Intracellular Protozoan Parasites: Trypanosoma cruzi and Leishmania

Part 1: Trypanosoma cruzi and Chagas’ Disease

00:00:01.27 So my name is Norma Andrews, I'm a professor at Yale University.
00:00:05.18 And what I'm going to be doing in the first segment of my lecture is to give an introduction
00:00:10.10 on this parasite, Trypanosoma cruzi and the disease that it causes in humans,
00:00:14.28 which is called Chagas' disease.
00:00:16.15 Trypanosoma cruzi is a protozoan parasite from the order Kinetoplastida,
00:00:22.09 and inside this order there's the family Trypanosomatidae
00:00:25.06 which includes several protozoan organisms of which 2 are very important medically
00:00:32.20 because they cause serious diseases in man.
00:00:34.29 And one of them is the genus Trypanosoma which includes not only Trypanosoma cruzi
00:00:40.10 but also the African trypanosome which causes sleeping sickness in Africa
00:00:44.03 and Leishmania which I'm going to talking to you about in the second part of this lecture.
00:00:49.25 So, Trypanosoma cruzi is also known as the American trypanosome
00:00:54.29 and actually should be called the Latin American trypanosome
00:00:57.17 because the disease caused by this parasite is found only in South and Central America.
00:01:04.16 And in this region, a large number of people carry currently the parasite--
00:01:09.28 between 16 and 18 million people are infected at present.
00:01:15.05 And the history of Chagas' disease is very interesting in the sense that,
00:01:19.19 unlike other infectious diseases, it was a single individual--Carlos Chagas--
00:01:23.24 a Brazilian investigator working practically alone in the field that made all the major findings
00:01:30.23 that led to this realization of not only a completely new infectious agent
00:01:37.10 but also the vector that was responsible for its transmission to humans,
00:01:42.01 the animal reservoirs in the region,
00:01:44.26 and also the living conditions that really favor transmission to people.
00:01:50.09 So, what Carlos Chagas noticed...
00:01:52.08 He was in this rural area in Brazil, working on malaria transmission, and he,
00:01:58.29 being trained as a medical entomologist,
00:02:01.17 he noticed that insects that he found heavily infesting these mud huts
00:02:06.27 very common in this area and still seen in many regions of South and Central America...
00:02:13.02 These houses were very heavily infested with insects like this one shown here,
00:02:17.26 which are Reduvids, these crawling bugs that, during the day,
00:02:23.03 they hide in the cracks of the walls of these houses and come out at night
00:02:27.05 to feed on the blood of people and domestic animals.
00:02:30.18 So, Carlos Chagas dissected these insects that he found in these huts
00:02:36.10 and he saw that they carried these very large forms that, it was clear to him,
00:02:45.13 that this was a new protozoan organism
00:02:48.16 that had not been identified before, and he observed mainly two forms.
00:02:52.24 So, there was this longer form and a smaller one with an undulating flagellum
00:02:58.22 which is what we know now to be the infectious form.
00:03:02.26 And the way he learned this was when he sent
00:03:06.10 some of these infected bugs to the laboratory in Rio,
00:03:09.09 and they allowed these insects to feed on monkeys.
00:03:12.22 And these monkeys very soon developed an infection with large numbers of these parasites
00:03:19.04 which have very similar morphology circulating in the blood.
00:03:22.23 Carlos Chagas at the same time, he was able to show that
00:03:25.24 the circulating blood of children which showed signs of infection--
00:03:31.14 high fever and also these swelling regions in the face--
00:03:37.05 these children, in the blood, had large numbers of these parasites in circulation.
00:03:43.20 So in this way, he identified the basic elements of the life cycle of Trypanosoma cruzi
00:03:51.06 which we now know, inside the insect, we have the epimastigotes replicating in the digestive tract
00:03:57.24 of the insect and then these forms transform into an infective stage, the trypomastigotes
00:04:05.23 and are released with the feces of the insect, so transmission actually occurs
00:04:10.02 by contamination of either the wound bite or the mucosal membranes
00:04:17.10 in the mammalian host.
