(L-R) Divenita Govender, George Lacey and Linda Grillova in the Parasites and Microbes Laboratories, Wellcome Sanger Institute.

Categories: Sanger Science31 October 2023

The dark matter of microbiology

Only a small community of scientists around the world study Treponema pallidum, the bacterium that cause syphilis. And it is only in the last few years that anyone has been able to grow the bugs in a laboratory. Now, a handful of places can cultivate them, including researchers here at the Wellcome Sanger Institute.

A recent report published by the UK Health Security Agency shows that this year in the UK, syphilis diagnoses were at their highest levels since 1948². Though infection with Treponema pallidum is usually easily treated with antibiotics, untreated syphilis can cause serious, irreversible and potentially life-threatening problems with the brain, heart, or nerves.

Linda Grillova is a molecular microbiologist at the Sanger Institute. She works in the Parasites and Microbes Programme and has recently been awarded a BBSRC Discovery Fellowship¹ to study the biology of Treponema pallidum, its pathogenesis and potential new treatments.

Linda spoke to us about her research, her passion for the unexplored, and why the Sanger Institute feels like home.

Why study Treponema pallidum?

Firstly, T. pallidum is able to cause different severe diseases in multiple hosts including rabbits, primates and humans. The most significant of these diseases is syphilis, which has a high prevalence with congenital syphilis representing a particularly major issue at the moment.

Secondly, despite the fact that we have known for over 100 years that T. pallidum is responsible for syphilis, we have almost no knowledge of the basic biology and pathogenesis of these bacteria.

And lastly, these bacteria have special characteristics that make them unique. For example, they have a spiral shape that enables them to force through any physiological barriers. This allows them to enter any tissue in the body and infect any organ including the heart, brain and placenta.

Another unusual feature of treponemes is their very small genome. Their minimal genome lacks various genes from several important metabolic pathways, indicating that they must rely on their host to provide them with multiple essential nutrients. As a result, it was not until very recently that a way to grow them in the laboratory was discovered.

Divenita Govender looking at Treponema pallidum under the microscope.

Wall in the laboratory with scanning electron microscope images of Treponema pallidum.

Divenita Govender looking at Treponema pallidum under the microscope and wall in the laboratory showing electron scanning microscopy images of the bacteria.

Getting started

I started studying T. pallidum 10 years ago during my PhD back in the Czech Republic where I grew up. Despite rigorous attempts to develop a culture system, there wasn’t one available. So at the time, the research was pretty much limited to genomics. But we still did quite cool stuff. For example, I was lucky enough to be part of the project - led by our collaborator Natasha Arora at the University of Zurich in Switzerland - where were able to sequence the genome of T. pallidum directly from clinical samples for the first time³.

Also, we developed a molecular typing scheme. This time and cost-effective tool helped us better understand the epidemiology of syphilis in multiple countries. Many people are now using it and it's actually really nice to see something you created six years ago in action⁴.

I was thinking at the time, there's nothing like it, and I loved it. I found spirochetal research super interesting due to the unique nature of the bugs. But I wanted to do more than genomics.

After my PhD, I moved to Paris to the Institut Pasteur for a postdoctoral position studying Leptospira, another spirochaete bacterium. While it was possible to grow these in the lab, it was still a fastidious pathogen that was hard to work with.

After two years in Paris, I was convinced I wanted to learn more techniques that were not possible to apply to these demanding bugs. I thought that I would have to change field, and I was about to accept a position at Cornell University. But then, in 2018, a research paper was published which changed everything.

Around this time, I also met Nick Thomson, Head of Parasites and Microbes research here at the Sanger Institute. He was giving a talk at Institut Pasteur in Paris and I was amazed by his work and his approach to science. The combination of the paper and the fact Nick was planning to work more on T. pallidum made me apply for a Marie Curie Fellowship. I was extremely lucky to be funded, and this enabled me to come to the Sanger Institute to work with Nick who offered me the incredible support and opportunity to set up the lab for the T. pallidum culture.

George Lacey working in the low oxygen chamber where the bacteria are kept in flasks. Electron scanning microscope image of Treponema pallidum (image credit David Goulding / Wellcome Sanger Institute).

Growing Treponema pallidum

The 2018 paper was published by researchers at the University of Texas, showing how T. pallidum could be grown in the laboratory⁵. It’s not perfect, it’s difficult, but it was a revolution. It has taken 100 years from discovering the bacterium to get to here.

To grow T. pallidum, it has to be in the presence of other mammalian cells – in this case, rabbit cells. Essentially, the mammalian cells are supporting the growth of T. pallidum. We don't know how, or how the cells interact, we know almost nothing about that - this is one of the questions we have. Setting up the culture is challenging. Because there are so many steps, there is a high risk of contamination.

Another difficulty is that T. pallidum is microaerophilic – oxygen is toxic to it, so you need a special lab set-up. It’s also time-consuming – it takes seven days for the bugs to replicate sufficiently to be able to divide them and grow more. And, because of their shape, you can’t see them under the light microscope, you need dark field microscopy, and this requires a little bit of training, as you need to know what you are searching for. The analysis is hard too, because you’ve got a mix of cells in there, and the mammalian cell genome is about 3,000 times larger than the bacterial one. Genetic material, metabolites, or proteins that the bacteria produce are all mixed up with the mammalian ones, and tiny.

