Bacteria on the run—An interview with Tara Moriarty and Rhodaba Ebady

Posted by Stephen Matheson


Lyme disease is caused by a bacterium, Borrelia burgdorferi. These nasty little bugs spread through bodies by entering the bloodstream and finding ways to grab hold of the blood vessel wall so they can crawl through and invade local tissues.

In a new Cell Reports paper, Tara Moriarty, Rhodaba Ebady, and their colleagues at the University of Toronto reveal some of the tricks used by the bacterium. Here are some of their reflections on the work, the lab, and the effort it takes to write a good paper.

Stephen Matheson: So you're studying the mechanics of bacteria in blood vessels. How did you arrive there?

Rhodaba Ebady: I love extracellular matrix biology. As a result, I ended up in Tara's lab, just after she'd joined the Matrix Dynamics Group at the University of Toronto, a group that studies topics related to the extracellular matrix and has a particular interest in the effects of force in this environment. It was Tara's idea to develop a flow chamber model system to study the mechanisms of these interactions and the role of BBK32 in this mechanism. The initial results that we got from studying the effects of changing shear stress on Borrelia endothelial interactions in flow chambers were what got us excited about the possibility that B. burgdorferi interactions with endothelia are strengthened by force. Also, since BBK32 was the only bacterial adhesion protein that was known to support vascular interactions under physiologically relevant shear stress conditions, it was even more exciting to study this topic, because we were obtaining some of the earliest insights into a new subject.

Tara Moriarty: I started working in this area when I was doing my post-doctoral training in the lab of George Chaconas. George is a major expert on the topic of DNA transposition and bacterial telomere resolution. I did my PhD training as a biochemist and molecular biologist studying the mechanism of the human telomerase enzyme. George studies telomeres in B. burgdorferi, and my first major post-doctoral projects were to develop methods for studying telomere biology in this bacterium. Along the way, I developed a fluorescent strain of B. burgdorferi that got us involved in a collaboration with the lab of the immunologist and intravital microscopist Paul Kubes. Ursula Norman, an amazing post-doc in Paul's lab, and I ended up developing the first intravital microscopy methods for visualizing individual bacteria in the bloodstream of living mice.

Then, since intravital microscopy is technically challenging, I knew that it would be smart for me to develop methods for studying this process that weren't as hard to learn, because I had to start my own lab working with pretty inexperienced junior students. When I was in George's lab, I started trying to establish flow chambers to study this process as well as trying to figure out if we could track individual bacteria in flow chambers and blood vessels and measure how fast they moved. I didn't get very far, but got far enough to know that I could probably get it to work once I started my own lab.

Once I moved to the University of Toronto, Rhodaba, who was the first person to start doing benchwork in my lab, and Alex, who was my first grad student, started working on the flow chamber system and optimizing it so that the interaction properties we saw in flow chambers were the same as the interaction properties we saw in the blood vessels of mice. In parallel, I and two undergrads in the lab, Nupur Gupta and Cornelia (Tian Tian) Tang, started trying to figure out how to track the bacteria. Rhodaba, Alex, and Anna took over the tracking part of the project with help from Gloria (Yae Ram), and Tanya finished up the tracking. This took about two and a half years. 

The remaining two and a half years were taken up by figuring out how to analyze and understand the resulting data, including learning how to write formulas in Excel to do this (I know—there are Matlab and R users all over who are cringing when they read this). Oh, and by writing the paper, which was truly brutal. Along the way, our collaborators Hui Zhi and Jon Skare helped out with the bbk32-deficient strains and writing, and our bioengineer collaborator Craig Simmons helped make sure we were on track with our formulas and calculations for the biomechanics part of the project. I really hope someone takes the time to read the Acknowledgments for this paper, because we had a small village of colleagues read it to help make the writing more accessible, including helpful but unacknowledged peer reviewers and journal editors. We also had to get a science writer, Farah Thong, to help us figure out how to reduce the jargon as much as possible and to make our ideas more broadly intelligible. She made us do the writing ourselves, and told us when it didn't make sense, and in the end is probably one of the most important people to have helped with this project. Figuring out how to write this was itself quite an education and exercise in perseverance.

SM: OK, but what about before that? What were your influences earlier in life?

RE: As a kid, I loved everything science related. My favorite shows were The Magic School Bus, Bill Nye the Science Guy, and Popular Mechanics for Kids. I think it influenced how I think now as a scientist in training. When I was volunteering at the first lab I ever worked in, I remember the graduate student I was working with having some trouble getting the results he was expecting from an experiment which they had conducted before. This graduate student and the lab technician spent hours troubleshooting. They were very frustrated. But when the experiment finally worked, they were so happy, and this was only for a control. I remember thinking then about how happy they were and how I wanted to do something like that where the smallest thing would make me happy. I've learned that you take the smallest wins when it comes to science.

