Scientist Stories: Sebastian Lourido, How Toxoplasma has Taken Over the World

Good afternoon, everyone. Welcome to today's webinar, The Patient Parasite: How Toxoplasma Has Taken Over the World. I'm Sini Shaharwatine, a member here at the Whitehead Institute, where my lab studies states of stasis such as hibernation as a means to harness the therapeutic potential of these biological adaptations. And I'm excited to be your host and moderator today. Today's webinar is a part of a series of talks that we've organized to share Whitehead Institute science and research with the wider community. And in a few moments, we'll hear from our presenter, my fellow Whitehead Institute member, Sebastian Larrido. First, I'd like to let you know that this webinar is being recorded for future viewing. Also, we'll be answering questions at the end of today's presentation, but you can submit a question at any time using the Q&A box at the bottom of your screen. With that, I'd like to introduce our speaker today, Sebastian Larrido. Dr. Sebastian Larrido attended Tulane University where he dual majored and earned degrees in both studio art and cell and molecular biology. After deciding that being a scientist offered much of the creativity he had sought through the arts, Sebastian went on to earn his PhD in 2012 from Washington University in St. Louis in the lab of Dr. David Sibley. Since 2012, Sebastian has been leading an independent lab at the Whitehead Institute, first as a fellow and starting in 2017 as a Whitehead member and as faculty in the Department of Biology at MIT. In 2013, he was awarded the prestigious National Institutes of Health Director's Early Independence award. At Whitehead, Sebastian's lab studies Toxoplasma gondii as a model to investigate the underlying mechanisms of how the Apicomplexia phylum of parasites infect and spread diseases such as malaria and toxoplasmosis. And today he'll discuss his lab's latest findings on how parasites establish long-lived chronic infections in their host. Sebastian, welcome. Thank you. We look forward to this.

