Intracellular Protozoan Parasites: Trypanosoma cruzi and Leishmania
Transcript of Part 3: Current Research: Strategies for Cell Invasion and Intracellular Survival
00:00:03.20 Hi, my name is Norma Andrews. 00:00:05.19 I'm a professor at Yale University, and what I'm going to do in this third part 00:00:09.20 of my lecture is to talk to you about insights that we have obtained in my lab 00:00:16.10 on the strategies used by both Trypanosoma cruzi and Leishmania to invade host cells 00:00:22.09 and then to survive inside the compartments generated inside these host cells. 00:00:27.04 Trypanosoma cruzi, as already detailed in the first part of this lecture, 00:00:33.11 is the causative agent of Chagas' disease, 00:00:35.29 a serious endemic disease in South and Central America 00:00:39.15 which is shown here in this infected child. And this is what is seen 00:00:43.20 in the blood of these patients during the acute phase of the disease 00:00:47.10 is this large number of these very highly motile parasites circulating in the blood. 00:00:55.28 This infective stage is the form that is deposited by the insect after the blood meal, 00:01:01.22 so it is contamination of the wound created by the blood meal 00:01:07.05 that transmits the parasite through the feces of the insect, 00:01:12.11 and this is the way they gain access to the mammalian host. 00:01:16.12 What is really the main characteristic of Trypanosoma cruzi is that 00:01:20.01 it is capable of invading and replicating inside a large number of different cell types. 00:01:27.04 This is the mechanism that we have studied for several years in my laboratory. 00:01:32.24 Our insights into this mechanism started with these scanning electron micrographs 00:01:38.01 taken by Edith Robbins at NYU. 00:01:40.11 What is shown here is this infective stage, trypomastigote 00:01:44.08 attached to a host cell. And what happens after this, 00:01:48.18 is that these parasites initiate this very dramatic process of entering the host cell. 00:01:54.21 You can see that these are large parasites, and what is surprising... 00:01:58.01 what surprised us in these first observations is this entry mechanism 00:02:03.25 was very different from phagocytosis. 00:02:05.29 And our assumption at the time was that these parasites should enter by phagocytosis 00:02:11.02 because they are so large, so they would, in principle, need actin polymerization 00:02:17.22 on the side of the host cell to really expand the membrane 00:02:21.01 to be able to accommodate a particle so large. 00:02:23.14 But these images were suggesting that something quite different was going on. 00:02:27.24 Just to illustrate here, this is what is seen in cells 00:02:32.10 when they are undergoing phagocytosis. 00:02:34.18 These are images of large particles being taken up by macrophages, 00:02:40.17 showing how you see this profusion of pseudopods 00:02:44.11 and this extension of membrane that gradually envelops the particle. 00:02:48.27 What happens with Trypanosoma cruzi entry is something quite different. 00:02:52.09 We were sure that this was the case when we just did these simple experiments 00:02:57.16 of staining infected cells. So, these cells were just exposed 00:03:00.23 to the parasites for very brief periods of time of just 10 minutes, 00:03:04.12 and then these parasites that are inside the cell... 00:03:08.17 The parasites had been stained with antibodies against surface proteins 00:03:12.17 (which is shown in red) and then the cell is stained with phalloidin, 00:03:16.14 in green, which is a protein that binds to polymerized actin. 00:03:20.13 And, you can see here how different it is, 00:03:23.17 the patterns seen with these parasites inside host cells 00:03:27.25 and what is seen in macrophages when they take up for example yeast particles, 00:03:32.15 in which you see this classical ring of actin around the particle, 00:03:37.12 and we don't see it around parasites that have successfully entered host cells. 00:03:43.03 Although the microfilaments of the cells are still present, 00:03:47.03 they don't seem to be altered significantly by this invasion process. 