Understanding the Circadian Rhythms of the African Sleeping Sickness Parasite

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00:00:13.01 We all know how tired we get
00:00:15.07 if we have a bad night of sleep,
00:00:16.24 and if we have a couple of those in a row
00:00:19.12 it really gets exhausting.
00:00:21.17 So, can you imagine if you had
00:00:23.27 to experience daytime sleepiness
00:00:26.17 together with nighttime insomnia
00:00:28.14 for months and years?
00:00:30.15 That's what people with sleeping sickness experience.
00:00:34.05 Sleeping sickness is actually an infectious disease
00:00:36.24 caused by a parasite called Trypanosoma brucei.
00:00:40.23 This parasite is transmitted
00:00:43.21 by the bite of a fly called the tsetse fly
00:00:45.26 that is endemic in 36 African countries.
00:00:49.16 But this parasite is not picky.
00:00:51.15 It not only is able to infect humans,
00:00:53.25 but it's also able to infect wild and domestic animals.
00:00:57.28 So, it makes it not only a health problem in these countries,
00:01:01.09 but because it affects their cattle,
00:01:03.18 it also becomes an economic burden.
00:01:05.25 But the World Health Organization
00:01:08.17 has done big efforts towards the elimination
00:01:10.27 of this infection,
00:01:12.23 and the number of cases has been dramatically reduced.
00:01:16.15 But there is still no vaccine
00:01:19.01 against this parasitic infection.
00:01:20.23 It's almost always lethal if left untreated.
00:01:23.27 And in fact, the treatment itself is quite complicated.
00:01:27.27 It requires intravenous treatment,
00:01:30.02 and it is extremely toxic.
00:01:32.14 It's believed that about 5% of the people
00:01:35.00 that have this treatment
00:01:36.20 die of complications.
00:01:38.25 And this is all caused by a tiny parasite
00:01:40.23 that is changing the sleep-wake cycles of its host.
00:01:44.14 This parasite is not making these people sleep more,
00:01:46.27 but instead is shifting the timing
00:01:49.14 at which their sleep occurs.
00:01:52.16 And the parasite does that by affecting
00:01:55.23 the circadian clock of its host.
00:01:57.20 But what do I mean by the circadian clock?
00:01:59.20 Well, by living in our very rhythmic environment,
00:02:04.15 with day and night cycles
00:02:07.03 due to the Earth's rotation,
00:02:09.04 organisms like us have evolved ways
00:02:11.17 to anticipate these rhythmic changes.
00:02:13.11 These are called our circadian rhythms.
00:02:15.16 Our sleep-wake cycles
00:02:17.18 are an example of those.
00:02:19.04 We have hormones and some signals
00:02:21.16 that make us feel sleepy
00:02:23.13 during the night time,
00:02:25.02 and the next morning a different set of signals
00:02:27.00 makes us feel active and hungry
00:02:29.04 and ready to enjoy our day.
00:02:31.01 These rhythms are repeated every day
00:02:33.08 and they last about 24 hours.
00:02:35.23 That's exactly where the word comes from,
00:02:38.03 from the Latin circa diem,
00:02:40.00 which literally means "around a day".
00:02:43.04 So, these rhythms are also very interesting
00:02:46.11 because they are endogenous.
00:02:48.00 What this actually means
00:02:50.10 is that they are not really driven
00:02:53.17 by the presence of these light and dark cycles,
00:02:55.19 but they are instead really coming from within.
00:02:59.02 And this was shown by an experiment with plants.
00:03:03.04 So, plants have circadian rhythms
00:03:05.20 on their leaf movement:
00:03:07.12 the leaves go up during the daytime
00:03:09.11 and they go down at night.
00:03:11.12 And a French scientist thought that
00:03:14.02 the plants were not sensing light and they're...
00:03:16.25 and thus putting their leaves up,
00:03:18.19 but instead, that the plants were anticipating
00:03:20.29 that the light was coming
00:03:23.02 and placing their leaves.
00:03:25.01 So, he did a beautiful experiment,
00:03:26.20 which consisted in putting one plant by the window
00:03:29.23 that would be exposed to both day and night
00:03:33.21 environmental changes,
00:03:35.19 and a second plant was placed in a cabinet underneath it,
00:03:38.08 so this plant was in complete darkness,
00:03:40.26 isolated from any environmental cue.
00:03:43.19 What he saw was that both plants
00:03:46.02 -- the one by the window
