Tryp/Leish Stickers!

It's been a busy year starting my lab... But in more important news, T. brucei, T. cruzi and Leishmania stickers!

How does a cell swim fowards?

“Why are they swimming backwards?” This is one of the most common questions I get asked whenever I show a video of Leishmania parasites swimming.

Swimming Leishmania at 200 frames per second (8× slower than actual speed)

If you look at Leishmana with a high speed video it's easy to see they tend to swim 'tail-first', with the flagellum sticking forward into the direction of travel. Sperm, probably the best-known swimming cells, do the opposite, and swim 'head-first'.

Sperm on the left, Leishmania on the right

Of course, if you could ask the Leishmania, they'd say that they're swimming forwards and it's the sperm which are swimming backwards. This raises the question of how does a swimming cell decide which direction it should swim? Which direction is forwards?

The direction a cell swims depends on the waves which travel down the flagellum. If they start at the base and go towards the tip then the cell will swim head-first. If they start at the tip and go towards the base then the cell will swim tail tail first. So how do they choose where the waves start?

We answered this question taking advantage of a useful behaviour of Leishmania. If you look closely at the video above you can see one cell is swimming forwards (the cell on the left) and the other is turning on the spot (the cell on the right). And if you look very closely you'll see that the waves in the cell on the left start at the flagellum tip, and the waves in the cell on the right start at the base of the flagellum.

Tip-to-base on the left and base-to-tip on the right.

This let us use genetic tools to try to break one direction of flagellum wave without affecting the other and tease apart how the flagellum movement might be chosen by the cell. To cut a long story short, we found differences in the motor proteins between the base and tip which seem responsible. Interestingly, similar differences turn up in many organisms, including human sperm, suggesting it might be a general pmechanism. You can read all about this in our recent paper: At the PNAS website or as a PDF.

https://doi:10.1073/pnas.1805827115

How to be a parasite

For an organism to become a parasite it has to adapt to live in a host. This might mean it needs to grow faster, invade cells or tissues, or avoid being killed by the immune system. It can also 'forget' how to live outside a host. It can forget how to search for food, how to survive the cold, and how to avoid drying out.

We can learn how parasites adapt to infect hosts by looking at the nearest free living relatives. What had the parasite lost relative to the free living cousin, because it just doesn't need it to survive in a host? Or vice versa, what has it kept because it's useful for infecting a host?

I've looked at this question for trypanosomatid parasites. They are protozoan (single cell) parasites, like the malaria parasite, and cause several deadly tropical diseases.

A few hundreds of millions of years ago, they weren't parasites at all. The ancestors of these parasites were free living, probably swimming in ponds, lakes and seas. Then, at some point, some evolved the ability to infect insects.

Millions of years passed. Then, several tens of millions of years ago, some managed to get transmitted from their host insect into a vertebrate host. And, most importantly, they survived and could get transmited back to the fly.

This adaptation to infect animals probably happened on three separate occasions. Those parasites kept evolving and adapting, and are now the three human trypanosomatid-caused diseases: Sleeping sickness, Chagas disease and leishmaniasis.

My most recent paper is all about this. Julius Lukeš has discovered a species of trypanosomatid that only infects insects, and looks like it hasn't changed much from the first ancestor ever to infect insects.

With Tomáš and Eva, we looked at what this species tells us about how these parasites adapted to infect flies: How does the cell grow, adapts its shape, and stick to surfaces? How does its internal organisation adjust to allow these changes? How does its metabolism change for different energy sources? And, how has the genome, which encodes the proteins that drive these functions, changed to achieve this?

Using this information, we could then get insights into what is important for human infective parasites. What aspects of shape, structure and metabolism adaptations have they 'forgotten'? And which have they kept? The ones they have kept are the ones important for infecting, and killing, people.

Want to read more? You can get a copy of the paper from my website: richardwheeler.net

Skalický T*, Dobáková E*,1, Wheeler RJ*, Tesařová M, Flegontova P, Jirsová D, Votýpkaa J, Yurchenkoa V, Ayalag FJ, Lukeš J (2017) "Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early-branching trypanosomatid" PNAS doi:10.1073/pnas.1712311114

Moving in a straight line - sounds simple, right?

One hundred years ago Asa Schaeffer blindfolded his friend and challenged him to walk in a straight line. He did three loops of a spiral, before tripping over a tree stump. This wasn't a cruel prank, it was an experiment, and all people are surprisingly bad at this simple challenge.

