campylobacter弯曲杆菌 in raw food and Listeria in fresh food or to reduce food spoilage bacteria. • In agricultural practice phages were used to fight pathogens like Campylobacter, Escherichia and Salmonella in farm animals, Lactococcus and Vibrio pathogens in fish from aquaculture and Erwinia欧文氏菌 and Xanthomonas in plants of agricultural importance. • The oldest use was, however, in human medicine. Phages have been used against diarrheal diseases 腹泻caused by E. coli, Shigella or Vibrio and against wound infections caused by facultative pathogens of the skin like staphylococci and streptococci. • Recently the phage therapy approach has been applied to systemic and even intracellular infections and the addition of non-replicating phage and isolated phage enzymes like to the antimicrobial arsenal（兵工厂，库）. However, actual proof for the efficiency of these phage approaches in the field or the hospital is not available.

34. Some of the interest in the West can be traced back to 1994, when Soothill demonstrated (in an animal model) that the use of phages could improve the success of skin grafts移植 by reducing the underlying Pseudomonas aeruginosa绿脓假单胞菌 infection. Recent studies have provided additional support for these findings in the model system. • Although not "phage therapy" in the original sense, the use of phages as delivery mechanisms for traditional antibiotics constitutes another possible therapeutic use. The use of phages to deliver antitumor agents has also been described, in preliminary in vitro experiments for cells in tissue culture.

35. Should we be worried about antibiotic-resistant microbes? • Absolutely! In 2000, an interagency task force including members from FDA, CDC, and NIH released this statement: • “The world may soon be faced with previously treatable diseases that have again become untreatable, as in the pre-antibiotic era.”

36. Bacterial infections are responsible for significant morbidity发病and mortality 死亡in clinical settings. Many infections that would have been cured easily by antibiotics in the past now are resistant, resulting in sicker patients and longer hospitalizations . The economic impact of antibiotic-resistant infections is estimated to be between $5 billion and $24 billion per year in the United States. Antibiotic resistance can be acquired genetically (e.g., via mutations in antibiotic targets) or result from persistence, in which a small fraction of cells in a population exhibits a non-inherited, phenotypic tolerance to antimicrobials.

37. Why use bacteriophage as antibiotic replacement? • Harness the potential of phage to quickly lyse/kill bacterial host • Once it infects its target, it quickly (about 30 min) generates 100s of copies of itself that search out additional microbes to kill • Because phages are ‘living’ organisms (unlike antibiotics), they can evolve as the bacteria evolve; so as bacteria evolve to resist phage, the phage evolves too • Infect only bacteria so they are harmless to mammalian cells and to non-target bacteria (unlike antibiotics), which leaves beneficial gut flora intact

38. Phage can lyse bacteria or be modified to express lethal genes to cause cell death • 1. Hagens S, BlasiU(2003) Genetically modified filamentous phage as bactericidal agents: A pilot study. Letters in Applied Microbiology 37(4):318–323. • 2. Hagens S, Habel AvAU, von Gabain A, Blasi U (2004) Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage. Antimicrob Agents Chemother 48(10):3817–3822. • 3. Westwater C, et al. (2003) Use of genetically engineered phage to deliver antimicrobial agents to bacteria: An alternative therapy for treatment of bacterial infections. Antimicrob Agents Chemother 47(4):1301–1307. • 4. Heitman J, Fulford W, Model P (1989) Phage Trojan horses: A conditional expression system for lethal genes. Gene 85(1):193–197. • 5. Brussow H (2005) Phage therapy: The Escherichia coli experience. Microbiology 151(Pt 7):2133–2140.

39. Potential benefits • Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. • It is conceivable that, although bacteria rapidly develop resistance to phage, the resistance might beeasier to overcome than resistance to antibiotics. • Bacteriophages are veryspecific, targeting only one or a few strains of bacteria. Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The specificity of bacteriophages might reduce the chance that useful bacteria are killed when fighting an infection.

40. Some evidence shows the ability of phages to travel to a required site — including the brain, where the blood brain barrier can be crossed — and multiply in the presence of an appropriate bacterial host, to combat infections such as meningitis脑膜炎. However the patient's immune system can, in some cases mount an immune response to the phage (2 out of 44 patients in a Polish trial).

41. A few research groups in the West are engineering a broader spectrum phage and also target MRSA treatments in a variety of forms - including impregnated wound dressings血衣, preventative treatment for burn victims, phage-impregnated sutures手术线. • Enzobiotics are a new development at Rockefeller University that create enzymes from phage. These show potential for preventing secondary bacterial infections e.g. pneumonia developing with patients suffering from flu, otitis中耳炎 etc.. Purified recombinant phage enzymes can be used as separate antibacterial agents in their own right. • Some bacteria such as multiple resistant Klebsiella pneumoniae have no non toxic antibiotics available, and yet killing of the bacteria via intraperitoneal腹腔, intravenous静脉 or intranasal鼻内 of phages in vivo has been shown to work in laboratory tests.

