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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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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