Toxoplasma gondii: A Model Apicomplexan

Part 1: Toxoplasma gondii: A Model Apicomplexan

00:00:07;07 Hi.
00:00:08;07 My name is Sebastian Lourido.
00:00:09;10 I'm a member of the Whitehead Institute and an Assistant Professor of Biology at the
00:00:14;13 Massachusetts Institute of Technology.
00:00:16;17 I'd like to give you an overview of this remarkable phylum of organisms called the apicomplexan parasites
00:00:23;11 through the lens of one of them: Toxoplasma gondii.
00:00:26;20 But before I do that, I thought it would be good to situate ourselves within the
00:00:31;17 Tree of Life, and remind ourselves through this diagram that was recently put together by
00:00:38;09 Jill Banfield and colleagues that the vast majority of life on Earth is microbial.
00:00:44;25 This is... so many of the species that are on this planet are either bacterial or archaeal.
00:00:53;02 And even within our own domain, the eukaryotes, whose genetic information is encapsulated
00:00:58;05 within membranes, even there, multi-cellular organisms are but a few of the species that
00:01:06;14 belong to that phylum, and the vast majority of its diversity is in microbial life.
00:01:12;23 And this is important because we often overlook these organisms, and really only become aware
00:01:18;19 of their existence because they make something good -- like beer or cheese --
00:01:24;05 or because they make something bad -- like disease.
00:01:27;08 In the case of apicomplexan parasites, what they do is they create a lot of
00:01:34;17 mortality and morbidity worldwide.
00:01:37;20 This is exemplified in the extreme by nearly 200 million infections from malaria each year
00:01:45;14 throughout the tropical areas of the world.
00:01:50;09 And this leads to nearly half a million deaths, mostly in young children.
00:01:57;01 At the same time, another apicomplexan parasite causes cryptosporidiosis which is the causative agent
00:02:07;08 of nearly a quarter of infant diarrheas.
00:02:12;11 And these diarrheal diseases account for nearly 10 percent of infant mortality globally.
00:02:18;19 So, it's another way in which these parasites are causing tremendous morbidity.
00:02:24;24 The last exemplar is a bit of an outlier in this sense, because most people who are infected
00:02:31;17 with Toxoplasma gondii, and get the disease toxoplasmosis, even though it's nearly
00:02:39;14 a quarter of the world's population by seroprevalence, very few of them actually become symptomatic
00:02:45;10 or even notice the disease.
00:02:46;24 It threatens, really, only the immune-compromised or pregnant individuals who can
00:02:53;10 carry the infection congenitally to the developing fetus, where it can be lethal.
00:03:00;28 So, these three very different diseases are all caused by representatives of the phylum
00:03:08;15 apicomplexa.
00:03:10;03 And here I'm highlighting them in blue, within the eukaryotic radiation, so that we can appreciate
00:03:18;04 that they're a very distinct branch of this radiation, very different from the
00:03:24;19 canonical models of molecular biology, such as fungi and metazoans and even plants.
00:03:32;26 And because of this difference, many of the lessons that we have learned from
00:03:37;28 studying the molecular biology of these model organisms can't be easily translated to
00:03:44;26 apicomplexan parasites and their molecular biology and, in fact, the principles through which
00:03:50;10 they're able to infect human cells, to replicate within them, and to cause disease.
00:03:58;07 Those molecular principles are difficult to ascertain except through the study of
00:04:05;17 the organisms themselves.
00:04:07;11 If we take a closer look at this apicomplexan radiation, the representative organisms
00:04:13;13 that I've highlighted are shown here, and they really span the entire phylum.
00:04:20;22 Throughout the evolution of this phylum, these parasites have adapted to virtually
00:04:27;19 every branch of the animal radiation, and they have co-evolved for about 500 million years along
00:04:34;21 with animals.
00:04:37;00 Their definitive hosts, which are the ones where the sexual cycle of these organisms
00:04:44;01 can take place, is highlighted for some of these representative organisms in red.
00:04:50;27 And it shows you that often times the cycle can go in that definitive host round and round.
00:04:59;00 And in some other cases you need an intermediate host, such as humans for malaria,
00:05:06;12 or most warm-blooded animals for Toxoplasma, in order to carry that infection from the definitive host
00:05:12;19 back to another definitive host.
00:05:18;06 Throughout this evolution, there have also been sophisticated adaptations to
00:05:23;03 different strategies of coexisting with the cells that they parasitize, because all of these organisms
00:05:30;17 depend on their ability to interact with the cells of the host in order to be able to get
00:05:38;27 the nutrients they need to replicate.
00:05:41;28 In other words, they are obligate intracellular pathogens.
00:05:47;02 And they need to get into the cells that they infect within the host to replicate.
00:05:53;15 And I'm showing you this progression because I think it illustrates how sophisticated these
00:06:00;06 methods of associating with host cells have become, from the really... a cellular vampirism
00:06:08;20 that is found between unicellular, close relatives to the apicomplexa, where they attach to
00:06:15;05 other unicellular organisms and suck out their cytoplasm through a process called myzocytosis,
00:06:22;28 to really sophisticated feeding organelles that are used to draw nutrients out of the endothelial cells
00:06:29;08 of the gut, and the complete encapsulation of the infectious agent within the cells of
00:06:37;15 either our blood, in the case of malaria and its relatives, or virtually any nucleated cell
00:06:45;16 in our body, as is the case with Toxoplasma gondii.
00:06:52;08 I'd like to illustrate that of the handful of organisms in this phylum that we can culture,
00:06:59;19 Toxoplasma has certain attributes that make it particularly well-suited for studying it
00:07:06;09 in the lab.
00:07:07;09 In particular, I'd like to note that it has a method of repairing its genome called non-homologous end joining,
00:07:15;18 which the other cultured organisms lack, and this allows us to perform some sophisticated
