Cat parasite Toxoplasma tricked to grow in a dish

Nick Petrić Howe

Welcome back to the Nature Podcast, this week: how to grow Toxoplasma without cats...

Shamini Bundell

…and how the world’s largest proteins might turn bacteria into killers. I’m Shamini Bundell.

Nick Petrić Howe

And I'm Nick Petrić Howe.

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Nick Petrić Howe

First up on the show, reporter Benjamin Thompson hears about a new way to culture a tricky-to-grow parasite in the lab…

Benjamin Thompson

Toxoplasmosis is a parasitic disease that affects huge numbers of people. Estimates suggest that perhaps 30% of the population across the globe either are, or have been, infected by Toxoplasma gondii, the single-cell parasite that causes the disease. For many, toxoplasmosis results in mild, flu-like symptoms, but for some — like unborn babies, or those with compromised immune systems, the results can be serious, even deadly. Like a lot of parasitic organisms, Toxoplasma has a complicated, multistage life cycle, but broadly it can be split into two parts: the asexual stages, and the sexual stages. While researchers know a lot about the former, the latter has proved somewhat tough to learn about, for one reason in particular.

Ali Hakimi

In the last 50 years, if you want to study any of the sexual stage you have to infect cats. You have to look, you know, what’s happening in the guts, and in the late 70s there is this seminal studies where they were able, you know, to examine the ultrastructures of all these stage, by infecting cat and killing cat. So for ethical reasons, you can understand very few labs, you know, start, you know, working on those stage just because of the fact that they had to use cats as a model organism.

Benjamin Thompson

This is Ali Hakimi from INSERM in France. He and his colleagues have a paper out in Nature this week laying out a new method to grow part of the parasite’s life cycle in the lab, without the need for cats. Now, cats are normally central to toxoplasma’s life cycle, which they pick up by eating an infected intermediate host, a mouse, let’s say. The parasite evades the mouse’s immune system by hiding, semi-dormant, in cysts. When the cat eats the mouse, these cysts make their way to cells in the gut, where the parasites wake up, and divide asexually inside cells in a phase of their life cycle where they’re known as merozoites. These merozoites are what later develop into the sexual phase of the life cycle, which reproduce, and ultimately come out in cat faeces that contaminate water and food, continuing the infection cycle. This pre-sexual, merozoite stage is normally very difficult to grow in anything but a cat but Ali and his colleagues wondered if there was another way.

Ali Hakimi

We start, you know, thinking of maybe finding a method, you know, to convert, or at least, you know, to culture this merozoite in vitro.

Benjamin Thompson

To do this the team went back to an earlier stage in the parasite's life cycle, when they’re known as tachyzoites. These tachyzoites also divide asexually, but can be easily grown in the lab, without the need for cat guts. Ali and the team reasoned that because there are specific genes turned on and off at each stage of the parasite’s life cycle, there must be a switch that controlled the transition between these two stages. If they could find it, they could artificially get the parasite to develop into merozoites, something that normally only happens inside cells in a cat's intestines. By combing through Toxoplasma gondii’s genome they found two switches that work together, both a type of protein called a transcription factor, snappily titled AP2XII-1 and AP2XI-2.

Ali Hakimi

Those proteins they are going to bind to DNA and to repress genes and the genes they are repressing are the genes that are specific to the merozoites. If you release a brake, you have the expression of the merozoite programme and you are going to make merozoites.

Benjamin Thompson

So having found the brakes that stop tachyzoites becoming merozoites the team went on to cut the brake cables using genetic tools like CRISPR-Cas9.

Ali Hakimi

We used different methods to lower down the quantity of those factors to create an artificial system where we are pushing forwards by inducing the conversion from tachyzoite to merozoite.

