University Place

The Toxoplasma Life Cycle

– Welcome everyone to Wednesday Nite @ the Lab. I’m Tom Zinnen, I work here at the UW-Madison Biotechnology Center. I also work for the Division of Extension and Wisconsin 4-H, and on behalf of those folks and our other co-organizers, PBS Wisconsin, the Wisconsin Alumni Association, and the UW-Madison Science Alliance, thanks again for coming to Wednesday Nite @ the Lab. We do this every Wednesday night, 50 times a year. Tonight, it’s my pleasure to introduce to you Bruno Martorelli Di Genova. He’s with the Department of Medical Microbiology and Immunology. He’s gonna talk to us about this interesting research that came out in the last few months on how folks in the lab that he works in figured out how to get the toxoplasmosis parasite to grow, not just in cats, but in mice. And I shouldn’t say grow, but to go through its sexual cycle in mice. Bruno was born Sau Paulo, Brazil and went to high school in Sau Paulo, and then he went to the University of Sau Paulo and studied chemistry as an undergrad. Then he stayed at the University of Sau Paulo and got his PhD in microbiology studying Giardia, which is one of my favorite parasites, which I picked up once in the boundary waters and I forgot to drop it off. [audience laughing]

He came here in 2016 to work in the laboratory of Professor Laura Knoll as a postdoc. And he and that team have done this research that really is pretty interesting stuff. We get to hear about this interaction between chemistry and the biology and the sexual cycle of the plasmodium parasite. So would you please join me in welcoming Bruno Martorelli Di Genova to Wednesday Nite @ the Lab. [audience applauding]

– So, hello everyone, it’s a pleasure to be here. My name is Bruno, and we’re gonna be talking about a protozoan parasite named Toxoplasma. And I’ve decided to start this talk with this very beautiful painting of a happy cat. And I hope by the end of this talk, everyone is smiling as this cat. It’s very cheerful, and I hope this is a very cheerful talk. So this is a very conceptual talk.

We’re gonna be touching a lot of different concepts, and I decided to actually build up those concepts before and in a way that everyone can actually appreciate what’s going on. So we’re gonna be talking about protozoan parasites. And the word protozoan is not exactly very common. We don’t actually hear that a lot. And the very easy way to talk about this is saying they are single-celled eukaryotes. What I’m saying with that is an organism made up by one cell. So we have billions of cells. This is another organism that instead of billions, it only have one. When we’re talking about protozoans, we can divide them in two different groups. The first one we call free-living, and by free-living means an organism that actually doesn’t depend on anyone else, so it’s independent, and it can fulfill all its nutritional needs by itself.

The example is Tetrahymena, that is a water protozoan pretty much living by itself, able to do everything by itself. That’s not actually the big picture; that’s actually a different group that is a pathogenic or parasitic group. So parasites, I have three examples, Trypanosoma and Plasmodium, they are very nasty parasites causing disease in humans. The difference between the protozoan group that is free-living and the pathogenic one is the pathogenic one needs a host in order to survive. So there’s intrinsic need for a host; that’s why they’re pathogenic. And we’re gonna be touching this in a little while. So parasite, what am I saying? So this is actually a very nice figure that exemplifies all the possible outcomes of two relationships between the host and the microbe. So the host is the organism that’s gonna be infected, and the microbe is those protozoans that we are studying. So here, we are classifying the outcomes of those relationships as positive, neutral, or negative. By positive is when the relationship is beneficial for the microbe or for the host.

Negative is exactly the opposite, so it’s negative for the microbe or the host. And neutral is when you don’t actually have a change in the outcome. When you’re talking about a relationship that’s negative for both parties, we’re talking about a competition. When you’re talking about a relationship that is positive for both parties, we’re talking about mutualism. Tonight, we are exclusively talking about parasitism. That is a relationship which the host has a negative consequence, so the outcome for the host is negative; however, for the microbe, it’s essential. And that’s very important for it to be very clear. The microbe needs the host. The host is really important for the microbe. That’s why it’s a positive outcome for the microbe.

So just to actually make it really clear, what is the host? Host is any organism that can be parasitized. Meaning that it’s any organism that can actually be infected by a pathogen and then basically, that’s it. So we have many, many different parasites can infect trees, humans, dogs, fish. So basically what I’m trying to say is, host is a very complex and abstract concept that pretty much means anything, any organism that can actually have a parasite. And when you think about hosts and parasite interaction, actually, this is a very complex and very dynamic interaction between the parasite and the host. So one important thing that is important, should be really clear, is a parasite may change behavior or the infection is differing between hosts. So you have parasites that are very deadly for one species; so for one specific host, cause very bad infection and leading to death. And the same parasite in a different host is completely negligible. So there’s no symptom and there’s no outcome for the disease. So this actually is a pretty common thing.