00:03:48.19 and the one inside the cabinet --
00:03:50.12 still maintained these leaf movements, up and down.
00:03:55.02 So, this doesn't mean that light is not important;
00:03:57.10 it's just not... it's not driving these rhythms.
00:03:59.10 But it's very important to synchronize them.
00:04:02.24 A simple way to imagine that
00:04:05.05 is through jetlag.
00:04:07.00 So, I am originally from Portugal,
00:04:08.21 but I live and work here in the US.
00:04:10.21 But every time I travel to Europe to see my family,
00:04:14.02 I have to travel about 6 hours difference
00:04:17.20 in time zone.
00:04:19.19 So, my sleep-wake cycles
00:04:21.15 take a couple of days to get synchronized
00:04:23.13 to these new light and dark cycles.
00:04:25.01 But light is important,
00:04:27.19 and eventually I become synchronized with the environment
00:04:30.02 in Portugal.
00:04:32.01 So, this means that we have circadian rhythms.
00:04:34.15 Plants have circadian rhythms.
00:04:36.16 But actually, circadian rhythms go all the way
00:04:39.00 through the Tree of Life,
00:04:40.29 to the simpler organisms, like bacteria.
00:04:44.07 And since I'm so interested in parasitic
00:04:47.00 and infectious disease,
00:04:49.03 I wonder if this was just something
00:04:51.22 that was common in organisms that are free-living
00:04:53.24 and thus exposed to light and dark cycles.
00:04:56.17 So, my question is,
00:04:58.22 do parasites also have circadian clocks?
00:05:01.18 If organisms like parasites
00:05:04.02 that are never really exposed to light and dark cycles...
00:05:06.24 if they also have developed -- evolved --
00:05:11.09 these circadian rhythms mechanisms.
00:05:14.06 It turned out that the parasite I was studying,
00:05:16.09 the sleeping sickness parasite,
00:05:18.07 was a really good model to answer this question.
00:05:20.04 And that's because this parasite
00:05:22.09 is a single-celled parasite,
00:05:23.26 but it's also extracellular.
00:05:25.18 So, as you can see, we can culture it in the lab,
00:05:28.04 and it's by itself.
00:05:29.26 So, no host cells involved.
00:05:33.02 So, whatever rhythms I can identify,
00:05:34.29 they're really coming from the parasite
00:05:37.06 and not from the host.
00:05:39.11 But where to start?
00:05:41.01 Well, I cannot just go and look at the leaf movement
00:05:43.19 in this parasite, right?
00:05:45.07 So, instead, I had to take an unbiased approach
00:05:47.04 and look at gene expression in these parasites,
00:05:49.27 this gene expression that can potentially
00:05:53.19 regulate all the downstream rhythmic behaviors.
00:05:57.09 So, in order to do that,
00:05:59.07 I would have to first synchronize the parasite population
00:06:02.22 to day and night cycles,
00:06:04.19 just like the light and day for us.
00:06:07.06 But for parasites,
00:06:09.01 I thought that they might not be really responsive to light.
00:06:12.06 They are actually always inside our bodies,
00:06:14.20 so they are not really exposed to it.
00:06:16.29 But instead, they may be exposed to temperature fluctuations.
00:06:20.10 Our temperature drops at night
00:06:22.29 when we are sleeping,
00:06:24.21 and it increases during the day when we are awake and active,
00:06:27.01 so parasites that are swimming in our blood
00:06:29.26 could potentially sense these temperature cycles.
00:06:33.04 So, to mimic those,
00:06:34.28 I actually cultured parasites
00:06:37.15 and shifted every 12 hours
00:06:40.12 in between incubators
00:06:42.08 so they would be exposed to day and night temperature rhythms.
00:06:46.00 Afterwards, I either placed them in the...
00:06:48.22 kept them in these alternating conditions
00:06:51.10 or placed them always at 37.
00:06:53.20 So, in the end, I was trying to
00:06:56.09 replicate the experiments that the French scientist did
00:06:59.09 with the plants, either by the window,
00:07:01.17 exposed to day and night cycles,
00:07:03.23 or at always 37,
00:07:06.21 which will be the plant inside the cabinets.
00:07:09.08 Okay. I tried to synchronize my parasite population,
00:07:11.25 so now I'm gonna go and check,
00:07:14.09 what is their gene expression?
00:07:16.23 But I don't know what genes to look for.
00:07:18.25 So, instead, taking advantage
00:07:22.07 of a technique used in biology called RNA sequencing,