00:04:20.03 When these parasites cross into the mammalian host,
00:04:24.24 they find host cells--large, different types of whole cells can be infected
00:04:30.21 and that's where they transform into the amastigotes
00:04:33.14 the form that replicates in the cytosol, and then the parasites at the end of the cycle are released.
00:04:40.09 They can either continue this cycle in the mammalian host
00:04:43.23 or they can be taken up by the insect during a blood meal.
00:04:47.23 So another important finding that was made several years later
00:04:53.08 is that there is an important difference between epimastigotes
00:04:56.29 these forms found in the gut of the insect
00:05:02.09 and the forms responsible for transmission which are the trypomastigotes
00:05:06.28 So the epimastigotes are lysed by the alternative pathway of complement activation
00:05:12.01 And this is the cascade of events that happens, initiated by hydrolysis of C3
00:05:18.00 which is cleaved into fragments, of which C3b has the capacity of cleaving C5,
00:05:24.16 and then C5b is a component of this membrane attack complex
00:05:31.27 that forms after association with C6, C7, C8, and C9,
00:05:36.24 forming a transmembrane pore that punctures the membrane of cells causing their lysis.
00:05:41.26 So this explains why the epimastigote form is lysed in mammalian serum
00:05:47.28 and also why trypomastigotes resist, because they have developed mechanisms
00:05:53.01 to avoid activation of this pathway.
00:05:55.14 So, here in this scanning electron micrograph, taken by Edith Robbins at NYU,
00:06:02.00 we can see a closeup of the trypomastigote -- the infective stage,
00:06:05.14 attached to the surface of a host cell.
00:06:08.15 And when these parasites enter the cells, just the morphology of this process indicates that
00:06:15.04 there is something very different and unique going on because this is a very large parasite.
00:06:19.24 It is more than 10 microns long,
00:06:21.17 but we can see that this happens with no extension of pseudopods of the host cell,
00:06:26.29 which is the usual mode of ingestion of large particles which is phagocytosis.
00:06:32.18 So in the last segment of this lecture, I'm going to get into some detail of what we learned
00:06:40.15 about the mechanism by which Trypanosoma cruzi enters mammalian cells.
00:06:45.05 So in this movie, made by Mark Rioult in my lab,
00:06:47.21 the movie starts with a parasite already half inside the cell and half outside,
00:06:54.04 and we're going to be able to see accelerated (this is going to show at 10x real time),
00:06:59.13 we're going to see the complete process of the parasite entering the cell.
00:07:04.07 So we can see here that it's the extracellular part that still has the very active motility,
00:07:11.04 and the parasite gradually slides into the cell, and we're going to actually see the moment here
00:07:16.29 in which the parasite enters the host cell and gets completely released into the cytosol.
00:07:24.23 And actually the parasite appears free in the cytoplasm, but we know that at this point,
00:07:29.11 it is surrounded by a membrane of host origin.
00:07:32.01 I'll also talk about this in the third part of this lecture.
00:07:37.15 We learned that this invasion process is actually quite unique,
00:07:41.17 and it happens by recruitment of intracellular membranes, mostly from lysosomes of the host cell.
00:07:47.19 So, after acquiring these membranes, Trypanosoma cruzi resides inside this vacuole
00:07:53.24 for some time, for a few hours, and then this vacuole is disrupted and it is free in the cytoplasm.
00:08:00.10 And then the next stage of development happens and the parasites replicate.
00:08:04.21 So these scanning micrographs here show the remarkable transition in morphology
00:08:10.21 that these parasites undergo while they are escaping from that initial intracellular vacuole,
00:08:18.11 and we can see that this involves a reduction in the size of the body
00:08:23.13 and a dramatic reduction in the size of the flagellum in which this form at the end,
00:08:29.05 amastigote, which is the one responsible for replication inside the host cell
00:08:33.26 has only a very short flagellum.
00:08:36.20 What I'm going to show you in this movie here, made by Hertha Meyer in Italy in the 60s
00:08:41.05 are the last stages of this transformation
00:08:44.11 of the intracellular parasite into the replicative amastigote.