And another important concept is that parasites, sometimes they need more than one host. So they have the need to go to different species of hosts in order to complete what we call a life cycle. So it’s very interesting that parasites, also, they have different development stages. So they are different cells in different hosts. So this tells us there’s a very complex and a very effective interaction between the host and parasites. Much more complex than a simple infection; there’s much more going on. And tonight, we are focusing more on the side of the parasite. So we are just seeing how the parasite actually interacts with the host and how the parasites change towards the infection because of the host is different. So there’s actually a way that it can classify hosts in two different groups, intermediate and definitive. So intermediate host, we have this picture.

It’s just saying so you have one cell, one protozoan cell, and then becomes two and then becomes four over time, so you have replication. So when you have the parasite only replicating, meaning expanding in number in a host and that’s the only function of the host, we classify this host as intermediate. So we don’t have any sexual recombination. You only have a simple expansion in number. So one becomes two, becomes four, eight, sixteen, and so on, so it’s exponential growth of the parasite. Another possibility of the host is what we call definitive. So definitive host is the only host which the parasite can form the gametes and have sexual recombination. So it’s very important to actually make a very clear difference because actually this is a very important concept for tonight’s talk. So you can have a host that pretty much the parasite uses to expand in numbers, so you have replication. So by replication meaning one cell becoming two and four, so the cells are multiplying.

And you have a host that the parasite can form gametes, so recombine its genetic material. And this is actually very important, and different parasites are gonna have different intermediate and different definitive hosts. So that’s actually, parasite to parasite is different, there’s no rule. And we’re gonna be exploring this difference in relationship with Toxoplasma. So the star tonight is Toxoplasma gondii. So it’s a protozoan parasite. So what is a protozoan parasite? It’s a single-cell eukaryote and needs a host to survive. So that’s what I’m saying when I say Toxoplasma’s a protozoan parasite. And the name is very weird, Toxoplasma. And the name comes from the Greek toxon, means bow.

And when you actually see the picture of a Toxoplasma cell, that actually makes sense. So we see here four different Toxoplasma cells, and they have this curved shape, that’s why they have this name. So the name of the parasite, Toxoplasma, basically means you have a cell with a shape that is curved like, banana-like. So even though the name of the protozoan is actually not common, the infection is actually very common. So we’re talking about a parasite that’s everywhere. So this is a map showing different countries and the rates of Toxoplasma infection among the population. And we can see we have as low as 10% in China, for example, and as high as 60% or more in Brazil. So of course, they are different countries, they’re gonna have different incidence of the infection, but overall, when you think about human population, we are thinking about a parasite that potentially infect a third of human population. So one, every three human beings probably carries Toxoplasmic gondii with themselves. So that’s actually a very big number, and as far as we don’t actually think about Toxoplasma everyday, Toxoplasma is there.

A third of human population is a lot of people. We’re talking about two or three billion people that possibly, they are carrying the infection right now. So one actually very interesting thing about Toxoplasma biology, Toxoplasma can infect any warm-blooded animal as an intermediate host. So again, taking back that concept. So Toxoplasma can multiply its numbers, so grow exponentially in any possible warm-blooded animals. So we have examples of Toxoplasma infection in fish, species of birds, in small mammals like rodents, in humans, infecting a third of human population. So it’s a very versatile parasite that actually can infect very different species with completely different lifestyles. This is a very remarkable parasite. It’s not exactly, this is the case for many of them. So Toxoplasma has this ability to infect many, many different species.

And one actually is very interesting about Toxoplasma. Toxoplasma is an intracellular parasite. So this might sound complicated, but it’s basically a cell that is living inside a host cell. So here in this diagram, we have this parasite in green, looking like a little avocado. So pretty much what happens in the beginning of the infection is, this little cell goes inside the host cell that is this bigger cell and then, after the invasion, you’re starting having replication. So then, it’s pretty much what it does in intermediate host, it starts multiplying in two and then you have four and so on. So it gets to the point where the parasites burst out one host cell and then they can re-invade and restart the cycle. So this actually happens in an intermediate host many, many, many times, and that’s actually the way the parasite has to go and disseminate infection throughout the host’s body. So that’s actually is a very important and remarkable characteristic of this parasite. A very interesting thing is parasite not only can infect a very wide range of species, Toxoplasma can infect any nucleated cell type.