42. Disadvantages • Phages must be refrigerated until used, and a physician wishing to prescribe them needs special training in order to correctly prescribe and use phages. • Phages come in a great variety. That diversity becomes a disadvantage when the exact species of an infecting bacteria is unknown or if there is a multiple infection. For best results, the phages should be tested prior to application in the lab. For this reason, phages are less suitable for acute cases. Mixtures consisting of several phages can fight mixed infections.

43. Bacteria can become resistant to treatments, and in this case they can mutate to survive the phage onslaught突击. However, evolution drives the rapid emergence of new phages that can destroy bacteria that have become resistant. This means that there should be an ‘inexhaustible’ supply. • Phages that are injected into the bloodstream are recognized by the human immune system. Some of them are quickly excreted and, after a certain period, antibodies against the phages are produced by the body. For this reason, it appears that one type of phage can only be used once for intravenous treatment.

44. Phage that are directly lethal to their bacterial hosts can select for phage-resistant bacteria in a short time. • 1. Hagens S, BlasiU(2003) Genetically modified filamentous phage as bactericidal agents: A pilot study. Letters in Applied Microbiology 37(4):318–323. • 2. Hagens S, Habel AvAU, von Gabain A, Blasi U (2004) Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage. Antimicrob Agents Chemother 48(10):3817–3822. • 3. Summers WC (2001) Bacteriophage therapy. Annu Rev Microbiol 55:437–451.

45. Combination therapy with different antibiotics, different bacteriophage, or antibiotics plus phage may reduce the incidence of phage resistance and/or antibiotic resistance. • 1. Lu TK, Collins JJ (2007) Dispersing biofilms with engineered enzymatic bacteriophage. Proc Natl Acad Sci USA 104(27):11197–11202. • 2. Bonhoeffer S, Lipsitch M, Levin BR (1997) Evaluating treatment protocols to prevent antibiotic resistance. Proc Natl Acad Sci USA 94(22):12106–12111. • 3. Chait R, Craney A, Kishony R (2007) Antibiotic interactions that select against resistance. Nature 446(7136):668–671. • 4. Levy SB, Marshall B (2004) Antibacterial resistance worldwide: Causes, challenges and responses. Nat Med 10(12 Suppl):S122–S129. • 5. Hagens S, Habel A, Bla¨ si U (2006) Augmentation of the antimicrobial efficacy of antibiotics by filamentous phage. Microbial Drug Resistance (Larchmont, NY) 12(3):164–168.

46. Application • Collection • In its simplest form, phage treatment works by collecting local samples of water likely to contain high quantities of bacteria and bacteriophages, for example effluent outlets, sewage and other sources. They can also be extracted from corpses. The samples are taken and applied to the bacteria that are to be destroyed which have been cultured on growth medium.

47. The bacteria usually die, and the mixture is centrifuged. The phages collect on the top of the mixture and can be drawn off. • The phage solutions are then tested to see which ones show growth suppression effects (lysogeny) and/or destruction (lysis) of the target bacteria. The phage showing lysis are then amplified on cultures of the target bacteria, passed through a filter to remove all but the phages, then distributed.

48. Treatment • Phages in practice are applied orally, topically局部 on infected wounds or spread onto surfaces, or used during surgical procedures. Injection is rarely used, avoiding any risks of trace chemical contaminants that may be present from the bacteria amplification stage, and recognizing that the immune system naturally fights against viruses introduced into the bloodstream or lymphatic system.

49. Phage therapy has been attempted for the treatment of a variety of bacterial infections including: laryngitis喉炎, skin infections, dysentery痢疾, conjunctivitis结膜炎, periodontitis齿根骨膜炎, gingivitis齿龈炎, sinusitis鼻窦炎, urinary tract infections尿道感染 and intestinal infections, burns, boils, etc. also poly-microbial biofilms on chronic wounds, ulcers溃疡 and infected surgical sites. • Reviews of phage therapy indicate that more clinical and microbiological research is needed to meet current standards.

50. Obstacles • General • The high bacterial strain specificity of phage therapy may make it necessary for clinics to make different cocktails for treatment of the same infection or disease because the bacterial components of such diseases may differ from region to region or even person to person. • In addition, due to the specificity of individual phages, for a high chance of success, a mixture of phages is often applied. This means that 'banks' containing many different phages are needed to be kept and regularly updated with new phages.