00:03:53.14 Drugs that disrupt the actin cytoskeleton 00:03:57.26 also had no inhibitory effect on Trypanosoma cruzi entry. 00:04:01.14 But, to our surprise, this invasion process was highly sensitive 00:04:06.24 to drugs that disrupt microtubules, 00:04:08.28 which is the second important cytoskeletal element in cells, 00:04:13.18 which actually are elements of the cytoskeleton 00:04:17.22 that provide the tracks where organelles move. 00:04:20.17 This observation and this second important observation that markers for lysosomes 00:04:28.12 (which is shown here in this immunofluorescence) 00:04:30.25 is that when we stain cells also exposed for brief periods of time 00:04:35.28 to infective stages of Trypanosoma cruzi, 00:04:38.03 we can see that these markers for lysosomes 00:04:42.05 (in this case, antibodies against Lamp-1, a major glycoprotein of lysosomes) 00:04:46.12 can be seen in this very tight vacuole that forms 00:04:50.09 around the parasites as they enter the cells. 00:04:53.10 This also looked very different from the classical maturation of phagosomes 00:04:58.07 that was known to take several minutes. 00:05:01.13 Lysosomes were known to meet particles after they were internalized, 00:05:06.16 and in this case, here, this appeared to be happening much faster 00:05:11.14 during the invasion process. 00:05:14.10 What was important about this observation is that we know 00:05:19.16 that lysosomes move on microtubules. 00:05:22.10 So these two findings that drugs that disrupt microtubules 00:05:27.12 inhibit Trypanosoma cruzi entry, 00:05:28.28 and the fact that you see markers for lysosomes 00:05:31.05 associated with the vacuole surrounding these parasites, 00:05:34.04 really indicated to us that probably the parasite had very unique mechanisms 00:05:40.10 to really recruit these organelles early in the invasion process. 00:05:45.04 This is what is shown in these images here. On the top is again immunofluorescence 00:05:51.08 in which antibodies against the parasite were added to these cells 00:05:56.07 before they were permeabilized. 00:05:57.25 Only the parasites outside the cell got stained in red. 00:06:01.09 Then these cells were permeabilized with detergent 00:06:03.27 and we added antibodies against lysosomal proteins, 00:06:07.09 so we can see here that the second parasite inside the cell 00:06:10.25 is actually stained by lysosomal markers 00:06:14.27 indicating that it's already inside the cell. 00:06:17.11 The important finding was that we see this dramatic pattern 00:06:22.02 of tight association of lysosomes, 00:06:25.00 which are inside the cell, just immediately underneath this parasite 00:06:29.18 attached to the surface, 00:06:30.21 indicating that there was a localized signaling process 00:06:34.25 recruiting these lysosomes exactly to the site of the invasion. 00:06:38.06 And this is illustrated perhaps better here in this transmission electron microscopy, 00:06:45.10 in which the lysosomes were loaded with horseradish peroxidase, 00:06:50.07 so this allows us to do this cytochemistry reaction and visualize these lysosomes, 00:06:54.28 and you can see that the parasite is still completely outside the cell, 00:06:59.07 but these lysosomes were recruited to the site of the invasion. 00:07:03.25 The next step in this process is that you can see images like this, 00:07:08.11 in which this part of the parasite is still out of the cell, 00:07:11.29 but the portion that is inside is surrounded by markers of lysosomes. 00:07:17.20 And the same thing is seen here. 00:07:19.12 This is a section through a parasite in the process of entering cells. 00:07:23.23 It's actually hard to distinguish the vacuole because this is a very tight vacuole, 00:07:28.20 but there are two membranes here, and this material that is coming from lysosomes 00:07:33.07 is actually delivered to this very tight vacuole 00:07:37.22 that forms around the parasite as it enters cells. 00:07:41.18 In this movie here, I'm just going to show you, 00:07:46.00 this is one fibroblast, in which the outline 00:07:48.02 of the cell is around here, and there are parasites attached. 