So if you just grow any nucleated cell type in a tissue culture room and you just add Toxoplasma there, Toxoplasma will grow. So Toxoplasma is a very versatile parasite. It’s a very– and Toxoplasma has this very amazing mechanism to infect new cells. So, and that’s why I’m bringing it up because I think this is very crazy. Like, there’s not exactly a lot of examples of parasites that actually are able to do that. So when you’re thinking about intermediate hosts, the infection has some steps and I’d like to go over. So pretty much here is represented in a rodent, could be a human, could be bird, could be a fish, as we know now. So the first step of the infection is when the host is exposed to an infectious particle. I will be going over what is an infectious particle in a moment. But by now, basically I’m saying when the parasite is exposure to the infection.

And then the first step of the infection is what you would call acute infection. Acute infection is when the parasite is spreading through the body. So pretty much what I showed last slide. So the parasites, they start to infect new cells, replicating numbers, burst out, go to new cells, restart invading, replicating, and then invading new cells. This cycle go for many, many cycles, spreading the infection throughout the body. So in the acute stage, the parasite goes everywhere. After, what happens is the parasites are growing so much the host has a response towards that. So the immune system of the host is starting to respond and kill off some parasites. This triggers the parasites to differentiate to what we call chronic stage. Chronic stage pretty much is tissue cysts.

So the parasite, instead of be growing and replicating the cells, the Toxoplasma parasite is starting to grow much slower and form cysts in the organs of the intermediate host, in a way to protect itself from the host and the immune system. So that’s actually a way that the infection goes. You have an acute infection, parasites growing very fast and growing everywhere. In the chronic stage, the parasite’s actually responding to that infection and differentiating to a stage that has a wall can protect them. So here’s a picture of a tissue cyst. So you can see, this is brain tissue. And by the center of this picture you have a very dense area, and this dense area is a tissue cyst. So why are the parasites doing that? The tissue cyst confirms a very important characteristic of the parasites, it makes them resistant. So the tissue cyst, the wall, will cover the parasites and protect them from the immune system. So they can be for a long time there without actually being noticed by the immune system.

In the intermediate host, the cysts mainly are in the brain, which is actually very important. It’s very important information and also, they can be found in muscles. So the acute stage can happen everywhere, and I told you Toxoplasma can infect any nucleated cell in the intermediate host. However, when you’re talking about chronic stage, we’re talking about mainly cysts in the brain and in the muscle. And that actually is one infectious stage. Toxoplasma has two, so that’s the first one we’re talking about is a tissue cyst. So very important information about them as well is they can be life-long. So after the parasite develops the chronic stage and you have a wall and they are protected, they can be living in the host as long as the host is alive. So we don’t actually have a lot of idea what happening in humans with very long infections, but as far as we know in rodent models, the chronic stage is persistent, and the parasite stays in the chronic stage for many, many years, possibly life-long. So what that means, means if you’re infected once with Toxoplasma, you’re gonna have Toxoplasma for the rest of your life.

So we talked a lot about intermediate host, and there’s a different host for Toxoplasma. So everything we are talking now is a little different, so we are switching gears to the cats. So Toxoplasma can infect anything, can infect any warm-blooded animal, but infection in cats is really different than what actually we saw before. So the infection in cats, when it starts with the tissue cyst, so this infectious stage that is in the muscles or brain of the intermediate host, the infection, instead of going to acute or chronic stage, it goes to the sexual stages. So in this picture, basically we are showing the differentiation of very complex sexual stages. We don’t need to go over the names of them, but pretty much what is happening is sexual recombination within the cat’s intestine, and by the end of the cycle, we have an oocyst being shed within the cat feces. So the sexual cycle of Toxoplasma gondii is restricted to felines. That means like, among all the possible hosts, that only definitive hosts are felines. So even though Toxoplasma can infect and replicate in fish, birds, in small rodents, in humans, only felines are the platform that Toxoplasma can actually undergo its sexual cycle. By sexual cycle, I’m mainly meaning genetic recombination.