00:08:50.14 So we can see as we play the movie, that this parasite that just entered these chicken retinal cells
00:08:59.09 is going to reorganize itself into the rounded amastigote form,
00:09:03.25 and as this parasite enters this replicative stage it starts undergoing binary fissions,
00:09:14.28 which we can see here in this cell,
00:09:17.10 that there are already several parasites replicating in the cytosol,
00:09:21.26 and we can see clearly also that the whole cell
00:09:24.23 remains quite viable throughout this process
00:09:27.14 and this is going to become obvious just by the observation of the fact that
00:09:31.20 these cells are capable of going through mitosis normally.
00:09:35.19 We can see here the condensed chromosomes aligning themselves
00:09:40.16 at the center of the mitotic spindle
00:09:42.26 and we're going to be able to see when
00:09:45.08 these chromosomes are actually pulled apart by the spindle
00:09:49.25 and then the cell rapidly enters cytokinesis.
00:09:54.06 And if we focus on these cytoplasmic parasites, it's possible to see that
00:09:58.14 one of them was actually delivered to one of the daughter cells
00:10:01.26 while the majority remained in the other cell.
00:10:04.06 So, this cycle continues, and this was Jim Dvorak at the NIH in the 70s
00:10:11.09 who really worked out clearly the details of this intracellular cycle
00:10:16.23 and what he learned is that they go through nine successive divisions,
00:10:21.00 so each parasite that enters the cell actually originates around 500 parasites
00:10:26.17 and then at the end of the cycle which is around four to five days after the original infection
00:10:32.17 they change back again into this highly motile trypomastigote form
00:10:37.05 that we can see here completely filling the cell at the end of the cycle
00:10:42.08 and at this stage, the cell degenerates rapidly, and we're going to be able to see actually
00:10:47.20 in this cell here that the nucleus is already quite degenerated,
00:10:52.11 and we're going to be able to see the moment in which the plasma membrane breaks down
00:10:57.16 and these parasites are released into the medium.
00:11:01.02 So this is how they reach circulation, and they can then be taken up by the insect during a blood meal.
00:11:07.21 So, this is exactly what happens during the acute phase of this infection.
00:11:12.23 This is a picture of another child with the classical swelling around the region of the eye,
00:11:19.00 which is a very common site of entry for these parasites in humans,
00:11:23.27 and this is the classical diagnostic picture of finding these highly motile trypomastigotes
00:11:31.05 in the blood of these patients.
00:11:34.05 So, the acute phase of the disease is characterized by this localized swelling at the site
00:11:40.03 of entry of the parasite, also very intense episodes of fever and enlargement of the spleen,
00:11:47.25 and this is actually very possible that death is an outcome of these acute infections
00:11:56.03 not only in children, but we have learned recently that even adults that had never been exposed
00:12:01.19 to the parasite in childhood can also die from the acute phase of Chagas' disease.
00:12:07.17 So, the largest number of people infected with Trypanosoma cruzi are actually in the chronic stage
00:12:16.24 of the disease because we know clearly now that immunity does develop against these parasites
00:12:23.07 and the immune system is capable of clearing the large majority of these parasites,
00:12:29.04 but they're never completely eliminated.
00:12:32.10 So these patients that carry the parasite...
00:12:35.02 a large fraction (around 40%) are actually asymptomatic throughout their lives,
00:12:40.05 but they still have the parasite and then there's a large fraction of around 45%
00:12:48.24 that have the more serious form of the disease which is the cardiomyopathy
00:12:52.26 which involves enlargement of the heart
00:12:57.03 and then a smaller fraction of these patients develop megaesophagus or megacolon
00:13:03.02 which is this dramatic enlargement of internal organs
00:13:06.13 that requires correction by surgery.
00:13:09.27 So, the serious form of the disease which is the cardiomyopathy is actually
00:13:15.18 responsible for sudden death in around 58% of the patients that have this form of the disease.
00:13:22.08 So it's the most common form of sudden death in these endemic areas for Chagas' disease,
00:13:28.13 and we can see here that... here is a picture of cardiomyocytes infected by the parasite,
00:13:36.03 and this movie that I'm going to play now down here just shows isolated cardiomyocytes
00:13:41.18 that contain a large number of parasites close to the end of the cycle,
00:13:45.27 and you can see this cell beating, showing that these were heart cells which are the cells
00:13:52.01 that are preferred by these parasites for infection in vivo
00:13:56.10 So another very important point with Chagas' disease is that even this large number of
00:14:02.05 asymptomatic patients, they carry the parasites
00:14:05.29 and they can transmit the infection through blood transfusions,
00:14:10.07 so it's something that is very important in the endemic area.
