Studying intracellular parasite DNA is like panning for gold

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The organisms I focus on – the microsporidians – are tricky to study. They are widespread microscopic parasites that are obligately-intracellular – which means they can’t survive outside a host cell. There may be a microsporidian specialised to infect every living invertebrate and maybe even every Metazoan, which is a complex animal. These creatures are probably the kings of parasitism; they have shed many typical eukaryotic features in order to maximise their parasitic abilities. One thing I find really fascinating is that microsporidia cannot even generate their own energy – they steal it from their hosts using transporters they previously acquired from intracellular bacteria.

I wanted to work on symbionts in mosquitos. Symbionts are organisms living in partnership with another organism. I had heard about Group Leader Mara Lawniczak, who had been involved in a paper about microsporidia in mosquitos. Mara has huge datasets of different mosquitoes from around the world so I reached out to her, and she encouraged me to apply to the Sanger Institute PhD programme.

There are so many questions still unanswered in this field! Microsporidian infection is quite unpleasant, and can even spell doom for its host. However, in mosquitos, a microsporidian infection is associated with a reduction in Plasmodium transmission – the genus of parasites responsible for malaria. What this means is that if mosquitos carry microsporidian parasites, they’re less able to spread malaria, which is a good thing. That’s why I wanted to study microsporidia in mosquitos. Studying symbionts that are parasites is fascinating – as is symbioses in biology in general. Instances of symbiosis can be found everywhere in the world around us.

How long have we known about microsporidia?

1838

1857

1980s

1990s

2020s

1838

Microsporidia are either early-diverging fungi, or very closely related to fungi. The earliest report of microsporidia goes back to 1838, where they were first described by biologist Theodor Gluge. He is credited with the first description of Glugea anomala, a microsporidian parasite found in fish.

Glugea anomala infection in a stickleback. Image credit: Archiv für mikroskopische Anatomie (1913). Public domain.

1857

The silk industry was collapsing globally in 1857. The Swiss botanist Carl Wilhelm von Nägeli identified the microsporidian Nosema bombycis as the causative agent of the disease the silkworms were experiencing. The French Government at the time turned to their microbiologist du jour, Louis Pasteur, to solve the silk industry problem. Amazingly, Pasteur found a way to identify infected silkworms and save the industry from ruin.

Left to right: Carl Wilhelm Nägeli, the lifecycle of the silkworm moth (Bombyx mori), and Louis Pasteur. Image credits: Borvan53 / Wikimedia Commons CC BY-SA 4.0, Daderot / Wikimedia Commons. CC0 1.0, and Paul Nadar Public Domain.

1980s

In the 1980s and microsporidia were found to co-infect people living with AIDs. With little to no immune system due to living with HIV, these people could not fight off these parasites – many of which were emerging as opportunistic.

Left to right: HIV virus and Microsporiodosis in humans. Image credits: BruceBlaus / Wikimedia Commons. CC SA 4.0, and CDC/Alexander J. da Silva, PhD/Melanie Moser. (PHIL #3411), 2002.

1990s

Microsporidia were linked to honeybee colony collapse in the 1990s, devastating industries reliant on these hard-working arthropods. Microsporidia are also well described as pathogens in fisheries, and constitute a growing problem for aquaculture industries worldwide.

Honey bee colony. Image credit: Benlisquare / Wikimedia Commons. CCA-SA4.0.

2020s

In the 2020s, two species of microsporidia that infect Anopheles mosquitos, with limited negative impact to the mosquito, were found to inhibit the spread of malarial parasites. Could this be the good press microsporidia have been waiting for? Using symbionts as a potential biological control is not a new idea and has been deployed with some success using Wolbachia bacteria.

Anopheles arabiensis mosquito. Image credit: James Gathany / CDC. Public Domain.