So pretty much, the genes of the parasites are recombined. And this is really important for the parasites to have variability, so that’s how the parasites actually can be evolved selectively and actually, this is a very important function of any organism actually, the sexual reproduction for sexual variability. And another important thing is the oocyst that is shed within the cat feces is at the other infectious stage. So we have two: we have the cysts from the tissues in the intermediate host, and we have the oocysts that only happens in felines and is shed within the feces. So this is the big picture that we call the life cycle. So we have here the representation of rodent, but it’s any intermediate host. So pretty much, you have intermediate host developing acute and then chronic stage, forming tissue cysts that can be fed to a cat, and the cat will develop the very complex sexual stages that will have sexual recombination, and by the end of this, you have an oocyst that is an infectious stage, can infect another rodent, and then so on. So when we’re talking about life cycle and completing the life cycle, we are saying the parasite was able to go over all the possible development stages, so that is actually what completing the life cycle means. So probably the question is, okay, so how do I get infected with Toxoplasma? And according to CDC, what is actually very alarming, Toxoplasma is the leading cause of death attributed to contaminated food in the United States. So we’re not only talking about an infection that is really, really common worldwide, we’re actually talking about an infection that is the leading cause of death attributed to foodborne illness.

And as Toxoplasma has two different infectious stages, the infections actually can happen two different routes. So when you think about oocysts that are only shed by cats, we are talking about oocyst contaminations in water or food. So meaning, if a feral cat sheds oocysts in the corn field, then you eat that corn, you can get infected. Or if you have a domestic cat that happens to be infected and you don’t wash your hands, you might get infected. So pretty much, the oocyst route of infection is by touching or interacting with feces that are contaminated. We’re gonna be talking a lot about this and there’s a lot of mysticism about this, and I want to clarify. The tissue cyst is completely different. So tissue cyst infections, they are not exactly by eating– they don’t have any relation with cats, so they are intermediate host. How this happens? If you got to a restaurant and have lamb that is poorly cooked and if the lamb has cysts, you get infected. And actually, the tissue cyst route of infection is the most common one.

So mainly, most of people that have an infection of Toxoplasma, they are infected by eating poorly cooked or raw contaminated meat. That actually is the biggest route of infection. So meaning that we need to cook our meat. So when we’re talking about prevention of Toxoplasma, we are basically talking about wash your hands. So most of times, wash your hands after handling any cat litter, wash your hands when you handle raw meat. So if you’re just cooking something, wash your hands. This not only prevents Toxoplasma parasites, but also prevents other possible pathogens that can be there. And again, the most common route of infection is eating raw meat that is contaminated, and we’re talking about lamb and pork. Those possibly are the very common species that actually are carrying Toxoplasma, especially pork and lamb. So very important for us, make sure when you’re eating them, the internal temperature is as high as expected for that specific meat.

This actually is pretty important; it’s a very safe measure that people should be doing. A very poor outcome of Toxoplasma infection happens during pregnancy. And this actually is a very problematic stage of Toxoplasma infection. So if a woman is pregnant and she never actually had contact with Toxoplasma, so she’s not infected by Toxoplasma prior to pregnancy and then she gets contaminated by Toxoplasma, the outcomes can be really poor. So what happens is, Toxoplasma can cross the placenta. So you do have congenital transmission of Toxoplasma, meaning the mother gets infected and the parasites are able to cross the placenta and go to the fetus. The outcome of this is usually really severe, and you do have severe brain damage in the fetus. Most of the case, this leads to abortions. So this only happens if the woman has never been exposure to Toxoplasma before. What also we know is if the woman was previously exposure to Toxoplasma, meaning the woman had an infection in the past and now she has the chronic stage, she is protected from infections of Toxoplasma, so this won’t happen.

So this only happens in the acute stage of Toxoplasma, meaning the first initial steps of the infection, and only happen if the woman was not exposure to Toxoplasma prior the pregnancy. The best way to actually be sure is when you know you’re pregnant, go to your physician, and it’s a very easy lab, it’s a blood test, and you know if you have been exposure to Toxoplasma, yes or no, and also just make sure you’re fully cooking your meat before you eat, and everything’s gonna be fine. So it’s really important to clarify a lot of things people talk about cats. And I think that actually is a very important information that I want to bring tonight, is cats can only be infected by Toxoplasma once in their lifetime. Even though they are definitive hosts, they do have an immune system able to prevent further infections when they are initially infected. So they’re gonna have a response, they’re gonna have antibodies in their blood, and they’ll be able to fight newer infections. This is actually very important information because that means a cat will only be infectious for 15 days. So if, for example, you adopt a cat from a shelter, probably the cat has been already infected, and probably the cat will never be infected again. So this is very important information. Another fundamental information is the oocysts that are shed within the cat feces are not infectious up to 24 hours.