Benjamin Thompson

Normally, inside cat intestinal cells a molecular signal would alert the parasite that this is where it needs to be, and this process would occur. But using this new method, the signal from the cat isn’t required. This allowed the team to grow merozoites in a type of human cell that’s commonly used in lab biology. When the team looked at them, it was hard to tell their parasites apart from merozoites grown in cat cells.

Ali Hakimi

So, this is a podcast, but I mean, if I had to show you a slide with pictures that were made like 50 years ago, our images they look pretty the same. And the whole process we describe, all the subcellular content of those merozoites basically they look the same. So, we’re quite proud for us to reproduce what our peers show in infected cats, but 50 years after they did. And using of course, again, this small trick instead of using cat, to process, you know, this differentiation.”

Benjamin Thompson

Not only could this method help avoid the need for using cats as model organisms, it should also help researchers delve into this part of the life cycle to better understand this complex parasite — but this isn’t the end of the story. Because after Toxoplasma reaches the merozoite stage, it differentiates into gametes: male and female cells, which are the sexual reproduction stage of the parasite’s life cycle, and working out how that transition happens is the next part of the puzzle.

Ali Hakimi

We are able just to grow the pre-sexual stage, and then they’re stuck. The idea now for us now is to find new brakes, or maybe activators to go to sex determination, to create male and female gametes. This will be a real also next breakthrough, and I hope, you know, next podcasts, and I would say we need maybe, you know, a decade to get those gametes.

Benjamin Thompson

Ali says that getting to this stage will have a number of benefits. For example, if researchers want to one day make a vaccine for cats to prevent transmission, they’ll need a way to easily culture gametes to understand more about this part of the parasite’s life cycle. Questions remain too about exactly how Toxoplasma moves from cats to intermediate hosts, and how it is able to infect so many different animals like rodents, pigs, birds and, of course, humans, who can pick up the parasite through handling cat faeces or eating unwashed vegetables or infected meat. And given that so many people around the world appear to be carrying Toxoplasma, developing a more straightforward way to grow gametes in vitro without the need for cats could help explain why some infections are more severe in humans than others.

Ali Hakimi

Most of the strain of the parasite right now in Europe and North America are strains that are well adapted to cats that were domesticated, but also to rodents that were domesticated. But when you get infected by strains that are coming from likely wild cats and wild rodents, those strains create several outbreaks in Brazil, in French Guiana, Uruguay, and so on. So, one way of studying those strains is to cross those strains, for example from wild cat with the one from domestic cat and study their virulence markers, and for that we need to culture this sexual stage in vitro. Of course you can make this, you know, by infecting cats, but if you don’t want to infect cat, the only way of doing it is to reach this level of having these gametes, and fertilisation, and so on.

Nick Petrić Howe

That was Ali Hakimi from INSERM in France. To read his paper, head over to the show notes for a link.

Shamini Bundell

Coming up, the mysterious giant proteins that may or may not actually exist. Right now though, it’s time for the Research Highlights, with Dan Fox.

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Dan Fox

Virtual reality might be about to suffer a pest problem, as scientists have developed tiny VR goggles for mice. Researchers can use VR systems to study how animal brains react to a simulated setting. But mice don't always fall for VR illusions: their wide-set eyes revealed the real world in their peripheral vision. Now a team has developed tiny lenses that fit over each of a mouse's eyes. For mice viewing a virtual maze on a curved screen, the goggles provided depth information and filled the animals’ entire field of view with the scene. Mice wearing goggles reacted faster to shapes than those watching on regular screens and also learnt more quickly when they were approaching a reward in the maze, licking their lips in anticipation. Analysing the animal's brains as they used a treadmill to ‘move’ through the maze, revealed that navigation neurons were activating, suggesting the researchers had successfully tricked them. Find your way over to that research in Neuron.

