Genomics and Cell Biology of the Apicomplexa
Transcript of Part 2: The Apicomplexan Plastid: Something Old, Something New, Something Borrowed, Something Green
00:00:01.01 Hello, my name is Davis Roos and I'm a Professor at the University of Pennsylvania in Philadelphia, 00:00:09.14 and in the second segment of this iBioLecture, 00:00:12.16 I'd like to talk to you about the discovery of the apicoplast and I think you'll see what I mean 00:00:18.13 by this somewhat whimsical title "Something old, something new, something borrowed, 00:00:22.26 and not blue, but something green." In the last segment we talked about 00:00:31.04 Apicomplexa parasites, a group of five thousand eukaryotic protozoa, 00:00:37.28 which are obligate intracellular parasites living inside the host cells 00:00:44.06 including humans and a wide variety of other organisms. These parasites include 00:00:50.00 the malaria parasites responsible for hundreds of millions of cases of disease globally 00:00:56.00 and on the order of two million deaths every year they include Toxoplasma, 00:01:01.07 a parasite that is even more widespread, infecting approximately a third of the world’s 00:01:07.15 population, normally without adverse effects, but with several significant exceptions 00:01:14.15 to that rule including the importance of Toxoplasma as an opportunistic pathogen 00:01:20.00 associated with AIDS and other immunosuppressive disorders, and particularly as a 00:01:24.16 congenital pathogen, the leading source of congenital neurological birth defects 00:01:28.24 in many parts of the world. This is a malaria parasite that we are looking at, 00:01:34.20 and we discussed the various organelles inside this parasite last time through. 00:01:40.22 We looked at the nucleus, the secretory organelles including the golgi apparatus here, 00:01:45.22 and the specialized organelles involved in host cell attachment and invasion. 00:01:50.10 We talked a great deal about the inner membrane complex, this double membrane 00:01:55.00 that is required for the assembly of daughter parasites inside the mother, 00:02:00.12 in a fascinating process known as schizogony. These are eukaryotic cells 00:02:05.19 and as such they harbor in addition to a nucleus, the mitochondrion, an endosymbiotic organelle 00:02:11.23 and we'll say a little bit more about endosymbiosis in general in a moment, 00:02:15.15 but the focus of this talk will actually be on the apicoplast, or Apicomplexa plastid, 00:02:22.04 an organelle with, I think you'll agree, a remarkable biological history, which lends 00:02:28.13 insights into the evolution of the eukaryotic cells in general and potential targets 00:02:34.01 for drug development. Indeed, this story got its start through stories 00:02:40.06 not on the cell biology of organelles, but on the mechanism of action of drugs 00:02:46.08 and the identification of candidate drug targets. It began a decade ago through 00:02:51.29 the work of graduate student Maria Fichera, who was interested in asking the following question: 00:02:57.04 Why do these drugs, drugs like chloramphenicol, clindamycin, azithromycin, 00:03:04.09 all well known as antibacterial antibiotics, all well known as effective antibiotics 00:03:13.02 because they inhibit protein synthesis on bacterial ribosomes, but not on human ribosomes. 00:03:20.09 Why is it that these compounds are effective against malaria parasites and Toxoplasma parasites? 00:03:28.15 Clindamycin is regularly used clinically to treat patients. Now the initial suggestion 00:03:36.15 was that perhaps there is something bacterial like about the synthesis of proteins 00:03:43.16 in Toxoplasma and malaria parasites, but Maria was quickly able to show that that is not the case. 00:03:49.20 Cytoplasmic protein synthesis doesn't appear to be bacteria-like, 00:03:54.18 it's certainly not sensitive to these antibiotics although it was difficult to look at 00:03:58.20 mitochondrial protein synthesis, certainly mitochondrial function 00:04:04.19 is unimpaired by these drugs. And yet, these compounds kill parasites and in a very peculiar way. 00:04:12.28 Let me describe this phenomenon which Maria defined as the delayed death phenotype. 