Hello, my name is David Roos and I’m a Professor of Biology at the University of Pennsylvania located in Philadelphia. Today I am here to talk about my favorite group of organisms, the Phylum Apicomplexa, a large group of protozoan organisms which are parasites and make their living inside their host cells. Just by way of example, here are four parasites in the genus toxoplasma and you can see their nuclei stained in blue and we will see some of the other organelles associated with them later on in the talk and by way of comparison, the length of these organisms from one end to the other is about 10 microns long. One one-hundredth-thousandth of a meter, and in contrast, a mammalian host cell, a human host cell in fact in this case is about 10 times that size. In this seminar series, we have three talks, and in the first I will be introducing the parasites, the organisms in question, and talking a little bit about how they grow, how they live, how they replicate, a little bit about their clinical relevance, and also a great deal about how they serve as fascinating windows into the biology of eukaryotic cells, enabling us to understand a little bit more about what are common aspects of cell biology, and what are novel aspects in cell biology that highlight the diversity of life on earth and also particular features which we might be able to as potential targets for therapeutic intervention in trying to target these organisms. So let’s look a little bit deeper at the phylum Apicomplexa. This is a schematic view of the tree of life divided into three great domains defined as the eukaryotes, the eubacteria, and the archaebacteria. Eubacteria and archaebacteria are as different from each other as either is from the eukaryotes. Both of these groups lack nuclei although they do of course have their own genetic material, and the vast majority of life on earth is in fact bacterial or archaebacterial life, and we won’t’ be talking about that today although there are other discussions in the iBioseminar series which describe these organisms. We’ll be focusing on the eukaryotes, nucleated cells that include the animals and plants, fungi, that are a part of our everyday existence. But you’ll note that the two great Linnaean kingdoms of animals and plants are just branches off on the edge of this tree, and in fact even among the eukaryotes, the vast majority of life is microbial diversity. Protozoans, unicellular or in some cases colonial unicellular organisms including many species that we care quite a lot about. All of those which are underlined here are human pathogens that are of concern. Some of you may have encountered giardia, for example, in drinking water from a mountain stream that perhaps you shouldn’t have. Some of these others cause more severe disease, and we will be talking today about the phylum Apicomplexa, indicated in red. A group that includes more than five thousand known species, although to be honest, I couldn’t tell you very much about most of them. This group does include many dozens, scores, perhaps hundreds of species in the phylum Plasmodium that is responsible for malaria. Five species of which cause malaria in humans and I urge those of you interested in further information about this to take a look at the iBioseminar by Joseph DeRisi that described some aspects of malaria biology. We’ll be talking a little bit more about malaria in the course of these talks. Plasmodium parasites infect red blood cells and only red blood cells in this stage of their life and only those in humans and a few species of relatively closely related monkeys exquisite tissue and species specificity and cause devastating disease, as in the case of this child with a coma, although this particular child survived his infection with no serious adverse effects. Many individuals do not. There are thought to be hundreds of millions of cases of malaria every year word wide, chiefly in Sub-Saharan Africa, South Asia, and in South America. And something on the order of two million people die every year from this disease, particularly from Plasmodium falciparum and particularly in Sub-Saharan Africa. Among the other five thousand species of organisms include many that are of importance, particularly in immunosuppressed individuals. Cryptosporidium for example, with something of the transmission cycle indicated here, something that you can pick up from contaminated water causes a devastating diarrhea in immuno-compromised patients, and for which we have no effective treatment. A serious problem in patients with severe HIV AIDS or with other immuno-suppressive disorders perhaps a treatment for cancer chemotherapy or for transplantation. Toxoplasma is also an opportunistic pathogen that causes problems in HIV patients, in this case the lesions shown in the CT scan of a patient with Toxoplasmic encephalitis. But this parasite is classically known as the leading source of congenital neurological birth defects throughout the world. Chronic infections are on the order of one third of the population are thought to be chronically infected in the US, in South America, in Europe, in Asia, in Africa, globally, a ubiquitous pathogen normally innocuous