00:14:13.18 Usually the blood banks screen the blood for the presence of Trypanosoma cruzi
00:14:18.01 but this is something that in many developed countries, actually,
00:14:20.28 there's not enough awareness for the possibility of blood infection with Trypanosoma cruzi
00:14:26.09 and this is increasingly more important with the high mobility of the human population.
00:14:33.13 So what is important and what is very good news with Chagas' disease is that it has been clear
00:14:40.06 almost since the beginning of the 19th century, Carlos Chagas had already pointed out
00:14:45.04 that transmission of the Trypanosoma cruzi to man can be interrupted
00:14:52.02 and this can be done by very simple measures
00:14:56.08 which involve just the control of the insect vectors.
00:15:00.01 So just simple spraying of the houses with insecticide
00:15:03.13 which is what is shown here in these images
00:15:05.24 can have a profound effect on the incidence of the disease
00:15:10.11 and also another very important factor is adequate finishing of the walls,
00:15:15.27 so not providing these cracks where these insects like to hide.
00:15:21.26 What is at the same time very disturbing is that, although this was known to be effective
00:15:29.27 since the 40s, it was only decades later that these programs
00:15:34.07 of controlling this vector have been implemented
00:15:37.06 throughout this region, and this slide here actually illustrates very well the problem
00:15:43.00 which what is shown here is the distribution of the various species of the insect vector
00:15:49.21 that are capable of transmitting Chagas' disease, and we can see, for example,
00:15:54.14 that here in the south of the US, there is the wild cycles, so wild animals are found easily
00:16:02.26 carrying Trypanosoma cruzi and there are insects
00:16:06.28 which are responsible for maintaining this cycle
00:16:09.25 but this does not cause human infections, and the sole reason is because the living conditions
00:16:15.22 are much superior in the US than they are in these poor areas of Central and South America.
00:16:24.10 So Chagas' disease is clearly a disease of poverty.
00:16:27.15 And it's a disease that has already been demonstrated that it could potentially be eliminated
00:16:33.09 just by a consistent program of surveillance and elimination of the domestic vector.
00:16:42.18 So a very important initiative in this sense is called the Southern Cone Initiative
00:16:48.10 that was created by the Pan American Health Organization (PAHO)
00:16:52.11 and by the World Health Organization (WHO) around 1991.
00:16:57.02 And you can see here the numbers here really look great in a short period
00:17:02.14 Chile, Uruguay, and large regions of Brazil and Argentina
00:17:05.24 have practically eliminated transmission to humans
00:17:09.10 and many other countries in this area are also reaching excellent results
00:17:15.29 and they are now in what is called the surveillance phase
00:17:18.21 which is just maintenance of this measure and to prevent reinfection of the homes.
00:17:26.25 So, this initiative has been cited as one of the 17
00:17:30.14 most cost effective international public health interventions
00:17:34.12 that have been done, and it's actually more than proven to be highly effective.
00:17:40.29 So it is a matter of political will, so it is pretty disturbing that in large fractions
00:17:48.02 of Central and South America, this has not been achieved yet.
00:17:54.04 We can see that these initiatives here in the Andean countries and in Central American countries
00:18:00.00 have only been initiated much more recently and it was only in 2001
00:18:04.11 that a Mexican initiative for the control of the domestic vector was put into place.
00:18:09.10 But this should rapidly progress if these initiatives are maintained
00:18:16.10 so the important point here is that Trypanosoma cruzi will never be eliminated from nature.
00:18:20.20 It is known that more than 100 vertebrate species can serve as hosts for this parasite in nature.
00:18:27.22 However, Chagas' disease can be prevented and this can be done very effectively
00:18:34.02 by an improvement in the social and economic conditions of the population,
00:18:38.18 so it is a disease of poverty and it is expected to improve dramatically with development.