00:07:25;03 methods of genome engineering, which I'll highlight in the second part of this talk series.
00:07:30;02 series.
00:07:31;26 We can also culture it continuously in the lab.
00:07:35;04 And we can perform different types of assays with those living organisms.
00:07:41;11 And we can label their amino acids or induce their motility, all aspects that allow us
00:07:47;08 to investigate key elements of the lifecycle of these organisms within this representative
00:07:54;17 apicomplexan parasite, Toxoplasma gondii.
00:07:59;09 by Charles Nicolle, who was working in Tunisia, who found it within these African rodents called the gundis,
00:08:06;12 and Alfonso Splendore, who was working in Brazil and found it killing rabbits.
00:08:14;08 called the gundis, and Alfonso Splendore, who was working in Brazil and found it killing
00:08:22;12 rabbits.
00:08:24;10 In both cases, because they found it within nucleated cells, they categorized it initially
00:08:30;05 as a Leishmania species, but later, through careful microscopic evaluation, realized that
00:08:37;09 the shape of these organisms was unlike that of Leishmania.
00:08:41;04 And Nicolle eventually called his species of Toxoplasma Toxoplasma gondii because
00:08:48;02 it had been found within a gundi.
00:08:51;27 This led to a flurry of papers where these organisms, or what people thought were related organisms,
00:09:01;18 were found in virtually every warm-blooded species that people were looking at.
00:09:04;18 This led to a flurry of papers where these organisms, or what people thought were related
00:09:11;10 In other words, they were indistinguishable by serology and in fact, now, we know,
00:09:18;08 virtually indistinguishable by their genetic makeup.
00:09:23;18 So, this is remarkable for one of these parasitic organisms, because it means that the
00:09:30;12 same species has the ability to infect virtually every warm-blooded animal that experimentally
00:09:33;25 So, this is remarkable for one of these parasitic organisms, because it means that the same
00:09:41;11 species has the ability to infect virtually every warm-blooded animal that experimentally
00:09:49;15 has been infected.
00:09:54;07 But in immune-compromised individuals, it can lead to inflammation of the heart, brain, and eyes,
00:10:01;01 and really severe tissue damage.
00:10:03;13 But in immune-compromised individuals, it can lead to inflammation of the heart, brain,
00:10:09;25 And as I mentioned, immune-compromised individuals are unable to control that infection,
00:10:13;12 and can succumb to it if untreated.
00:10:19;28 Healthy individuals, especially in areas where there's high incidence of Toxoplasma,
00:10:25;10 such as in Latin America, can get ocular lesions.
00:10:27;17 Healthy individuals, especially in areas where there's high incidence of Toxoplasma, such
00:10:34;02 in more detail, but these ocular lesions, because we're only able to treat the
00:10:38;25 acute form of the infection, lead to sequelae that can [unknown] and oftentimes end up in
00:10:44;09 loss of eyesight within the infected eye.
00:10:50;05 form of the infection, lead to sequelae that can [unknown] and oftentimes end up in loss
00:10:59;04 of eyesight within the infected eye.
00:11:03;24 I mentioned that Toxoplasma can infect nearly every warm-blooded animal.
00:11:12;01 And this leads to different cycles of propagation of the infection throughout the environment.
00:11:19;06 So, cats can shed infectious stages, that I'll get to, that we can consume orally accidentally,
00:11:26;24 But there's a second way in which carnivores can get infected with Toxoplasma,
00:11:34;06 and that's by consuming the flesh of infected animals.
00:11:35;22 But there's a second way in which carnivores can get infected with Toxoplasma, and that's
00:11:39;25 by consuming the flesh of infected animals.
00:11:44;07 Because once Toxoplasma finds itself within these intermediate hosts, it can develop into
00:11:51;10 latent stages that persist for the life of the host.
00:11:55;20 So, an individual that can be seen to be seropositive, because they have antibodies against Toxoplasma,
00:12:04;20 most likely has... somewhere within their bodies, typically in vascularized tissues
00:12:09;14 Of course, because we're rarely consumed by other carnivores, we ourselves are
00:12:15;10 dead-end hosts for this infection.
00:12:20;17 I've noted that felines are on the top of this life cycle of Toxoplasma because
00:12:27;08 they play a very important role as the definitive hosts of the infection.
00:12:30;06 I've noted that felines are on the top of this life cycle of Toxoplasma because they
00:12:38;11 play a very important role as the definitive hosts of the infection.
00:12:44;07 And what that means is that when cats consume the tissue cysts that are found within latently
00:12:51;10 infected animals, say because they hunted a cat... they... they hunted a mouse that
00:12:57;28 was infected with Toxoplasma, they can then have an intestinal infection where Toxoplasma
00:13:06;16 can differentiate into all other stages within their sexual cycle.
00:13:14;04 And at the conclusion of that sexual cycle are these very environmentally resistant oocysts,
00:13:20;24 which are shed within the feces of the cat.
00:13:23;17 And they're probably the reason many of you have heard about pregnant women not changing
00:13:29;23 the kitty litter.
00:13:31;03 That's because they can... these oocysts are extremely, extremely infectious to us and
00:13:37;18 To go into a little bit more detail, within the intestine of the... of the cat, or any other feline,
00:13:39;20 these tissue cysts can rupture and give rise to invasive forms that
00:13:43;10 enter the cells of the intestine, replicate within them.
00:13:50;10 other feline, these tissue cysts can rupture and give rise to invasive forms that enter
00:13:57;06 the cells of the intestine, replicate within them.
00:14:01;26 They can replicate asexually, leading to more and more of these infectious forms, or they
00:14:07;18 can differentiate into these sexual forms, that form male and female gametocytes.
00:14:15;12 And then the zygotic product of that... of that mating can lead to these oocysts,
00:14:20;12 which then sporulate -- essentially go through meiosis -- within the feces of the cat and become
00:14:22;02 And then the zygotic product of that... of that mating can lead to these oocysts, which
00:14:31;23 then sporulate -- essentially go through meiosis -- within the feces of the cat and become