00:07:53.01 And what was done here, is that this cell was preloaded 00:07:56.16 with albumin complexed to colloidal 00:07:59.24 gold so the lysosomes in this movie appear as these small black dots. 00:08:04.22 And when we play this movie, we can see that actually, it's possible 00:08:09.04 to visualize microtubules as these elevated regions 00:08:12.06 and it is possible to see lysosomes actually moving in these very large steps 00:08:17.17 towards the site where this parasite is going to start to invade the cell. 00:08:21.28 So then, analyzing these movies... 00:08:26.01 taking the position of each lysosome in each time frame, 00:08:29.11 it is possible to construct these diagrams in which it becomes very clear 00:08:34.20 that just in regions slightly removed from the invasion site, 00:08:39.07 microtubules are showing 00:08:40.18 the known behavior which is this saltatory movement which is bidirectional. 00:08:45.11 But, when it comes close to the site where an invasion event is going to happen, 00:08:50.25 there are these dramatic directional steps 00:08:53.10 which can be seen perhaps easier here with these arrows 00:08:56.19 indicating that this directional movement is happening 00:09:00.20 in this area of influence of the parasite, 00:09:02.29 but before invasion starts. 00:09:06.02 This is really what led us to then study in detail the signaling process that was involved 00:09:13.17 in this process. And what we learned is that the parasites produce an agonist 00:09:19.24 that interacts with receptors in the host cell, activates phospholipase C 00:09:24.01 and the production of IP3, which is a mediator 00:09:27.25 for the release of calcium from intracellular stores. 00:09:31.09 There is an elevation in intracellular calcium, 00:09:33.29 which is what this movie up here is showing. 00:09:35.28 This cell was loaded with a calcium-sensitive dye and exposed to the parasites. 00:09:41.00 And we can see how these cells continuously flash 00:09:44.00 as they are being stimulated by the parasite, 00:09:46.13 showing transient elevations in the cytosolic, free calcium. 00:09:50.14 We showed in a series of experiments that this calcium elevation is necessary 00:09:58.09 for this recruitment of lysosomes to the site of parasite invasion. 00:10:02.29 What we learned through this process 00:10:06.03 (this is a very good example of the unexpected findings 00:10:08.12 that can happen) By studying this exotic parasite from South America, 00:10:14.06 we actually understood something quite basic 00:10:16.03 about the behavior of conventional lysosomes in mammalian cells. 00:10:20.07 When we decided to take the trypanosomes out of the picture 00:10:23.22 and just look at the effect of elevations in cytosolic calcium, 00:10:28.14 we learned that actually many different cell types... 00:10:31.22 Lysosomes, which were originally viewed, 00:10:34.27 (or is still very consistently viewed) 00:10:38.26 as terminal compartments of the endocytic pathway... 00:10:41.23 Actually, these organelles can be transformed 00:10:46.22 into secretory and regulated secretory organelles, 00:10:49.16 just by elevating the cytosolic calcium concentration. 00:10:53.11 And I'm not going to get into this in this lecture, but we have learned, in my lab, 00:10:58.11 that this process is very important in the physiology of mammalian cells. 00:11:02.00 For, for example, the repair of mechanical wounds on the plasma membrane, 00:11:07.19 and also for phagocytosis, as a mechanism 00:11:11.13 for adding more membrane at the sites of phagocytosis. 00:11:16.01 What I'm going to show you, 00:11:20.01 in collaboration with Sandy Simon at Rockefeller University, 00:11:22.28 is how we have been able, recently, to directly visualize 00:11:26.23 this process of lysosomal exocytosis. 00:11:29.11 For this, we use this powerful technique of total internal reflection microscopy. 00:11:34.21 Which, what is indicated here, in this technique, the cells are illuminated 00:11:40.25 from the bottom with a laser beam, and the laser is directed to the cells at an angle 00:11:47.08 in which most of the light is reflected away, 00:11:50.03 so the small amount of light that penetrates 00:11:54.10 these cells does so at a very narrow field which is called the evanescent field. 