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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.

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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