More modern experiments showed it is lack of external reference points that trips people up. Blindfolded in a desert? You walk in circles. Not blindfolded in a desert? Straight lines. Forest on a sunny day? Straight lines. Forest on an overcast day? Circles. Current Biology, DOI: 10.1016/j.cub.2009.07.053

People need some external reference point, a distant hill, the sun, or even the direction of shadows, to manage a straight line. Why they drift into circles without a reference isn't clear. Is it asymmetric leg strength? A handedness bias? Or some psychological miss-correction? What is clear is that it is a universal problem.

Navigation without reference points is always difficult. This is why spin stabilisation is widely used to stop flying objects, from bullets to rugby balls, curving off course in the air. Even advanced tools like inertial navigation systems, which use dead reckoning from measuring acceleration, always drift off course. 

So how about swimming cells? Many swimming cells and microorganisms have the ability to swim in a straight line. Most have no ability to look at a reference point, they can only perceive the liquid they are in immediate contact with. They are also typically top small to be affected by gravity, so have no way of using up or down for reference. They are essentially blind.

The trick for straight line swimming in cells seems to be some kind of spin stabilisation. Way back in 1901, H.S. Jennings noted that many microorganisms spin as they swim, and thought this could be a spin stabilisation somewhat like a spinning bullet. The problem is it can't be. Rugby balls, bullets and spacecraft use spin stabilisation which depends on rotational inertia to keep them spinning and stable, similar to a spinning top. If you made a top that was the size of a cell, and span it in water, it would stop spinning immediately; there is too much friction. The American Naturalist, 35(413):369-379

It turns out the mechanism is just geometry. A person walking is a back-forward/left-right, a 2D, situation. If you curve the walking path it makes it into a looping circle. For a cell swimming there is another direction to think about; in 3D there are two ways to curve the swimming path. The first which curls the path into a circle, and a second which twists the circle to elongate it into a helix-shaped path. Elongate the helix far enough and it turns into a straight line, with the cell rotating as it swims.

The interesting property of the helical swimming paths is their stability. If a cell deliberately twists its swimming path into a helix then small asymmetries (the cellular equivalent of having one leg stronger than the other) won't bend the swimming path into a circle. Instead it just slightly alters the shape of the helix. It can ensure the cell swims in a dead straight line.

My latest paper is all about how cells manage straight line swimming, looking at trypanosome and Leishmania human parasites. Each aspect of swimming has been looked at before, but I believe has never previously been put together as a full story: from mutants with altered cell shape (to add more or less twist), measuring the effect on swimming, and matching this to simulation of how cells achieve their straight line swimming. PLOS Computational Biology, DOI: 10.1371/journal.pcbi.1005353

There are still big unanswered questions though: Why do parasites need to swim in a straight line? And what are they swimming towards? This is an area of active research, with several major trypanosome research groups (especially Kent Hill and Markus Engstler) interested in addressing these questions.

Molecular Cell Biology of Protozoan Parasites - Ghana 2017

Things change fast in Ghana! Three years ago, I helped teach a course for young African scientists in the University of Ghana in Accra. This January I got the chance to do the same again, and it has been fantastic. The University of Ghana is becoming a centre of great science in West Africa, with huge contributions by Gordon Awandare and his West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) course, funded by the World Bank.

This time around, our teaching course was made possible by our TrypTag project, funded by the Wellcome Trust. We applied for extra funds so we could transfer the genetic engineering technologies we developed for TrypTag into the hands of African researchers; to get the research techniques to the parts of the world that really suffer from parasitic diseases.

Our course focused on tools for analysing parasites and what makes them tick, particularly using genetic tools. We mostly looked at Plasmodium (malaria), Trypanosoma (sleeping sickness) and Leishmania (leishmaniasis). To teach the malaria side of the course we had the excellent Kirk Deitsch (Cornell University, New York) and Oliver Billker (Sanger Institute, Cambridge). On the trypanosome and Leishmania side we had Keith Gull (University of Oxford) and Sue Vaughan (Oxford Brookes University), along with the TrypTag team: Jack Sunter, Sam Dean and me!

So what was the course all about?

We focused on the tools to help young African scientists (starting their Master's or PhDs) take control of their research - from learning about free genome data and bioinformatics experiments, to computational and genetic tools to make discoveries about parasite biology.