00:12:00.01 And it's called the evanescent field 00:12:02.05 because it has this very interesting property of decaying 00:12:05.03 exponentially away from the coverslip where the cells are lying. 00:12:09.13 If you just add a fluorescent marker to your vesicles of interest, 00:12:14.09 it is possible to visualize these vesicles becoming brighter and brighter 00:12:18.19 as they travel through this evanescent field 00:12:21.17 and then the moment of fusion is quite obvious 00:12:24.29 because there is high intensity fluorescence and then diffusion of the label. 00:12:31.04 In these studies, Jyoti Jaiswal and Sandy Simon observed 00:12:37.04 something quite interesting that we didn't know when we started these studies. 00:12:41.12 Lysosomes were known to concentrate in cells in the perinuclear area, 00:12:47.02 which is shown here is this classical epifluoresence microscopy. 00:12:51.15 And what this technique of TIRF microscopy showed is that, 00:12:57.15 in addition to this perinuclear population, 00:12:59.06 there is quite a significant number of lysosomes 00:13:02.05 that can be seen in this very membrane-proximal region, 00:13:07.12 which is just 80 or 100 nanometers close to the plasma membrane. 00:13:12.20 This was an important observation because when these fusion events were analyzed... 00:13:18.25 I'm going to show you one example, here, in which this is an embryonic fibroblast. 00:13:23.22 Again, like in the previous pictures, the lysosomes were loaded 00:13:27.21 by chasing a fluorescent molecule, dextran, 00:13:30.25 throughout the endocytic pathway, and then accumulating in lysosomes. 00:13:36.14 And this movie is going to show, when it starts playing, now 00:13:39.08 this cell being stimulated by a calcium ionophore. 00:13:43.03 And we're going to see these puffs of fluorescence, 00:13:46.12 which reflect the luminal dextran being released. 00:13:49.23 I'd like you to focus on this lysosome here that is getting brighter and brighter, 00:13:53.21 and we're going to see the moment in which this 00:13:55.22 lysosome fuses with the plasma membrane, 00:13:57.12 releasing its contents. 00:13:59.22 When Jyoti Jaiswal and Sandy Simon analyzed these events, 00:14:04.11 what became clear is that, in cells either stimulated with a calcium ionophore 00:14:09.16 or these two more physiological agonists, thrombin and bombesin, 00:14:14.17 the large majority of the lysosomes that were seen fusing with the plasma membrane 00:14:20.07 were originating from this membrane-proximal population. 00:14:27.03 Very few, shown here in red, were recruited from deeper regions of the cell, 00:14:32.20 meaning that they were first invisible and they entered the illuminated field. 00:14:37.12 So, this gave us a very important piece of information 00:14:41.03 that this peripheral population of lysosomes 00:14:44.25 (that had really not been described before) actually is functional and is actually 00:14:51.11 responsible for the majority of these fusion events that are modulated by calcium. 00:14:57.08 This got us interested in the molecular machinery controlling lysosomal exocytosis, 00:15:05.27 and shown here in this diagram from a review of Ed Sheckman, 00:15:10.05 it is quite understood now 00:15:12.27 because of the work of Ed Sheckman and several others that this class of molecules, 00:15:17.25 the synaptotagmins, are very important in coupling calcium 00:15:21.17 to membrane fusion events. 00:15:23.05 So what is indicated here are the SNARE molecules, in which you have 00:15:27.18 the v-SNAREs on the membranes of vesicles 00:15:30.10 and the t-SNAREs on the acceptor membranes. 00:15:34.00 And these molecules form these tight, coiled-coil bundles, 00:15:38.13 which are believed to be responsible for promoting membrane fusion. 00:15:43.16 This process can be made sensitive to calcium when synaptotagmin molecules interact 00:15:54.16 with the SNARE proteins after binding calcium, 00:15:58.28 and they bind calcium through these two domains 00:16:02.04 that they have in their cytoplasmic region, 00:16:04.10 which are the C2 domains. 