After the Paper – Tackling trypanosome biology takes a lot of guts

In 2019, we published a paper about how cAMP signaling in the flagellum of the parasite Trypanosoma brucei is required for them to move throughout their tsetse fly host. These parasites cause African sleeping sickness, which is a deadly disease if left untreated.

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In our study, we wanted to understand how cAMP signaling was involved in or required for their progression through the tsetse fly. We found that it is in fact required, and specifically, T. brucei needs to use cAMP signaling to migrate across the peritrophic matrix, which is this chitinous barrier between the midgut lumen and the ectoperitrophic space. We then used mutant analysis of a specific gene phosphodiesterase B1, or PDEB1, to show that disturbing the cAMP signaling pathway also disables the parasites in the fly from progressing/transitioning from the midgut lumen to the ectoperitrophic space.

In this After the Paper post, Sebastian Shaw and I discussed the impact of our paper, which was published in Nature Communications in February 2019. We have lightly edited our conversation for length and clarity.

What impact did our paper have?

Sebastian Shaw: One great experience was working with the tsetse flies because not everybody has a lab that is equipped to work with tsetse flies. I mean, a tsetse lab isn't something very special. It’s just a room with some nets, so the flies cannot fly out, right? But I think it was still great to work with them.

Stephanie DeMarco: It was a really unique experience. I remember coming back to the US and people being like, 'oh my gosh, you worked with tsetse flies. That's so crazy!' I'd show them pictures, and they'd say 'they're so big!' I feel like people think that they're like Drosophila – like they're tiny, but tsetse flies are sizable flies! 

It was also cool because we got to look at their tissues with the parasites and see where they were relative to the different tissues inside the fly.

Sebastian: Yeah, the combination of staining tissues and doing microscopy with fluorescent parasites – that was cool. We established an advanced standard of studying the parasites in vivo. Earlier work in tsetse flies was concentrated on infection rates in the midgut and the salivary glands, sort of where they first establish an infection and where they sit to get transmitted to the next host. Then, scientists also considered looking into the proventriculus, a tissue that trypanosomes have to infect and colonize before they reach the salivary glands. We zoomed in even more and reached enough resolution to distinguish between parasites sitting in the midgut lumen and the ectoperitrophic space. That was important for our findings.

Stephanie: Exactly, yeah,

Sebastian: But it has made it more complicated for people that want to study parasite transmission through the tsetse fly now.

Stephanie: I know, I feel sort of bad. Now everyone's going to have to do all of the crazy dissections that we did!

This paper was also impactful because it helped me think about how my follow up paper, recently published in mSphere, fit into the context of parasites in the tsetse fly. It made me think about what might be some of the outside cues that the parasites are responding to inside their fly host. Using social motility (SoMo) as an in vitro assay for how parasites behave when they interact with surfaces, we saw that parasites engaging in social behavior are attracted to bacteria and potentially molecules that bacteria are secreting into the environment. There are three species of symbiotic bacteria in the tsetse fly, so it's possible that maybe the bacteria in the fly are producing some of these molecules.

Sebastian: I think the different and sometimes surprising findings in our paper made us realize that knockouts might be better than knockdowns. This motivated me further to establish our own CRISPR/Cas9 cell line and a system to use CRISPR/Cas9 transiently, which was published last year in BMC notes. I believe that this only happened because I realized that I need knockouts rather than knockdowns to analyze signaling in trypanosomes.

And as you just said, I think for me using SoMo as an assay to study environmental cues was a cool side project. Testing all sorts of different solutions, chemicals and conditioned medium guided me towards the story about pH taxis in trypanosomes. We have just submitted a manuscript about this work.

Stephanie: I wanted to ask you, what was the response when you presented this work at a conference? How did people react?

Sebastian: I was able to present our PDEB1 story at the Molecular Parasitology Meeting 2018. There were some people there that are skeptical of social motility. But I had fruitful chats with some of these people after my talk, and I think that I could convince them that it has a biological relevance and that it's not just about dividing cells on the plate. We know that they migrate one centimeter per day which corresponds to 200 body lengths. So there's more going on than just dividing cells.

I think this paper is a strong driver for the biological relevance of SoMo.

