Insights into the elusive RNA bacteriophages

Issue 64 | February 21, 2020

8 min read

Capsid and Tail — Bioinformatics. Photo by Rich Tervet on Unsplash

This week, Julie Callanan, a PhD student at APC Microbiome Ireland and University College Cork, introduces us to RNA phages, why they’re so elusive, and how her research is beginning to unravel their secrets.

What’s New

Alexandra Petrovic Fabijan (Westmead Institute for Medical Research, Australia) and colleagues published their safe IV treatment of 13 critically-ill, septic patients with a GMP-prepared S. aureus phage cocktail in Nature Microbiology. Jon Iredell and Ruby Lin, who led the project, take us behind the scenes with two blog posts: Jon’s perspective as a physician on where phage therapy needs to go, and the team’s plans to build a phage biobank to supply matched phages to clinicians and researchers.

Vivek Mutalik (Lawrence Berkeley National Lab) and colleagues have published a new preprint on high-throughput mapping of the phage resistance landscape in E. coli. Through systematic, genome-wide loss-of-function and gain-of-function studies (14 phages, 2 host strains), they confirmed previously described phage receptors and uncovered new host factors that confer phage resistance.

Rogelio Rodriguez-Gonzalez (Georgia Institute of Technology) and colleagues published a new paper in mSystems on quantitative modeling of phage-antibiotic combination therapy. Their findings highlight the role of antibiotics and the immune system in promoting phage therapy effectiveness.

Mohammadali Khan Mirzaei (McGill University, Montreal, Canada) and colleagues have shown that phages isolated from growth-stunted Bangladeshi children are different from those in non-stunted children. They show these phages are infectious in vitro, and suggest they may contribute to the bacterial communities seen in the guts of stunted children.

Phages are extremely diverse at the genetic level, but not so much at the structural level… why? In an informative new review, Moïra Dion (Université Laval, Canada) and colleagues have explored phage diversity at the structural, genomic and community levels, and given an up-to-date analysis of the complex evolutionary relationships between phages.

Are there really 10^31 viral particles on Earth? Here’s a new minireview by A. R. Mushegian (National Science Foundation) on how that number (“The Hendrix Product”) was derived, and whether it needs to be revised.

Submit an abstract for the Monash-UCSD Bacteriophage Symposium (Mar 30, 2020, Melbourne, Australia).

ISME (International Symposium on Microbial Ecology, August 9-14, 2020, Cape Town, South Africa) will have a virus/phage session this year. Submit abstracts by March 6; propose round table topics by Feb 29!

Latest Jobs

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Betty Kutter at The Evergreen State College in Olympia, WA seeks a motivated full-time post-doc to investigate the adaptation of phage infected cells to stationary phase and anaerobic growth.

University College Cork has an opening for an MSc student to study the role of anti-phage antibodies in the gut of mammals.

A lab at the University of Michigan is hiring a full-time research technician to support projects on host-parasite interactions that use model communities of E. coli and phages.

Intralytix (Columbia, Maryland) is seeking a Research Scientist and a Clinical Trial Specialist.

Phagelux (Montreal, Canada), is seeking a medical writer to prepare documents for the FDA as they move their phage products toward approval.

Community Board

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Anyone can post a message to the phage community — and it could be anything from collaboration requests, post-doc searches, sequencing help — just ask!

Phage Methods Q&A

What is the best way to search an assembled phage contig against human metagenome datasets? Regular blastn at NCBI doesn’t seem to include these, do we have to download the data to search locally or is there an online resource? — @jason_j_gill

Check out the responses on Twitter here!

Insights into the elusive RNA bacteriophages

PhD Research Scholar

APC Microbiome, University College Cork, Cork, Ireland

Twitter @julie_callanan

I’m a PhD student, part of Professor Colin Hill’s phage lab in the APC Microbiome Ireland. During my penultimate undergraduate year, I was lucky enough to do an 8-week summer studentship in this lab, where my interest in these entities began. Since then my research has progressed to studying the RNA phage consortium of the phage population as they remain understudied in many ecosystems. This particular research was carried out with Dr Stephen Stockdale, a post-doctoral researcher in our lab. In order to verify the presence/absence of ssRNA phages from samples, we developed a method to detect these entities.

The delight of finding Wally amongst the crowd of people at the funfair is similar to that when you detect a rare RNA bacteriophage in metagenomic or metatranscriptomic samples. These phages were originally identified in 1961 by Loeb and Zinder when they isolated a phage which had an RNA genome as opposed to the typical DNA. Since their initial discovery, RNA phages have served as important molecular models for understanding some of biology’s most intricate molecular pathways such as gene regulation, transcription and translation. They have also been central to many molecular milestones such as the first gene (the coat gene) to be sequenced in 1967 and the first entire genome to be fully sequenced in 1976.

A little insight into these little phages

Despite their historical significance, there is very little known about RNA phages, with only two families described in the literature, the Cystoviridae, with dsRNA genomes, and the Leviviridae, which have ssRNA genomes. Cystoviridae have tri-segmented genomes, which generally range from 12.7 to 15.0 kbp in length, enclosed in a protein envelope. The three segments are organised to encode different functional units. In the latest ICTV report, there is only one recognised genus, Cystovirus, with a single species. Additionally within the latest ICTV report, there are six more phages belonging to Cystoviridae, with several more being associated with this family.

Leviviridae have a positive-sense, single-stranded genome of approximately 4,000 bp. This family is currently separated into two genera, Levivirus and Allolevivirus, primarily based on whether they have three or four genes. There are 25 complete and partial ssRNA phage genomes belonging to these two genera in the latest ICTV report, and 32 more sequences recognised as potential Leviviridae.

Importance of RNA phages

General interest in all things phage has exploded in recent years, as studies have uncovered the important role they play in shaping and structuring bacterial communities. Another important feature is their potential application as therapeutics in a post-antibiotic era. RNA phages have been found to target a variety of bacteria, including those listed by the World Health Organisation (WHO) as some of the deadliest pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

Problems with trying to find them

However, RNA phages have remained somewhat enigmatic in phageome studies, where extraction and isolation methods may be biased in favour of DNA phages. Another problem associated with these phages is the delicate nature of RNA and the ubiquitous presence of RNases in environments. It may also be that bioinformatics protocols during downstream processing do not capture RNA phage sequences. These issues have limited the expansion in the knowledge and diversity of these interesting groups of phages. The development of an optimised protocol for the isolation and characterisation of RNA phages should significantly improve our attempts in this field.

Just the beginning

A recent study by Krishnamurthy et al. in 2016 showed that RNA phages may be more abundant than previously believed as they noted the partial genomes of five cystoviruses and 138 leviviruses through the mining of metatranscriptomic datasets. This expansion also revealed novel hosts including a Gram-positive bacterium. This was one of the first papers I read as I began my PhD journey and it was where my interest in these unusual and understudied phages peaked. How can we progress with our understanding of the human phageome without taking RNA phages into account?

To address the potential issue of bioinformatics protocols, Dr. Stephen Stockdale and I built a specific ssRNA-phage search tool. Applying this method to a variety of metatranscriptomic samples, we expanded the number of available ssRNA phage sequences by 60-fold, and this enabled us to examine their genome structure and phylogenetic relationships.

Given that eukaryotic RNA viruses make up the largest portion of the total human virome, it seems unlikely that there would be so few RNA phages associated with us and our microbiome. The search for these phages and their roles is just beginning in an RNA phage-based renaissance.