00:16:05.12 These calcium sensor molecules associated with membranes 00:16:09.18 were very interesting candidates 00:16:11.19 for also mediating lysosomal exocytosis, which 00:16:15.11 we learned was a process clearly controlled by elevations of calcium in the cytosol. 00:16:22.09 We got interested in one specific isoform, synaptotagmin VII 00:16:26.26 because it is ubiquitously expressed, 00:16:29.18 it is evolutionarily conserved (so it is one of the isoforms conserved in Drosophila 00:16:36.03 and other lower organisms, including C. elegans) 00:16:39.04 and it has a very wide distribution, which could be consistent 00:16:44.22 with a lysosomal localization. 00:16:46.25 This was confirmed in several studies in our lab. 00:16:52.16 I'm just showing here one example here of immunofluorescence, 00:16:55.11 in which antibodies specific to synaptotagmin VII in red 00:16:59.26 were used to stain a cell that is also infected with Trypanosoma cruzi. 00:17:04.08 And we can see that in addition to the lysosomes, that over here, (stained in green 00:17:10.15 with antibodies against Lamp-1) we can see a very good overlap, 00:17:15.22 which is shown in the yellow image down here, 00:17:17.24 showing that the synaptotagmin VII localizes to lysosomes in mammalian cells and is 00:17:25.09 also delivered to the vacuole surrounding Trypanosoma cruzi right after invasion 00:17:29.11 just like other lysosomal markers. And then we went on to show that 00:17:35.19 deficiency in this molecule, synaptotagmin VII can inhibit not only lysosomal exocytosis 00:17:42.04 induced by calcium, but also invasion of these cells by Trypanosoma cruzi. 00:17:47.16 Additional work allowed us to identify the partners of this fusion reaction: 00:17:57.08 the SNARE molecules (in which VAMP7 present on lysosomes 00:18:02.00 interacts with syntaxin 4) and SNAP-23. 00:18:06.25 And synaptotagmin VII associates with these molecules in a calcium dependent manner, 00:18:12.12 which led us to propose that it is also similar 00:18:16.24 to what has been shown in synaptic vesicles 00:18:21.15 in neuronal cells that this would be how this process becomes calcium regulated. 00:18:28.12 These lysosomal fusion events actually have proven 00:18:32.19 to be critical not only for Trypanosoma cruzi 00:18:36.03 invasion, but they're interestingly in more recent work from the lab. 00:18:38.23 We understood that this event is very important for retaining 00:18:42.16 these highly motile parasites inside host cells. 00:18:45.17 So, after Trypanosomes invade, as I already showed you, 00:18:48.28 there is during the invasion process, 00:18:51.02 there is this gradual fusion of lysosomes, 00:18:54.13 and we know that lysosomes are associated with 00:18:57.09 these microtubules tracks through the molecular motors. 00:19:01.24 And we think that this process is very important for keeping 00:19:05.27 these parasites inside the host cell 00:19:08.00 for completion of the cycle. 00:19:09.27 Part of the evidence for this is just indicated in this diagram here, 00:19:13.10 that when we prevent lysosomal fusion with different procedures, 00:19:18.17 these parasites can still enter cells by invaginating the plasma membrane, 00:19:23.04 most probably propelled by their very active motility. 00:19:27.08 But very interestingly, this entry can be reversible, so if there is no lysosomal fusion 00:19:33.04 and there's no tethering to the microtubule network, 00:19:37.15 these parasites actually can exit the cell, 00:19:40.16 and this invasion process actually can be reversible. 00:19:44.19 So this is an interesting concept that I believe will prove 00:19:47.23 to be true for other motile pathogens, 00:19:51.13 in which they have not only to be able to invade cells 00:19:56.03 and to create an intracellular compartment, 00:19:58.00 but they have to be able to be retained inside host cells. 00:20:01.06 What this image here is showing, is a summary of what we learned. 00:20:05.21 The signaling process induced by trypanosomes in host cells 00:20:10.16 triggers this calcium elevation in the cytosol, 00:20:13.28 which drives this fusion of lysosomes at the site of invasion 00:20:18.27 This provides the parasites with membrane of host cell origin, 00:20:23.13 in which they form this initial compartment. 