Thank you so much, Suneesha. It's really a pleasure to be able to share our work with so many people affiliated and outside of our communities. So to start, I'd like to really kind of take a step back and highlight some of our motivations for studying parasites. I think for me, when I imagine an ecosystem, I typically consider the relationships between free-living organisms as some of the key drivers of adaptations like camouflage and claws and social behavior. However, as free-living organisms thrive, they themselves, and us as some of those free-living organisms, become ecological niches. And many surveys throughout the world have shown that the numbers of these free-living organisms are in fact dwarfed by those of the species that parasitize them. One survey in the jungles of Central America found over a dozen parasite species living in the intestines of jaguars, identified here by some of the eggs and oocysts typically used to classify them. Most of the parasites that are found in these jaguars, just like the jaguar, are animals, and they offer us a glimpse into a much broader pattern. That parasitism is impressively common amongst animals, shown here as that kind of radiation of the tree of life, particularly the branch that comprises the animal phyla. It has evolved independently 223 times amongst this group in 15 out of the 35 large groups of animals, with 5 animal phyla completely devoted to the process of parasitism. The question is more challenging to address when we look at the broader eukaryotic radiation, so all of the organisms that have a nucleus like our own. But there too, we appreciate a similar pattern, that parasitism has independently evolved many, many times and that there are entire phyla that are devoted to the parasitism of animals. This is of course a more complex picture even if we were to consider how some of these organisms in fact are parasites of plants. So parasitism is undoubtedly a dominant strategy amongst eukaryotic species and a key driver of many of the adaptations that we observe in nature. Adopting a parasitic lifestyle is of course often considered a moral failing or at best a simplification of free-living complexity. But today I want you to think about it as one of the great transitions in biology, which like the evolution of multicellularity, all of a sudden affords a new venue for natural selection and the development of complex systems of regulation to navigate those trade-offs that are inherent to this lifestyle. There are incredible adaptations here, like identifying and establishing a niche to replicate in inside another living organism, the ability to manipulate the host defenses of the organisms being parasitized, or for instance, the balancing of replication of the parasite with the need to maintain viability of those hosts in order to ensure transmission to another host. These incredible adaptations have, of course, evolved along very different paths, and our understanding of host-parasite interactions is often crippled by the challenges of studying organisms that only replicate in very specific contexts inside other creatures. You can imagine how hard it is to bring that into the lab. Our own lab seeks to understand the molecular hallmarks of parasitism through the lens of parasites belonging to the phylum Apicomplexa, one of these groups. These single-celled organisms replicate exclusively inside their animal hosts and include several species that are recognized pathogens of humans. These include perhaps the best known of the causative agents of malaria, the parasites belonging to the Plasmodium genus, which continue to cause hundreds of millions of cases each year and nearly half a million deaths. About 10 years ago, a landmark study in The Lancet found that other apicomplexans belonging to the genus Cryptosporidium comprised the second leading cause of diarrheal diseases after rotavirus. And Toxoplasma gondii, the theme of today's lecture, it has been found to chronically infect nearly a quarter to a third of the world's population. And despite being largely asymptomatic, can cause ocular lesions in about 1% of infected individuals and life-threatening diseases in immunocompromised patients or when acquired congenitally. We can also find Toxoplasma throughout the animal kingdom, infecting anything from alpacas to birds to jaguars. We have chosen to study Toxoplasma as a representative of this phylum because of several experimental advantages, including the availability of a continuous culture system that in human cell lines in the lab allows us to perpetually grow the parasites for experimental purposes. They also survive extracellularly, although they don't replicate. And so we can study their biology in isolation from the host cells that they would require to replicate in. And we have a fully competent mouse model of infection where we can replicate many of the phenomena associated with the pathogenesis of toxoplasmosis. Additionally, Toxoplasma has several features that make it easy to genetically edit and thereby test genetic hypotheses as to the function of its genes, which will become relevant throughout today's talk. With this in mind, we can take a picture of where Toxoplasma exists in the environment. It is maintained by transmission between intermediate hosts in which parasites replicate asexually, which can be pretty much any warm-blooded animal on the planet, and definitive feline hosts like cats or jaguars where the parasite can replicate sexually. The product of sexual— of the sexual cycle are these environmentally resistant oocysts that are extremely orally infectious. The reason pregnant individuals should not change the kitty litter is they risk acquiring Toxoplasma and transmitting it to the fetus. Transmission can also occur between intermediate hosts or back into the definitive host by consumption of chronically infected tissues. Within the intermediate hosts, the parasites have the ability to infect and replicate within pretty much any host cell. The process begins with invasion, where parasites direct formation of a specialized compartment inside our host cells, in which they ultimately replicate. Over multiple replication cycles, they form dozens of parasites before ultimately lysing the compartment to disseminate and infect new host cells. These repeated cycles of invasion, replication, and host cell lysis are what destroy the tissues that Toxoplasma infects and cause much of the pathology associated with Apicomplexan infections. If we look at similar trends throughout other diseases caused by Apicomplexans. However, under certain conditions, parasites can opt for a different fate within host cells and in fact replicate into these slow replicating stages that persist intracellularly for weeks or months as these large