So if you change your cat litter every day and you wash your hands, nothing will come back from that. So the most important thing to say is if you’re pregnant and you have a cat, you don’t need to give the cat away. Many people do that, and actually that’s a very common thing. That is a very common practice of pregnant women, and I do understand that you are afraid of having any problems, but if you’re changing your cat litter every day and you wash your hands, you’re fine. Especially when you’re talking about domestic cats that don’t have actually any contact with the outside, it’s completely safe. So the cat actually is not the villain. And again, when we’re talking about new Toxoplasma infections, we’re talking about consuming poorly cooked meat, most of times. So it’s more important for you to make sure you’re fully cooking your meat than actually freak out because of your cat. So we talked a lot about the acute stage and what actually can happen. The chronic stage is a little different.

And so, I told you that a third of human population are possibly contaminated, possible infected, by Toxoplasma. And you might be thinking, okay, but no one says anything, so how are not a third of human populations not sick? Right, that’s an important thing, so how this make sense? And it makes sense because in healthy individuals, there are no symptoms. So once the parasites reach the chronic stage and it’s the brain cysts, it’s not doing much. And your immune system is controlling that infection in a way that nothing actually can happen further the chronic stage. So pretty much, you are protected and the parasite is in this dormant stage, so nothing is actually happened, there is no symptoms from that. This is a little different when we are talking about immuno-compromised or immuno-depressed individual. So if the individual is going through a treatment or has a different medical condition that is depressing the immune system, so pretty much you have the response is not actually working anymore. You’re taking medication that is depressing immune system, the parasite can reactivate, and the reactivation of the parasite will usually lead to brain damage or ocular toxoplasmosis. So as I told you before, the cysts are mainly in the brain and in muscles, right? So if the parasites start sensing that the host’s immune system is not working anymore, they start growing fast again, and this can lead to brain damage. And actually, that’s a very poor outcome of the infection.

And it’s important as a side note to say that up to now, up to today, we don’t have any drug against the chronic stage of Toxoplasma. So we don’t have any drug able to cross the blood-brain barrier, go to the brain and effectively kill the parasite. So this is still a problem, and we still need to have a lot of like investment and a lot of studies to actually find something that actually can prevent this very poor outcome of the infection. So this actually is a very fun slide. So when we think about cat-mouse relationship, we learned from cartoons that mice are usually really afraid of cats, right? We have this image of mice being afraid and hiding and going somewhere ’cause they actually want to avoid the interaction with cats as much as they can. What actually has been showed is when a mouse is infected of Toxoplasma, this mouse potentially loses fear against cats, and it starts to be more vulnerable. So what we know, and it has been shown in this study, is infected mice are less scared. And what actually is proposed is actually there’s a way, as you know, the parasite’s in the brain, and this actually is a change of behavior. And this change of behavior would possibly result in more cats being infected. As the mice are less scared, they’re gonna be easily prey, and then restarts the infection in the cat.

So that, I think, a very interesting relationship and how actually the parasite’s changing behavior in a host. I think that’s very fascinating. So by now, I hope everyone knows that Toxoplasma is very common, that Toxoplasma is transmitted by contaminated food, and everyone is gonna be cooking their meat very fully, and the cats are a very different host when we’re talking about Toxoplasma infection. And that cats allow Toxoplasma gondii to go through its very complex sexual stages and promote sexual recombination in the parasite. And when we’re talking about Toxoplasma infection, we are talking about this parasite always changing. So when you think about the infection by the parasite side, we are thinking about the parasite going from acute to chronic stages in the intermediate host and going through sexual stages in the cat. So what I’m saying with that is the parasite’s not the same. So the different stages, they have completely different growth rates, they have infection rates, they are different. They’re expressing different proteins. They have different lifestyles.

Thy are found different places. So this is very important information because this is where we’re gonna build up from now, just knowing those different development stages. So the big question that I actually wanna talk to you tonight is why cats are the only definitive host. It’s a very complicated question because if you think about it, like how this parasite can infect so many species, so many different cell types, is so limited to felines. What is going on there? So here I’m showing you, without scale, a lot of different species that can be infected by Toxoplasma. So all of those can possibly get infected. However, somehow, the parasite is able to identify the three felines: domestic cat, a bobcat, and a lion, and the sexual cycle only happens in those three. Again, all of those, and the parasite knows somehow, can identify, those three, and it will be very different outcomes of the infection. So this actually is like, what’s going on there? And then we try to work with that in the lab. So in a way to study that without actually using animals, we developed this cat cell model.