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Dan Fox

A coffee might be just what you need to give your morning a jumpstart, but an unwanted charge could make your preparation less efficient. During grinding, coffee particles accumulate electric charge. This can make the particles clumped together and stick to the grinder, leading to beverage inconsistencies and wasted product. To tackle this, researchers investigated how the charge accumulation is affected by the properties of the coffee beans being ground. They found that less charge built up when they ground beans that had been roasted to a specific degree of darkness or when using a coarser grinding setting. But the largest decrease in charge came when they added a small amount of water to the beans before grinding them. In the case of espresso, this addition also boosted the intensity and reproducibility of the final beverage’s flavour. The team say that the findings could have an economic impact on the coffee industry and might have implications for material science, geophysics and engineering – fields in which similar charge accumulation is being actively studied. Get yourself a hot drink and read that research, in full, in Matter.

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Shamini Bundell

Finally on the show, it’s time for the Briefing Chat, where we discuss a couple of articles that have been highlighted in the Nature Briefing. So, Nick, what have you been reading this week?

Nick Petrić Howe

Well, I've been reading a story in Nature this week about not one of our favourite topics, but maybe one of our very-often-talked-about topics on the Nature Podcast, which is sort of publish or perish. And in this particular case, some authors have kind of taken this to an extreme–

Shamini Bundell

–extreme publishing–

Nick Petrić Howe

–exactly, no, no, no, genuinely, exactly that. So this is a story about extremely productive authors. And so some of these really, really productive authors are producing on average a paper every five days, and that includes weekends, such an almost constant publication of papers.

Shamini Bundell

That seems like a lot of work, to me.

Nick Petrić Howe

It is indeed a lot of work; a lot of work generally goes into making scientific papers. Which is why some researchers are a little bit concerned about these authors that are producing a lot of papers, as they're worried that some sort of duplicitous actions may have gone into getting so many papers so quickly. So this article concerns a preprint, that has been analysing these very sort of highly productive authors. And they've sort of determined like, how many highly productive authors like this there are and where they're from. And so, they found in 2016, there were around 387 authors around the world that were publishing, like one paper every five days — publishing this really large amount. And then in 2022, they found that there are 1266 authors publishing one paper every five days. So, a real big jump since then.

Shamini Bundell

Oh okay, so was that several, several times more just in the last six or seven years? And is that the sort of suspicious thing? Not that anyone could possibly manage to do so much work but how this number sort of gone up so much?

Nick Petrić Howe

Yeah, it's a cause for concern, because perhaps there could be some fields or some authors who are able to produce, you know, a lot of papers. And for this analysis, for example, they didn't include physics authors, because in physics, they have very different sort of ways of authorship. And you can be an author on a lot of papers in physics quite easily. But this is–

Shamini Bundell

–I was ‘gonna say it's not, it's not first authors. It's not like me, by myself, you know, writing a whole paper every five days, this is sort of, including teams and collaborations and things like that.

Nick Petrić Howe

Yeah, exactly. So it could just be that there's been a rise of sort of collaborations and sort of push towards that. But the fact that these numbers have sort of skyrocketed is the thing that's raised concerns, and also, because a previous analysis showed that the number of these highly productive authors had sort of like stabilized in 2014. So it wasn't going up at that point, but then suddenly, in the past six, seven years, it seems to have really gone up and there's some places where it seems to have gone up quite a lot. So a lot of this article talks about Thailand, so Thailand in 2016 had one such highly productive author and now there are 19 highly productive authors. And Thailand was an interesting case, because there's a few different things that seem to be going on to actually cause that. So in Thailand, they've tried to focus more on interdisciplinary teams. So, there's more funding–

Shamini Bundell

–ah–

Nick Petrić Howe

–for bigger teams. So that could be a part of it. But there is also a focus on University Rankings there. And these rankings are sort of underpinned by the numbers of publications. And so to try and get these rankings higher, there are actually cash incentives for researchers to publish a lot. So you could earn up to 28,000 US dollars in a year just by publishing. And you know, maybe because of this, there have been like paper mills, which we know are these sort of nefarious firms that produce fallacious papers, these paper mills have started to propagate in Thailand, and that sort of thing. And there was an investigation that was done on some authors that had these suspiciously high number of published papers. And they found that 33 researchers at 8 universities had paid for authorship.