cysts containing hundreds of parasites. Parasites in this chronic state are resistant to clearance by the immune system and clearance by all available antiparasitic therapies. So Toxoplasma, like herpes and many other chronic infections is for life once it is acquired. This outlines then two distinct cell states for the parasite, this acute stage tachyzoite on the left and the chronic stage bradyzoite. And these prefixes we use, tachy and brady, are for the fast and slow replication of the zoites, the cells of the parasite. And so we've been wondering and working in our lab to try to understand the defining features of the adaptations that have allowed Toxoplasma to undertake these remarkable transitions across its life cycle. And of course, biologists have gotten very good at reading out the genetic code encrypted within DNA, and in part through efforts and by, for many decades, by investigators at Whitehead and the use of incredible model organisms like yeast and worms and flies and mice, we really have gotten very good at doing this. We now know the sequence of the 60 million letters that make up the Toxoplasma genome. And using computer algorithms, we can divide those letters into the functional segments we call genes, each encoding the message to make a different parasite protein. Parasites have about 8,000 such protein-coding genes, and the reality is that we have no idea what most of those genes are doing. And in particular, we have very little information about which of those genes are meant to sustain parasitism, and because they have no counterparts in those free-living organisms, that most of biologists have spent studying. And so we need new tools to be able to dissect the Toxoplasma genome and identify those particular host-pathogen interaction genes that interest us. Several technologies have come into the field over the past years, and in particular within our lab, the efforts of two scientists, Saima Siddiq and Diego Huet, were instrumental in developing a genetic system that would quickly allow us to en masse interfere with each one of the 8,000 or so Toxoplasma genes to create this population of parasites that now contained individual mutations in each one of those genes and were in fact deficient in each one of those particular genes. These populations could then be taken through a particular selective pressure which in the case of some of our early experiments was simply their ability to grow inside mammalian cells. And as these populations made their way through that selection, the ones that were— had lost their ability to infect cells or survive within them were cleared out of the population and were lost to us. We could then take particular sequencing technologies to identify which mutations were in fact, necessary for parasite replication to, in a sense, calculate the genetic contribution to parasite fitness for each one of those 8,000 genes and thereby understand which genes are necessary for Toxoplasma survival inside of human cells. This has granted us a really unprecedented look at the genetic contributions to parasite fitness, within human cells. In a single experiment, we were able to recapitulate the findings of dozens of individual gene studies, sorting along this ranking of fitness genes considered essential from those considered dispensable. We can distinguish those from one another. These screens have provided incredibly useful— have proven incredibly useful in the curation of metabolic pathways together with the lab of Dominique Soldati, in determining pathways of drug resistance and susceptibility, identifying pathways that contribute to resistance against the antimalarial compound artemisinin, and finally, the identification of factors involved in immune evasion together with the lab of Yeroon Tsai. Taking a closer look at some of those parasite-specific genes that participate in the process of infection, Saima Siddiq and Diego— and Dylan Vallieu zeroed in on a gene we came to call CLAMP. When we knocked out CLAMP in parasites, which are shown here in green, we observed something really phenomenal, that those parasites were no longer able to complete their cycle and invade cells. You can see them attaching to the host cells, but ultimately struggling to get in. This is a complex process that involves many different steps and cellular pathways, and we can start zeroing in on precisely which defect, clamp disruption is causing And we've started appreciating that it is in fact this middle step where host cells are being recognized by parasites in a manner that is mediated by CLAMP itself, meaning that they can no longer commit to entering those cells in which they ultimately replicate, a defect that is in fact lethal to the parasites. We, however, collaborated with Jacqueline Niles to investigate whether this gene that we had discovered in Toxoplasma was indeed necessary for Plasmodium falciparum, the causative agent of the most severe forms of malaria. And they, with their genetic tools, engineered Plasmodium parasites where they could conditionally downregulate the expression of Clam shutting it off to examine its consequences. What you can see in the yellow line is that upon reduction of CLAMP expression, those parasite cultures completely crushed, they crashed. So the ability of Plasmodium to survive in red blood cells absolutely depends on the availability of CLAMP in this parasite. This then suggests that we can identify features common to the entire phylum, means in which they are using their adaptations to infect diverse types of host cells by discovering those features initially through our screens in Toxoplasma, something that has us really excited about this technology. However, for the rest of today's talk, I'm going to instead switch to a phenomenon that allows Toxoplasma, as I had introduced, to persist within our bodies for years and perhaps for our entire lifespan. And this project was undertaken by a graduate student, Ben Waldman, who decided to use some of our screening technologies to ask questions about which genes were necessary to regulate that developmental switch between the acute infection and the chronic infection. He devised a really clever strategy where he engineered parasites to become green fluorescent when they upregulated the gene expression program of the chronic stage. So the chronic stage parasites became green. This allowed us to use a special type of cell sorting to be able to isolate from the population those parasites that were green fluorescent. And in the context of our genetic screens, to distinguish which mutants were being lost in the green population compared to a population that had not been induced to differentiate. What stress means in this context is an abiotic stress that is used to induce differentiation in cell