00:04:19.18 Here's the way it works. We can treat parasites with up to 00:04:24.04 10,000 times the lethal dose of drug, here using two different antibiotics, 00:04:31.02 one a protein synthesis inhibitor, one a fluoroquinolone 00:04:33.29 that blocks the replication of bacterial DNA, 00:04:38.19 and they grow like there is no tomorrow, through 48 hours, 00:04:43.05 or 6-8 cell cycles in tens of thousands of times the lethal dose of drug. 00:04:51.04 They escape from the host cell, survive extracellularly, and invade a new host cell without difficulty, 00:04:58.24 and there, inside that new host cell, they die or more precisely, they don't actually die. 00:05:06.17 These parasites grow, but they grow more slowly, and they grow more slowly 00:05:12.16 to an extent that is determined by the duration and concentration 00:05:17.27 of drug treatment that they saw way back 6 cell cycles earlier. 00:05:22.17 Now I must confess I can't give you an explanation for the reason for this, 00:05:27.13 but this very peculiar phenomenon is a characteristic of all of these compounds, 00:05:32.12 structurally unrelated compounds, that have in common 00:05:35.28 only the fact that the inhibit transpeptidation on bacterial ribosomes. 00:05:40.29 And so that made it seem very unlikely that these drugs would have unusual 00:05:49.25 different off-target activities in malaria parasites, suggesting that they must be 00:05:56.08 inhibiting protein synthesis, but not the protein synthesis that we knew of 00:06:01.00 and that drew our attention to a mysterious set of ribosomes that was identified 00:06:07.24 many years ago, many decades ago, in both Plasmodium and Toxoplasma parasites. 00:06:14.18 On an episomal DNA, a 35,000 nucleotide circular DNA originally thought to be 00:06:23.23 the parasites mitochondrial genome, but when the true mitochondrial genome 00:06:28.04 was discovered in malaria parasites and later in other Apicomplexa parasites as well 00:06:33.25 that left this 35 kb circle as a molecular biological mystery, 00:06:41.05 a mystery without a function, without a home, but the one thing that we knew about it 00:06:45.20 from work chiefly done by Ian Wilson's laboratory in the UK, was that these DNAs 00:06:52.26 contain ribosomal genes, and what’s more those ribosomal genes 00:06:57.16 had sequence characteristics that suggested that they might be susceptible to macrolide antibiotics 00:07:04.25 drugs like those that we've seen before. So to investigate this further we cloned 00:07:10.06 and sequenced the entire sequence from these parasite genomes 00:07:16.02 and taking advantage of predicted open reading frames, 00:07:19.06 particularly that for the elongation factor Tu gene, a very widely sampled gene 00:07:25.04 that's know from throughout life, we can begin to get 00:07:28.28 an idea of what this mysterious episomal DNA might be. And those studies 00:07:34.19 yielded several findings, the first of which was that all of the 00:07:42.01 Apicomplexa, Plasmodium, Toxoplasma, the poultry pathogen Eimeria are monophyletic, 00:07:48.13 they form a single group and that of course was no surprise it was no surprise at all. 00:07:55.07 The second was that among the bacterial world, the region from about here downwards, 00:08:03.28 the sequences look not at all like mitochondrial genomes indicated in yellow, 00:08:10.00 down at the bottom of your screen, and in fact they look more closely related 00:08:16.19 to cyanobacteria, blue-green algae, in fact the ancestor of chloroplasts in plants and algae, 00:08:22.26 and this wasn't much of a surprise either because we and others had previously noticed 00:08:28.00 that this episomal DNA bore many similarities to chloroplast DNA 00:08:35.28 and so we suspected that just as the ancestor of all plants and algae had acquired 00:08:42.19 an organelle from a blue-green algae, cyanobacterium, giving rise to modern day 00:08:49.27 chloroplasts, similarly these parasites might have acquired another cyanobacterial endosymbiont, 00:08:59.13 giving rise to a novel organelle. What was actually a much greater surprise though 00:09:05.12 was that these parasites look not just a little bit like the chloroplasts associated 00:09:11.13 with plants and algae, but a whole lot like them, as if we hadn't cloned a bit of 00:09:17.19 parasite DNA at