Molecular Virology - Bacteriophages and Phage Therapy

Bacteriophages and Phage Therapy

What is Bacteriophage?

• Bacteriophages are viruses that infect bacteria and archaea.
• They have diverse morphologies and genetic compositions.
• Structure:
  • Head: Contains the genetic material.
  • Collar: Connects the head and tail.
  • Core: Central structure.
  • Sheath: Contractile structure used for injecting DNA.
  • Baseplate: Structure for attachment to the host cell.
  • Tail fibers: Help in recognizing and attaching to the host cell surface receptors.

Viruses Infecting Bacteria and Archaea

• Bacteriophages are a diverse group of viruses infecting bacteria and archaea.
• They can have:
  • Nucleic acid: ssRNA, ssDNA, dsRNA, dsDNA.
  • Genome structure: Linear or circular.
  • Envelope: Enveloped or non-enveloped.
  • Morphology: Icosahedral or filamentous.
  • Genome: Segmented or non-segmented.
• Examples of families:
  • Myoviridae: Nonenveloped, contractile tail, linear dsDNA. Example: T4 phage.
  • Siphoviridae: Nonenveloped, noncontractile tail (long), linear dsDNA. Example: λ phage.
  • Podoviridae: Nonenveloped, noncontractile tail (short), linear dsDNA. Example: T7 phage.
  • Lipothrixviridae: Enveloped, rod-shaped, linear dsDNA. Example: Acidianus filamentous virus 1.
  • Rudiviridae: Nonenveloped, rod-shaped, linear dsDNA. Example: Sulfolobus islandicus rod-shaped virus 1.
  • Ampullaviridae: Enveloped, bottle-shaped, circular dsDNA.
  • Bicaudaviridae: Nonenveloped, lemon-shaped.
  • Clavaviridae: Nonenveloped, rod-shaped, circular dsDNA.
  • Corticoviridae: Nonenveloped, isometric, circular dsDNA.
  • Cystoviridae: Enveloped, spherical, segmented dsRNA.
  • Fuselloviridae: Nonenveloped, lemon-shaped, circular dsDNA.
  • Globuloviridae: Enveloped, isometric, linear dsDNA.
  • Guttaviridae: Nonenveloped, ovoid, circular dsDNA.
  • Inoviridae: Nonenveloped, filamentous, circular ssDNA. Example: M13.
  • Leviviridae: Nonenveloped, isometric, linear ssRNA. Examples: MS2, QB.
  • Microviridae: Nonenveloped, isometric, circular ssDNA. Example: QX174.
  • Plasmaviridae: Enveloped, pleomorphic, circular dsDNA. Example: L2.
  • Tectiviridae: Nonenveloped, isometric, linear dsDNA. Example: PRD1.

Short History - Virology

• Bacteriophages were independently discovered by:
  • Frederick Twort (English) in 1915: He investigated the nature of ultra-microscopic viruses.
  • Félix d’Herelle (French) in 1917: He described an invisible microbe antagonistic to dysentery bacteria.
• The term “bacteriophage” was coined by d’Herelle.
• There are some disputes regarding d’Herelle's honesty.
• Ivanosky (Russian) reported in 1892 the presence of a new pathogen that could pass through the Chamberland filter.
• Beijerinck (Dutch) recognized the significance and called the pathogen “virus” in 1898. This is known as tobacco mosaic virus.

Short History - Phage Therapy

• D’Herelle promoted phages as the main antibacterial agents.
• Before the widespread use of antibiotics, Eli Lilly and Abbott Laboratories commercialized phage preparations against Streptococcus spp. and Staphylococcus spp.
• This practice is now called “phage therapy”.

Short History - Molecular Biology

• In the 1940s, phages were used as a model system for understanding molecular events inside a cell.
• This ushered in modern-day molecular biology.
• Erwin Schrödinger’s book, What Is Life? The Physical Aspect of the Living Cell, influenced this.

Lecture Overview

• For phage λ:
  • Lysis-lysogeny decision
  • Host lysis
  • Miscellany
• For phage T4 and T7:
  • DNA Entry
  • Miscellany
• Etymology of phage families:
  • Myoviridae: Myo- (Greek) meaning “muscle”.
  • Podoviridae: Podo- (Greek) meaning “foot”.
  • Siphoviridae: Sipho- (Greek) meaning “tube” or “siphon”.
  • These are all “tailed” phages.

Phage λ

• Phage λ is a dsDNA phage.
• It has a $T = 7$ icosahedral capsid and a $48,502$ bp genome.
• It belongs to the Siphoviridae family, characterized by a non-contractile tail.
• The virion is moderate in size (~135 nm length, ~63 nm width).
• The original isolate has side tail fibers (Stf), but most lab strains do not.