TM: I did a lot of successful science fair projects as a kid and teenager, and I have a story about a beloved pet mouse called Pinky who ran through Lego mazes for one of these projects, and came to school with me in the Toronto subway system nestled in the pocket of my school uniform blazer with bits of Kleenex, cheese, and vegetables of course (I ensured that Pinky was fed what I thought was a balanced diet). However, I only tell the whole Pinky story to those who know me well, because I usually get quite teary, and this embarrasses everyone, particularly the students in my lab, and I really shouldn't be crying too too much in public. After all, people might get the wrong idea about what female scientists are like, even if we work on topics like biomechanics.

Editor's note: we didn't ask Tara for the whole Pinky story, but we did succeed in getting the picture below. That's Pinky in the Lego maze circa 1985.

Pinky the mouse and friend, photo by Christine Lalonde

Ummm ... now that I think of it, Rhodaba was with me in the office when I opened the letter that told me I'd received the first grant of my independent career, which helped us get the data for our first stable operating grants, and I know for sure I did some crying then. Poor Rhodaba, she's really had to be understanding with me over the years. Rhodaba was the first person to start volunteering in my lab when I started out, so we've grown up in our respective roles together. So, Rhodaba and I have been through a lot since the beginning, as have the other students from our lab who are on the paper—Alex, Anna, Nupur, Gloria (Yae Ram), Tanya, and Cornelia (Tian Tian). Every one of them has better control of their crying in public than I do, sadly. Hmmm. My students are also much much better than me at staying on topic. What was the original question again?

SM: What are the big questions and challenges right now in your field?

RE: The biggest question in our field right now still is how bacteria disseminate. The biggest challenge is studying bacterial interactions under dynamic conditions as well as mixing multiple disciplines. With our kind of work, you're always going to lose someone—meaning that the biologists find the physics challenging, and people from the physical sciences often don't have a lot of biology training. The biggest change driving development of this field is the amount of "real-time" technology that’s becoming available to study mechanisms such as those supporting bacterial-endothelial interactions in a dynamic environment, often in the complex context of whole, live cells.

TM: I think that force of all kinds is intrinsic to biology and the way biological forms function and that our biggest challenge as biologists is to imagine and understand how these forces drive biological processes. We need to train ourselves to think about force, and this is now happening in many sub-fields of biology. What we most need is to cross-train students across physical and biological sciences, because there is so much insight that is within relatively easy reach if you can cross over between these major disciplines. And because most of these students' supervisors were trained in one discipline or the other, we really need help from people who are more diverse than us!

SM: Any other interesting stories about this work? Setbacks? Epic experiments?

RE: This work took five years to complete. There were a lot of setbacks. The ability for bacteria to form catch bonds and tethers, like leukocytes, was very unexpected in my opinion. There were plenty of mistakes. As someone who only worked with fibroblasts before this project, which are very easy to culture, it took me a while to get the hang of working with primary endothelial cells and even longer to get the hang of getting a fully confluent monolayer. At the beginning, only about half of the experiments would be good because we couldn't keep the endothelial cells happy in the flow chamber.

TM: Rhodaba didn't tell you that for the first couple of years we didn't have a microscope which was fast enough to do these experiments, so for every experiment done for this paper, Rhodaba and Alex would pack up their endothelial cells and flow chambers in pre-warmed thermoses, styrofoam boxes, insulating packing materials, and even little beds of hot wet paper towels and run (or walk very, very fast) to a building a couple of blocks away to do the imaging at a microscopy facility where the cells could be kept warm in incubators. There were some bad periods in the coldest months of the year when it was really hard to get data because they couldn't keep the cells warm enough for the run to the core facility. There were a lot of "experiments" conducted just to get the transport right. And they got very, very good at never forgetting the materials they needed to do the experiments. Toronto is not a particularly cold Canadian city, but no one wants to run back and forth on wind-swept streets in January to get a piece of tubing that got left behind.


SM: What would you like non-scientists to know about your work?

RE: Bacteria are amazing, and for Borrelia to actually cause Lyme disease and colonize a wide range of tissues in the body, they must overcome a lot of obstacles. I almost feel that for the amount of work it takes for bacteria to get to these tissues, they deserve to settle and stay there. That's a very bacterio-centric view of the world, but it helps to try to understand the world from their point of view and not from the point of view of our own bodies and the bodies of other animals they can colonize and infect.