all, but had inadvertently contaminated our cultures 00:09:22.08 with some pond scum from the pond outside of our laboratory. But no, we didn't 00:09:29.13 contaminate the cultures, this is indeed genuine parasite DNA 00:09:34.10 that looks for all the world like it came from a plant. So this raises a bit of a paradox, 00:09:42.25 because we know a great deal about the evolution of these parasites, 00:09:47.28 we know for sure that they are not plants and algae, they diverged off of the common 00:09:54.15 eukaryotic lineages shown to the left here, prior to the divergence of animals and fungi, 00:10:00.23 probably prior to the divergence of animals, fungi and plants, they are most closely related 00:10:05.25 to ciliates like paramecium and to dinoflagellates like the organisms that cause 00:10:12.00 red tide and poison the shellfish industry. And yet they harbor what appears to be a plant chloroplast. 00:10:23.28 There's really only one possible resolution to this paradox, 00:10:28.26 and that comes from considering the phenomenon of endosymbiosis. 00:10:32.22 It's now well-known universally accepted that virtually all, perhaps all eukaryotes 00:10:42.20 harbor a mitochondrion or at one point harbored a mitochondrion 00:10:50.16 subsequently lost, and that that mitochondrion was acquired 00:10:54.11 by a horizontal transfer event when the ancestor of all eukaryotes ate a bacterium, 00:11:00.19 an alphaproteobacterium in fact, giving rise to the ancestor of mitochondria, 00:11:06.18 surrounded by a double membrane and containing that mitochondrial genome 00:11:10.19 and similarly we know that the ancestor of all plants and algae acquired a cyanobacterium 00:11:17.01 by a similar sort of horizontal transfer event, an invasion of the ancestor 00:11:21.22 or an engulfment of the bacterium depending on your perspective 00:11:26.04 giving rise to the chloroplast, a distinctive organelle, again, surrounded by a 00:11:31.26 double membrane and harboring the DNA that was acquired from that cyanobacterial ancestor. 00:11:38.08 So either everything else we think we know about the origin of these parasites 00:11:44.17 is wrong, and in fact the Apicomplexa should branch off of the plant lineage 00:11:49.22 or instead maybe they just picked up a bit of plant lineage by horizontal transfer, 00:11:57.02 as indicated by this diagram. The process of secondary endosymbiosis 00:12:03.24 argues that an ancestral parasite ate a eukaryotic algae, which had previously acquired a 00:12:12.04 chloroplast from the engulfment of a cyanobacterium. These parasites have 00:12:18.02 maintained that plastid organelle despite having dispensed 00:12:27.06 with most of the functions we think of as associated with chloroplasts, photosynthesis for example 00:12:34.01 does not take place in the parasites we work with. Here's another cartoon 00:12:39.19 version of what you might think of old video game aficionados 00:12:43.16 as the Pacman model of organelle evolution in which an ancestral eukaryote 00:12:49.04 ate a unicellular algae giving rise to the chloroplast surrounded by a double membrane 00:12:55.11 and along comes the ancestor of these parasites which then engulfed 00:13:02.21 that eukaryotic plant or algae giving rise to modern day Apicomplexa 00:13:10.27 parasites harboring an endosymbiotic organelle, which we know is essential 00:13:15.17 as the target for these various drugs. Consistent with this model, 00:13:22.13 the organelle that we now know as the Apicomplexa plastid, 00:13:26.22 or apicoplast is surrounded by four membranes, which we can see in these Toxoplasma parasites, 00:13:33.01 organelles distinct from the golgi apparatus, the mitochondrion, the nucleus. 00:13:39.07 Now you might wonder how an organelle as striking as this could have been missed 00:13:45.06 by cell biologists for all these years, and the answer of course is that it wasn't missed 00:13:49.29 at all, this organelle had been seen many times and had been the subject of much debate, 00:13:54.22 but given a variety of uninformative names, the spherical body, 00:13:58.20 the golgi adjunct, or depending on your linguistic affinity 00:14:03.12 in France called the organelle plurimembranaire, or in Germany, the Hohlzylinder 00:14:09.05 but what we can now say is that the answer to this cell biological mystery, 00:14:13.25 what is this organelle, is it a distinctive organelle, is the same as the answer 00:14:19.13 to our molecular biology mystery, what is this episomal DNA, 00:14:23.21 and the answer to our pharmacological mystery, how is it that these drugs, 00:14:29.05 normally active only against bacterial species, are active against organisms such as Toxoplasma and plasmodium. 