culture, a process which naturally occurs during the process of infection in mice. And so we could then appreciate that a single factor was lost from these populations and deemed to be necessary for the induction of the differentiation program, in a sense, for entry of these parasites into that chronic state. We called this one gene BFD1. For Bradyzoite formation deficient one. Investigating what this gene did, we then created a set of strains that would allow us to test whether the gene was indeed necessary for differentiation. Looking at its sequence, we could appreciate that it was a DNA binding factor with a specific domain capable of binding DNA highlighted there in blue that was analogous to a DNA binding domain of a transcription factor called C-MYC in humans. You can see the structure of one such transcription factor binding the DNA double helix in gray in the bottom right. And so we went into the parasite genome and using our engineering tools, we created a clean deletion of that BFD1 locus such that we could create parasites that no longer had BFD1. And then we also tried to reconstruct that locus and fix it with a new copy of BFD1 to validate that anything we saw in the BFD1 knockout was indeed caused by the loss of BFD1, a common control in genetics. And so when we looked at parasites that were under this alkaline stress that induces differentiation, we could now appreciate the formation of these chronic stages, only in the wild-type parasites or in these complemented strains. But we absolutely never observed this in the parasites that were lacking BFD1, demonstrated then that indeed, in parasite cultures, BFD1 is necessary for the process of differentiation, which we could then very carefully quantify over multiple experiments to be sure of the robustness of this result. We then asked whether under conditions of animal infection, BFD1 was indeed also necessary for this process. And so, we took those 3 strains that I outlined, the wild type, knockout, and complemented strain, and we infected mice intraperitoneally to initiate that process of infection. We could appreciate by the weight loss of all of these infected animals that the acute phase of infection, which triggers weight loss, was normal in all of these different strains. And so it was unaltered by the process— by the knockout of BFD1. However, when we looked at the chronic phase of infection starting around 30 days post-infection, we could appreciate that these chronic stages, which are characterized by these large vacuoles replete with parasites seen as the individual magenta dots. Those were only visible in the brains of mice that had been infected with wild-type parasites or with the complemented strain. But again, absolutely never with the parasites that were knocked out for BFD1. This told us that this factor is necessary for entry into the chronic stage not only in cell culture, but also in the mouse model of infection, and most likely in us. We then wanted to ask a slightly different question of whether the factor is indeed sufficient for differentiation. And to do this, we used another method which allows us to very specifically express BFD1 at will. This depends on a fusion protein of BFD1 with a degradation domain, which is intrinsically disordered and destroyed by the parasite cell upon synthesis. However, in the presence of a small molecule called SHIELD-1, this unfolded domain is stabilized, and you get accumulation of whatever factor is linked to it, in this case, BFD1. We could see by several approaches, Western blot, immunofluorescence, that indeed BFD1 in this system was expressed exclusively when we added SHIELD-1 to the media. The consequence of that expression was a near-uniform expression of chronic stage markers in the population under conditions that would normally not trigger differentiation. This was incredibly exciting to us because up till now, We've been using these very rough stimuli like alkaline stress to induce chronic stage differentiation. But now we essentially have a genetic switch with which we can just upregulate differentiation at will and study its consequences. Something very exciting to the understanding of that biological state. We wanted to delve deeper into the molecular biology of this factor. Again, I mentioned that it's a DNA binding factor. And so a logical question is to ask where in the Toxoplasma genome this factor is binding. And to do that, we used a technique called CUT&RUN, which was pioneered in our lab by Dominik Schwartz. CUT&RUN isolates the nuclei with all of those proteins and factors of parasites, and then infuses into them a nuclease that is targeted to the locus of interest, in this case, any place in the genome that's bound by BFD1. This nuclease then excises the specific piece of the genome, which then comes out of those nuclei, and we can then sequence and map throughout the Toxoplasma genome where it is that this factor was bound. When we do that, we can appreciate that BFD1 binds right at where gene expression starts for many of the genes that are upregulated during stress-induced differentiation, which are shown here by the green peaks. And so we can consider BFD1 as a positive regulator of that gene expression program that allows the parasites to survive within us for long periods of time, those chronic stages. This also outlines that not all of the genes that are being upregulated during this chronic differentiation are indeed under the control of BFD1. We can hypothesize that there are secondary waves of regulation that mediate the full expression of all of the genes that are required for chronic stage differentiation. With this in mind, Haley Lakan, when she joined our lab, wanted to find that second wave of gene regulation that was mediating chronic differentiation. And to do so, she selected a set of genes that were upregulated following alkaline stress or BFD1 expression in those experiments I showed you. They were— their promoters were bound by BFD1. And so they were likely candidates of the first wave of gene expression. And they themselves encoded domains known to bind DNA or RNA, implicating them in the control of gene expression within these parasites. There were 5 such factors, and she used again these genetic systems that we have to conditionally degrade each one of them. She tagged them with an mNeon green cassette that allowed us to see them under the microscope, and a minimal auxin-induced degron that upon addition of the small molecule auxin now induced degradation of these proteins, allowing us to study what happens in their absence. And so with this method, she went through all of these different factors and found a single one that behaved similarly to BFD1. We called it BFD2. And so now analyzing the function of BFD2, we could appreciate