but under certain circumstances, for example, during pregnancy a serious problem. Now, despite the very different diseases that these organisms cause, they all share a common ancestry and exhibit many similar features. For example, all of them, as unicellular organisms infect a cell in the case of Plasmodium that may be a red blood cell, in the case of Toxoplasma, it may be a nucleated cell in the muscle or in the brain. In the case of Cryptosporidium it may be a cell in the gut. They then differentiate to form a different kind of cell, a cell that you wouldn’t even think to look at it was the same cell at all, and are transmitted often from one organism to another. Plasmodium, malaria parasites, are transmitted by mosquitos. Theileria, an important veterinary pathogen of cattle are transmitted by ticks. Toxoplasma are transmitted by cats and this is the reason why pregnant women are often told not to empty the kitty litter box. And so this allows us to overlay their lifecycles one on top of the other and to take advantage of experimental opportunities for example in things that we can study in Cryptosporidium to gain insights into what’s happening in Plasmodium. To study Plasmodium, to understand what happens in Toxoplasma, this concept of model organisms, biological concept of the guinea pig, allowing us to use a guinea pig or a fruit fly or a parasite to understand aspects of the biology of organisms we care more about is a fundamental principle of biology, and one that we will explore further. Most of the research in my laboratory focuses on Toxoplasma, and for the specific reason that this organism has turned out to be the most experimentally tractable of all of the Apicomplexan parasites. It’s easily cultivated in the laboratory. We have excellent models for human disease, a serious problem for some of these parasites, where, for example, malaria parasites that cause disease in birds or in mice or in lizards may provide at best an incomplete model for human disease. We can carry out genetic crosses, much as Mendel did with his pea plants and in the case of Toxoplasma those crosses need to be done in cats, doesn’t bother the cat, but it certainly not the most convenient way to do experiments, but at least it’s something that we can do if we are interested in re-assorting genes, putting together genes from one mutant with another. And fortunately we are not restricted to doing our genetics in cats because this parasite is readily amenable to molecular genetic analysis. Toxoplasma exhibits extraordinary ultrastructural resolution as you can see from these transmission electron micrographs and we’ll talk more about that in just a moment. The complete genome sequence is known for actually several of the isolates of Toxoplasma, and we have a wide range of functional genomic and bioinformatic tools, which we will talk about in the third of the sessions in this seminar series. All of these parasites are obligate intracellular parasites, they live inside host cells, and within those host cells, they establish a unique compartment, the parasitophorous vacuole, which you can see surrounding these two parasites living inside in this case a human fibroblast. And that vacuole, which we know relatively little about, is the key factor in mediating communication between the parasite and its host cell. Despite being so greatly divergent from animals, plants, fungi, more familiar eukaryotic cells, Toxoplasma and all of these parasites, harbor a virtually complete set of canonical eukaryotic organelles, that we have come to know and love from introductory cell biology. They have a nucleus, they have a golgi apparatus and other components of the secretory pathway, and many other organelles including two endosymbiotic organelles. But interestingly, they have only one of each of these organelles, so we can think of Toxoplasma as a minimalist eukaryote, stripped down to its barebones minimum, an organism which has all of the organelles that we might be interested in studying in a way that we can study genetically, that we can study cell biologically, and yet without such a wide range of diversity that we can hope to try to make sense of what’s going on where. So for example, imagine we were to consider the host cell side cytoplasm here, the host cell mitochondrion, a little bit of ER, here is a ribosome. This ribosome here is presumably making protein, but I have no idea what protein that ribosome is engaged in manufacturing. Whereas in contrast if we take a look at the ribosome on a parasite, let’s say this ribosome down here, we have good reason to believe that this ribosome is likely to be making a secretory protein that will enter into the single interconnected endoplasmic reticulum network, pass up via the nuclear envelope to the single golgi apparatus up at the apical end of the cell, and from there to the apical secretory organelles that define the phylum Apicomplexa. So let’s take a closer look at some of those organelles. So all of these organelles that we’ve described, the nucleus, the golgi apparatus, the endoplasmic reticulum, mitochondria, even plastids are generic organelles that we see throughout the eukaryotic domain. But