00:14:39.25 So the apicoplast or Apicomplexa plastid is a novel organelle acquired 00:14:46.05 by secondary endosymbiosis, harboring its own genome and essential for parasite survival, 00:14:52.14 quite an exciting find. It's not every day that we discover a new organelle, 00:14:58.00 but the big question of course is what does the apicoplast do? Now we've already 00:15:05.24 cloned its genome, sequenced it in its entirety, we know every gene associated 00:15:11.18 with that organelle or genome on the order of thirty protein coding genes, 00:15:16.11 another thirty RNA genes encoding ribosomal RNAs and transfer RNAs, but unfortunately 00:15:24.23 that organellar genome which was so informative for telling us about the origin 00:15:29.23 of the apicoplast, about the mechanism of action of drugs such as 00:15:36.15 fluoroquinolones and macrolides and rifampicins against these parasites 00:15:41.25 is completely uninformative in terms of what we presume must be 00:15:46.23 the metabolic functions that make it essential for parasite survival and that probably 00:15:52.29 shouldn't be much of a surprise because we know that all endosymbiotic organelles 00:15:57.15 chloroplasts of plants, mitochondria of eukaryotes in general, 00:16:02.05 encodes some proteins in their own genome but most of the proteins associated 00:16:07.28 with their function are actually have been transferred to the nuclear genome 00:16:12.00 and are translated on cytoplasmic ribosomes, and post-translationally 00:16:17.25 imported into the organelles. And so if we are going to gain some insight 00:16:22.17 into what the metabolic pathways might be and whether those perhaps could be targeted 00:16:27.18 as the target for antibiotic treatment, we are going to need to look into the 00:16:34.20 nuclear genome of these organisms themselves. 00:16:38.14 We can do so and we can find those genes, we can screen through available genome 00:16:46.18 sequences, at that time a fairly limited number of genes, we can identify 00:16:52.03 proteins associated with the ribosomes, a ribosomal protein S9 for example 00:17:00.13 is a protein that is essential for ribosomal protein function which is readily 00:17:06.00 identifiable as bacterial type, or eukaryotic type, and which in this case this particular gene 00:17:15.10 possesses a long amino terminal extension in the predicted gene, suggesting 00:17:21.18 that there might be targeting information responsible for translocation 00:17:25.19 across those multiple membranes of the apicoplast. And indeed, in work done 00:17:30.26 in my colleagues laboratory in Australia, Ross Waller, a graduate student 00:17:35.04 of Jeff McFadden in Melbourne, raised antibodies to these proteins and showed 00:17:40.25 on western blots that in fact they bind to proteins of a molecular weight consistent 00:17:47.13 with a processing of this large precursor into a smaller version, 00:17:51.13 which would be the mature ribosomal protein. And the same is true for other proteins 00:17:55.29 for Toxoplasma and Plasmodium. Now this gives us the tools that we need 00:18:03.00 to be able to explore not only what these proteins might be doing, certainly we 00:18:09.14 already knew that there were ribosomes associated with the apicoplast, it's not that surprising 00:18:14.08 to find ribosomes encoded in the nucleus and imported into the apicoplast, 00:18:19.02 but this gives us tools which we can use to explore how proteins might traffic 00:18:24.10 across those many membranes of the organelle. We know that those proteins 00:18:31.09 are in fact targeted to the organelle, and we know that from in situ hybridization experiments, 00:18:37.25 binding studies done on fixed samples in Dr. McFadden's laboratory, 00:18:43.15 where we can see in Toxoplasma and in Plasmodium in living parasites fused 