Phage λ: Lysis-Lysogeny Decision

• Phage λ can choose between two life cycles: lytic or lysogenic.
• The decision-making process in phage λ has been extensively studied.
• Mark Ptashne's book provides a well-written description of this process.
• The study of phage λ lytic/lysogenic decision serves as a template for understanding how a bi-stable genetic switch functions and how it is maintained.

Phage λ: Genome Organization

• The genome encodes about 60 proteins, arranged in clusters (modular) according to their functions.
• In the virion, the genome exists in a linear form with 12-nt 5’ overhang cohesive ends (cos).
• The genome circularizes upon infecting the E. coli cell.
• Transcription occurs in seemingly contradictory directions, which becomes clearer in the circular form.

Phage λ: Decision Making

• Upon infecting an E. coli cell, phage λ has a “decision” to make: lytic or lysogenic.
• The “decision” is the result of a tug-of-war between leftward and rightward transcriptions, modulated by cell conditions.
• This decision is made at the regulatory region.
• The lytic pathway results in host lysis, while the lysogenic pathway results in the integration of the phage genome into the host cell genome.

Phage λ: Initial Infection

• The basic control logic involves termination and anti-termination.
• There are various promoters that E. coli RNA polymerase can access, but only three are strong and constitutive: $P<em>L$, $P</em>R$, and $P_R'$.
• Downstream of these promoters are terminators – $t<em>{L1}$, $t</em>{R1}$, and $t_{R}'$ – that would stop transcription.
• The result is that the N protein is made from the initial left transcript, Cro from the right, and a 6S RNA that is not translated to a protein.

Phage λ: N and Q Antiterminators

• N is an anti-terminator that modifies the RNA polymerase.
• Once enough N is accumulated, it anti-terminates at various terminators, allowing transcription to proceed.
• This prepares the phage for both the lysogenic and lytic pathways.
• For the lysogenic pathway, proteins for recombination and genome integration/excision are produced.
• For the lytic pathway, proteins for DNA replication and the anti-terminator Q for late transcript are produced.
• When enough Q is accumulated, it modifies the RNA polymerase and anti-terminates at $P_R'$, thus making a long, late transcript.

Phage λ: CI and Cro

• CI (the repressor) and Cro are competitors at the same regulatory region, influencing the lytic or lysogenic decision.
• One consequence of N-anti-termination is the production of CII and CIII proteins.
• CIII is an inhibitor of HflA (high frequency of lysogenization).
• Sufficient amounts of CII and CIII will result in transcription from the $P_{RE}$ promoter and production of the CI repressor.
• CII is unstable, easily degraded by the HflA protease.
• The $P_{RE}$ (repressor establishment) is a weak promoter that requires CII for activation.
• The $P_{RE}$ transcript is also anti-cro.
• In the meantime, the Cro protein (control of repressor and other things) is also made.

Phage λ: Lytic Pathway

• When Cro dominates:
  • The $P_{RM}$ promoter (repressor maintenance) is blocked, so there is no CI production, thus ensuring the lytic pathway.
  • Cro and CI have opposite affinities toward various operator sequences (OL and OR).
  • High Cro concentration blocks all operators, shutting off early gene transcription.
• Cro is constitutively expressed, while CI’s concentration depends on CII’s and CIII’s concentrations.
• Initially, both Cro and CI are present.
• Both Cro and CI form dimers.
• Both Cro and CI can bind to OR’s and OL’s, but with different affinities.
• The affinity for operators is as follows:
  • Cro: Low affinity for OL1, OL2, OR2, OR1; High affinity for OL3, OR3
  • CI: High affinity for OL1, OR1; Low affinity for OL2, OL3, OR3, OR2

Phage λ: Lysogenic Pathway

• When CI dominates:
  • CI first binds to OR1 and OL1.
  • Cooperative binding between CI dimers helps the formation of a CI tetramer, thus fully blocking $P<em>R$ and $P</em>L$ promoters.
  • RNA polymerase can then get access to $P_{RM}$, thus only CI is made.
  • Enough CI means CII is present in high enough concentration as well; besides $P<em>{RE}$, $P</em>{int}$ and $P_{AQ}$ also need CII for activation, further entrenching the lysogenic pathway.

Phage λ: Positive Feedback Loop and High CI Concentrations

• There is a positive feedback loop with CI production – more CI is made, the more $P<em>R$ and $P</em>L$ are shut off, and the more the $P_{RM}$ promoter is accessed, resulting in more CI production.
• When the CI repressor concentration is high, even less preferred operator sites like OR3 and OL3 will have repressors occupying them.
• Cooperative binding among CI dimers on OR3 and OL3 prevents RNA polymerase from accessing $P_{RM}$.
• Octamerization helps maintain the looped $3.8$ kb DNA structure in place
• There is a competition between a CI dimer and RNA Polymerase.