00:20:26.08 In the case of Trypanosoma cruzi, they escape from this compartment very soon, 00:20:32.06 a few hours after invasion in some cell types, 00:20:35.17 and complete the process in the cytoplasm, 00:20:37.17 as I already discussed in the first part of this lecture. 00:20:42.21 We were interested in, in addition to this entry process, 00:20:45.15 in understanding how the parasites survive in these lysosome like compartments 00:20:50.03 which are so degradative and, in principle, should be promoting destruction 00:20:56.26 and not survival of these pathogens. 00:20:59.25 For this question, we turned to Leishmania 00:21:03.10 which I also already introduced in the second segment of this lecture, 00:21:08.07 which is a parasite transmitted... it's closely related to Trypanosoma cruzi. 00:21:12.26 It is transmitted by sand flies, and when these infective stages 00:21:18.24 are introduced into the mammalian host, 00:21:21.06 they invade macrophages and they replicate inside these lysosome-like compartments. 00:21:27.19 This is here showing a picture of a patient with a cutaneous form of the disease 00:21:32.29 which is a consequence of the inflammatory process 00:21:37.11 that develops when these parasites are replicating inside macrophages. 00:21:42.04 So, what is interesting about Leishmania is that they are adapted, (as shown here, 00:21:47.00 examples of several species of Leishmania) in which in all of them, 00:21:50.25 the vacuole that is formed around this parasite 00:21:54.18 and where they replicate really has markers of lysosomes. 00:21:59.12 There are many studies in which this was shown, 00:22:03.04 mostly from Jean-Claude Antoine in the Pasteur Institute in Paris, 00:22:07.22 to be lysosomal compartments, so the question became 00:22:13.26 how do they survive inside these degradative compartments. 00:22:18.19 So, this leads us to a very interesting pathway in mammalian cells, 00:22:24.22 which is the mechanism by which cells acquire iron. 00:22:28.28 Mammalian cells have receptors for transferrin, 00:22:32.05 which is the carrier protein for the oxidized form of iron, Fe3+. 00:22:37.15 This is very understood that the receptors for transferrin are endocytosed 00:22:42.29 and inside these early endosomes, when the pH drops, 00:22:47.08 the iron is released from transferrin 00:22:50.19 and then transferrin is recycled back out of the cell, 00:22:55.01 and the iron that is released gets translocated into the cytosol. 00:23:01.10 The way this happens is by reduction into Fe2+ by reductases inside the endosome, 00:23:08.02 and then there are specific transporters, 00:23:10.02 which in the case of early endosomes, this transporter is known as Nramp2. 00:23:17.19 And it is responsible for translocation of a large fraction 00:23:23.26 of the iron trafficking through this pathway into the cytoplasm, 00:23:27.16 where it becomes accessible to the metabolic processes of the mammalian cell. 00:23:32.11 What was a subject of discussion for a long time, but is recently becoming more clear 00:23:39.13 is that another transporter, Nramp1, works in a similar manner as Nramp2, 00:23:45.08 but it is deeper in the endocytic pathway, so it is found mostly in lysosomes. 00:23:50.14 What is very interesting about Nramp1 is that it is widely known 00:23:57.22 as a susceptibility gene for infectious diseases. 00:24:00.21 So, for quite some time, it has been clear that mutations in Nramp1 00:24:06.13 promote susceptibility to several pathogens 00:24:09.17 like Leishmania, salmonella, and mycobacteria, 00:24:12.18 which replicate inside the endocytic pathway. 00:24:16.22 This creates some very interesting questions and also illustrates the fact 00:24:22.08 that this mechanism of gradually depleting the endocytic pathway in iron 00:24:28.06 is a mechanism of resistance against pathogens 00:24:32.04 because this severely limits the access of these intracellular organisms to this key 00:24:39.08 metal that is needed for many important enzymatic reactions. 