these parasites also harbor a variety of unique organelles, most notably the apical complex that gives the phylum its name, up here at the apical end of the parasite where invasion occurs includes a variety of specialized organelles known as rhoptries, here are smaller organelles, the micronemes, that play a key role in invasion. So let’s take a closer look at these organelles, these apical complex organelles, that are responsible for secreting proteins essential for parasite invasion. We’ll take a look at those, and we’ll take a look at the involvement in invasion in a beautiful time-lapse video sequence taken in real time by Gary Ward at the University of Vermont. Here you can see a single parasite as it moves along gliding over the surface of cells, and now watch it stops and at this point it would be secreting proteins out of the rhoptries, as it penetrates into the host cell through this narrow constriction of a moving junction, establishing that intracellular parasitophorous vacuole, within which the parasite will live and replicate. Here are two more parasites living in the progeny of one parasite that had invaded, living within this cell. Now this raises a number of interesting points. These parasites are obviously dividing more rapidly than the host cell itself. One parasite has invaded giving rise to two, and while they are still within a single host cell, and this process of proliferation is of course key to the pathogenesis of the parasite. And we’ll take a look at structures that are involved in that pathogenesis or that are involved in the replication of parasites as a potential means of understanding the diversity of eukaryotic replication, but also as a potential target for interfering with the replication and survival of these cells. So let’s look back at the morphology of these parasites. We’ve discussed the nucleus and the golgi apparatus and generic secretory structures. We’ve discussed the micronemes and rhoptries, parts of the secretory pathway that are critical for invasion. The apical complex also includes a variety of cytoskeletal structures. The conoid here, a fascinating spiral organelle whose function is quite And underlying the entire parasite, the inner membrane complex, a series of flattened vesicles which are sutured together in a patchwork associated with cytoskeletal structures, such that the surface membrane of the parasite consists of a plasma membrane, but also these inner two membranes and those cytoskeletal components, which are essential for parasite survival and replication as we shall see. All of these organelles can be labeled in living parasites if need be, with fluorescent protein reporters in any color of the rainbow. We can study the secretory of the organelles, both parasite specific and generic. The cytoskeletal structures including generic structures such as microtubules and parasite specific organelles such as the inner membrane complex, the endosymbiotic organelles, and so on. And, being able to study these in living parasites, being able to manipulate them, allows us to study both pathogen specific processes, which we might use to interfere with parasite survival, as well as the evolution of eukaryotic organelles in general, studying the biogenesis of the golgi apparatus for example or the structure of microtubules in addition to the beautiful structure of the coronoid organelle or parasite replicative processes as we will be discussing here, and indicated as daughter parasites that are assembling within the mother. For the next few minutes, I’d like to concentrate on the process of parasite replication, a process that is normally quite familiar, one cell goes to two goes to four, but which we will discuss because it is critical to the pathogenesis of these parasites. It is after all the frank tissue destruction which is responsible for neurological birth defects as parasites in the fetus destroy tissue before they come under control. It is the tissue destruction that is responsible for lesions like that encephalitic lesion we saw in the brain of a HIV patient. And this is a common feature of many pathogenic microorganisms although there are certainly organisms that cause disease by interfering with say normal cellular signaling pathways. It’s actually the replication of the organisms themselves which is responsible for disease in many other organisms and in this way we can think of the problem as very much analogous to that of cancer cells where it is not the mutation in an individual cell which is responsible for disease, but the uncontrolled proliferation of cells and therefore cancer chemotherapy is typically targeted at blocking that proliferation in much the same way, much antimicrobial therapy is targeted specifically at blocking the replication of parasites. So if we understand more about that replication process and particularly novel features that we might be about to specifically target, we may be able to interfere with them in useful ways. Here we see a micrograph of host cells which have been infected with a single parasite, and that parasite is divided once, twice, giving rise to four parasites living within that parasitophorous vacuole. Here’s