00:07:25.29 I'll be able to not only count the number of molecules of RNA
00:07:30.09 being expressed through time
00:07:32.23 but also to which proteins they can potentially encode.
00:07:36.04 So, this is... can be simply explained here,
00:07:39.12 by this diagram.
00:07:41.00 As you can see, at zero hours,
00:07:43.08 this plant has only two molecules of RNA:
00:07:46.09 one that is red, one that is blue.
00:07:48.18 But at 12 hours, the blue molecules are now four.
00:07:51.29 So, if I plot this data,
00:07:54.04 I can actually identify that
00:07:58.29 the red RNA encodes for a protein called protein A,
00:08:00.29 and there... there's the RNA.
00:08:03.14 It's called RNA A.
00:08:05.08 And it's constant through time.
00:08:07.05 Whereas the RNA blue,
00:08:09.02 it actually has a higher expression at 12 hours,
00:08:11.26 and then it drops again.
00:08:13.28 So, these actually have these rhythms
00:08:16.06 in gene expression.
00:08:18.00 So, when I take a look at,
00:08:20.07 what is the gene expression in my parasites?,
00:08:22.24 I'll be specifically searching for genes
00:08:25.19 that have the gene expression pattern
00:08:28.05 like this RNA blue.
00:08:30.02 So, when I first looked at what happened in the parasite population
00:08:33.23 that was alternated between these incubators,
00:08:36.29 I was very happy to see
00:08:39.07 that we were able to synchronize the parasite population,
00:08:41.15 and that's because we indeed found genes that were cycling,
00:08:44.27 like here, this gene A.
00:08:46.22 So, you can see that the gene was highly expressed first,
00:08:49.15 and then it got dropped,
00:08:51.09 and then it went up again.
00:08:53.08 And this was repeated on these two days that I measured.
00:08:57.05 And this was not the case of only this gene A,
00:08:59.13 but also for these 1,500 genes.
00:09:03.29 And actually, here, in these plots,
00:09:06.17 the genes are representing one row for each,
00:09:09.16 and the color code represents their gene expression level.
00:09:13.16 So, you can see that, in the beginning,
00:09:16.11 for example, gene A is marked as red,
00:09:19.12 and then it becomes blue, and then it gets red again,
00:09:23.00 and blue, and so on,
00:09:24.23 which will be an example of, here,
00:09:26.25 one of the few first rows in these heat map plots.
00:09:29.17 So, this was what happened when the temperature cycles were present,
00:09:33.12 so I cannot really be sure
00:09:35.23 if these are circadian rhythms of the parasite
00:09:38.11 or just genes that are cycling
00:09:41.20 because they are responding to the temperature fluctuations.
00:09:44.03 So, instead, to really be sure
00:09:46.19 that parasites have circadian rhythms,
00:09:48.29 we have to look at what the gene expression
00:09:51.02 of the parasites inside our 37 incubator looks like.
00:09:55.22 So, excitingly enough,
00:09:58.18 parasites had gene expression cycling
00:10:01.19 even in the constant conditions --
00:10:03.25 even in the absence of any environmental signals.
00:10:07.28 And this was not only for gene X,
00:10:10.29 but actually for 1,000 genes
00:10:13.09 that were cycling in these constant conditions.
00:10:15.23 So, these are indeed circadian rhythms,
00:10:17.28 the really endogenous rhythms
00:10:20.15 coming from this Trypanosoma brucei parasite.
00:10:23.11 So, this was very exciting.
00:10:25.12 This really proved that parasites
00:10:27.18 can indeed have circadian clocks.
00:10:29.24 But the question now is,
00:10:31.19 what are these clocks for?
00:10:33.24 Why are the parasites putting so much effort
00:10:36.03 in making 1,000 genes cycling throughout the day?
00:10:41.03 What are these rhythms actually encoding for?
00:10:44.22 So, to answer this question,
00:10:46.28 we went and specifically looked at,
00:10:48.26 what are the genes that are cycling throughout the day?
00:10:52.10 And what we found was that
00:10:54.20 most of these genes are encoding for metabolic pathways.
00:10:57.16 They are either involved in the metabolism
00:11:01.08 of sugars or carbohydrates.
00:11:03.21 They're involved in the metabolism
00:11:06.02 of lipids or fats,
00:11:08.07 or in the amino acids of proteins.
00:11:10.10 But this was all at the gene expression level,
00:11:13.05 so I needed to validate if this was really true