Microsporidia can only live and grow inside the cells of another organism. In their spore forms, they can be found distributed worldwide in the soil and water. The spore contains a coiled spring-like structure that is released when the spore germinates – a process likely triggered by water – piercing the host cell with its harpoon-shaped edge. It then uses this harpoon like a syringe, injecting the ingredients it needs to thrive directly into the host cell and taking it over. Eventually, it needs to leave again to allow the cycle to start over, so the mature microsporidia are released to start the cycle all over again.

The difficulties in studying microsporidia are due to their small size – spore length can be as small as one micron – and the need for high-resolution technology to observe them. In comparison, the average human hair is around 100 microns in width.

Genomics offers a solution. I can study the DNA of microsporidia without ever needing to visualise them. The microsporidia field has a rich history of morphological descriptions, thanks to high-resolution technology and the dedication of wonderful experts. But this doesn’t give us a full picture, and the genomics layer can allow us to build up a detailed biological understanding.

For my PhD research, I really wanted to apply genomics to mosquito-microsporidia symbiosis. As part of this, I naturally found myself studying microsporidian genomes in more detail, trying to understand their evolution and why microsporidian species are different from one another. However, I quickly realised there are even bigger challenges – we had little data and many basic questions that needed answering before it could even be dreamed as a biological control measure. So, cue panning for gold amongst genome sequences.

I found the DNA of microsporidians as a by-product of an effort to sequence all complex life in the British Isles and Ireland: the Darwin Tree of Life project. As a partner of this project, the Sanger Institute is sequencing all sorts of creatures sent by sample partners from all over Britain and Ireland.

Typically, when you sequence an organism, you also sequence its lunch; you sequence things living on it and its parasites. So, I started looking for microsporidians in just the arthropods genome sequences. Arthropods are invertebrates with hard outer bodies and jointed appendages. I focused on these since we know microsporidians are associated with insects like bees and mosquitoes.

I panned for my version of gold – the DNA of infected individuals. I pulled out the microsporidian DNA, and then assembled the microsporidian genomes to build up a bigger picture of how the genomes fit together. Ultimately, fitting the DNA puzzle together and understanding a genome helps us to answer big questions like how microsporidians reproduce.

It’s taken me over a thousand arthropod genomes, but so far, I’ve managed to piece together 40 genomes of microsporidia. This is literally a gold-rush of new information for the field.

I am not one to shy away from a challenge, which is good, because looking for microsporidian DNA in arthropod genomes ended up feeling like panning for gold. But the data I have managed to track down and analyse allowed me to shed some light on the mechanisms of reproduction in microsporidia and their evolution.

I’d say one of my big findings was that tetraploidy – a fancy way of saying an organism has four copies of its DNA instead of the usual two – is widespread in microsporidia.

Discovering this tetraploid state in microsporidia was really a catalyst for all that came next and the kinds of questions I started asking. Essentially, some microsporidia have one nucleus, and some have two nuclei. The possibility of tetraploid microsporidia having two nuclei was something speculated about before in the literature. So this was a big aha moment for me; genomics was getting us closer to biology!

We also see evidence of recombination both within and between the two compartments, providing evidence for a sexual cycle in microsporidia. All of this new information has allowed us to propose a new life cycle model, confirming some of the earlier hypotheses proposed by colleagues years ago.

Tetraploidy is pretty unusual and costly for an organism to persevere with. It also can make for a really complex genome architecture for researchers to unpick, with all the extra repetitive DNA bringing new challenges for genome assembly. As part of my PhD, I have also developed a variety of bioinformatic tools to overcome such challenges, which I hope will have broader applications for other tetraploid organisms.

This is a very exciting time. As more microsporidian data keep coming out from all around the world, with many interesting observations and hypothesis, we could potentially see a transformation in the field.

I’m keen to apply my genomics expertise to new areas of research, with different, but hopefully equally wacky organisms. I have been able to see a variety of different genomes whilst panning for gold amongst the data deluge from the Darwin Tree of Life project, and now I am ready for a new challenge!