Phage λ: Decision Summary

• The occupancy at the operator and activity of the promoter dictates the decision for Lysis or Lysogeny.

Phage λ: Decision Implications

• Mathematical models can describe conditions under which certain decisions are made.
• Stochastic modeling allows us to get a general sense of how a population may respond and how variable the responses may be.

Phage λ: Consequences of Strategy

• Unlike phage T7, phage lambda prefers dithering, which comes with a penalty:
  • T7: Latent period ~11 min; burst size 120 – 300 pfu/cell
  • Phage λ: Latent period ~50 min; burst size of 100 – 150 pfu/cell
• The “indecision” is beneficial in gathering cellular information to inform the lytic/lysogenic decision.

Phage λ: Food for Thought

• Under different concentrations, the CI repressor can either stimulate or repress its own production.
• On average, only a low amount of the homeostatically maintained CI repressor is needed to maintain the lysogenic state.
• There are two consequences of such an arrangement:
  • The lysogen has a built-in immunity against homologous phages still floating around in the environment.
  • Low repressor concentration can sometimes lead to spontaneous induction due to stochastic distribution of the repressor molecules in each daughter cell.
• Questions to consider:
  • How tight should the repressor binding be so that it can effectively repel infecting phages without affecting its own chance of breaking out?
  • How frequent should the spontaneous induction be so that the phage can always maintain some progeny in the prophage state and some in the virion state? (basically, bet-hedging)

Phage λ: Prophage Induction

• The process of a prophage reactivating its lytic pathway, resulting in lysis of the lysogen, is called induction.
• Damaged DNA is a good indicator that the host may be in trouble.
• CI mimics LexA by using the activated RecA* to enhance CI self-cleavage.
• The presence of RecA* is an indicator of bad condition.
• The SOS system is activated to repair damaged DNA. It is initiated by the activated RecA protein (RecA*) that, when bound to the LexA repressor, will enhance self-cleavage of LexA, resulting in the de-repression of the SOS genes.

Phage λ: Late Transcript

• Genes for lysis, head, and tail biogenesis are transcribed from the genome during the late stage of the lytic cycle of the lambda (λ) phage.
• These genes are transcribed from the $P_R ʹ$ promoter and are regulated by the Q late transcript.

Bacteriophage Lysis: General Strategies

• There are two general strategies for bacteriophage lysis:
  • Single lysis gene: Used by small genome phages.
  • Holin-endolysin: Used by large dsDNA phages.

Bacteriophage Lysis: Saltatory Nature

• Host lysis often occurs within a short period.
• Different mutations in the holin gene can affect the timing of lysis.

Bacteriophage Lysis: Holin-Endolysin System

• Used by large dsDNA phages.
• The holin creates pores in the cytoplasmic membrane.
• The endolysin degrades the peptidoglycan layer.

Bacteriophage Lysis: Real-Time Sensing - Lysis Inhibition (LIN)

• Besides the normal holin-endolysin system, phage T4 also has a real-time sensing mechanism called lysis inhibition (LIN).
• LIN is a phenomenon where the lysis of the host can be actively inhibited, depending on the conditions.
• In a single-phage infection, the antiholin RI will be degraded by the periplasmic protease DegP after spontaneous release into the periplasm; cell lysis occurs at 25 min.
• In a superinfection, the DNA of a superinfecting T4 phage generates a “signal” to stabilize RI, which leads to the formation of the T-RI-RI-T heterotetramer, which then facilitates the binding of the cytoplasmic antiholin RIII to the N terminus of T.
• The consequence of LIN is that the burst size is increased from 130 to 1,000 phage progeny per infected cell.

Phage Miscellany: Genome Mosaicism

• The genome mosaicism is not limited to phage λ’s relatives (lambdoids), but also many other dsDNA phages.
• The break points are usually found at the gene borders or sometimes domain borders.
• There are many horizontal gene transfers among phages, sometimes even distantly related.
• This makes it difficult to trace their lineages.