00:18:48.27 to a fluorescent protein reporter that proteins are targeted to the apicoplast 00:18:53.25 at the apical end of the parasite nucleus. In Plasmodium we can watch the process 00:18:59.17 of replication as a ring stage parasite proceeds to this segregating schizont 00:19:07.01 that will then burst out as sixteen daughter merozoites via this 00:19:11.01 remarkable structure that mediates the segregation of organelles among the 00:19:17.05 various daughters, and an even more extreme example in work recently carried out 00:19:23.01 by Boris Striepen at the University of Georgia, we can see the segregation 00:19:27.05 of the apicoplast in another Apicomplexa parasite Sarcocystis neurona 00:19:33.01 an important pathogen of horses as it is segregated among the scores of daughters 00:19:39.17 that are developing in those parasites. We can map out the targeting signals 00:19:45.02 that are responsible for this in much greater detail by cut and paste molecular genetics. 00:19:51.03 When we look at that long N-terminal extension, suspected to mediate targeting 00:19:57.09 to the apicoplast, we can see that indeed it does if we take the apical extension from one protein 00:20:03.05 and cut off the mature part of the protein indicated in yellow here, 00:20:07.04 and fuse that to a fluorescent protein reporter, it goes into the apicoplast. 00:20:12.27 But the N-terminal signal itself doesn't look very much like a classical chloroplast 00:20:20.15 targeting signal, in fact its extreme N-terminus doesn't look anything like 00:20:25.21 a chloroplast targeting signal, it looks for all the world like a secretory signal sequence, 00:20:31.07 the kind of signal responsible for secreting pancreatic enzymes for example, 00:20:36.29 into the small intestine. Indeed, if we take that small N-terminal region, 00:20:44.15 the hydrophobic region indicated here in blue, and fuse it to a fluorescent protein reporter, 00:20:49.10 it behaves like a secretory signal sequence, it secretes a fluorescent protein 00:20:54.11 outside of the parasite, you can see four Toxoplasma parasites here, silhouetted in black. 00:21:01.25 The rest of this long N-terminal extension on its own mediates no targeting at all, 00:21:08.06 as we see in these parasites, nicely stained within the cytoplasm. But that region 00:21:18.07 can be replaced with a genuine chloroplast targeting signal from a pea plant 00:21:23.23 or from the experimental organism, Arabidopsis, indeed we can make a completely 00:21:30.01 synthetic nuclear encoded plastid protein by taking a pancreatic signal sequence, 00:21:36.20 fusing that to a chloroplast targeting domain from pea plants, 00:21:42.22 and fusing that to a fluorescent protein derived from a jellyfish and bingo, 00:21:47.26 it goes directly into the apicoplast in both Toxoplasma and Plasmodium 00:21:55.23 so faced with the daunting problem of how to target proteins into these organelles, 00:22:03.22 these parasites have evolved a remarkable mechanism. Fusing what we normally 00:22:11.16 think of as completely distinct targeting pathways, the blue bit, 00:22:16.16 the secretory signal sequence, with the green bit, a plastid targeting domain, 00:22:22.11 and we can map out the nature of those plastid targeting signals in great detail. 00:22:27.19 Experiments here carried out by post-doctoral fellow Omar Harb, 00:22:31.16 in which we can distinguish between proteins that target to the apicoplast, 00:22:36.04 and those that target to the membrane of the apicoplast as opposed to the lumen, shown here in red, 00:22:41.13 and we can follow the processing of those proteins on western blots as the targeting signal is cleaved off. 00:22:47.22 Those that have lost the ability to target apicoplast but which still harbor 00:22:53.12 secretory signal sequences, and those that have lost all targeting information as well. 00:22:59.18 We know the targeting to the chloroplast of plants and targeting to the apicoplast 00:23:07.22 of these organisms is quite complex and involves a variety of redundant signals, 00:23:13.11 for this particular targeting signal there are in fact one, two, three, 00:23:19.04 completely distinct plastid targeting signals, any one of which is sufficient 00:23:24.22 to mediate targeting to