TM: By this point in the interview, non-scientists are going to think that our work is mainly about fits of crying interspersed with stubbornness and that we only really care about the obstacles and forces faced by bacteria in the bloodstream and not the diseases that dissemination of bacterial infection can cause. Although, I have to say ... it's really useful to try to imagine life as a bacterium in that environment, instead of just thinking of how to stop dissemination from happening from the point of view of the bodies bacteria infect.

What I want non-scientists to know about this work is that, despite the large number of differences between the bacteria that cause Lyme disease and the cells of our own bodies, these bacteria manage to stick to blood vessel walls using mechanisms that are remarkably similar to those used by our own cells. So, even though this work is important because it provides tools to study how many other disseminating bacteria cause serious infection and disease, in the end we may find out that other bacteria and the cells of our body are not so different when it comes to overcoming forces in the vasculature. This may or may not be true, but if it is, then strategies which have been developed to limit how our own cells stick to blood vessel surfaces might possibly be useful for blocking the spread of bacterial infection via blood vessels. There's potential to rediscover new uses for clinically established drugs as well as discovering new ones.

SM: What was the hardest thing about this project?

RE: The hardest part was trying to communicate the biophysical aspect of the study to the general scientific community. There's a lot of physics that is hard to explain clearly and compellingly to scientists whose primary expertise is not in physics.

TM: Writing the paper over and over and over and finding something new that we realized we had to understand or explain each time we'd re-written it. We hung on and kept doing this until we knew we had a coherent and plausible model to explain what we were seeing. This model seems so obvious now (at least to us!), but it was like unravelling about 80 tangled balls of the same colored yarn for an infinity of time. We just had to keep finding little ends of the data that we thought might lead somewhere and then patiently (sometimes desperately) untangling bit by little bit. If we'd had training in engineering or physics, it probably would have been a lot easier, but going through this process as biologists also probably helped us think about the problem in a different, perhaps simpler way. And even once we'd figured it out, we still had to explain it in writing.

SM: All of you started on this project five years ago as undergraduates and fresh MSc students (with the exception of Tara, who was just starting her independent lab). Do you think being a young and inexperienced team was an advantage or a disadvantage? Why or why not?

RE: I think it was an advantage. We were venturing into the unknown not knowing whether the results would be significant, but as a young researcher, that's something that excites you. I think sometimes more senior trainees and scientists are more careful about taking risks and evaluate projects with an eye to their feasibility and prospects for producing papers quickly. I really hope I can continue to work on challenging projects in the future.

TM: From my point of view, I think it was an advantage to do this project as a young team. Rhodaba is right when she says that more senior trainees have to be careful about taking risks. Also, I think that the naive optimism that's a little bit inherent in being a young team, and the crazy and probably out-of-proportion sense of achievement that went with each problem we solved, carried us a long way. Post-docs and research associates might have got through some steps faster, but I'm not totally sure about this, because all of us, including me, had to teach ourselves what we needed to know almost from the bottom up, and in a way it was probably just as hard for me as it was for the students on the team. We actually did reading classes about this topic together, which made an enormous difference to the project. There's also more opportunity for a supervisor and students to learn together because the training process is much more direct and detailed with junior students, and we really worked as a team. 

SM: Apart from the science, what are the biggest professional and personal lessons you learned from this project? What will you always remember as you move on?

RE: Being smart about how you work and working hard pay off. I hope I remember that in the future.

TM: I am verklempt! That is the lesson I'd most want students in my lab to learn, and Rhodaba has clearly learned it!

Moriarty lab members, left to right: (front row: Rhodaba Ebady, Helena Pĕtrošová, Alex Niddam, Nataliya Zlotnikov, Zoha Anjum, Michele Bergevin); (back row: Azad Eshghi, Anna Boczula, Tara Moriarty, Wei-Chien (Andrew) Kao, Tian Tian (Cornelia) Tang, Nupur Gupta, ghost of Dr. John G. Fitzgerald, Peng Song)

Rhodaba Ebady is a PhD student in Tara Moriarty's lab. As an undergraduate studying biology and chemistry at the University of Toronto, she became fascinated by the extracellular matrix in which host cells and microbes live. She is now interested in vascular dissemination mechanisms of cells that travel through the bloodstream, including metastasizing tumor cells.

Tara Moriarty was born in Canada, began conducting (non-invasive) experiments with family members and beloved pets at an early age, and has always liked understanding how things move and work. She studied at McGill University, followed by post-doctoral training at the University of Calgary. She has been a faculty member at the University of Toronto since 2010, is up for tenure and funding renewal in one month, and has about 18 papers to submit in the next four weeks.

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