00:24:43.15 What we decided to do is that, to understand how Leishmania acquires iron 00:24:51.15 in these compartments, since it was clear that this is the type of compartment 00:24:56.24 where they replicate and unlike bacteria, 00:24:59.29 nothing was known about how Leishmania could acquire iron intracellularly. 00:25:06.02 In my lab, this work was done by a very talented investigator, Chau Huynh 00:25:11.09 who asked this question: How does Leishmania acquire iron 00:25:15.05 in these very specialized lysosome-like compartments 00:25:18.27 of which an example is shown here, these highly expanded compartments 00:25:24.06 that are typical of Leishmania amazonensis. 00:25:26.29 What Chau found, just doing homology searches in the recently completed Leishmania 00:25:36.02 genome, he found a protein which he called LIT1 for Leishmania Iron Transporter 1 00:25:41.15 that showed significant identity of 30% and 54% similarity 00:25:48.09 with a known iron transporter from Arabidopsis. 00:25:52.13 This is the structure of this transporter. It has 8 transmembrane domains, 00:26:00.06 and these residues which have been shown to be critical 00:26:03.05 in iron transport are highly conserved 00:26:05.17 in the Leishmania protein. 00:26:08.03 Chau postulated that this would be a member of this ZIP family of metal transporters, 00:26:14.11 and what he did initially was to produce antibodies 00:26:18.07 specific against this Leishmania protein. 00:26:21.23 And interestingly, he could not detect this protein by immunofluorescence 00:26:24.24 on parasites that had recently invaded cells. 00:26:27.16 He could not see it either in the extracellular stages of Leishmania. 00:26:34.06 So what is shown here in blue is just the parasite... 00:26:37.08 the DNA of the host cell and of the parasites, 00:26:39.29 and we can see that there is no staining with the antibody which is shown in green. 00:26:44.09 But if he just waited 24 hours... So this parasite still has not started replicating, 00:26:49.17 but you can see that the compartment has greatly expanded, 00:26:53.18 and they are going to start to replicate soon. 00:26:56.28 At this stage, there is clearly a signal with the antibodies against this LIT1 protein, 00:27:04.13 and in this enlarged portion here, we can see that the pattern is what we would expect. 00:27:09.06 It is distributed around the surface of these intracellular parasites. 00:27:13.24 What Chau went on to do is to really obtain evidence 00:27:18.18 that this could function as an iron transporter. 00:27:21.03 He first did experiments in which he introduced this LIT1 gene from Leishmania 00:27:27.16 into strains of yeast that are defective in iron transport. 00:27:32.10 What is shown here is that if you grow these strains in iron-rich media, 00:27:38.10 they can still grow, 00:27:39.16 although they lack two main pathways of iron transport. 00:27:43.17 But, when iron is chelated in the medium, they cannot grow. 00:27:49.11 But, just introducing the LIT1 gene allows us to rescue the growth of these strains, 00:27:56.26 really providing strong evidence that LIT1 can function as an iron transporter. 00:28:01.16 What is shown down here is the direct demonstration that LIT1 00:28:06.05 functions as an iron transporter in Leishmania. 00:28:09.16 So, Chau created a null mutant (a knockout mutant) lacking LIT1 00:28:15.26 and compared it with parasites that had been complemented with this gene. 00:28:20.27 This curve up here is uptake of radioactive iron by these complemented parasites, 00:28:27.18 and you can see that it's very different from the very low levels 00:28:31.10 observed in these knockout forms. 00:28:34.06 This knockout was constructed by this standard technique in the field 00:28:40.07 of homologous recombination. 00:28:41.23 LIT1 is actually encoded by two genes, LIT1A and LIT1B, 00:28:47.06 but these genes are close enough that they can be eliminated 00:28:52.05 (deleted) in one step of homologous recombination 00:28:55.20 by replacement with a selectable marker 00:28:58.01 and in Leishmania this only requires that it's done in two steps 00:29:01.24 with two different selectable markers, because these are diploid organisms. 