that the changes in gene expression during that chronic state were nearly identical when we deleted BFD1 or when we deleted BFD2. They suggested that those two operate together in a single genetic circuit to regulate the process of chronic differentiation. Once again, we used our genetic tools to very finely dissect this problem and ask what BFD2, which has several RNA binding domains, might be doing in this process. We created a line that had knocked out BFD2 cleanly and analyzed its consequences once again. During the process of chronic differentiation. What we observed was that once again, the ability of parasites to upregulate these chronic markers under alkaline stress conditions was compromised. They no longer formed this nice expression of chronic stage markers shown in green or to the right in gray. However, in some rare instances, we saw somewhat sputtering incipient formation of some of these cysts, suggesting that the phenotype was not quite as complete as when we knocked out BFD1. Taking this into the mouse model of infection, we observed that this was not sufficient to form chronic stages. Once again, the acute phase of infection and the weight loss in these mice was normal, suggesting that the parasites replicate normally in the acute tachyzoite state. However, when they transition into the chronic state, they're no longer found. We see formation of nice cysts in the wild-type infected animals, but absolutely no cysts in the deltaBFD2 knockout infected mice. Using a lot of different molecular tools, we've come to really understand how BFD2 and BFD1 are operating in unison to control the process of chronic stage differentiation. And the model we've come up with is thus: BFD2 is always present at a low level in unstressed parasites. However, when a— during stress conditions, it has the ability to help the message BFD1 gets translated to make protein and function. And so the BFD1 protein then can go on and bind the promoter of BFD2, induce the upregulation of BFD2 expression, which then makes more BFD2 to help out with making more BFD1. This is what we call in biology a positive feedback loop. Just to show you some experiments that we've used To make these conclusions, we can appreciate that in stress and unstressed conditions, there are similar levels of BFD1 message, of BFD1 mRNA. However, we only find the BFD1 protein under stress conditions. Moreover, when we look at parasites under unstressed or stressed conditions, we see some BFD2 there in the unstressed parasites., as shown there in green or to the right in gray. But the amount of BFT2 definitely increases when the parasites are stressed. And then finally, we see that the ability of parasites to accumulate BFD1 inside their cells depends on the presence of BFT2. To the right in the warmer colors, if BFT2 is absent, you no longer have that robust accumulation of BFD1 in the parasite nucleus. And then finally, we can appreciate by isolating BFD2 and looking at what messages are bound to it, that it is indeed bound to the BFD1 mRNA, however, only under conditions of stress, suggesting that It is the binding and the association of BFD2 with the BFD1 message that is governed by stress and in fact reading out stress in ways that we are really intrigued to figure out. This then comes together into a model that translates stress in these parasites to induce BFD1 expression and play in this positive feedback loop between BFD1 and BFD2 to ensure the translation, to ensure the movement of these parasites into that chronic state that allows them to persist within ourselves. I've shown you that the genetic approaches that we've devised can help us investigate and identify new factors and that this new factor CLAMP is in fact required for invasion in Toxoplasma and essential for the survival of Plasmodium in blood stages. It really brings to light this question of whether we could block parasite replication by inhibiting CLAMP. I've also then shown you that the regulation of chronicity is mediated by this genetic circuit that we've uncovered between BFD1 and BFD2,. And we are really intrigued to know whether if experimentally inhibiting this circuit, we could in fact reactivate Toxoplasma to treat it with the drugs that are effective against the acute stages of the infection. And then finally, we can take a look back and wonder whether the BFD1 regulation helps in fact balance parasite replication and host pathology. Because as parasites move into this chronic state, they stop damaging the tissues of their host, allowing them to preserve the well-being of those host cells that they depend on. And so could stress-responsive differentiation in fact help Toxoplasma persist within a wider range of hosts as they're able to, in a sense, read out the stress around them and realize that they are causing too much damage in a molecular sense. This plays into an equation that we've become really familiar with, which is the basic reproduction number. We've thought about this as we think about the transmission of COVID-19. It estimates how many new cases will result from a primary infection and governs the fate of infectious organisms within host populations. This number needs to be above 1 for an infection to persist. It's influenced, of course, by transmissibility, the probability of an infection to occur given contact between a susceptible individual and an infected individual, the number, the average number of contacts between those susceptible and infected individuals, and finally by the duration of infectiousness. Of course, we pay a lot of attention to all of the things that we can do to reduce this basic reproduction number. We start wearing masks, we attempt physical distancing, we treat infections to prevent transmission and as well as to cure the infected host. However, pathogens are also fighting back. They increase their tissue tropism to be able to generate kind of higher numbers of organisms in a greater diversity of tissues. They, in some cases, affect host behavior and enhance the number of contacts that might occur. And then finally, the way in which Toxoplasma has in fact hacked this equation, they promote latency and chronicity. Dilating this final factor and increasing the basic reproduction number to mediate their persistence within the planet. It's this final variable that I believe has promoted Toxoplasma as this global pathogen. To end, I'd like to acknowledge our collaborators, who have helped us apply many of these cutting-edge approaches to Toxoplasma, our funding sources, in particular the NIH, the Burroughs Wellcome Fund, and the Smith Family Foundation, and all of the wonderful members of our lab who I've acknowledged along the way, who make it really a blast to work every day. With that, I'd be happy to take any questions.