Phage Miscellany: Genes with Foreign Origins

• Comparative genomic analyses showed that dsDNA phages usually carry “accessory” genes that are likely to be of foreign origin (e.g., different GC content, expression orientation etc).
• Some of these genes are virulent factors, e.g., the Shiga toxin and possibly the bor gene in lambda.

dsDNA Phages: T4 and T7 - T-even and T-odd

• T (type) phages have an historically important place in modern molecular biology.
• 7 phages were chosen, T1 to T7, to focus on:
  • T1: 48,836 bp; 77 genes
  • T2: 163,793 bp; T4-like
  • T3: 38,208 bp;
  • T4: 168,903 bp; 279 genes
  • T5: 121,750 bp; 162 genes
  • T6: 168,974 bp; T4-like
  • T7: 39,937 bp; 22 genes
• T-even phages (T2, T4, and T6) are all related to each other and belong to the Myoviridae. The most famous one is T4.
• T-odd phages are quite different from each other. T1 and T5 have long tails (Siphoviridae); T3 and T7 short tails (Podoviridae).
• All are lytic phages (as opposed to the temperate phages).

dsDNA Phages: Entry - T4 and T7

• T4 and T7 have entirely different ways of injecting their genomes into their host, E. coli.
• T4 adopts the strategy of “molecular syringe”, and T7 uses a “molecular winch” to pull its genome in.

dsDNA Phages: Entry - T4

• T4 recognizes the receptor (LPS and/or OmpC) via its long tail fiber (LTF).
• LTF is composed of several proteins and requires two phage-encoded chaperones to assemble and fold correctly.
• Although T4 is often seen as a lunar landing module, in reality, the LTFs are most likely retracted and retained by gpwac, which is hypothesized as a sensing device for the environment.
• Presumably, thermal fluctuation will every now and then allow one or more LTFs to extend and make contact with cell receptors.
• Once $≥3$ LTFs are bound, presumably thermal fluctuation triggers conformational changes along the LTFs, which in turn trigger baseplate change.
• Short tail fibers (STFs) extend from the baseplate, resulting irreversible binding.
• The syringe tip (gp5) extends from the baseplate, ready to penetrate barriers.
• The tail sheath contracts, pushing the gp5 tip through the OM.
• The gp5 tip falls off, leaving the lysozyme domain of gp5 ready to digest the peptidoglycan cell wall.
• The tail tube can now penetrate further and allow the T4 DNA to enter the cytoplasm.

dsDNA Phages: Entry - T7

• Tail fiber recognizes the LPS and is made of homotrimer gp17.
• After “walking” across the cell surface to find the receptor for the tail, all fibers rotate downward to contact the outer membrane.
• Commitment to infection occurs after internal core proteins are ejected from the virion and the extended tail forms.
• Gp14-15-16 forms the T7 core proteins that translocate phage DNA into the cell.

dsDNA Phages: Entry and Transcription - T7

1. During initial DNA entry, only the first 850 bp is injected into the cell, thus exposing T7’s Class I promoter sequences to E. coli RNA Pol.
2. E. coli RNA polymerase engages the T7 genome and transcribes the first few genes, thus pulling in more sequences.
3. Transcription and translation are coupled, just like the expression of most bacterial genes.
4. The DNA is pulled in at ~40 – 60 bp/s (~3 min). The E. coli RNA polymerase acts like a molecular winch.
5. However, transcription terminated at the TE terminator, at ~7.5 kb mark.
6. The newly translated T7 RNA polymerase recognizes the exposed Class II promoter sequences, thus continuing transcription and pulling in the rest of the T7 genome (at ~250 bp/s; about 2 min; cf. 40 – 60 bp/s). The T7 RNA polymerase also acts like a molecular winch.

• E. coli cells over-expressing dam gene (encoding Dam, deoxyadenosine methylase, converting GATC → GAMeTC).
• GAMeTC is susceptible to DpnI digestion.
• Using 32P-labeled T7 genomic fragments as a probe.

dsDNA Phages: Unique Features of T7

• One interesting (unusual) feature of T7 plaque morphology is that it can continue to expand in size when most other phages would have stopped the plaque expansion.
• Its unusual biology, a much more independent requirement of host machinery (shutting down host synthesis, using its own RNA and DNA pol and other accessory phage proteins), may have contributed to the phenotype.
• T7 infection of cells containing F factor is abortive, i.e., no phage progeny is produced, and the infected cell is dead (an “altruistic” behavior of F).
• It is mediated by the pifA gene in the F plasmid (phage inhibition by F factors).
• The corresponding phage proteins are gp1.2 (inhibitor of dGTPase) and gp10 (capsid protein).
• Evolutionary interests among various genetic elements.

dsDNA Phages: Applications of T7

• T7 is one of the phage systems commonly used for experimental evolution.
• Besides serial passage experiments, evolution of phage strains with scrambled gene orders is also tested.
• A darling in synthetic biology.

Phage Therapy

• The rise of antibiotic-resistant bacteria, or