00:14:39;20 For the rest of my talk, I'm going to focus on the asexual cycle of Toxoplasma,
00:14:42;14 because it's the source of most of the tissue damage that's associated with the infection.
00:14:50;18 This asexual cycle starts with the invasion process, which is a process that's
00:14:55;24 common to all apicomplexans, which they use to enter and create their own niche within the cells
00:15:00;27 This asexual cycle starts with the invasion process, which is a process that's common
00:15:06;15 to all apicomplexans, which they use to enter and create their own niche within the cells
00:15:13;25 that they infect.
00:15:17;03 egress from those cells, rupturing and destroying the cell as they... as they move out and
00:15:22;21 try to find new cells to invade and fulfill the cycle again.
00:15:31;08 This entire cycle within the lab can be completed within human fibroblasts in 40-48 hours,
00:15:38;14 with one cell division -- one parasite going to two -- every 6-10 hours.
00:15:44;01 This entire cycle within the lab can be completed within human fibroblasts in 40-48 hours, with
00:15:53;15 one cell division -- one parasite going to two -- every 6-10 hours.
00:16:03;03 To understand how apicomplexan parasites are able to invade host cells, it's important
00:16:10;09 to understand their cell biology.
00:16:12;26 host cell to invade, we'll find many of the same organelles that you'd find in
00:16:15;26 any of our own cells.
00:16:21;15 host cell to invade, we'll find many of the same organelles that you'd find in any of
00:16:26;14 our own cells.
00:16:28;03 You'll find a nucleus, an endoplasmic reticulum, a Golgi apparatus, and a mitochondrion.
00:16:34;09 A single mitochondrion in the case of Toxoplasma.
00:16:36;27 But you'll also find some peculiar organelles that are more specific to this phylum or to
00:16:44;17 its close relatives in the Alveolata.
00:16:50;17 Amongst these organelles is the apicoplast, which is an organelle that originated from
00:16:57;28 between a proteobacterium, over here, and some sort of eukaryote at the base of the radiation.
00:17:00;25 Many of us are familiar with the source of mitochondria being an endosymbiosis that occurred
00:17:08;18 between a proteobacterium, over here, and some sort of eukaryote at the base of the
00:17:16;26 radiation.
00:17:18;11 And that gave rise to the mitochondrion.
00:17:22;00 And similarly, plastids within plants and green al... and green and red algae originated
00:17:28;07 from the engulfment of a cyanobacterium.
00:17:31;25 Those are primary endosymbiotic events.
00:17:35;19 And the closest free-living ancestor of apicomplexans still carries this apicoplast in its photosynthetic state,
00:17:44;26 as shown here by that pigment within this relative, Chromera velia.
00:17:50;04 And that red algal cell gave rise to this organelle called the apicoplast.
00:17:57;12 And the closest free-living ancestor of apicomplexans still carries this apicoplast in its photosynthetic
00:18:06;25 state, as shown here by that pigment within this relative, Chromera velia.
00:18:13;20 And if we look by electron tomography, here in a beautiful study by the Frischknecht lab,
00:18:20;24 we can see four membranes, which are the remnants of all of those endosymbiotic events that
00:18:27;18 gave rise to the plastid itself.
00:18:33;06 And it's essentially a set of flattened vesicles that are stitched together and subtend
00:18:43;00 the entire plasma membrane.
00:18:44;22 It's called the inner membrane complex.
00:18:47;04 And it's essentially a set of flattened vesicles that are stitched together and subtend the
00:18:52;13 entire plasma membrane.
00:18:54;23 with a particular fluorescent protein that shows you how these parasites form daughter cells
00:19:01;24 within the mother cell, essentially forming all of the structures except for the
00:19:11;15 And what I'm showing here in this video is actually the inner membrane complex marked
00:19:17;17 with a particular fluorescent protein that shows you how these parasites form daughter
00:19:24;01 cells within the mother cell, essentially forming all of the structures except for the
00:19:30;02 plasma membrane, which invaginates and gives rise to those daughter cells at the very end
00:19:36;22 of replication.
00:19:37;27 To illustrate some of the other ways in which they can replicate, and in which Toxo,
00:19:40;05 in fact, can replicate within the gut of the cat, I have this illustration here.
00:19:47;05 And it's just one of the ways in which apicomplexan parasites can replicate themselves.
00:19:55;00 but in fact other organisms like Plasmodium, the causative agents of malaria,
00:19:59;15 can form multiple cells from a single division in a process called schizogony.
00:20:07;05 I already went through endodyogeny, that formation of two daughter cells within the mother cell,
00:20:13;24 but in fact other organisms like Plasmodium, the causative agents of malaria,
00:20:20;23 can form multiple cells from a single division in a process called schizogony.
00:20:27;10 And other apicomplexans yet, like Sarcocystis, can duplicate their genome several times before
00:20:36;05 dividing their nucleus in a process called endopolygeny.
00:20:40;20 So, you can appreciate the tremendous variety of cell cycles that are possible within the
00:20:50;14 apicomplexan phylum.
00:20:53;05 Another feature of this inner membrane complex is that, in conjunction with secretory vesicles
00:21:00;13 called the micronemes, which are secreted at the apical end of the parasite through
00:21:05;12 the conoid, which is a rigid structure that allows the cell to penetrate hard tissues...
00:21:13;03 these micronemes can release adhesins, which get translocated to the posterior of the cell
00:21:19;13 by actomyosin motors that are anchored within the inner membrane complex.
00:21:25;11 The motors... the anchored motors are shown here in blue, and the adhesins are shown,
00:21:30;28 secreted at the apical end, engaging that motor through actin, and translocated to the posterior.
00:21:38;04 And it's that treadmilling of adhesins that propels the parasites forward in a process
00:21:44;12 called gliding motility.
00:21:45;28 Again, this is a really unique adaptation that allows parasites to invade host cells
00:21:52;05 and to move through tissues.
00:21:54;20 And you can see it here, in this experimental injection of parasites into a mouse ear, where,