00:11:16.16 for the parasite population --
00:11:18.18 if the parasites were somehow in a different metabolic status
00:11:21.06 throughout the day.
00:11:22.19 So, I could potentially go and just
00:11:25.26 look for the expression of these proteins,
00:11:28.00 but instead I looked at ATP levels.
00:11:30.06 So, ATP can be seen as
00:11:32.18 the ultimate energy molecule
00:11:35.04 that will be the result of all these metabolisms.
00:11:38.14 So, when I measured the ATP's in this parasite,
00:11:42.20 I found that the ATP levels
00:11:45.22 are going up and down throughout the day.
00:11:47.23 And in fact, this happened in the conditions
00:11:50.27 that were in alternating incubators,
00:11:53.07 but also in the condition that's always at 37.
00:11:57.03 So, you can also see that
00:11:59.25 the timing of these higher ATP concentrations
00:12:02.12 also matches the higher...
00:12:05.06 that you have more genes being expressed
00:12:07.20 of these metabolic pathways.
00:12:09.12 So, there seems to be a big importance
00:12:11.17 of circadian rhythms in regulating
00:12:15.03 the metabolism of these parasites.
00:12:17.21 This was very interesting
00:12:19.22 because of course I'm very passionate
00:12:22.06 in understanding how the biology of these parasites work,
00:12:25.02 but also, in the end,
00:12:27.10 I'm interested in trying to find ways to fight them.
00:12:29.14 And because we know that most of the antiparasite drugs,
00:12:34.11 they actually act on these metabolic pathways,
00:12:37.07 it could be that just by
00:12:40.23 simply administering the drug at specific times of the day,
00:12:43.13 we might find the parasites more susceptible
00:12:45.25 to these treatments.
00:12:47.19 So, I set out exactly to test this question,
00:12:49.21 to try to answer it.
00:12:51.20 And I first started by... go and...
00:12:54.07 synchronizing this parasite population,
00:12:56.22 again, with our temperature cycles.
00:12:58.19 Then, to each of these cultures,
00:13:01.25 I gave an increasingly higher drug concentration
00:13:05.24 and hoped for the increasing higher drug
00:13:09.01 to kill more parasites,
00:13:10.21 and that's exactly what I... what I saw.
00:13:13.00 So, if I give very little or no drug,
00:13:15.11 100% of the parasites remain alive,
00:13:18.27 but as I increase the drug concentration,
00:13:22.05 these parasites start dying.
00:13:23.27 And this was a drug called suramin,
00:13:25.23 which in fact is known to act upon the glycolytic pathway,
00:13:30.03 and it's used to treat sleeping sickness parasites in the field.
00:13:34.18 So, this was very exciting -- the assay was working.
00:13:37.24 So, I could then ask whether these...
00:13:40.19 exact death of parasites through this suramin drug
00:13:44.00 was similar throughout the day.
00:13:46.28 So, when I looked at what happened at 4 hours,
00:13:50.15 I was very disappointed,
00:13:53.07 because it was exactly the same.
00:13:55.01 You can see that these curves look the same
00:13:56.29 whether I treat parasites at 0 hours
00:13:58.26 or whether I treat them at 4 hours.
00:14:01.13 So, this could have just meant
00:14:04.07 that my hypothesis was wrong altogether,
00:14:06.22 that there are no benefits in treating parasites
00:14:09.28 at different times of the day,
00:14:11.26 and that their metabolic pathways changing
00:14:14.27 is not really correlated with the treatment.
00:14:17.27 But instead, I didn't take that as a no,
00:14:20.20 because these are just two time points
00:14:23.02 in the 24-hour day.
00:14:25.08 So, instead, I went and look at what happened at 8 hours.
00:14:28.20 And as you can see,
00:14:31.15 I was extremely excited,
00:14:34.09 because there is now a shift.
00:14:36.22 So, parasites are requiring
00:14:39.20 a higher drug concentration
00:14:42.07 in order to be killed.
00:14:43.28 So, for killing the same amount of parasites,
00:14:46.01 now the drug needs to be at a higher dose.
00:14:48.04 This was very exciting.
00:14:50.12 And so, when I finished...
00:14:52.23 for all the time points throughout the day,
00:14:55.01 I can then transform this plot
00:14:57.20 into the dose that I require to treat half of the parasite population.
00:15:02.07 So, you can see here that if I treat at 0 or 4 hours,
00:15:05.22 I need half of the amounts of drug