00:18:44.19 The critical issues are, of course, the effective and sustained surveillance of these regions
00:18:51.13 and to prevent re-infestation of the homes and also treatment -- better drugs and less toxic drugs
00:18:58.15 for treating the very large chronically infected population that exists presently.
00:19:04.12 So, thank you for your attention and in the next segment
00:19:08.04 I'm going to also introduce a related parasite which is also very important medically
00:19:14.17 and causes serious infections in poor areas of the world which is Leishmaniasis.

Part 2: Leishmania spp and Leishmaniasis

00:00:02.04 My name is Norma Andrews, I'm a professor at Yale University,
00:00:05.18 and what I'm going to be talking to you about in this second part of my lecture
00:00:09.12 is about Leishmania, this parasite, and the disease that it causes in humans
00:00:15.00 which is called Leishmaniasis.
00:00:17.06 This is also a protozoan parasite from the order Kinetoplastida,
00:00:23.04 family Trypanosomatidae, and inside this family, we find several protozoan organisms,
00:00:30.09 and the two groups that are medically important because they cause serious diseases in man,
00:00:35.26 Trypanosoma which includes Trypanosoma cruzi that I already talked about
00:00:39.23 in the first part of this lecture.
00:00:42.03 And also the African trypanosome that causes sleeping sickness in Africa, Leishmania,
00:00:47.08 which I'm going to introduce to you now is closely related to trypanosomes,
00:00:53.09 and it's also an intracellular parasite.
00:00:58.12 So, here in the distribution of Leishmania throughout the world,
00:01:02.25 we can see that it's not only restricted to South America like Chagas' disease,
00:01:07.21 but is also found in many regions of Africa, the Middle East, and also around the Mediterranean.
00:01:15.09 So, similar to Chagas' disease, Leishmaniasis is also largely a disease of poverty.
00:01:21.07 It is mostly found in underdeveloped regions of the world,
00:01:27.22 but it has some characteristics that actually make it very serious in the sense that
00:01:34.17 development is proving not to be sufficient to stop transmission,
00:01:39.01 and actually there's several signs that the disease is recently moving
00:01:44.23 from rural areas to urban areas of the world.
00:01:48.18 So, this is a very serious development that indicates that this number of people
00:01:54.19 which are currently around 12 million that are already infected by this parasite...
00:01:58.15 There are 350 million people at risk worldwide.
00:02:02.17 So, the instance of Leishmaniasis may actually increase over time.
00:02:07.28 Here we see an example of one of the clinical manifestations of Leishmaniasis.
00:02:14.27 This is a child with a cutaneous lesion, which... The form of the disease in Leishmaniasis
00:02:22.10 is directly associated to the species of Leishmania that causes the infection.
00:02:27.07 So, these three species here, L. major, L. tropica, and L. mexicana
00:02:31.18 are frequently associated with the cutaneous form of the disease
00:02:35.07 which can be very disfiguring, although these lesions most of the time, they are self-healing.
00:02:41.10 There's a more serious form of this cutaneous disease
00:02:45.21 which is the form that attacks the mucosal membranes
00:02:49.06 in which this can really be seriously disfiguring and this is usually
00:02:53.09 associated with some of the species, for example Leishmania braziliensis.
00:02:58.06 The most severe form of Leishmaniasis is by far the visceral form
00:03:03.24 which is caused by these three species, mostly,
00:03:08.28 Leishmania donovani, Leishmania infantum, and Leishmania chagasi,
00:03:12.21 and this disease is characterized by periodic fever,
00:03:18.13 severe enlargement of the liver and the spleen, and also weight loss and anemia.
00:03:25.19 This is significant cause of morbidity and mortality
00:03:30.02 in the endemic areas for visceral Leishmaniasis.
00:03:33.27 This disease was already known to be present.
00:03:38.02 It was called early on Calazar, but what was not known is what was the organism causing it.
00:03:46.27 It was not even clear that it was an infectious disease.
00:03:49.10 So, it was this British physician, William Leishman
00:03:54.03 which analyzed the material from patients in India and also simultaneously,
00:04:01.26 by the Irish investigator Charles Donovan,
00:04:05.26 they realized that macrophages present in the spleen
00:04:10.11 of these patients were heavily loaded with these organisms
00:04:15.26 which they recognized as similar to trypanosomes.