00:22:04;07 by two-photon microscopy, you can see the movement of parasites within these tissues.
00:22:11;07 And the characteristic shape of Toxoplasma, along with this treadmilling, gives rise to
00:22:18;13 corkscrew motion through the tissues.
00:22:22;20 A final feature that I'd like to highlight is the combination of organelles that
00:22:31;15 give rise to the process of invasion.
00:22:34;07 I mentioned that adhesins are secreted through the apical end, but then how do they stick
00:22:39;26 to the host cell to effectuate the process of invasion?
00:22:44;13 In fact, a second class of organelles called the rhoptries, which are these club-shaped organelles,
00:22:49;24 are secreted only at the moment of host cell recognition and place
00:22:56;03 within the membrane of the host cell the receptor for some of those adhesins.
00:23:04;09 The structure of these has been solved.
00:23:06;25 In the case of the microneme protein AMA1 and the rhoptry protein RON2, they form
00:23:14;02 a very tight complex that allows parasites to form the tight junction through which
00:23:20;10 they can propel themselves into the host cell being infected.
00:23:26;11 We can see the process of RON2 deposition by tagging it with a fluorescent a protein.
00:23:34;18 And you can see that during the process of invasion -- in this beautiful video by Isabelle Tardieux --
00:23:40;00 you see the formation of a ring that follows the surface of the parasite
00:23:46;09 all the way until it's inside the host cell.
00:23:50;24 Rhoptries, in addition to their important role during invasion, have a second role,
00:23:57;10 which is in the secretion of effectors, along with a second set of organelles,
00:24:02;24 the dense granules.
00:24:05;06 These effectors can have many different effects, and they are a very active area of Toxoplasma research.
00:24:13;08 They are secreted directly or indirectly into host cells.
00:24:17;17 Some of them require specialized export machinery.
00:24:21;20 They are involved in host manipulation
00:24:24;18 such as mitochondrial recruitment or transcriptional regulation.
00:24:30;00 And they are necessary to evade innate and adaptive immune responses.
00:24:36;06 Differences in these effectors can lead to dramatic differences in the virulence of
00:24:41;24 different Toxo strains, and some think that they might be responsible for the highly virulent strains
00:24:49;17 that are found in Latin America.
00:24:54;24 An important part of deploying all of these organelles, allowing the parasites
00:25:02;02 to move and eventually invade, is signaling.
00:25:06;09 And in the case of motility, one of the primary messages within the parasite
00:25:13;22 that tells the parasites to get going is calcium.
00:25:18;08 Calcium is used by our own cells -- for instance, in neurons secreting neurotransmitters,
00:25:24;11 or even in our muscles contracting -- but the ways in which Toxoplasma untangles those calcium signals
00:25:31;17 and changes its cellular behaviors are very different from our own.
00:25:38;05 But I'd like to give you a few examples of how calcium is working.
00:25:43;17 During invasion, we can observe calcium by having parasites that are expressing
00:25:50;03 a green fluorescent sensor that becomes brighter when the cytosol is experiencing more calcium.
00:25:58;06 And you'll see the cell oscillate in its calcium concentrations, prior to a peak of calcium
00:26:07;21 right before they invade, and then a dramatic decrease in the total cytosolic calcium concentrations
00:26:15;00 once the cell has invaded.
00:26:16;22 So, you can imagine how these modulations of calcium are allowing the cells to respond
00:26:24;02 to the interaction with host cells or to produce the secretion of adhesins that's necessary
00:26:30;06 to propel motility.
00:26:32;17 On the other side of the life cycle, we can artificially stimulate motility with
00:26:38;09 different agonists.
00:26:40;02 Shown here are intracellular parasites.
00:26:43;24 They're green because of this calcium reporter.
00:26:46;14 And they're also secreting a red fluorescent protein that fills up that parasitophorous vacuole
00:26:51;13 so that we can monitor the permeabilization of that vacuole in relation to the initiation
00:26:58;14 of motility.
00:27:00;05 And when we stimulate parasites, what you can see is a dramatic increase in intracellular calcium
00:27:05;20 prior to the permeabilization of the parasitophorous vacuole, and that initiation
00:27:11;00 of motility.
00:27:12;27 And so we know that calcium is a necessary signal for motility, both at its onset,
00:27:20;24 when the parasites are egressing, and during the invasion process.
00:27:29;08 Unlike in our own cells, where calcium signals are transduced by calcium/calmodulin-dependent
00:27:37;06 protein kinases, apicomplexans, like plants and other protists, have a completely different
00:27:43;24 class of enzymes that are transducing those signals.
00:27:48;09 And those are the calcium-dependent protein kinases.
00:27:51;27 These are different because they intrinsically have the ability to sense calcium.
00:27:58;02 And that's unlike our own kinases, which require calcium sensing components external to them
00:28:04;28 to activate them.
00:28:07;08 And so this leads to structures that are exquisitely responsive to calcium signals.
00:28:14;28 Shown here is one of these kinases, in fact, the one that regulates that secretion of adhesins.
00:28:21;09 And you see it here in the absence of calcium, where the active site of the kinase is occluded
00:28:28;23 by this regulatory domain, in orange.
00:28:31;27 And that domain can be changed dramatically, by binding to calcium, and restructure itself
00:28:41;18 on the other side of the kinase, inducing activation of the kinase, which then allows it
00:28:49;14 to change other proteins which are participating in this process of motility and invasion.
00:28:58;08 And this is just one example in which apicomplexan parasites do it differently.
00:29:05;03 And that's why it's so important to study their activities locally, and to understand
00:29:11;01 their cell biology within the context of a model apicomplexan organism.
00:29:17;24 And in my second talk, I'll discuss the methods that we're taking within our own lab to
00:29:25;11 address this question, and get genetic tools to really investigate apicomplexan parasitism.
00:29:32;04 Thank you.