Shamini Bundell

So that was that was a separate investigation that sort of looked into individuals.

Nick Petrić Howe

Yep.

Shamini Bundell

But this preprint article you're talking about, it has just sort of looked at the numbers, and I guess, looking at the individual countries, then helps you kind of figure out, is this a broad thing across science across a field? Or are there sort of particular causes that vary geographically?

Nick Petrić Howe

Yeah exactly. So in the preprint, they found that pretty much everywhere, the number of these highly productive authors has doubled. But in some places such as Thailand, it has gone up by quite a lot. And the highest absolute numbers were in Saudi Arabia. And the article doesn't exactly go into why that might be. But because of this sort of general increase in highly productive authors, one of the authors of the preprint said that they suspect that questionable research practices and fraud may underlie some of these quite extreme behaviours.

Shamini Bundell

And so are these researchers going to sort of dig into this any further? Is there more to sort of figure out about this sort of situation?

Nick Petrić Howe

Yeah I mean, understanding why it is in some places that this is happening, or why this is happening more broadly across the world. Is it things that we talked about that are maybe just the way science is changing, more focus on interdisciplinary teams, bigger teams, more authors, that sort of thing? Or is it some of this more duplicitous sort of thing? Like paper mills and paying for authorships, and that sort of thing? I mean, the same author that I quoted from before, says that they think that we should focus more on the quality rather than the quantity of publications that scientists do. Publish or perish is a real phenomenon and researchers are, their careers kind of depend on their publications. So perhaps moving towards quality would be better. Although I would caution that with saying that how exactly we determine the quality of a publication is also quite a tricky thing. So yeah, I'm sure we'll be talking about this a lot more though in the future.

Shamini Bundell

No, no easy answers to that one. I'm going to tell you about my story now, which is also not really a simple answer. But we're going back to some basic science here. And I have been reading about the largest protein in the world, maybe, possibly, could be.

Nick Petrić Howe

So I'll confess, I don't know a great amount about determining the size of proteins. But why is it that you're saying ‘maybe’ in such a way? Like, surely if it's the biggest protein, it's quite easy to spot.

Shamini Bundell

When you put it like that, it sounds like it should be. So let's go back to some fundamentals of biology, little primer here. Proteins are made of strings of amino acids. Amino acids are encoded for by sequences of DNA or RNA. So what this story is about, so I've been reading about this in Nature, and the reason, maybe, is that they haven't actually spotted a giant protein and gone ‘oh, my god’, look, there it is, it's huge, you know, looking down the microscope found it, what they've been doing is looking through genome sequences–

Nick Petrić Howe

–right–

Shamini Bundell

–finding these big genes, which potentially encode for like potentially the longest ever protein. So like the biggest number of amino acids, and we're talking huge here, actually so well, compared to the previous world record. So current world record, there's an amino acid protein found in muscles called Titin, and that's been for ages the ‘World's Biggest Protein’, it's 35,000 amino acids that make up this protein. But theoretically, the ones they're kind of maybe looking at now 85,000 amino acids–

Nick Petrić Howe

–woah–

Shamini Bundell

–so more than double the length of the previous longest protein.

Nick Petrić Howe

So what might this potentially enormous protein be used for then?