A major part of the course was tools for handling DNA: PCR for detecting genes in a sample, amplifying DNA to clone it into a plasmid, and working with software (ApE) to design cloning strategies for gene tagging, deletion and RNA interference/siRNA knockdown. Teaching how to design a PCR or cloning experiment, rather than just teaching how to do the experimental technique, was very popular.

We also taught how to use the sequence resources you need for working with DNA: How to get the most from genome databases, like PlasmoDB for malaria and TriTrypDB for trypanosomes and Leishmania. The course also covered bioinformatics experiments, thinking how to test a biological hypothesis using existing data from genome data. The students quickly recognised the power of this approach, particularly given genome sequence resources are free! Many were immediately applying these ideas to their areas of research.

We made sure there was a big push towards critical thinking. The student's loved critical reading of articles in the journal clubs, and thinking about how to apply this critical assessment to their own experiments to make them as good as possible.

We also tried, for the first time ever, using the TrypTag.org website as the start point for a bioinformatics experiment. The students were challenged to start with a protein localisation patterns to identify protiens likely involved in particular aspects of parasite energy metabolism, then test whether any of these were unique to trypanosomes making them a potential drug target.

This was the perfect stress test for the new TrypTag.org website and server. It coped with up to a page view per second, and downloads of 10 images per second, with no problems. All over a slightly unreliable internet connection in Ghana! Many thanks to the scientific computing at the Sir William Dunn School of Pathology in Oxford for helping make this happen.

Overall the course was a great success, with very positive feedback from the students and local research staff. The students were smart, engaged and hard-working. It will be exciting to see what these young people can achieve over the next few years.

Cell Biology of Infectious Pathogens - Ghana 2013

For the last four years there has been a cell biology workshop in West Africa, organised by Dick McIntosh, an intense two week course aiming to help young African scientists around the master's degree stage of their careers. This course ran again this year, and was the first organised by Kirk Deitsch (malaria expert and a regular from the previous courses) and I was fortunate enough to be invited to teach the trypanosome half of the course. For its fifth incarnation the course returned to a location where it has previously been held, the Department of Biochemistry, Cell and Molecular Biology in the University of Ghana, and was organised with Gordon Awandare.

[Interactive 360° Panorama]

The Department of Biochemistry, Cell and Molecular Biology - University of Ghana, Accra.

The focus this year was teaching basic cell biology and the associated lab techniques, emphasising how this helps understand and fight some of the major parasitic diseases in Africa: African trypanosomiasis (sleeping sickness), leishmaniasis and malaria. All three of these diseases impact Ghana and the surrounding countries and these diseases are of enormous interest to students embarking on a scientific career in Africa.

[Interactive 360° Panorama]

The teaching lab in The Department of Biochemistry, Cell and Molecular Biology.

Of the three diseases we were teaching about malaria is by far the most well known, both locally and internationally. It is caused by Plasmodium parasites (which are single cell organisms) which force themselves inside the red cells in the blood to hide from the host immune system. Malaria is often viewed as the iconic neglected tropical disease, however in the last 10 years or so the understanding of the disease and efforts to find a vaccine and new drugs has improved vastly. Unfortunately it is still very common (we had one case in the participants on the course in the two weeks), drug resistance is rising, and it places a huge cost and health burden on the affected countries. It also impacts a huge area; almost all of sub-Saharan Africa is at risk.

Looking at Leishmania. One of the lab practicals was making light microscopy samples from non-human infective Leishmania using Giemsa stain.

Leishmaniasis and trypanosomiasis are caused by two related groups of parasites, Leishmania and trypanosomes (also single cell organisms), and if malaria is a neglected tropical disease the these are severely neglected tropical diseases. The two parasites live in different areas in the host, trypanosomes swim in the blood while Leishmania live inside macrophages, a type of white blood cell that should normally eat and kill parasites. In comparison to malaria fewer drugs are available, the drugs are less effective and several have severe side effects. Even diagnosis is thought to often be inaccurate. The impact of these diseases is less than malaria; human trypanosomiasis is thought to be relatively rate and leishmaniasis is confined to a semi-desert band just to the south of the Sahara. Trypanosomiasis does have a huge economic impact though, as it infects cattle and prevents milk and meat production, and cases of leishmaniasis are probably under-reported.

Staining trypanosomes. One practical was making immunofluorescence samples. In this sample the flagellum of trypanosomes was stained fluorescent green using the antibody L8C4.

So what did we teach?