So here we have this very round sphere, and like here, and pretty much what this sphere is, they are intestinal cat cells that they grow in this particular shape, and we call them like test tubes. So that’s actually the platform we have to study these questions without using animals. So when I’m saying in vitro in the presentation, I’m meaning I’m studying this question without actually using or harming any animals. So this is pretty much cell culture that grows in a plastic. So the first experiment we want to do in order to understand what’s going on with cats, the rationale is, well, something is happening. There’s something special in cats, somehow Toxoplasma identifies it. So if you got the chronic stage, meaning the brain stage, and we add to our cat cells’ test tube, we hypothesize that it would be able to fully have sexual development in vitro because that makes sense, right? And that actually wasn’t the case. So actually this, what I’m showing you in gray here is the cat cell, so the picture of the cat cell, and in black is the absence of the sexual markers. So as I told you, the parasite has those many different stages, and we do have to identify which stage is present. So if we have any sexual stages initially, they would be lighting up and we would see colors.

Black means absence. So actually, that was weird, and it was like, oh, so what is missing? If we have a cat cell, intestinal cat cell, and in fact of like the chronic stage, pretty much reproducing what happens in nature, why I can’t see any sexual stage? What is happening? And I will tell you that this question has been going on in labs since 1971 or ’72, so it has been awhile because that actually is the date that when the Toxoplasma was discovered to be a cat parasite. Since then, people have been working actively on that, and they couldn’t find what is going on. So I tried, many other people before me also tried, and we couldn’t actually understand what’s going on. So we go back. So what is different? If I have a cat cell, why is that not enough? So actually, when you think about cats, they are very, very different. So first important thing is cats are obligator carnivores. So they actually, they have a need to eat meat. It’s not only a choice, they actually have biochemical and nutritional needs to eat meat. So yeah, I even make like a joke, cats are not small dogs.

So what it means, you cannot feed a cat dog food. That will harm the cat, and why that? Because cats, they have unique dietary requirements, so they do need something that is present in the cat food specially. And one of those things is called arachidonic acid, that is an essential fatty acid. So basically, cat food is supplemented with this special arachidonic acid that is an essential fatty acid. And this is one of the reasons why cats, they must eat their diet. So you cannot feed cats anything. You cannot feed cats rice, and they get sick. So what are fatty acids? Fatty acids are nutrients. So they are commonly found in food. They are lipids, so fat.

They are one of the possible lipids you can have from the diet. And there are many types of fatty acids, and many of them are considered to be essential. When I’m saying a fatty acid’s essential, I’m saying that this fatty acid must be acquired from the diet. So you must eat it in order for your, because your body cannot produce it. So in the way of cats, they actually, one of the essential components of their diet is arachidonic acid that is an essential fatty acid for cats because they need to get this from the diet. So fatty acids, they can be used in two different ways. They can be used as energy source. Pretty much, you eat something, it give you energy to you keep alive and keep functioning. Or they can be transformed by an enzyme in another molecule. So that’s a little more abstract.

So pretty much, that means we have enzymes, enzymes are proteins that they can take a molecule and transform chemically into another molecule. So fatty acids, they can either be just simple energy, or they can be transformed into something else. So another important fatty acid is actually the big star of tonight. It’s called linoleic acid. So linoleic acid is a very common fatty acid, and usually every oil or plant-based oil is pretty much 50% or more linoleic acid in composition. So it’s very common. Olive oil is pretty much linoleic acid. And linoleic acid is one fatty acid that is transformed to arachidonic acid by an enzyme called delta-6 desaturase that I’m shortening for D6D. So what I’m saying is linoleic acid is a fatty acid, you have this enzyme, and this enzyme will convert linoleic acid to arachidonic acid. And an important thing is arachidonic acid is a very important fatty acid because it controls and has many roles in the immune system.

So arachidonic acid is actually very important not to have the energy role, but actually, arachidonic acid’s controlling our response against pathogens, for example. So this is everything I showed you in the last slide, but in a more chemistry representation. So we have linoleic acid, and you have this enzyme that depends on iron. So D6D will convert linoleic acid to arachidonic acid through a pathway. So what actually we know, since the ’70s as well, is cats, they don’t have this enzyme in the small intestine. They don’t for some reason, for some evolutionary reason, they don’t have this enzyme in their guts. So what happens if you don’t have this enzyme? You have an accumulation of linoleic acid. So if cats are carnivores, they’re eating a lot of linoleic acid from the diet for the meats they are eating. And this increase of linoleic acid and not having a functional D6D will make them have a decreasing arachidonic acid. That’s exactly the reason why they need to get from the diet.

