Engineering phage ΦX174’s genome: a recap of November’s PHAVES talk

Issue 103 | November 27, 2020
7 min read
Capsid and Tail

This month, we hosted episode 10 of PHAVES, a talk on phage engineering by Dr. Paul Jaschke, Lecturer at Macquarie University! Here’s the recording, along with a recap written by Stephanie Lynch, PhD candidate at La Trobe University (and our amazing volunteer PHAVES coordinator)!

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What’s New

Ameneh Khatami (University of Sydney) and colleagues published a new preprint showing therapeutic monitoring of the host-bacterium-phage interaction in a child. They describe how they treated a 7-year-old with a chronic osteoarticular Pseudomonas infection, and how monitoring phage and bacterial kinetics and the human host response allowed them to adjust dosing early on, leading to a strong therapeutic effect within two weeks. Phage Directory is proud to have helped source phages for this effort, and especially proud of the 12 labs who responded to our alert last summer.

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Lorraine Draper (APC Microbiome Ireland) and colleagues published a new paper in BMC Biology showing the impact of faecal viral transfer (FVT) on the mouse microbiome after antibiotic perturbation. They saw that FVT helped the mice get back to a pre-antibiotic microbiome, but only when the FVT contained viable phages.

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Roberto Molina-Quiroz (Centro de Estudios Científicos, Chile) and colleagues published a new paper in mSphere showing that prophage-dependent neighbor predation in Vibrio cholerae leads to enhanced horizontal gene transfer by natural transformation.

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The WHO’s 4th Clinical Antibacterial Pipeline Advisory Group Meeting took place this week. Haileyesus Getahun, MD (Joint Secretariat on AMR at WHO) stated via Twitter that for the first time, available data on phage therapy was set to be evaluated.

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Adaptive Phage Therapeutics (APT) is working toward a phage-based COVID vaccine — a phage engineered to express epitopes of SARS-CoV2 on the surface of the capsid. Phage-based vaccines are attractive because they are considered safe in the body and self-adjuvanted. APT is exploring the potential for an oral delivery system in the form of a lozenge.

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Dr Andrew Millard (University of Leicester) is hiring a PhD student to study the development of phage-based vaccines for animal health.
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Join Jean-Paul Pirnay, PhD, Gina Suh, MD, and Nicholas Chia, PhD Nov 30, 2020 12:00 PM Central time for an engaging virtual discussion on phage therapy as a tool in the fight against antibiotic-resistant infections. The talk is entitled “Bacteriophages: It’s a Medicine, Jim, But Not As We Know It”.

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Engineering phage ΦX174’s genome: a recap of November’s PHAVES talk

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PhD Candidate
La Trobe University

I am a third year PhD student from La Trobe University, Melbourne, currently isolating and researching the use of bacteriophages for skin infections in animals. I have a background in Animal & Veterinary Bioscience and hope to continue research of bacteriophages as therapeutics within the veterinary or livestock sector. I am also currently developing and optimising the use of animal-alternative models for safety and efficacy trials of phage therapy. I am always willing to chat about phage research and would like to connect with phage biotech companies as I am interested in jobs within the industry sector.

This month, we hosted Dr. Paul Jaschke, Assistant Professor at Macquarie University, Sydney, Australia, and CSO of Hyperdrive Science for our November episode of PHAVES; check out the recap below!


Learning how to engineer genomes by building phage

YouTube video of PHAVES 10 (1:00:24)

Highlights of Paul’s talk

  • Throughout his career, Paul has extensively studied a phage that belongs to the Microviridae family known as ΦX174. This phage is known to infect specific strains of Escherichia coli and Salmonella spp.
  • Interestingly, throughout its infection process, ΦX174 creates a protein known as ‘Protein E’ which is an antibiotic-like lysis protein which disrupts peptidoglycan synthesis, which may be of interest from a therapeutic point of view.
  • However, Paul’s work (at Macquarie University, Sydney, Australia) has focused on the genome of ΦX174, which is a circular ssDNA genome with extensive gene overlap. The overlap allows the 11 genes to only span across 5,386 bp. Does this overlap account for a message from extra-terrestrial intelligence? Unfortunately, probably not. However, importantly, a genome size >5kb does result in a lowered infectivity rate!
  • Due to the structure of the ΦX174 genome, Paul and his team are interested in using this phage for synthetic genomics, which is the “extensive and intentional genetic modification of a replicating system for a specific purpose”. Why phage genomes for synthetic genomics?
  • Phages are self-contained genetic programs with a constant operating system, therefore easier to tell if changes affect points of the phage lifecycle
  • Phage lifecycles share characteristics such as DNA replication with other self-replicating systems
  • Understanding how to engineer phage genomes may help accelerate them as next generation antimicrobials
  • However, as mentioned, ΦX174 has gene overlaps, therefore, Paul begun by decompressing the genome of ΦX174 to remove all overlaps. Characterisation of decompressed ΦX174 found that it had smaller plaque sizes, smaller burst size, poor attachment and a downregulation of two proteins involved in scaffolding and replication in comparison to wild type ΦX174. Despite this, CryoTEM revealed that the structure of decompressed ΦX174 was indistinguishable from wild type ΦX174, and decompressed ΦX174 lysed cells at a similar efficiency in vitro at a higher MOI. This highlights the important trade-offs between removing overlaps and phage fitness.
  • Paul finished up by talking about the cryptic genes in ΦX174. When conserved genes among the Microviridae family were erased (resulting in phage kleenX174), plaque size was reduced and therefore phage fitness was reduced, confirmed by creating chimeric genomes. This reduction in fitness was shown to be due to a nucleotide mutation in the ‘H protein’. Reverting the single nucleotide mutation back to wild type allowed the doubling of protein H production.
  • Coming soon(ish), Paul and his team have some very exciting studies up next. One that particularly interested the audience was the creation of a Plaque Quantification Tool by Master’s Student Ellina Trofimova – stay tuned!

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