the organelle, so in quite an unusual story, 00:23:30.05 very different from what you may have learned in introductory cell biology, 00:23:34.15 we take two targeting signals, normally thought of as quite distinct, 00:23:39.11 the secretory signal sequence, which mediates co-translational 00:23:43.17 translocation across the endoplasmic reticulum where upon 00:23:47.03 signal peptidase processes off the signal sequence, the pink bit, 00:23:52.06 to expose a sub terminal domain, the yellow bit, normally thought of 00:23:57.20 as a post-translational targeting signal mediating translocation 00:24:03.03 across the membrane of the chloroplast, now this yellow bit is exposed 00:24:08.10 within the lumen of the endoplasmic reticulum and as that protein 00:24:12.03 winds its way through the secretory pathway, mediates translocation across 00:24:18.29 the remaining membranes into the apicoplast where upon a pitrilysin protease 00:24:23.15 cleaves that to expose the mature protein indicated in green. 00:24:29.20 We know that this process proceeds through the classical secretory pathway 00:24:34.17 by a number of studies carried out by Manami Nishi in this one experiment, 00:24:39.11 we use a fluorescence photobleaching study to follow the timing of targeting 00:24:45.15 to the apicoplast, in this case two parasites labeled in red 00:24:51.02 with a marker for the inner membrane complex, the apicoplast labeled 00:24:54.29 in green, we've photobleached one apicoplast just prior to parasite division 00:25:05.10 and you'll notice that it recovers through the production of new protein 00:25:09.00 very rapidly within a few minutes. We'll then take the same cells 00:25:13.24 and photobleach the other apicoplast here, and now this has been done 00:25:20.05 just at the time as parasite replication begins, subsequent to the division 00:25:26.17 of the golgi apparatus, here you can see the developing daughter parasites 00:25:30.13 within the mother as they replicate now and extend over a subsequent 80 minutes, 00:25:36.24 no recovery of protein trafficked into the apicoplast indeed, five hours later 00:25:42.07 we still see no protein targeted to the apicoplast and we won't until those parasites 00:25:47.11 start to divide again. Trafficking to the apicoplast occurs only over this very 00:25:52.26 narrow window of time indicated here by the white triangles as the apicoplast 00:26:00.04 is itself dividing, and as the golgi apparatus is dividing 00:26:03.24 and most active as well. We can see this process and the organelles 00:26:08.24 that are involved in an electron micrograph with distinctive large vesicles, 00:26:16.12 here the dark electron dense vesicles, distinct from the COPI and COPII vesicles 00:26:24.21 associated with trafficking to and from the golgi apparatus 00:26:29.17 and indeed in these larger vesicles we can stain protein destined for the apicoplast. 00:26:35.17 But I raise this problem not as a cell biological question of distinctive 00:26:41.09 targeting of the apicoplast, but as a problem of really 00:26:44.19 fundamental importance if we are interested in identifying targets for parasite survival. 00:26:51.09 Remember, this is an essential organelle, it’s likely to harbor a variety 00:26:55.26 of metabolic functions, what are those functions and can we target that 00:27:00.28 with new drugs. Now you might imagine a variety of strategies 00:27:08.28 that one could take to explore the functions of a novel organelle. 00:27:13.26 We know quite a lot about the function of chloroplasts for example, 00:27:18.11 and we know that through, for example, enzymological studies on purified 00:27:23.02 chloroplasts, but here we suffer from the difficulty in obtaining 00:27:28.18 large numbers of parasites. If we worked on the chloroplasts of pea plants, 00:27:34.05 or spinach plants, we could go down to the market or field and purchase 00:27:38.15 a truckload of spinach and isolate grams or kilograms of plant chloroplasts 00:27:47.12 for enzymological studies. Similarly, if we were interested in taking more 00:27:51.22 modern proteomics approaches, we would want to have large amounts of material, 00:27:56.17 and yet for these parasites, we can obtain at best a gram of parasites 00:28:03.19 and of course