So what actually we know is, cats, they don’t have this enzyme, and not having this enzyme, that can change pretty much the behavior of the cat because there must be fat meat in order to survive. This actually is an essential fatty acid, so they do need to have it. So actually, this exemplifies why they have a very different diet. So what is the important message from this is cats are accumulating linoleic acid. So every meal they have, they accumulate and this will go everywhere. So they’re gonna have a lot of linoleic acid, for example, in the bloodstream. And then, this is our second hypothesis, so if cats have so much linoleic acid and is definitely the most different species of mammals according to fatty acid composition that we can think of, is linoleic acid what we’re missing in our model? So we go back to our initial experiment. So here is exactly what I showed you last time, and when we add linoleic acid, we can see colors, meaning we can see sexual stages. So we have two different markers. We have this red one and the green one.

The names don’t matter, what matters is saying just by addition of linoleic acid, we can see the sexual markers being expressed in tissue culture. So this is still being from our cat test tubes, so our cat cells that are growing on a plastic dish. So this actually was pretty exciting. So it seems that somehow, just giving the parasites linoleic acid, you are signaling them that the host is a feline. So a cat cell without linoleic acid doesn’t support sexual development. However, just by adding this nutrient, you somehow are signaling the parasite that this host is a feline, and the sexual development starts. And so, this was actually very interesting for us because we were not predicting that actually this would happen. And in a way, to take this hypothesis further, our question was, well, what about other species? So we know the cats are like, they don’t have D6D in the guts. What about other species? Could we reproduce this just by adding linoleic acid? Could we change D6D expression? Could we do something to try to reproduce this in other species? If this actually is the important signal that the parasite needs in order to know this is a cat? So one thing we started doing, this is the same diagram, is another possibility now we are switching to mouse, by the way. So now we are using mouse cells.

We can actually add an inhibitor. So what is an inhibitor? An inhibitor is a small molecule that will inhibit the function of an enzyme. So when you have D6D plus an inhibitor there’s no activity, so the enzyme doesn’t work anymore. So if you take a mouse cell that has a functional D6D and we add the inhibitor, what will happen? Increase of linoleic acid, decrease of arachidonic acid, and the cell will be cat-like. So our second hypothesis, okay, if that actually is the important thing, if that is the signal that is changing Toxoplasma development, I should be able to reproduce this in a different species because that is the only thing that is different, right? So here is a mouse cell now. So here the gray is the mouse cell, the sexual markers. So if I just in fact took a mouse cell with the parasites without actually adding an inhibitor, just adding linoleic acid, linoleic acid would be converted to arachidonic acid by the functional D6D pathway, nothing will happen. However, what we observed is by adding the inhibitor, I do have the same sexual markers now in a mouse cell after adding linoleic acid and inhibitor. The inhibitor prevented D6D function, and you definitely will have an increase in the concentration of linoleic acid that is signaling the parasite, “This is a feline,” even though it’s a mouse cell. So it seems that only the inhibition of this enzyme is enough to show the parasite this host is feline, so that’s the host that it should have sexual development.

And this is very exciting. That suggests that this pathway is the only difference between a cat and all the other possible species that Toxoplasma can infect. So we decided to actually take this finding further and go to a full mouse. We were feeling very lucky and, okay, we can do this in a tissue culture, what about in animal? So this is a little more complex, but I’ll go over. So pretty much here, we are quantifying those sexual markers. So instead of being with an image that I’m showing you, I’m showing you a graph. So here we have in the y-axis, the highest it is, it means that more is there. So they either can be asexual or sexual. Asexual is all the other stages that are not cat stages. So when we have a mouse, an animal, being fed linoleic acid and infected with the parasite, and then we quantify what is happening there, we can only get asexual parasites, meaning no sexual, so the chronic and acute stages, right? However, if we also feed the mouse inhibitor for this specific enzyme, we now are getting this red bar.

The red bar tells us that those parasites are now sexual. So they are expressing, they are showing those sexual markers as a cat. So this is showing us that our findings in tissue culture, they are actually consistent and they are reproducible in mice. And this is very exciting because we can not only get those markers, but in an animal, we can get an oocyst from the feces of the mouse. So pretty much, we infect a mice, infect a group of mice and they are inhibited with the inhibitor for D6D, we can get an oocyst. And why this is important? So here is a representation of the sexual cycle. So we have the infection, and then the parasite will start developing different stages. You have the gametes and the oocyst. So by us finding oocysts in the feces of a mouse inhibited with the D6D inhibitor, this tells us that the parasite fulfilled a complete life cycle. So it’s pretty much the mouse with inhibitor behaves for the Toxoplasma as a cat.