00:04:19.13 So this was the first indication that this was a parasitic disease,
00:04:24.03 and another very important finding in the history of Leishmaniasis
00:04:29.00 was made by Rogers and Nicolle a few years later,
00:04:32.28 in which they took this material from the spleen of the patients and put it into culture
00:04:37.26 and they were able to show that a second form of the parasite appeared in these cultures
00:04:43.19 indicating directly the existence of a life cycle and most likely an insect vector.
00:04:49.17 So this actually was only decades later that the first hint for which could be
00:04:56.09 the insect involved in this transmission was made by John Sinton when he realized that
00:05:02.05 the incidence of visceral Leishmaniasis in Eastern India
00:05:05.01 actually coincided with the distribution
00:05:07.26 of a specific type of sandfly, the silvery sandfly.
00:05:12.05 This rapidly led to a lot of work in which this picture is what emerged
00:05:19.14 in which these regions that I already showed
00:05:22.08 that we can see Leishmaniasis transmitted to humans.
00:05:27.22 In each case, a specific species of Leishmania
00:05:30.05 is associated with a specific species of the sandfly
00:05:33.06 which is responsible for the transmission.
00:05:35.15 And it was only more recently, decades later after these original observations,
00:05:42.09 that there was a formal demonstration that sandflies
00:05:46.25 could transmit Leishmania to humans.
00:05:49.04 Although earlier, it had already been detected in sandflies
00:05:53.13 that had been feeding on an infected animal, but also in humans.
00:06:02.07 It was then only this time around the 40s that
00:06:08.01 the life cycle of Leishmania was really understood,
00:06:12.21 with this replication of the parasites happening inside the sandfly
00:06:17.14 and this is similar to Trypanosoma cruzi in the sense
00:06:23.03 that these parasites also don't gain access to the salivary gland.
00:06:25.23 They are transmitted in this case by regurgitation.
00:06:30.06 So, after a blood meal, this fly delivers these parasites
00:06:36.06 which replicate abundantly in its digestive tract,
00:06:40.06 and this is how they penetrate the mammalian host,
00:06:43.26 which in addition to humans, dogs, and rodents play a major role in this cycle,
00:06:49.17 and it is inside macrophages of the mammalian host that the intracellular replication occurs.
00:06:56.03 More recently, David Sacks at the NIH made another very important observation
00:07:02.23 and contribution to the understanding of the biology of Leishmania parasite
00:07:08.00 which was by identifying an infective stage.
00:07:10.26 So, similar to what I discussed in the first part of this lecture, for Trypanosoma cruzi,
00:07:16.00 Leishmania also has an infective stage, so the parasites that replicate inside the insect vector,
00:07:22.22 they replicate into these infective stages which David Sacks called metacyclic promastigotes.
00:07:29.07 During this differentiation, they also acquire that resistance to lysis by complement,
00:07:34.26 which allows them to survive inside the mammalian host.
00:07:38.01 David Sacks and other groups have made
00:07:42.13 very important contributions in linking this differentiation
00:07:46.13 into the metacyclic form to this surface molecule, LPG,
00:07:50.10 which is a major component of the surface coat of these parasites.
00:07:54.09 What is shown here in this immunogold electron microscopy,
00:07:59.23 these black dots that we can see here in this enlarged segment
00:08:03.20 represent antibodies reacting with this surface molecule
00:08:08.07 and what David Sacks and others showed
00:08:12.05 is that these molecules are lipid-anchored,
00:08:16.05 and they have these repeated disaccharide-phosphate units...
00:08:20.03 This molecule undergoes significant elongation during differentiation
00:08:25.11 and these side groups which are usually terminated in galactose
00:08:29.00 in the epimastigote insect form
00:08:31.13 acquire these side chains terminated by arabinose.
00:08:36.06 This is a key change in one of the specific species, Leishmania major
00:08:41.28 that David Sacks showed that is responsible
00:08:46.01 for a very important part of this transmission cycle,
00:08:48.24 which is the detachment of the parasites from the midgut of the insect,
00:08:54.04 allowing them to be delivered to the infected host.
