How a prophage controls fly reproduction

Issue 255 | March 8, 2024
12 min read
Capsid and Tail

Photo by Chris Curry on Unsplash

This week we’re highlighting a brand new paper out of the Bordenstein lab, out yesterday in Science, that unravels the mechanism by which prophage-encoded proteins control whether Drosophila flies reproduce or not.

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How a prophage controls fly reproduction

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Phage microbiologist and co-founder of Phage Directory
Co-founderPostdoctoral Researcher
Iredell Lab, Phage Directory, The Westmead Institute for Medical Research, Sydney, Australia, Phage Australia

Phage characterization, Phage-host interactions, Phage Therapy, Molecular Biology

I’m a co-founder of Phage Directory and have a Ph.D in Microbiology and Biotechnology from the University of Alberta (I studied Campylobacter phage biology). For Phage Directory, I oversee community building, phage sourcing, communications, science, and our awesome team of volunteers.

As of Feb 2022, I’ve recently joined Jon Iredell’s group in Sydney, Australia as a postdoctoral research scientist for the Phage Australia project. I’m diving back into the lab to help get Phage Australia’s country-wide phage therapy system up and running here, working to streamline workflows for phage sourcing, biobanking and collection of phage/bacteria/patient matching and monitoring data, and integrating it all with Phage Directory’s phage exchange, phage alerts and phage atlas systems. I’m also delving into phage manufacturing and quality control.

This week things take a different turn (and I found myself googling/ChatGPT-ing insect sperm…). You may not think about insects much if you’re a phage researcher, like me, but hear me out! A paper that just came out shows that a prophage actually controls a major eukaryotic process… reproduction (or more specifically, whether an embryo lives or dies).

If you’re like me you’ve probably fielded many questions over the years about whether phages could do anything to human/animal cells. Well, apparently they do something to fly sperm development! Let’s dig in.

The paper

Rupinder Kaur (Pennsylvania State University/Vanderbilt University, Bordenstein Lab) and colleagues found that a prophage interferes with Drosophila reproduction. It came out yesterday in Science.

Rupinder Kaur, Angelina McGarry, J. Dylan Shropshire, Brittany A. Leigh, and Seth R. Bordenstein. (2024) Prophage proteins alter long noncoding RNA and DNA of developing sperm to induce a paternal-effect lethality. Science. DOI: 10.1126/science.adk9469

You may know this group as the ‘Wolbachia group’ (at least I do!). They’ve been longtime phage community members, but they’re the special (awesome!) ones over there studying insects.

What the team has described here is how specific nucleases encoded by a prophage that lives within a symbiotic bacterium that lives inside the fly act as the switch that tells a new fly embryo whether it lives or dies. 🤯

Phage genes active in eukaryotic cells?

I know there’s a ton to take from this paper (and I’m sure many an article will be written about it!). For this post, I’m going to give a bit of a primer to save you some googling, share my understanding of what they did, and talk a bit about why I think it’s interesting to the phage field.

One of the pressing things I feel like the phage field wants to know right now is whether/to what extent phages interact with eukaryotes. For those of us working toward putting phages into people and farms and crops, it seems that the world especially wants to know. Are phages really only interacting with bacteria? Are there safety concerns we haven’t yet mapped out, that we should be worried about?

For example, Jeremy Barr’s group has found that phages stick to mucus and translocate into eukaryotic cells. But are they doing anything in there?

When I get asked if phages are likely to impact ‘our’ cells, I mostly say something like ‘they mostly don’t seem to, and if they do it seems pretty benign, but we should probably check’.

And when I think about what research to do in this area, I usually think about phages and human gut epithelial cells. Maybe the blood brain barrier.

Meanwhile, some phage researchers in the community are working with insects like Drosophila, the fruit fly many of us studied in intro genetics, and of course one of the major model systems for animal biology. It’s been a while since I’ve thought to peek over at what they’ve been up to!

A bit of a primer on Drosophila, every bio undergrad’s favourite fruit fly

Drosophila are one of the most well known model organisms for eukaryotic biology. Many of us probably remember counting sleepy flies with a paintbrush in intro biology class. But you may not have known that Drosophila has a bacterial symbiont called Wolbachia.