Part 2: Genetic Approaches to Study Toxoplasma gondii

00:00:08;02 My name is Sebastian Lourido.
00:00:09;21 I'm a member of the Whitehead Institute and an Assistant Professor of Biology at the
00:00:14;09 Massachusetts Institute of Technology.
00:00:15;27 I'm going to tell you about the genetic approaches that we're using in our lab to study
00:00:21;13 Toxoplasma gondii.
00:00:24;00 In my previous lecture, I introduced the life cycle of Toxoplasma and gave some of the highlights
00:00:29;28 of what we know about how these organisms fulfill this really remarkable feat of
00:00:37;17 infecting human cells, replicating within them, and coming back out.
00:00:42;17 But I also mentioned that there's very little that we actually know about them because
00:00:47;14 they're very different from the canonical models of molecular biology.
00:00:53;15 And so I'd like to tell you about what we're doing to really get a broad view of the
00:01:00;10 genetic makeup of these organisms, and understand what we don't know about them.
00:01:06;16 So, before I get going, I can give you an example of why we believe that Toxoplasma's genome
00:01:15;16 is largely cryptic, or why we really understand so little.
00:01:21;01 If we take the genes of Toxoplasma -- and we can do this exercise for any of the other apicomplexans --
00:01:26;16 we can categorize genes by whether they are conserved in all of the eukaryotes
00:01:34;19 or only in the most closely related ones, or only in apicomplexans, and we can do this
00:01:42;04 to generate sort of bins of genes that are of increasing depth of conservation.
00:01:50;18 And if we do this and we look at the composition of these bins, we can see that the more deeply
00:01:59;10 conserved a gene is -- for instance, in this eukaryotic category -- the better annotated
00:02:05;26 it is.
00:02:06;26 In other words, fewer of those genes are categorized as hypothetical proteins, which means that
00:02:14;00 most of those genes have cognate homologues or orthologues in other organisms.
00:02:21;09 But the closer we get to apicomplexan-specific features or to genes that are
00:02:26;09 exclusively found within Toxoplasma, the less we know about them.
00:02:31;04 So that, of the 1200 or so genes that are only found within Toxoplasma and have no correlates
00:02:38;03 outside of the species, nearly 90% of them are categorized as hypothetical.
00:02:45;15 And that is evidence for how little we really know about even the genes that we really care about,
00:02:50;24 such as the ones that are conserved throughout the apicomplexan phylum.
00:02:56;08 And so we need better tools to dissect them and to understand what these genes are doing.
00:03:02;04 And so, when I started my lab at the Whitehead Institute, I was captivated by the possibility
00:03:08;19 of adopting the latest genetic engineering strategies, which were being implemented in
00:03:16;11 mammalian biology.
00:03:18;13 And what I'm talking about is CRISPR/Cas9, which is this remarkable system that has really
00:03:24;12 taken over genetics because of its remarkable efficiency and portability.
00:03:31;28 In a single plasmid, illustrated here, we can code for the guide RNA, which instructs
00:03:38;25 the nuclease, coded for in the same plasmid, where to bind, where to go
00:03:44;11 within the genome of choice, and, based on 20 nucleotides of homology to the site that we want to cut,
00:03:52;22 we can really tell this nuclease where to go, where to cut, and where to generate
00:03:59;16 a double-stranded break.
00:04:01;06 And this is important because generating that local double-stranded break is key to efficiently
00:04:09;26 disrupting a given gene or modifying that particular site within the genome of an organism.
00:04:16;24 And this worked remarkably well in Toxoplasma.
00:04:19;24 So, what had taken about six months when I was in graduate school, now we could
00:04:26;25 do within a matter of days by transfecting this one plasmid that contained the nuclease and the
00:04:32;25 address for where... where we wanted to disrupt.
00:04:36;02 And what you see here is the disruption of a surface antigen called SAG1.
00:04:42;26 SAG1 is not essential within the culture conditions that we typically keep the parasites under,
00:04:51;09 and we have an antibody against it, which is why we targeted it.
00:04:54;22 And what you can see is that, in the absence of any selection, just by introducing through transfection
00:05:01;15 this one plasmid, we could get vacuoles of parasites that lacked SAG1 staining.
00:05:10;00 And in fact, nearly 20% of the vacuoles in the resulting population were disrupted
00:05:16;21 for this surface antigen.
00:05:17;28 This is a remarkable efficiency in the absence of any selection and really made us very,
00:05:25;08 very eager to try it in different settings.
00:05:28;17 One thing I will note is that this rate of disruption depends on the fact that Toxoplasma
00:05:35;05 fixes its genome through non-homologous end joining.
00:05:39;00 That means that it can take two broken ends of DNA and just splice them together
00:05:46;02 through this method that generates small changes that disrupt the coding of different genes.
00:05:54;27 And we knew that this was the case because if we removed a protein that's necessary
00:06:00;16 for the process of non-homologous end joining the, KU80 protein, the rates of SAG1 disruption
00:06:08;10 plummet.
00:06:09;26 And this is the case because those parasites are unable to repair their genomes, and die.
00:06:17;09 This in fact ends up being useful in other settings, where we want the genome to be fixed
00:06:23;00 only in the presence of a repair template, so that we can dictate what changes
00:06:28;23 should be made to the genomic sequence.
00:06:34;06 That's what I'm illustrating here.
00:06:36;18 So, the locus that we targeted in this case was the end of the coding sequence of
00:06:44;02 one of these calcium-dependent protein kinases that I talked about in my previous presentation,
00:06:50;18 a... the protein CDPK3.
00:06:54;08 And we'd... directed the cut right at the very end of the coding sequence, where we
00:06:59;09 could introduce an epitope tag by, in addition to the CRISPR system, including a repair template.
00:07:08;05 This was a synthetic repair template that had 40 base pairs of homology on either end
00:07:14;22 that were homologous to the cut site and could be, through the homologous recombination machinery,
00:07:21;24 incorporated into that genomic sequence, and include, in tandem to the protein sequence
00:07:26;26 of CDPK3, an epitope tag that allows us to visualize it with this particular antibody.
00:07:34;27 And what we observed was that, in contrast to what we had seen for gene disruption,
00:07:40;24 in the absence of non-homologous end joining we actually had an increased rate of
00:07:46;19 homologous recombination-mediated repair and of the incorporation of the desired rearrangement.
00:07:53;17 So, these are very powerful tools to study the genomes of apicomplexan organisms
00:08:00;26 like Toxoplasma gondii.
00:08:03;27 And so we thought, well, we can apply this to particular dedicated settings.
00:08:10;09 Why don't we try to apply it to the entire genome?
00:08:14;06 There are about 8,000 protein coding genes in Toxoplasma gondii, and scientists
00:08:21;16 had only really studied a handful of them.
00:08:24;20 There had been some genome-wide approaches previously, based on genetic crosses,
00:08:31;24 classical genetic crosses, but these require, as you'll know from my previous lecture,
00:08:38;14 the definitive host, which is the cat, which is difficult to use,
00:08:44;17 and they require probing of pre-existing variation within two parental strains that are crossed.
00:08:53;27 And then those changes, or the source of that variation, is disambiguated based on
00:08:59;24 classical genetics.
00:09:01;17 So, that's difficult to implement and has several restrictions in terms of understanding
00:09:06;28 what the entire genome is doing.
00:09:10;22 And as I mentioned, we had targeted approaches that allowed us to, in a very dedicated fashion,
00:09:16;24 manipulate one gene at a time.
00:09:18;26 But these were difficult to implement at scale.