00:29:06.03 Once these parasites are generated, Chau could show here, 00:29:12.01 again by immunofluorescence, 00:29:13.21 that this protein is detected on the surface of the parasites only in the wildtype strain, 00:29:20.12 but it is not present in the mutant. 00:29:24.05 What was interesting was the behavior of these forms inside macrophages. 00:29:30.04 This here shows how Leishmania amazonensis grows inside macrophages, 00:29:38.01 forming these extremely enlarged compartments 00:29:40.16 that contain the lysosomal protein, Lamp-1, 00:29:42.26 which is shown in green. 00:29:44.07 Over here in blue is DNA staining showing the parasites replicating 00:29:49.03 associated with the membrane of these vacuoles. 00:29:53.10 When the same timeframe was analyzed in cells 00:29:58.06 infected with these parasites lacking the LIT1 protein, 00:30:02.00 we could see that the initial stages were very similar. 00:30:05.04 The parasites actually can infect the cells, they can expand initially the vacuole, 00:30:12.18 but then they don't divide. 00:30:13.17 This is indicated here by the DAPI staining showing that the number of parasites 00:30:19.24 remains similar throughout several hours that this infection was followed. 00:30:26.18 This is the quantification of the process, 00:30:29.01 really showing that we see wild-type parasites increasing in numbers, 00:30:35.11 as they replicate inside the host cells. 00:30:37.28 And in black, how these knockout parasites do not grow, 00:30:42.15 but if the LIT1 gene is reintroduced into this knockout strain, growth is rescued. 00:30:48.26 This was the formal demonstration that actually LIT1 is an essential requirement 00:30:53.12 for intracellular growth in macrophages, 00:30:56.03 really linking this pathway of iron acquisition as a critical requirement for Leishmania 00:31:02.06 to actually make it in this iron-poor, deprived compartment 00:31:07.07 of the late endosomes/lysosomes. 00:31:10.06 With the help of David Sacks at the NIH, we also analyzed the capacity 00:31:16.24 of these knockout parasites to cause pathology in mice, 00:31:21.05 and this cutaneous form of Leismaniasis can be reproduced in mice 00:31:26.12 by injecting the parasites into the footpad. 00:31:29.08 This has a very clear pattern when the animals are injected with wildtype organisms 00:31:37.24 that a lesion develops in the footpad. 00:31:40.26 Consistent with what was observed in the macrophages in culture, 00:31:45.26 these parasites did not form lesions in the mice 00:31:50.07 even after being injected in large numbers 00:31:52.24 and being followed for several months in the animals. 00:31:55.20 The only observation was that, despite the complete absence of pathology, 00:32:01.23 these mutant parasites lacking the LIT1 protein could be recovered 00:32:06.24 from the tissues of these mice, indicating persistence. 00:32:10.26 This is a very important and interesting point which I would say 00:32:15.12 is a very critical question for future research, 00:32:18.22 which is How does Leishmania survive and persist in host tissues 00:32:25.22 even after effective immune responses are developed? 00:32:31.21 It is known in physiological conditions, when small numbers of parasites are injected 00:32:36.20 that the lesions usually heal but the parasites persist, 00:32:41.00 and it is not known in what cell type 00:32:45.09 these parasites are persisting and how they survive and prevent elimination 00:32:52.04 when their main host cell (at least the major host cell that is well known) 00:32:59.07 is the macrophage. 00:33:01.06 This is the point that I hope that we're going to get to understand better in the future 00:33:06.18 when we start to go deeper into what are the requirements for Leishmania survival 00:33:12.09 in the mammalian host. 00:33:14.20 I would like to acknowledge a large number of very talented people 00:33:19.02 (post-docs and students) 00:33:21.11 that over the years have contributed to the work that I mentioned here, 00:33:25.14 and also I would like to acknowledge important collaborators in this work 00:33:30.21 and also the sources of funding which were mostly from the NIH, 00:33:35.01 the Burroughs Wellcome Fund, and the Human Frontiers Science Program. 00:33:38.24 Thank you.