the plastid organelle is going to be only a small fraction of that gram. 00:28:08.14 We can imagine other approaches as well, we could devise genetic screens, 00:28:13.16 saturating the parasite genome with insertional transgenes and perhaps 00:28:19.00 selecting for genes that have acquired plastid targeting signal by virtue 00:28:24.13 of screens for targeting to the apicoplast, and indeed, we've pursued all 00:28:29.27 of those approaches, enzymological studies, proteomic studies, genetic studies, 00:28:35.17 in my laboratory and in other laboratories as well, and in sum total 00:28:39.27 we've learned a little bit about the function of the apicoplast, 00:28:43.06 we've identified some viable candidates, but the most successful approach 00:28:48.07 by far has been an informatics approach, and I'd like to try to describe 00:28:54.12 that to you just briefly. So this illustration indicates what you might imagine 00:29:02.20 as a conceptual schema, a strategy for identifying nuclear encoded 00:29:10.06 apicoplast proteins through genome database mining. We'll go to the 00:29:16.28 genome sequences that are available, any sequences, complete, 00:29:19.29 incomplete, genome sequences, EST sequences from any organism that has an apicoplast 00:29:25.28 and we will troll through those sequences asking a variety of 00:29:31.02 pretty simple minded questions. Asking for example, for any genes 00:29:37.27 that shows some level of similarity with proteins known to be associated 00:29:42.20 with plants, or with chloroplasts. Now there are going to be many many 00:29:48.29 false positives in this kind of search, after all, many proteins are associated 00:29:53.15 with plant chloroplasts that won't necessarily be associated with the apicoplast. 00:29:59.04 There will be false negatives there will be proteins that we simply 00:30:03.15 can't recognize because they're too divergent. Similarly, we can look 00:30:08.24 for proteins that have either one or the other of those distinctive targeting signals, 00:30:14.07 we can look for proteins with a secretory signal sequence for example. 00:30:17.24 But once again, we may not be able to identify all signal sequences 00:30:23.25 particularly in organisms for which the structure of the genes is not well characterized. 00:30:29.29 Moreover, by searching for signal sequences, we are undoubtedly 00:30:34.12 going to identify proteins destined for the endoplasmic reticulum 00:30:38.10 or the golgi apparatus, or the secretory organelles important for invasion 00:30:42.19 or the plasma membrane or the parasitophorous vacuole or the host cell beyond, 00:30:46.26 so false positives, false negatives, and similarly for all of the various 00:30:52.24 screens that we imagine carrying out. But all of the screens listed on this chart, 00:30:57.12 I would suggest harbor two features in common, and those that 00:31:02.24 they hold in common with all successful computational approaches. 00:31:06.29 The first is that they are hopelessly non-specific, but the second is that they 00:31:15.07 are all computationally tractable, and therefore easy to do, and if we do 00:31:18.27 a search like that we identify many proteins with secretory signal sequences 00:31:24.02 or similarity to plants or cyanobacteria or with N-terminal extensions, 00:31:28.11 but what we are looking for are those candidates that harbor two or ideally three 00:31:33.12 of those and in our first set of screens we identified several 00:31:37.28 hundred proteins that might be associated with the apicoplast. 00:31:43.25 A single approach, an informatics approach, undoubtedly many 00:31:48.14 false examples, but certainly hypotheses that we can test experimentally at the lab bench. 00:31:54.18 Here's what one such protein looks like, a protein with a N-terminal 00:31:58.27 hydrophobic signal sequence a subterminal region rich in charged 00:32:04.05 amino acids, one of the hallmarks of plastid targeting, 00:32:07.09 at least in Plasmodium parasites, although Toxoplasma parasites look 00:32:11.04 a little bit different, and most importantly in this case, a N-terminal region 00:32:15.20 that shows unequivocal similarity to ferredoxin the terminal electron acceptor 00:32:21.14 in photosynthesis and while these parasites are 00:32:24.00 not photosynthetically active, they've retained two proteins, 00:32:29.10 vestigal relics of photosynthesis in the ancestor of the apicoplast. 