00:09:22;22 And they are very gene-dependent -- so, some genes are amenable to some strategies but not others,
00:09:28;12 and so they're very difficult technologies to implement in a genome-wide approach.
00:09:37;28 And I should also note that apicomplexans lack RNAi, which had been used previously
00:09:45;05 in many organisms to perform genome-wide screens.
00:09:51;17 But in apicomplexans they just can't be implemented because the machinery that is required by them
00:09:57;21 is simply not there.
00:09:59;26 So, we needed to boost the efficiency of CRISPR/Cas9, and we did this by expressing Cas9
00:10:08;19 within parasites.
00:10:10;02 And once we had strains that stably expressed Cas9, we could introduce the instructions
00:10:15;24 for which particular gene to disrupt in a single plasmid that also had
00:10:24;15 a selectable drug resistance marker, shown here as the DHFR/pyrimethamine resistance cassette.
00:10:31;20 And when we selected for the stable integration of those instructions into a population,
00:10:37;02 now the rates of gene disruption skyrocketed to nearly the entire population.
00:10:43;09 We estimate that 97% of the parasites that stably incorporate this plasmid disrupt
00:10:50;25 the gene that is targeted by the guide RNA encoded in it.
00:10:56;06 And so these are remarkable rates of gene disruption that are now efficient enough
00:11:02;16 to implement at scale against the entire genome.
00:11:07;06 So, the strategy is illustrated here.
00:11:11;20 It's a pooled genetic screen.
00:11:14;00 We can synthesize, using a highly parallel oligo synthesis approach, ten different guide RNAs
00:11:25;20 against each one of the 8,000 genes in the Toxoplasma genome.
00:11:30;03 That's 80,000 different sequences or so that can be cloned all as one into an array of
00:11:37;20 vectors that target each one of those genes.
00:11:41;09 And we can put those in, through transfection, into parasites that carried Cas9.
00:11:47;17 And by doing that, we generate a really diverse population of mutants, where different genes
00:11:55;20 have been disrupted.
00:11:58;14 And the identity of the disrupted gene within each one of those organisms can be...
00:12:06;14 can be uncoded by reading out the sequence of the guide RNA, because that guide RNA
00:12:13;16 gives us the instructions for which gene is disrupted.
00:12:17;23 And so this diverse population can be placed in a culture with human fibroblasts.
00:12:25;21 And we can allow the parasites to grow as they would normally, in culture,
00:12:30;22 by invading those human fibroblasts, replicating within them, egressing.
00:12:35;20 Over several cycles, in fact, three different cycles in our initial experiments.
00:12:42;04 And what will happen is that when an essential gene or a really important gene is disrupted,
00:12:49;01 those guides will be lost from the population.
00:12:52;17 And the reason they'll be lost is that they'll be out competed by guides against genes that
00:12:58;11 are dispense... that are dispensable within these given conditions.
00:13:03;06 And so, what you'll have at the end is the ability to amplify the guide RNAs from these
00:13:09;07 resulting populations and compare them to the guide RNAs in the initial population
00:13:15;26 in order to estimate which genes are fitness-conferring, which genes are important for the
00:13:22;23 fitness of these parasites under the tested conditions.
00:13:26;04 And this can help us... through averaging the loss of all 10 guide RNAs against each gene,
00:13:34;25 this can help us identify those genes that are fitness-conferring.
00:13:42;05 So to test this, we used something that we've known since the 1970s, through the work of
00:13:48;10 Elmer Pfefferkorn, which is the ability of parasites to incorporate from the environment
00:13:55;27 a different pyrimidine.
00:13:59;14 And these parasites utilize this so-called salvage pathway, which is shown here, to incorporate
00:14:08;19 cytidine derivatives from the host cell, but we can trick them and introduce
00:14:16;22 fluorinated versions of these, which end up being toxic when the parasites incorporate them into their
00:14:22;10 dUMP pools.
00:14:25;21 In the absence of that salvage pathway --and in fact in the absence of a particular gene
00:14:32;10 called UPRT within that salvage pathway -- parasites stop importing those toxic compounds and
00:14:40;03 rely exclusively on the de novo synthesis pathways that they have within them.
00:14:47;17 And so they don't depend on import, and they're now resistant to compounds like FUDR.
00:14:53;28 And so the hypothesis was that if we have this diverse population of parasites and
00:14:59;05 we place them under the pressure of FUDR selection, that guides against UPRT are going to be
00:15:10;02 selected for because they disrupt the import pathway and make the parasites resistant to these compounds.
00:15:17;25 And that is in fact what we found, both at the guide RNA level, as shown there by
00:15:22;24 the increase in abundance of those guides against UPRT in the presence of FUDR, and then,
00:15:29;09 when we summarized those scores, at the gene level we saw UPRT, distinct from
00:15:36;15 all of the other genes in the Toxoplasma genome, as solely responsible for resistance against FUDR.
00:15:45;04 This was remarkable because in the course of a single experiment we could identify
00:15:52;06 genes involved in drug resistance, which typically would have taken convoluted crosses or
00:16:02;28 really sophisticated genetic approaches to identify.
00:16:07;07 In the course of this experiment, we also were collecting the data for identifying
00:16:15;02 which genes are fitness-conferring.
00:16:17;01 And so these are plotted here.
00:16:19;07 Each one of these bars is one of the 14 chromosomes of Toxoplasma gondii, and the colored lines
00:16:28;18 are individual genes, those 8,000 genes that I mentioned.
00:16:32;24 And what we have here is a heat map for the genetic contributions to parasite fitness
00:16:40;26 by each one of these parasite genes.
00:16:44;22 And if we zoom in to different regions, we can for instance see that the pantothenate kinase
00:16:49;20 is particularly important during these growth conditions,
00:16:53;26 whereas this ubiquitin ligase is particularly dispensable.
00:16:58;17 And so, you can already see how these approaches are giving us remarkable information about
00:17:05;26 the genetic makeup of Toxoplasma gondii and, really, a roadmap for what the most important
00:17:13;11 genes within this genome are.
00:17:16;07 And I'd like to emphasize that this is of course under the growth conditions tested.
00:17:21;21 You can imagine that remaking these... these pools of parasites and retesting them in animals
00:17:31;05 would give a different set of fitness-conferring genes, as the parasites are challenged
00:17:40;07 by the immune system or with... or challenged by other conditions that they encounter
00:17:46;27 under those settings.
00:17:50;04 If we take a look at the ranking of these genes, and correlate that to 40 genes
00:17:57;10 that had been previously studied and shown to be dispensable, and 40 genes that had been
00:18:02;21 previously studied and shown to be fitness-conferring, we see that our phenotype scores on the y-axis
00:18:09;08 give us a very nice sorting of those two populations, showing us that our data is well-correlated
00:18:18;05 by the literature.
00:18:19;26 And in fact, the one outlier, which was this RAB4 gene, we could later confirm was
00:18:25;20 a misannotation of the literature, and it was in fact dispensable where it had been reported to be essential.
00:18:34;15 There are other broad metrics of... of genetic importance, such as expression and conservation
00:18:43;22 and gene ontology, that we can look at to see whether they correlate with the
00:18:50;23 fitness scores that we calculated.
00:18:53;20 And in fact, what we see is that, for instance, categories such as the ribosomal proteins
00:18:58;22 -- which are important, in fact, essential in every eukaryotic cell --
00:19:04;24 are collected at very low phenotype scores, as shown in the red line, indicating that they are
00:19:11;01 in fact fitness-conferring within the screen.