Thank you, Sebastian. As always, it's a true pleasure to hear about your work. And I'd like to invite everyone to submit questions that they have to the Q&A. In the meantime, I'll start with one. What are some ways, I'm curious, that Toxoplasma avoids the immune system of the, of the organism to establish itself?

Yeah, so let me take a step back because I think that We've, we've thought a lot about this question in infectious diseases for generations, and we've in a sense started with some of the most simple infectious agents, viruses, and learned so much about our immune system, about our cell biology, as we investigate that interplay between those two parties, and later kind of molecular bacteriology and it's been hard to get to find purchase in these much more complex organisms, right? Organisms that now have complexity that mirrors our own. And the advent of these molecular tools, and in part some of the work that we've done, but work that our entire field has been doing over the past decade, has allowed us to now start understanding how these parasites, how these much more complex cells in fact deploy an arsenal of approaches to manipulate us and ourselves in various ways. So in terms of Toxoplasma and the immune system, from these organelles that the parasite is releasing upon infection, it's releasing a number of proteins that interfere with innate immune pathways. So these would be pathways that would normally be present within our cells to attack invading organisms. Those are disabled. By Toxoplasma. They can manipulate ways in which the immune system would otherwise recognize them. Of course, their lifestyle itself, hiding inside of our host, of our own cells, and replicating in that protected niche, protects them from other immune responses. And so in a sense, many different layers and many different aspects of parasitism collude to allowing these organisms to subvert our immune responses.