00:15:10.28 to kill the same number of parasites.
00:15:13.04 So, if I treat them at 8 hours instead,
00:15:15.11 I would need 2.5 times more drug.
00:15:18.00 And this really means that there is
00:15:21.20 a potential for improving the treatment
00:15:24.02 by simply administering at different times of the day.
00:15:27.01 So, altogether,
00:15:29.13 I was able to show that Trypanosoma brucei parasites
00:15:31.17 have circadian rhythms
00:15:34.20 and that these rhythms regulate the parasite metabolism,
00:15:36.22 making it more susceptible to drug treatments
00:15:39.11 at different times of the day.
00:15:41.18 But as you can imagine,
00:15:43.17 parasites probably didn't evolve these circadian rhythms
00:15:46.22 in order for us to use it against them.
00:15:49.05 I can think of that parasites
00:15:52.00 have evolved these rhythms
00:15:54.19 to anticipate when the vector is coming to bite at the end of the day,
00:15:57.28 to be prepared and thus enhanced transmission.
00:16:01.08 But I can also think that maybe parasites
00:16:03.20 have evolved circadian rhythms
00:16:06.13 in order to predict when the strong immune response
00:16:09.10 is gonna happen,
00:16:11.04 because our immune system
00:16:13.07 is also cycling throughout the day.
00:16:15.08 I can think that parasites may have evolved circadian rhythms
00:16:17.26 in order to anticipate when the nutrient influx
00:16:20.20 is gonna happen --
00:16:22.14 we only eat during the day, right?
00:16:24.29 So, there are many different reasons
00:16:27.15 for why circadian rhythms may be so important
00:16:30.12 for parasites.
00:16:32.07 But what we know now is that they have them --
00:16:34.26 Trypanosoma brucei has circadian rhythms --
00:16:37.25 and there... and there is a potential to use the metabolic rhythms
00:16:41.25 that it has to treat them
00:16:44.29 at specific times of the day.
00:16:46.26 So, I think it's worth it to confirm
00:16:48.28 that our results from the cultures
00:16:51.15 really translate into differences in mouse models.
00:16:56.00 And what we found for suramin
00:16:58.19 may be also true for other, different drugs available to treat this infection.
00:17:03.10 Also, it's worth it for us to go and look at,
00:17:06.13 what are the genes that actually
00:17:09.06 regulate these circadian rhythms?
00:17:10.26 What are the few genes that regulate
00:17:12.26 the expression of these cycling 1,000 genes?
00:17:16.09 And if we identify them,
00:17:19.18 can we actually use these genes as potential drug targets
00:17:22.01 to disrupt the circadian rhythms of these parasites?
00:17:26.06 Finally, what about other parasites?
00:17:29.28 So, do parasites have clocks?
00:17:32.23 It's a very general question.
00:17:35.07 We should look at what happens
00:17:37.20 in circadian rhythms
00:17:40.19 in malaria parasites, in Leishmania, in Toxoplasma,
00:17:44.00 all these other infections
00:17:46.08 that are very bad for both humans and animals.
00:17:49.06 Do these parasites also have circadian clocks,
00:17:52.14 and can we use that against them?

This Talk

Speaker: Filipa Rijo-Ferreira

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: May 2018

Talk Overview

Trypanosoma brucei, the parasite that causes African sleeping sickness, acts by disturbing the circadian rhythms, and therefore the sleep, of its host. But does T. brucei also have circadian rhythms? Dr. Filipa Rijo-Ferreira shows that yes, the African sleeping sickness parasite does have circadian rhythms and that they regulate its metabolism. She demonstrates that African sleeping sickness parasites have an intrinsic circadian clock that is independent of the host, and which regulates parasite biology throughout the day.

This talk is part of the Young Scientist Seminars, a video series produced that features young scientists giving talks about their research and discoveries.

Speaker Bio

Filipa Rijo-Ferreira

Filipa Rijo-Ferreira received her bachelor’s degree in Molecular and Cellular Biology from Nova University of Lisbon. For her MSc she joined the laboratory of Dr. Charles Bangham at Imperial College London where she studied immunology and infection. As a graduate student at the University of Porto, she became fascinated with Molecular Parasitology and Circadian Rhythms,… Continue Reading