The teaching was a mixture of lectures, small group discussions, lab practicals and lab demonstrations and we taught for 14 hours a day for 11 days; we could cover a lot of material! All the teaching was focused on linking basic cell biology to parasites and to practical lab techniques. Topics taught included how parasites avoid the host immune system, molecular tools to determine parasite species, light microscopy techniques, using yeast as tool to analyse cell biology of proteins from other species, host cell interaction of parasites, and many more.

Detecting human-infective trypanosomes. This gel of PCR products shows whether the template DNA was from a human-infective or non-human infective subspecies of T. brucei. If there was a DNA product of the correct size (glowing green) then the sample was human-infective.

A great example of how all the teaching tied together was polymerase chain reaction (PCR) to determine species. Human-infective trypanosomes have a single extra gene which lets them resist an innate immune factor in human blood which would otherwise kill them, and I taught about why this is important for understanding the disease and how it was discovered. This gene can be detected by PCR, and this technique is used to tell if a particular trypanosome sample could infect people. We ran a practical actually doing this in the lab.

PCR is a simple, adaptable and easy technique for checking any parasite for a particular species-defining or drug resistance gene, and we also taught how to use online genome sequence data to design PCR assays. We even worked through PCR assay design for many individual participant's personal research projects, really transferring the skills we were teaching to their current research. Finally we looked at papers using PCR techniques to critically analyse the experiment and assay design to help people avoid pitfalls in their own work.

This was a great demonstration of how basic cell biology and lab techniques can have real practical application with medical samples and help with surveillance of a disease. We designed all of the teaching to have this kind of practical application.

7x speed timelapse video of fish melanophores responding to adrenaline.

One practical with massive visual impact was the response of fish melanophores to adrenaline/epinephrine. Fish normally use these cells to change colour in response to stimuli and melanin particles (melanosomes) inside specialised cells (melanophores) run along the microtubules which make up a large portion of the cell cytoskeleton. We used it to demonstrate signalling; adrenaline can be used to stimulate movement of the melanosomes towards the centrosome.

This is really flexible experimental system for demonstrating the functions of the cytoskeleton, motor proteins and signalling pathways because the output (movement of the pigment particles) is so easy to observe with a cheap microscope or even a magnifying glass. This experiment was particularly chosen as it is a useful and accessible teaching tool for cell and molecular biology, and many of the course participants had teaching obligation in addition to their research.

Western blots in Western Africa. 100x timelapse of loading and running a SDS-PAGE gel.

We aimed to cover all the major molecular and cell biology techniques and had practicals doing microscopy, immunofluorescence, growing a microorganism (in this case yeast), PCR, agarose gel electrophoresis SDS-PAGE and Western blotting. The yeast practicals were particularly cool; using genetically modified cell lines the students analysed the function of p53, a transcription factor with a major role in recognising genetic damage and avoiding cancer, and how well it promotes transcription from different promoter sequences. These practicals taught growing yeast, temperature sensitive mutants, several types of reporter proteins in yeast and Western blotting, all concerning a transcription factor with huge clinical relevance in cancer!

Exploring DNA and protein structures through PyMol in a bioinformatics session.

Practicals weren't just limited to lab practicals though. We also ran interactive bioinformatics sessions looking at the kinds of data which are freely available in genome and protein structure databases online. These were also very popular, especially as so much data is available for free online.

All in all the course was a great success. The participants were all extremely enthusiastic, hard working and scarily smart! Feedback so far has also been very positive. I feel that courses like this can have a huge impact on the careers of young African scientists, and I sincerely hope that funding can be secured to continue running this type of course in the future.

You can also read more about this course at the ASCB website.

Software used:

ImageJ: Image processing and timelapse video creation.

Tasker for Android: Timelapse video capture.

Pymol: Protein structure analysis

Cell Picture Show

One of my scanning electron microscope images from my research into the division of Leishmania parasites is currently featured in the Cell journal picture show on parasites and vectors. You can check it out (image number 11) here.

A montage of seven scanning electron micrographs of L. mexicana.

"Leishmania mexicana promastigotes, which normally inhabit the gut of the sand fly vector, are shown at different stages of the cell cycle. Cells are arranged by cell-cycle progression, increasing in a clockwise direction. L. mexicana mainly causes a mild form of cutaneous leishmaniasis, forming ulcers at the bite site of an infected sand fly."

Software used:

ImageJ: Micrograph processing