Yeah, that's awesome. Really fascinating. So there is a set of questions that are about the BFD1/BFD2 system from Scott Landfear here and Ed Skalsnik, you know, congratulating you on your brilliant work and asking, on one end, how does the BFD1 protein bind the BFD2 promoter? Then separately, how does— well, let's start with that one.

That's a question that we answered or we tried to answer early on as we started dissecting where in the genome BFD1 was binding. When you take all of those sequences, you can analyze them, of course, for their positional information and which genes it's likely regulating. But you can also look at the sequence where BFD1 appears to be binding and ask of those sequences whether there's any particular motif, right? Any series, repeating series of letters that create the binding site. And we definitely find a predictive binding site for BFD1. And what's been really comforting in work that's occurred since in collaboration with Manuel Linas' group is that if we recombinantly generate just that DNA binding domain and then we use it to sample a complex pool of sequences, we retrieve exactly the same binding motif, suggesting that indeed it is recognizing a particular DNA sequence and binding to it.

Awesome. And now the second part of this kind of loop is how does BFD2 impact BFD1's messenger RNA? Is it stabilizing it? Is the effect there at the level of translation? And how come that this loop doesn't indefinitely keep on going?

What stabilizes it? I think you're asking the exact same questions that we are currently asking in the lab, and they were very exciting to excited to engage in. So we have some evidence now for ribosomal footprinting. This is measuring the machines that make up, that create proteins, right, on the basis of those RNA messages. We can actually monitor where in the entire set of RNAs inside the parasite they are sitting. And we can observe that in fact, under stress conditions, they start attaching to the BFD1 message, but under unstressed conditions, they don't. And so this suggests that BFD2 might be actually managing the binding of ribosomes to the mRNA, to the BFD1 mRNA. In other words, it's regulating the translation of BFD1. This is a somewhat unusual mechanism, although not unheard of, of course, in molecular biology, but unusual mechanism for this type of RNA-binding proteins. And so we're really intrigued to continue digging into the molecular details of this phenomenon.

No, that's awesome. There are a handful of questions that people were asking a little more on perhaps the medical side on the relevance. There is one person in the audience who said that they're diagnosed with toxoplasmosis during pregnancy and asking, can physicians do physicians differentiate between perhaps acquiring Toxoplasma recently as kind of an acute infection versus something that was pre-existing?

Yeah, so I should preface with saying that I'm not a physician, but of course I'm in the field and so have learned a bit from the physicians around me. The general principle for diagnosis of Toxoplasma, it's sort of one of these where the timing of infection really matters because it's so prevalent, you can actually differentiate with certain types of assays whether the infection was acquired recently, and that would be characterized by an IgM response, a certain kind of antibodies, or an IgG response, which is consistent with a more distant infection.. And so, there are serological tests that distinguish between those two and should be performed in kind of any pregnant individual who's newly diagnosed as being toxoplasma positive to make that distinction. There are also drugs that can be used to treat the acute phase of infection, as I mentioned. And that in certain cases might be prescribed by clinicians following such a diagnosis.

Awesome. And thinking along somewhat similar lines, there's a person who's wondering if there is an interaction between toxoplasmosis or toxoplasma and reproductive hormones, I assume at different stages of pregnancy itself.

But yeah, so I don't know of anything that has led us to that conclusion. Of course, that, you know, as scientists, we never want to discredit any kind of potential avenue. The connection with pregnancy seems to be simply the susceptibility of the fetus, right? And the ability of parasites, as I mentioned, to enter cells and enter pretty much any cell in the body, including the cells of the placenta or the cells of the blood-brain barrier. And by doing so, in a sense, cross those particular barriers that are meant to protect sensitive areas of our bodies. And so it is the entry of the parasite into those protected areas where the immune system is not as active or as active in different ways. That then creates a scenario where the parasite can rapidly replicate outside of the control of the immune system. And that's what happens in a developing fetus. It can proliferate and cause a lot of damage before inflammation takes place.

Awesome. Yeah. So back to, back to some questions with regards to BFD1 and BFD2. One of the questions was, are there specific post-translational modifications on BFD2 that allow it to bind BFD1 messenger RNA?

Yeah, I think that that's a great hypothesis. That would be perhaps one of the ways in which stress can be translated into this biochemical activity of binding the mRNA. We have some evidence that under certain conditions, BFD2 is phosphorylated, which is one of these post-translational modifications that can be used to regulate proteins. And we are currently pursuing that hypothesis, whether in fact phosphorylation is changing the behavior of BFD2. I think that it's sort of a key question how we're translating that information from stress to the molecular biology. So one, we're very excited to kind of keep digging.

Awesome. Yeah. And a question by Troy Whitfield is, apart from the RNA binding domain of BFD2, what orthologous proteins have the greatest similarity to BFD2? And can you get any insights by knowing how similar proteins function in other contexts?