00:08:57.12 So this change in the structure of this LPG molecule
00:09:01.22 is what mediates detachment and then facilitates transmission
00:09:07.10 during or after the blood meal by the sandfly.
00:09:11.10 Another very important point that emerged recently
00:09:16.13 also from the work of David Sacks at the NIH
00:09:18.28 is that the vectorial competence
00:09:21.06 --the ability of these sandflies to transmit specific species of Leishmania--
00:09:26.18 is related to polymorphisms on this LPG molecule, so only now, very recently,
00:09:33.25 a much more complete picture has emerged of what are the molecular properties of this parasite
00:09:40.16 that really create these very specific associations between vector
00:09:46.19 and cycles of transmission throughout the world.
00:09:50.24 What is the next frontier now, now that we really understand
00:09:55.20 quite well what is happening inside the sandfly
00:09:58.03 with the delivery of these infective stages,
00:10:00.17 the next frontier is understanding what's happening in the mammalian host.
00:10:04.15 We know that Leishmania mostly replicates inside macrophages,
00:10:08.14 since the original observations of Leishman and Donovan,
00:10:12.12 but we don't understand completely how the parasites actually survive in these cells.
00:10:18.10 Macrophages are cells that are actually designed to destroy intracellular pathogens,
00:10:23.01 and the entry of Leishmania into these macrophages
00:10:26.09 is known to be mediated by phagocytic receptors
00:10:29.01 so in this case, different from Trypanosoma cruzi.
00:10:32.11 There's no indication of an active mechanism of invasion,
00:10:35.26 but it is pretty clear that at least the complement receptors,
00:10:40.14 CR1 and CR3 play an important role
00:10:42.26 because these parasites are not lysed by complement,
00:10:46.06 but they are opsinized by complement,
00:10:48.05 which allows them to be efficiently internalized by macrophages.
00:10:52.03 Inside the macrophages, the really remarkable aspect of the life cycle of Leishmania
00:10:58.04 is that these parasites can survive in compartments
00:11:01.12 that have many properties of lysosomes.
00:11:03.26 They are acidic, they contain a full set of hydrolases
00:11:08.23 and also of lysosomal membrane proteins,
00:11:11.16 and we don't really understand how the parasites are able to spend their whole cycle
00:11:16.28 which also lasts several days--they have a doubling time of around 12 hours--
00:11:22.01 and what is indicated in this slide here is that even MHC class II,
00:11:27.02 which is a protein expressed by activated macrophages
00:11:31.01 is present on the membrane of these vacuoles containing the parasites
00:11:35.14 that replicate in spite of these harsh conditions.
00:11:40.09 In addition to this remarkable adaptation to survive inside cells of the immune system,
00:11:47.12 which in normal circumstances actually play a role in the elimination of pathogens,
00:11:51.19 another very challenging aspect of Leishmaniasis
00:11:56.22 is the difficulty in limiting exposure of humans to these insect vectors.
00:12:02.11 In this case, this is a very small insect.
00:12:06.16 What this picture here shows is one sandfly sitting on the knuckle of a finger,
00:12:11.24 just to give an idea of how small they are.
00:12:14.24 The simple measures that have been shown to be so effective
00:12:17.25 in containing malaria transmission, for example,
00:12:22.00 just bed nets, really are not effective
00:12:25.11 because these are very small flies that can go through these regular bed nets
00:12:30.15 that are normally used in these endemic areas.
00:12:33.23 What is very clear for Leishmaniasis is that there is a strong need
00:12:37.17 for development of new drugs
00:12:40.01 and new drugs with less toxicity, because again here the drugs currently in use
00:12:44.25 in large parts of the world are still very toxic.
00:12:49.19 The good news is that the recent developments are leading rapidly
00:12:54.19 to the identification, implementation,
00:12:57.10 and clinical trials of new and better drugs
00:13:00.25 with several philanthropic organizations and also non-profit institutions participating actively
00:13:08.00 in this concerted effort to make this very important need in public health met
00:13:17.21 by taking advantage of current state-of-the-art technology.
00:13:24.03 Thank you. In the next segment, I'm going to discuss more the cell biology of infection
00:13:30.07 of both Trypanosoma cruzi and Leishmania.

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.