Wolbachia lives in the ovaries and testes of flies (and tons of other arthropods!) (Fun fact: 40-50% of arthropods have Wolbachia, and around 80% of all animals are arthropods…! 🤯). So Wolbachia is quite prevalent! (It’s also been called the most successful pandemic; I heard this in Seth Bordenstein’s interview for the Meet the Microbiologist podcast).

Where do phages come in?

Wolbachia, this fly-loving bacteria, has its own endosymbiont: a prophage. (I am picturing Russian nesting dolls, and I hope you are too)

Apparently this prophage makes proteins called CifA and CifB that degrade Drosophila sperm DNA and RNA.

In this new paper, the Bordenstein group figure outs what they’re doing in there, and thus unravels the mechanism of how a phage is able to control whether a fly embryo develops properly or dies.

First, some definitions

Cytoplasmic incompatibility (CI): a mating incompatibility reported in arthropod species caused by parasites such as Wolbachia*,* which reside in the cytoplasm of host cells and modify host sperm in a way that leads to embryo death (unless this modification is ‘rescued’ by the same bacteria in the eggs).

Long non-coding RNA: RNA that are longer than 200 nucleotides and are not translated into protein.

Histone-to-protamine transition: something that has to happen during sperm development (it relates to chromosome packing)

What they did in the paper

  • First, they showed that CifA and CifB are nucleases (CifA degrades DNA and RNA, and CifB degrades only DNA). They show that they work in vitro and in vivo (in developing sperm cells)
  • Then they showed that CifA depletion of long non-coding RNA (lncRNA) in spermatocytes strengthens ‘cytoplasmic incompatibility’, the phenomenon that leads to embryos not developing. Specifically, CifA RNase activity recognizes a specific sequence in the long non-coding RNA, degrades it, and the more this happens, the fewer new flies hatch.
  • They showed that this happens through impairment of ‘histone-to-protamine transition’ (which is essential for condensing sperm DNA into a compact form for successful fertilization). This leads to DNA damage in embryos.
  • Then they showed that DNA damage is linked to altered sperm development (the embryos suffer from and succumb to this DNA damage)
  • So overall, prophage genes make nucleases, that get into the fly sperm cell nucleus, and ultimately lead to embryos not hatching (unless the female egg cell also has the right Wolbachia strain… a story for another day!)

Overall, I thought this was really impressive and exciting work — kudos to the authors for uncovering this strange process and figuring out how it works at the molecular level! I had heard of the Wolbachia phage story a little bit, but to see that phage proteins are getting into the eukaryotic nucleus and doing things (not just things, but very important life-or-death things for animals) is intriguing. As evidenced by DNA, restriction enzymes, CRISPR, all the other defense systems, and now this, I think we’re probably still at the beginning of what we’ll find if we keep studying phage molecular interactions with their hosts (and grand-hosts(?)).

Of course, we are talking about prophage genes within an endosymbiont here, and not about a free phage floating into a eukaryotic cell and exerting a function there. Still, it doesn’t seem like too much of a stretch that we might find examples of the latter in coming years (why not?).

(My) remaining questions

Do other animals have prophage genes in charge of major processes like their reproduction? Are there other phages in other endosymbionts? If so, what might they be doing? Do we need to start worrying about this if we’re doing phage therapy/applications? Presumably we could check for homology to these genes, but I’m betting they look like a lot of other nucleases… (don’t quote me on this, I am not a bioinformatician!). And even if we don’t biologically have to worry, will we need to worry about explaining whether ‘phages kill sperm’ is true or not when we talk to patients, family members and regulators?

Beyond phages (because not everything is about phages, Jessica), another interesting aspect this paper brings up is on the biocontrol level more broadly. The authors mention vector control, and I wonder how this discovery might apply to how we control insect populations, since it seems there’s increasing interest in this lately. (For instance, have you heard of gene drives? Excellent podcast episode interviewing their inventor, Kevin Esvelt, here)

Don’t forget, authors are people too!

Thanks for reading! I hope this post inspires you to check out the paper (because I barely scratched the surface here!) and to send your favourite Bordenstein lab member an email telling them what you learned from it/what you thought of it. (Have you ever got one of these? I still think fondly of the ~3 kind humans who emailed me about my papers from grad school; I will never forget them!).

Or better yet, invite or nominate someone on the team for the next phage conference you get convinced to help organize! I know I will.

Further reading

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