So we complete breaks the species barrier just by adding an inhibitor of one enzyme. I just wanna actually show our findings in a more graphical way. So when you think about cats, classically, they are infected with tissue cysts. They don’t have D6D, that’s what our finding. So that’s why the parasites, they undergo the sexual development in the guts, and by the end of this, they will shed an oocyst within their feces. The oocyst would then be able to infect an intermediate host, and in the intermediate host, you do have a functional D6D pathway, so the linoleic is converted to arachidonic acid, so the levels of linoleic acid are low, and the parasite go to the sexual cycle, forming the chronic tissue cysts that can actually infect a new cat. So our findings are showing us the only difference between an intermediate and a definitive host is the absence of delta-6-desaturase activity in the guts. Another very important finding is if we take an intermediate host that has the functional D6D pathways and we use the inhibitor, we are completely changing the fate of infection. So we are going through asexual acute chronic stage to sexual cycle and the production of oocysts. So that’s telling us the mechanism of species specificity in Toxoplasma gondii.

So now we know why cats are different. So the important message of tonight and I think is a very interesting thing for us to think about is, only the difference in one nutrient is enough to change the life cycle of a parasite. So our data supports that only the difference of linoleic acid, a very common fatty acid that’s everywhere, olive oil or any other sort of like a plant-based oil, is enough to change the life cycle of a parasite. And that’s actually is a very important thing because we don’t think about this. We think about protozoan parasites, or parasites in general, are very complex, but it’s just simply one nutrient that actually is changing completely the parasite infection. And this also show us that the host and the pathogen relationship is very close. So the metabolic needs of a cat, they change the parasite lifestyle. So this is very complex and everything is interconnected, and this, for us, is very interesting to see the relationship, the very intimate relationship, between the host and the pathogen. So we now, where we are at, we know how it starts. So we do know about the linoleic acid levels, and our data supports that linoleic acid levels is actually what drives parasites towards its sexual cycle.

But the question is, how can we prevent that? So how can actually prevent this to happen? How can we stop the parasites before they form oocysts? For example, develop vaccines. So that actually is our next step, and that is where we want to understand the sexual cycle and a way to stop it. And a very important thing, we were talking, for example, about Wisconsin farmers, is the problem that actually Toxoplasma causes in sheep farms. So sheep are very susceptible to Toxoplasma. So we humans can build up our immune system response enough to only be infected once in our lifetime. So we’re gonna only have one acute stage. Sheep can be reinfected many, many, many times. What is the problem with that? You have many, many cycles of abortions. So the frequency of abortions in sheep flock is really high because Toxoplasma can reinfect sheep many times. So these actually cause a huge economical burden for farmers, and that actually is one of the main reasons why sheep have abortions.

So for example, if we develop a vaccine that can prevent feral cats from shedding oocysts and stop the infection, we are potentially helping sheep farm. So it’s a very interesting way to see how actually a very abstract research can change day-to-day life. Another actually very interesting thing to say is Toxoplasma is not alone. So Toxoplasma has many parasites, many pathogens, that are in the same family. So they are the next cousins of Toxoplasma that we call coccidian subgroup. So the name is actually very complex, but basically what it means is there are many other species of pathogens intimately related to Toxoplasma. For example, Eimeria. Eimeria is a huge problem for chicken industry, so poultry industry loses a lot of money, billions of dollars every year because of Eimeria, and they’re intimately related to Toxoplasma. Neospora causes abortions in bovine. So Neospora, as Toxoplasma does cause abortions in sheep, Neospora does this to bovines.

Cyclospora is a very, very bad human pathogen; it causes very bad diarrhea. So our goal is also, now we understand how one of them actually works and how the cycle actually is built up and its need of fatty acids. We want to take those findings further and also be able to study those other pathogens that are not only changing and affecting humans, but also affecting chicken and bovine. So we want to expand this as further as we can. And on that note, I’d like to thank everyone and thank my lab, the Knoll lab, the Striker lab, also I have a lot of help from the Hehl lab in Zurich. I’d like to thank my funding sources: Morgridge Institute, I am a fellow there, thank you for funding me. NIH, and of course Wisconsin, and thank you everyone that is here, thank you. [audience applauding]