00:32:35.24 Here's the other such protein, ferredoxin NADP reductase 00:32:40.09 and in this case we've taken this protein, fused it to a 00:32:46.13 yellow fluorescent protein reporter, transfected it into Toxoplasma parasites 00:32:52.14 where we can readily carry out transient transfection studies in an overnight experiment 00:32:57.17 with proteins derived from either Plasmodium or from Toxoplasma, 00:33:01.26 demonstrate by co-localization with a validated apicoplast protein 00:33:06.18 that indeed this particular protein is targeted to the apicoplast. 00:33:13.10 The net result of these studies is what we believe to be a complete metabolic pathway 00:33:19.15 map for the apicoplast. We know that this organelle harbors its own DNA 00:33:25.02 and the machinery necessary to replicate it. It harbors its own transcriptional machinery 00:33:31.29 and those transcripts are translated into proteins using proteins encoded 00:33:37.12 on the apicoplast genome and additional proteins that are imported from the nucleus 00:33:43.12 And we now know through nuclear encoded proteins found to be associated 00:33:47.25 with the apicoplast, that a variety of other metabolic processes 00:33:53.07 take place within the four membranes of the apicoplast as well. 00:33:56.22 We know that this organelle is involved in lipid biosynthesis, using a fatty acyl synthase 00:34:04.12 that is significantly different from the fatty acyl synthase 00:34:08.03 of animal cells and humans in particular. The type II fatty acyl synthase 00:34:14.28 consists of multiple subunits which are assembled together in a process 00:34:21.09 that is the target of effective anti-microbial antibiotics 00:34:25.07 that might be candidates for treatment for Toxoplasmosis or for malaria. 00:34:30.18 We know that the apicoplast carries out isoprenoid biosynthesis, 00:34:36.07 the precursors to making cholesterol, although in this case not converting 00:34:44.27 into cholesterol isoprenoid units that are used certainly for modification of 00:34:51.01 transfer RNAs and possibly for other functions as well. 00:34:53.29 We know that the heme biosynthetic pathway involves not only the 00:34:58.22 cytoplasm and the mitochondria associated in the standard C-4 heme pathway 00:35:04.00 in for example, mammalian cells but also the apicoplast in an interesting process 00:35:10.26 that we don't understand yet in great detail. And we can dive down 00:35:15.11 into further detail to identify all the various components associated 00:35:19.22 with these pathways including several that are particularly attractive 00:35:23.10 as drug targets, some of which have compounds that are in clinical trials 00:35:27.25 as anti-malarials as we speak. But the point that I'd like to make today 00:35:34.24 is not just that malaria parasites stole a plastid from an ancestral plant, 00:35:45.03 and maintained that plasmid as a non-photosynthetic organelle 00:35:48.23 that's essential for parasite survival and therefore attractive as a drug target. 00:35:53.15 True though that may be, the real point that I wanted to make 00:35:57.13 and that I would like you to take from this segment of the iBioLecture 00:36:03.24 is that its these computational bioinformatics data mining approaches 00:36:09.10 that have been most successful and are really transforming the way we think 00:36:13.18 about doing biological experiments. In this area of parasitology 00:36:17.11 as in all areas of biomedical research. And in the next segment of this iBioLecture 00:36:24.29 we'll talk a little bit further about that, taking advantage of the genome sequences 00:36:30.04 now available for malaria parasites, as well as the mosquito vectors 00:36:34.22 and the human hosts and posing the challenge of whether we can design 00:36:40.00 and mine genome databases to take those genomes, identify the genes, 00:36:45.25 and develop diagnostics and therapeutics, drugs and vaccines 00:36:50.21 that might be effective against these organisms. And I look forward 00:36:55.00 to being able to tell you more about that in the next segment of this lecture series.
