Intracellular Protozoan Parasites: Trypanosoma cruzi and Leishmania
Transcript of 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.