Yeah, so it's a pretty big protein, and the only homology to other proteins in genome databases comes from the zinc finger domains, which are these RNA-binding domains, which are of the C3H type, which is sort of what leads us to calling it an RNA-binding protein aside from the empirical evidence. So we can't really find what else it's similar to outside of that region. And this ends up being kind of a common trend. BFD1 is similar where it has a DNA binding domain. We can find similarity to that DNA binding domain in other organisms, but then these large extensive regions with no characterised domain structure, which might be flexible regions that basically aid in attachment or recruitment of other factors through low affinity interactions.

We have lots of people actually asking about this really master regulator that you've discovered and all the excitement about both understanding the basic biology of it and the potential application. Several people are asking, how are you thinking about possibly therapeutically targeting this axis? Do we have the ability to regulate these genes in some way?

We're often caught in this problem of having to work our way up to a point that's treatable, where there— where an intervention is possible. And I think that we're getting there, particularly with this hypothesis that post-translational modifications or kind of enzymatic functions within the cell are in fact regulating the ability of BFD2 to bind BFD1. Many of those enzymes that we could hypothesize could be targeted by small molecules. And so, we could perhaps disrupt the circuit through an interference at a higher level. It is true that RNA-binding proteins and DNA-binding proteins are difficult to drug, but there are also some attempts underway in now thinking about other systems as cancer to interfere with nucleic acid binding proteins. There could be some opportunities there. That's probably a hard way to go. And so we're looking better kind of upstream at some of the modifying enzymes to see whether those would be good candidates for drug development.

Yeah, makes sense. And I was— the person that previously was asking about the mechanism of BFD2 regulation was also kind of expanding on this and asking, you mentioned stress, but is it clear how that initiating I guess, what initiates the BFD1-BFD2 axis?

Yeah, it turns out that experimentally, a lot of different stresses can induce differentiation. And then within the mouse model of infection, and in fact, in cell culture, certain cell types will induce differentiation. And so parasites infecting neurons or muscle cells in isolation will spontaneously differentiate at a higher rate than those infecting fibroblasts or macrophages or some other cell type. So there seems to be some signal that the parasite is getting that is indicating its context. And my ambiguity about that signal indicates that we don't know exactly what it is. It could be metabolic. It could be something else. It could be reactive oxygen species or things that are associated with the immune responses. When you have inflammation, a lot of physiology changes, right? We associate it with the immune system, of course, but you also have depletion of certain nutrients within cells and you have certain oxidative agents being produced by immune cells. And so there are many places where the immune system could interface and stress could interface and the identity of the cells could interface. And the complexity is to disentangle this in a system where you have these two coupled biologies, the biology of the parasite and the biology of the host that it's replicating within. So that's sort of our challenge for the next few years, but one that we're prepared to undertake.

Yeah, we have time for just one more question, actually. And I was thinking about, you've said that Toxoplasma can stay for life, and now you're talking about the different tissues in the body that it can hide in or be embedded in. And one of the people, frankly, was asking, does it produce some type of you know, byproducts that the immune system should recognize. And I'm thinking, or is it dormant in some ways and staying in there largely inactive for long periods of time in specific cells?

And that's how it persists. Yeah. So I keep using this word chronic and not so much latent because in my mind, kind of latent means in some of the viral cases, these very, very silent processes where they don't produce many of the viral proteins or they're just in stasis. And in the case of Toxoplasma, it's more complicated than that. They are definitely not destroying the cells, but they're still replicating a little bit, sometimes less, sometimes more. And, and then every once in a while, it appears like one of these cysts breaks open and they infect new cells and, and establish new chronic stages around. And so it really seems like It is much more of a dynamic process and much more of an active process. So, I think they are definitely producing molecules. They need to kind of cohabitate. There are influences on the immune system that our colleagues studying those aspects have identified as being consistent with the chronic infection. And so, Many things are being changed, and I think it is very kind of tempting to speculate about the effects of those changes in really sensitive tissues like the brain, where we would find these parasites.

Yeah, really beautiful biology, really, really fascinating. So this does, however, conclude our presentation today. We don't have time to answer more questions at this time, but thank you all for joining us today, and special thanks to you, Sebastian, for sharing your work with us.