Shamini Bundell

Yeah, it's a particularly good question when you have sort of started out with like, just like a stretch of DNA and a stretch of amino acids, because sort of famously, it's, it can be quite tricky to work out what the protein at the other end actually looks like from that. So this particular sequence has been found in a phylum of bacteria called Omnitrophota they’re really small bacteria, like really, particularly small cells, even for bacteria, that are already known for having loads of these giant proteins. And there's sort of two ways the researchers have tried to sort of figure out more about what these proteins could be doing. One of it is by trying to reconstruct the shape of the final protein from the amino acid sequence, which is particularly hard because it's so big. So we've chatted about AlphaFold on the podcast before. So this is what it's for predicting protein structures from the sequence. This is Google DeepMind’s sort of AI system. But it's, it's unsurprisingly, not really overly familiar with proteins like that big. It's not, it's not really equipped for proteins larger than a couple of thousand amino acids. So apparently, if you put in a sequence too long, the author of this preprint, where they've talked about this giant protein, and the author says, if you make it too long, alpha fold will just kind of give up at some point and give you a bowl of spaghetti.

Nick Petrić Howe

That's so funny. That's exactly what I would do as well.

Shamini Bundell

And so the way they got around it was they kind of split it up into overlapping sections, and then put each section in and then tried to like, see what those sections do. Sticking them all back together again. And they found sort of functional segments from doing that. So they've got like cell wall, binding regions, regions that look like enzymes that attach to and break up sugars, and other biomolecules found on cell walls. So potentially, what they're thinking is some sort of predatory protein, if I can put it that way, something to do with these bacteria, yet basically predating on and eating other microbes. And there's another strand of evidence that sort of backs up that which is that these Omnitrophota bacteria are actually really hard to grow in the lab. But some people in Germany last year, reported that they've been sort of slow growing, incubating these Omnitrophota in the lab, and then analysing it. And they did some electron micrographs, which seemed to show them attacking and devouring other bacteria and archaea. And this could be what a bunch of their giant proteins that this this phylum has, is for. So seems kind of plausible. Another researcher describes these sort of giant proteins as “sophisticated weapons, wielded by the diminutive microbial hunters in pursuit of bacterial and archaeal prey”.

Nick Petrić Howe

That is very a very cool way to describe it. But yeah, I guess as you say, it's going to be hard to sort of untangle all the bits of this and see how it would eventually form together to be a fully functioning protein. So I guess, what do they need to do next to sort of prove or disprove this hypothesis that it could be an eating device?

Shamini Bundell

Yeah, well, I mean, that's not the only thing they need to prove, actually it's, it's whether that protein, that sequence of 85,000 amino acids, does, in fact, make a single protein.

Nick Petrić Howe

Ah, so they might, they might have a little bit where it stops or something, it might be a few proteins together.

Shamini Bundell

Exactly, if you get snipped up after the fact, just because the gene is there doesn't mean the protein is all in one go. One researcher describes, you know, many of these giant proteins as just imaginary because we don't — we can't see them — we don't we don't know for sure that it exists. It could be yeah, you could start with some sort of big protein chain and then chop it into smaller pieces. And each sort of section does a different job. So that's, I guess that's the next step on this. And the first author of the paper, he is getting his PhD soon and you know, very busy doing lots of things. But there's a quote here, that he wants to see what they actually look like, right? Maybe cryo electron microscopy or something that you can actually map the proteins in cells. And he says, “I just really want to see it and get a ground truth of what it really is, it would be such a cool photo.”

Nick Petrić Howe

I mean, that would be a very cool photo and a great way to round off a thesis like oh, by the way, here's the thing.

Shamini Bundell

And here's a lovely photo! So, sans photo for now we have got the links to those will stick both of those stories in the show notes, so you can check them out and also, as always, you can sign up for the Nature Briefing. We'll have a link to that and get all these kind of cool science stories in your email inbox.

Nick Petrić Howe

That’s not quite all for this week from the Nature Podcast. Stay tuned to your podcast feed as tomorrow they’ll be another extra podcast from us about some new research on COVID vaccines.

Shamini Bundell

Brilliant, well, that is it for today's episode. However, if you want to get in touch with us, then you can. We’re on X, @NaturePodcast, or just send us an email. We are podcast@nature.com. I'm Shamini Bundell…

Nick Petrić Howe

… and I’m Nick Petrić Howe. Thanks for listening.