The result – only a few labs in the world can do it, and we’re the only ones in the UK. But now, finally, being able to grow it opens up huge opportunities for us. Most of our observations are new – not been seen before. Really, I want to do everything! Every time I give a talk people ask about using more techniques that would answer our questions from different perspectives. I would love to! But the day has only 24 hours, and we have to focus on one thing at a time. Focusing on just one thing has been the biggest challenge for me so far.

“It’s a new challenge working within a microaerophilic chamber, as we’ve not worked in them before. We are working out the logistics, but it will enable us to run more experiments. Previously, we were limited to 30 minutes of working time outside of the incubator – anything after that and the bacteria become stressed.”

George Lacey,
Technical Specialist in the Parasites and Microbes Programme, Wellcome Sanger Institute

The fellowship

One of the goals of the BBSRC Discovery Fellowship is to culture T. pallidum from clinical samples taken from patients. No one has done this before. The bugs we culture represent historical samples that have been collected in the last century (the oldest one in 1912) and have been kept alive in rabbits since then.

It is going to be difficult because clinical samples will be from swabs taken from genitals. There are going to be a lot of other bacteria in there – I think that’s going to be a problem. But we are working on how to deal with it. Growing the bugs from clinical samples will give us the representation of modern strains that we need. This is going to be vital for vaccine development.

Another goal is to understand what is happening inside the cell culture in the laboratory. This is not only going to help us to enhance the culture but also help us to understand how the bacteria survive there.

Many genes in T. pallidum are hypothetical, and we have no idea about their function.

We are planning to combine genomics and transcriptomics [gene activity data] with subsequent phenotypic profiling to model metabolic pathways and identify the metabolic interaction network between the mammalian cells and the bacteria. Based on these observations, we hope to identify the essential nutrients and requirements that are crucial for their survival in the lab.

Cholera and syphilis captured in BioArt

How historical artefacts can highlight the progression of science and medicine.

Teamwork

I think that the Sanger Institute is a highly supportive and high-achieving scientific working environment with many people who are excited about science. I am more than grateful to be part of it and lucky enough that it has been recognized that my project is not a one-person job. The laboratory work is supported by George Lacey and Divenita Govender, and William Roberts-Sengier will soon join us to support the computational projects.

“I've always been fascinated by genetics and was introduced to the Wellcome Sanger Institute during my undergraduate degree. Since then, it has always been a dream to work here! I joined from a research institute in South Africa, where I primarily worked on TB . It is a change of pace working on T. pallidum as they are very slow growing, and require careful planning due to their specific needs.”

Divenita Govender,
Advanced Research Assistant in the Parasites and Microbes Programme, Wellcome Sanger Institute

I feel like we are a team of excited people. There are also high-throughput genomics and transcriptomics teams who we collaborate with across the Sanger Institute. We also work closely with Dave Goulding, a microscopy expert. It is a real privilege to be able to use such a great microscopy facility as the one we have here and it is great working with everyone.

Essentially, I enjoy being in the lab, talking with people and asking questions. It’s about being result-driven. I would like to know what is happening! What’s working? What does it mean? What do we see? If it doesn’t work can we figure it out? I have to work out how I am going to answer these questions. I really like to do new things all the time.

I enjoy the excitement of everything new that we are discovering – big or small. We are really in a position to discover something really crucial. Right now, with all the ideas that we have, I'm sure that I can sketch out the next 20 years of experiments!

Tell us more about T. pallidum

I think one of the most interesting things is how it infects its host. How is it possible it can live in any organ? How is it possible that it can stay in your body for such a long time without the immune system being able to recognise it?

Answering these questions cannot only help us to combat the disease but help us to understand bacteria in general. I think this is very important. A lot of translational research – with application to health for example - is based on biology like this.

The dark matter of microbiology

It’s not just T. pallidum that isn’t really understood. We’ve known for a long time that there are a huge number of bacteria out there, but for about 99 per cent of them, we just can’t grow in a laboratory using conventional techniques. This phenomenon has been referred to as the "great plate count anomaly." Scientists have observed that the number of bacterial cells seen under a microscope is often much higher than the number that can be grown on agar plates in the lab. They’re known as the dark matter of microbiology. We know nothing about them. They are sometimes called "unculturable" but it doesn't mean that these bacteria can never be cultured. Instead, it often means that we haven't yet identified the specific conditions or nutrients required for their growth in the laboratory.

From the one per cent we can grow – we’ve learned so much. Studying bugs gave us invaluable insights into numerous aspects of biology, medicine, and technology from the discovery of antibiotics and the CRISPR/Cas System -the revolutionary genome-editing tool for genetic engineering and gene therapy to the production of vitamins and probiotics production.

Can you imagine what we might discover in the 99 per cent of bacteria we currently can’t grow? Who knows what else we are going to find when we learn to culture more bacteria. We’ve put effort into T. pallidum because it causes a horrible disease, but understanding all life is vital.