another parasitophorous vacuole another parasite infected maybe a little bit earlier, replicated three times, giving rise to eight parasites, and yet another with sixteen parasites. As we follow over time, over the next 24 to 48 hours, those parasites will replicate much more rapidly than the host cell, swell up like a fat sausage, and a few hours later burst out so that there is no residue, no evident cellular material here at all. And if we were to look inside an encephalitic lesion, this is precisely the kind of thing we would see. Destruction of tissue and perhaps inflammation that is a result of that tissue destruction. Now this process is quite different from the process of replication described in the text books for Plasmodium, or at least superficially, so after all Toxoplasma divides from one to two to four to eight the way mammalian cells, plant cells, bacterial cells do, in contrast Plasmodium parasites, as illustrated in these beautiful images drawn by Laurie Bannister of the UK a single Plasmodium parasite, showing all of the features that we looked at in Toxoplasma, infects the cell, in this case a red blood cell, and undergoes a process of de-differentiation, turning into what is known as a ring stage parasite responsible for setting up that intracellular home within which the parasite will live for the remainder of its tenure inside the red cell. Within the red cell, it specializes to a trophozoite form parasite, which engulfs hemoglobin, degrading the protein and detoxifying the heme as it is polymerized into a para-crystalline structure, and finally, multiple parasites are assembled, bursting out in the lysis of the red blood cell. Superficially, a very different process, but in fact a process that is more similar than one might originally think. Here’s the process again in Plasmodium, this time in actual images of parasites labeled with a fluorescent protein reporter. Parasites invading, developing ring stage parasites, maturing into trophozoites, we can see the beginning of that crystal of heme shown in a shadow starting to segment to produce the schizont that will then rupture out completing the cycle and going on to infect a new series of red blood cells. This process is difficult to study in malaria parasites for a variety of reasons, including the fact that red blood cells are inhospitable environments for a variety of cell biological studies, and the fact that this complicated process is very difficult to follow particularly in real time. In contrast, we can look at Toxoplasma parasites, in this case labeled with a fluorescent protein reporter linked to a histone protein, providing, incidentally, a quantitative marker for DNA content in these parasites, and what you can see is eight parasites living inside this vacuole, with the eight nuclei labeled in green. And as we start the movie, we can follow the replication of the parasites as the nuclei grow and divide and now we see sixteen nuclei but only eight parasites. If we continue to watch though, for a few minutes longer, what we will see is the emergence of the daughter parasites from the mother, leaving off this vestigial material, which will not be incorporated into the daughters. Waste material that is left behind as the parasites go on to mature. So this uncoupling of nuclear replication from cell division is a little bit unusual, and if we look in closer detail, we can see that in fact that process is more akin to the process of schizogony that we know from malaria parasites and the key to doing this has been the labeling of the inner membrane complex, and this particular set of experiments carried out by graduate student Ke Hu, we can label the inner membrane complex in such a way that it is most brightly visualized as it’s starting to assemble daughters and so we can define as time zero, these parasites that are beginning to divide. There are two parasites and two bright dots in each, as the new inner membrane complex starts to assemble. Over the next few minutes, you see those grow further until they expand and bud out of the mother, picking up the plasma membrane as they go and sloughing off residual material. This process takes about two hours, and at the end of that we will see no more changes in the inner membrane complex for an entire cell cycle. Eight hours later, we see the process repeated in the same cells, now four cells each of which develops two bright dots which grow and elongate and expand and so in contrast to the binary fission that we see dividing cells in half in mammalian cells, virtually all animal cells, in most fungal cells, in plant cells, in bacterial cells, this process is a little bit different. Conceptually more akin to the assembly of viruses within an infected cell. Two daughters are assembled within the mother and then they emerge and we know that this is the case quite clearly because we can even see rare cases of what one might imagine as schizont. Here’s a case of four Toxoplasma parasites, three of which are making two daughters, but one indicated in the red arrows, is actually making four daughters. Here’s another case of vacuole consisting of not sixteen, but seventeen parasites, so somehow in the