Stephanie: Yeah, absolutely. It was nice to show that something we see in vitro isn't just a weird thing about surfaces or something that happens in vitro. It actually looks like it's predictive or at least associated with a defect in vivo, which is really cool.

A dissected tsetse fly midgut ready for microscopy.

What were some challenges that followed the publication of the paper?

Sebastian: I am not sure about the challenges. It will be important, but also challenging to find additional candidates that are key for cAMP signaling, SoMo and the progression through the fly in trypanosomes. For me, it was really the realization that there is a lot going on at very specific locations in a cell, like microdomains. It made me realize that even though T. brucei is a unicellular parasite, right, there is the parasite’s body, the flagellum, the tip of the flagellum and it matters to look at the right spots. 

Stephanie: Yeah, that's so true. Because here we looked at specifically flagellar cAMP.

How did the paper affect our careers?

Sebastian: I'm now doing a postdoc in Boris Striepen's group, and there I work on Cryptosporidium, which is also a unicellular organism. I intended to do something totally different and it is indeed totally different to work on Cryptosporidium. I had to learn and still am learning loads of new stuff, but there might be a problem or a situation in this new environment where ideas will pop up because of my PhD.

Cryptosporidium is an apicomplexan, so it's a relative of Plasmodium. It only has one host where you can find asexual and sexual stages, and it infects the small intestine. There exist very similar strains that behave differently in the same host an I am very much interested in the question by what these differences are driven on a genomic level.

Stephanie: That's really cool!

Sebastian: But, yeah, it's really different. For instance, there's only one selection marker - whereas you have five or six in trypanosomes.

Stephanie: Exactly. Oh no!

Sebastian: And you can generate transgenics but they are not clonal, you have to work with mice… It's really challenging, but I am learning so much. I think that's a good thing.

Stephanie: Definitely. That's really cool. I am also now doing a postdoc, still in parasites. I'm working in Elissa Hallem's lab at UCLA, and I'm studying the mechanisms of skin penetration in skin penetrating parasitic worms, specifically the Strongyloides species. I was actually sort of inspired by the tsetse fly work we were doing in Bern. When we were infecting the flies with parasites, we would put the blood down on a hot plate, and then put down the silicone matrix that they would then bite through. I thought it was really interesting that  they liked to have the silicone matrix there to represent human skin. It got me thinking about the parasitic worms that I’m studying. They don't have an insect to help them get into the skin by biting. They have to bury through it themselves. So I was curious about what sort of processes and mechanisms regulate how they do that. What genes and neurons and sensory cues help them identify a human host, like, “Oh, I found human skin, so now I need to bury through it and infect the host”?

What’s new for me about working with these parasites is that all of us have to do “critter duty”, which involves going to the vivarium in the evenings and putting our rats or gerbils on a wire rack. Their poop collects overnight, and then someone collects the poop in the morning to get the parasites.

Sebastian: We do something similar.

Stephanie: Oh, that's good!

Sebastian: Actually, the two of us went on to become poop farmers.

Stephanie: Yes! Exactly! We deal with a lot more poop than people realize!

Feeding time! Blood spread onto a metal plate covered by a silicone membrane. Tsetse flies bite through the membrane to take a blood meal.

What further opportunities followed the publication of our research?

Stephanie: I feel like it might still be a little too early to say because our paper only came out a little while ago. But I think we moved the research forward and showed that this step between T. brucei getting out of the midgut into the ectoperitrophic space is actually an active barrier that they have to overcome. It's an active process. 

Sebastian: Yeah, yeah I feel the same. Something that just popped into my mind is that in liquid culture something can be totally fine, and it doesn't look as if there is something odd about it. But then if you do the SoMo assay you see, “Ah, okay. There is actually a difference.”

Stephanie: Yeah, I like what you brought up about the suspension culture versus on a plate versus in a fly, because I think, yeah, that's super important. In fact, that's where we first saw the PDEB1 defect. In liquid culture they look totally fine, but then, on a plate, that's where it's like “Whoa, what's going on?”

Sebastian: There are not so many genes involved in social motility, right? And you want to make sure that the ones that you have really show something in the fly as well.

Stephanie: Exactly!

You can find our paper: Flagellar cAMP signaling controls trypanosome progression through host tissues here.

Stephanie DeMarco

Postdoctoral Researcher, University of California, Los Angeles