00:19:14;04 And yet other categories that are likely highly redundant, because they are large families
00:19:21;08 of surface proteins or not expressed within the stage that we're assaying at all,
00:19:27;08 are collected late in the blue and green lines, indicative of their dispensability.
00:19:34;02 So, broad measures, broad genomic trends seem to correlate well with the fitness scores
00:19:40;28 that we are identifying.
00:19:43;02 So, let's go back to this question that I had posted at the beginning, which is,
00:19:49;24 can we identify within these categories of apicomplexan- or Toxo-specific genes those genes that are
00:19:56;19 most important, and which we might have not pursued because of their lack of correlation
00:20:02;13 with known systems of molecular biology?
00:20:06;25 If we take a look at each one of these categories, we see that with an increased depth of conservation
00:20:12;27 there's an increased proportion of each one of those categories that is comprised by
00:20:17;15 fitness-conferring genes.
00:20:19;18 And in fact, within the eukaryotic category, nearly 70% of the genes are fitness-conferring
00:20:27;18 and expected to be quite important.
00:20:31;01 This highlights a particularly interesting category right here, in the belly of the apicomplexan set,
00:20:37;20 which we termed the indispensable conserved apicomplexan proteins, or ICAPs just for short,
00:20:45;22 which were a set of proteins that we might have not pursued previously,
00:20:50;26 but which we now knew to be important for the completion of the Toxoplasma lytic cycle.
00:20:59;14 We used that trick with CRISPR of trying to manipulate genes within parasites that
00:21:07;13 lack non-homologous end joining, where we could integrate epitope tags -- tags that we could
00:21:13;11 recognize with known antibodies -- within the coding sequence of 17 different ICAPs.
00:21:21;24 And we localized them within the really defined structure of the Toxoplasma cell.
00:21:28;04 And what you see here is the diversity of patterns that resulted from this experiment.
00:21:34;14 We found some genes that localized to the nucleolus.
00:21:38;20 We saw others that were more generally distributed.
00:21:42;12 And yet many others that were localized to the mitochondrion.
00:21:46;26 I'd like to highlight a couple that were localized to these secretory organelles that I introduced
00:21:52;28 in my previous lecture, the micronemes.
00:21:57;17 And that's because micronemes are known to participate in motility and invasion.
00:22:02;26 And we were particularly interested to know whether any of these ICAPs participated in
00:22:08;24 these processes.
00:22:09;28 We did a secondary screen and in fact identified one of them, ICAP12, as important for invasion,
00:22:18;11 most likely.
00:22:19;11 And so we decided to take a closer look.
00:22:21;23 And what you see here is a Toxoplasma gondii where ICAP12 has been tagged, endogenously,
00:22:31;15 with a fluorescent protein that allows us to visualize it.
00:22:35;02 And you see it localized to the apical end of the protein.
00:22:38;26 And during the invasion process, you see it localize to the moving junction and then concentrate
00:22:45;11 at the posterior of the parasite, in a manner reminiscent to the localization of
00:22:51;09 the rhoptry proteins that form the tight junction.
00:22:58;12 We took a closer look at its localization, and in fact confirmed that it was
00:23:03;16 localized to micronemes along with known micronemal markers.
00:23:07;19 And topological predictions suggested that it had structural similarity to claudins,
00:23:16;18 which are the proteins that hold our cells together.
00:23:19;26 And this was particularly remarkable, not only because CLAMP, as we called it, was conserved
00:23:26;13 throughout the apicomplexan radiation -- in fact, one copy in each apicomplexan genome --
00:23:32;17 but also because, during invasion, that tight apposition of the plasma membrane of the host cell
00:23:39;24 with the plasma membrane of the parasite resembles structurally the
00:23:45;19 tight junctions that are maintained by claudins within our own cells.
00:23:53;01 We performed some genetic tricks to try to shut down the expression of CLAMP
00:23:59;02 in a regulated manner.
00:24:00;22 And what you see here, in green, are parasites that are deficient in CLAMP, where we've
00:24:06;25 tuned down its expression, and in red parasites that still have it.
00:24:11;20 And when they try to invade host cells, you see that the parasites that lack CLAMP
00:24:18;03 try to invade but are ultimately unable to do so, despite their several attempts.
00:24:25;07 We can quantify this, of course, and see a really dramatic loss of invasion in parasites
00:24:32;26 where we've downregulated CLAMP expression.
00:24:40;28 And so we are fascinated by this protein, because it provides one of the first proteins
00:24:48;10 that is completely conserved throughout the phylum and participates exclusively in
00:24:56;02 the invasion process.
00:24:57;02 There were previously other proteins, like actin and myosin, which are of course conserved,
00:25:04;25 but those also participate within general motility.
00:25:09;15 CLAMP-deficient parasites, however, taught us that CLAMP is involved only in the invasion process
00:25:16;11 and not in the motility process.
00:25:18;27 And this is interesting because, at a macro scale, invasion in Toxoplasma looks very much
00:25:26;26 like invasion in malaria, where parasites squeeze themselves slowly into erythrocytes,
00:25:34;09 or, in fact, invasion in Cryptosporidium, where these tiny little parasites also
00:25:42;18 squeeze themselves into the cells that they infect.
00:25:46;12 So, there is the possibility that through studying CLAMP we can identify features of
00:25:53;06 this invasion process that are in fact conserved throughout the entire phylum.
00:25:58;26 And although we haven't tested it in the most distantly related culturable parasite, Cryptosporidium,
00:26:06;18 we decided to take a look at Plasmodium, the causative agent of malaria.
00:26:13;25 In collaboration with Jacquin Niles' lab and, in fact, Suresh Ganesan and his lab, we performed
00:26:20;04 another genetic trick that allowed us to tune the expression of CLAMP within these
00:26:27;19 malarial parasites by... a trick that required
00:26:32;28 anhydrotetracycline in the media for the expression of CLAMP.
00:26:36;09 So, if we remove an anhydrotetracycline, this has no effect on the parental strain.
00:26:42;05 But when we tested the mutant, we could see that that removal of anhydrotetracycline and,
00:26:47;25 in fact, the removal of CLAMP expression, crashed the populations, shown here in yellow.
00:26:54;23 And essentially obliterated the infection.
00:26:58;23 This indicates that CLAMP is as important for malarial parasites as it is for Toxoplasma,
00:27:05;25 and gives us great hope that its study can tell us more about this fascinating process
00:27:13;11 of apicomplexan invasion.
00:27:16;15 And with that I hope that over the last couple of courses I've given you a sense of how
00:27:21;23 we can utilize Toxoplasma gondii to investigate the central hallmarks of apicomplexan parasitism
00:27:30;12 and understand the unique molecular biology of this important phylum.
00:27:35;16 With that, I'd like to acknowledge my funding sources -- the National Institutes of Health,
00:27:41;10 the Whitehead Institute, and the Massachusetts Institute of Technology --
00:27:45;20 and the members of my lab.
00:27:47;19 In particular, Saima Sidik and Diego Huet,
00:27:52;09 who led the efforts to apply the genome-wide CRISPR approaches.
00:27:58;06 Saima's work was also to investigate the function of CLAMP in the second part of this talk.
00:28:06;12 I have had a remarkable number of collaborators, too many to mention, but it's worth highlighting
00:28:13;13 Tim Wang from our neighboring lab, the Sabatini lab,
00:28:17;08 who helped us implement these genome-scale approaches in... in Toxoplasma.
00:28:25;14 I mentioned Jacquin Niles, our collaborator in malaria.
00:28:30;04 And Vern Carruthers and Mae Huyhn helped us with some of the genetic manipulations in
00:28:35;04 Toxoplasma.
00:28:36;26 And I'd like to thank you for your attention.