last replicative cycle, one of the mother parasites gave rise to not two, but three daughters for a total of seventeen and in this case we can see five daughter cells that are giving rise to three daughters each for that next round. So we can say in conclusion that the Toxoplasma replicates like Plasmodium, using the process of schizogeny, known in Toxoplasma as endodyogeny, but endodyogeny and schizogeny are really the same sort of thing, assembling daughters within the mother here we can see the daughter inner membrane complex schematically shown in yellow in contrast to that of the mother and as the daughter scaffolding develops, it will then emerge from the mother, picking up its plasma membrane and maintaining that key apical polarity that is essential for parasite invasion. Similarly in Plasmodium, we can see the assembly of the inner membrane complex, but in this case producing not two, but typically sixteen daughter parasites as they grow. So these provide landmarks for us to assemble a picture of the cell cycle of Toxoplasma and by analogy Plasmodium as well. And in work carried out by graduate student Manami Nishi, we now know a great deal about this process. We know that the key first step is in fact not the assembly of the inner membrane complex, but another cytoskeletal structure the centrioles of these proteins, which begin apically oriented, migrate to the basal end of the cell, where they then divide, migrate back up to the apical end, and associate with other organelles starting to put together in a concerted fashion, all the components that are essential for a daughter cell, associating with the golgi apparatus, or plastid organelle, and the nucleus and other structures as well. Last on this list is in fact the mitochondria. Watch this remarkable process. Here we see the assembly of the inner membrane complex, two daughters that are developing as bright green dots that then grow, grow further, and start to emerge from the mother so now we have four daughters emerging from the two mothers, ready to go, but wait, no mitochondrion. All the mitochondrion is left in the residual part of the mother and now in the space of ten minutes, that mitochondrion attaches probably to microtubules associated with the inner membrane complex and zips up to the top of the parasites which then proceed to pick up the plasma membrane and bud out of the mother. So in some studies like these have allowed us to put together a complete timetable that is rigorously adhered to for organellar replication in these parasites, beginning with the replication of the centrioles and successive packaging of the golgi apparatus, the plastid, the nucleus, assembly of this daughter scaffolding which then picks up the endoplasmic reticulum and the mitochondrion and finally the specialized secretorial organelles, the rhoptries and micronemes are assembled de novo in each parasite. So in answer to the question that we started with of how do we build a parasite, we do so with a process that’s significantly different than the familiar cell cycle processes that have been defined in yeast cells and in mammalian cells, where the hallmark marker of S-phase, DNA replication, is completely subsumed within the process that we normally think of as associated with M-phase, organellar replication, mitosis, cytokinesis, this large region indicated here in pink and encompassing approximately 80% of the parasite cell cycle, including the process of DNA replication. This argues certainly that there are likely to be significant modifications of the familiar checkpoints associated with cell cycle control, and changes that will be interesting to explore as we characterize the biology of these parasites further. So in answer to the question of how we build a parasite cell? The answer appears to be that we hang everything onto the cytoskeleton, building the parasite from the top down in a process that ensures the maintenance of polarity that is so essential to parasite survival and has a number of other interesting implications as well, because everything in the daughter parasite is put there by choice Residual material, waste products for example, are sloughed off behind, allowing the parasites to survive without classical lysosomes. So I hope that this tour through the biology of Toxoplasma parasites and the ways in which we use this as a model organism to study the biology of Apicomplexa parasites in general, has given you some insight into the fascinating cell biological processes that are involved both parasite specific features and features that are more general to eukaryotes, and I hope that will encourage you to read more about the biology of these organisms, perhaps to work on these organisms yourself. And in the next lecture I’d like to take you through the biology of one particularly interesting organelle, this organelle, the plastid, or apicoplast, an organelle that reveals some remarkable aspects of organellar evolution in eukaryotic cells. Here in a malaria parasites, the nucleus, golgi apparatus, secretory structures, inner membrane complex that we described mitochondrion and finally the apicoplast which will be the subject of the next iBioLecture.