Videos in this Talk

Part 1: Toxoplasma gondii: A Model Apicomplexan
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Genetic Approaches to Study Toxoplasma gondii
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Sebastian Lourido

Total Duration: 0:58:50

Recorded: November 2017

All Talks in Microbiology

Talk Overview

Apicomplexa are evolutionarily distinct eukaryotes that play an important role in human diseases such as toxoplasmosis and malaria. So how do scientists study their unique biology? Dr. Sebastian Lourido explains that his lab uses Toxoplasma gondii as a model to study the phylum Apicomplexa. In part one of his talk, he explains the complex life cycle of T. gondii and he describes the unique organelles found in apicomplexa and used to facilitate invasion, survival and replication inside host cells. He highlights research from his lab demonstrating the importance of calcium signaling for T. gondii invasion.

Since little is known about the Toxoplasma gondii genome, in his second iBiology seminar, Lourido explains how his lab developed CRISPR tools to study apicomplexan biology. His lab designed a strain of T. gondii constitutively expressing Cas9 that can be used in conjunction with guide libraries to identify biologically significant genes. Lourido explains how his lab used this system to identify genes encoding proteins necessary for apicomplexan invasion. These include a claudin-like protein, that they are calling CLAMP, that is conserved across apicomplexa and is necessary for invasion by both toxoplasma and the parasites that cause malaria.

Speaker Bio

Sebastian Lourido

Dr. Sebastian Lourido is a faculty member at the Whitehead Institute and an assistant professor of biology at the Massachusetts Institute of Technology. His lab uses Toxoplasma gondii as a model to study the underlying mechanisms of how the apicomplexan phylum of parasites infect and spread diseases such as malaria and toxoplasmosis. As an undergraduate… Continue Reading

Comments

JEYASHRI R says

June 22, 2020 at 10:31 am

The talk was very informative and I got to understand many concepts pertaining to the study in apicomplexan parasites.

Reply
Jhoel says

November 7, 2022 at 5:07 pm

Estoy inimaginablemente agradecido

Reply

Part 1: Toxoplasma gondii: A Model Apicomplexan

Part 2: Genetic Approaches to Study Toxoplasma gondii

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Sebastian Lourido

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