Medical Research Council and University of Exeter

The Hidden World of Killer Fungi

Meet the people behind the microscopes

Fungal infections kill more around three million people worldwide each year and billions suffer from fungal infections across the globe. These infections are becoming harder to treat, due to antifungal resistance, where fungi have evolved to evade the medicines used by clinicians to treat patients. Some fungi, like Candida auris, have become resistant to multiple antifungal drugs, and have been classified by the World Health Organisation (WHO) as a serious global health problem.

Despite the high fatality rates and ill-health that fungal diseases cause, their devastating impact is not widely appreciated. Nestled in the beautiful campus of the University of Exeter, is the Medical Research Council Centre for Medical Mycology (MRC CMM), where more than 90 researchers are working tirelessly to improve the diagnosis, treatment and wider understanding of deadly fungal infections.

In 2022 the MRC CMM’s laboratory expanded to include a state-of-the-art microscopy lab for medical mycology. We call it the MYCOscopy lab. Five bespoke microscopes were set up to capture 4D living images of host-fungus interactions, fungal cell biology and drug discovery. The technologies within the imaging suite enables CMM researchers to control the environment that fungi grow, such as conditions close to the human body. In the last year the microscopes have been used for just under 10 000 hrs by over 50 medical mycologists. The CMM’s Senior Experimental Officer Dr Darren Thomson built and manages the MYCOscopy lab to support scientists who capture fungal cells as they’ve never been seen before.

To highlight the work that we do, we were commissioned in 2024 to provide a range of fungal ‘artworks’ for an exhibition on The Hidden World of Killer Fungi at the Queen’s Exhibition Space at the University of Exeter.

Darren Thomson, the MRC CMM’s Chief Experimental Officer:

Upon arriving to the Centre in 2022, I was excited to work with top microscopists in medical mycology (MYCOscopers) to make more in-roads to understanding how these killer pathogens cause disease. The MYCOscopy lab has been a hub for discovery science in the CMM and attracted industrial partnerships to visualise antimicrobial interactions and infection biology. So much so, that we decided that we must show these outputs to the world and not keep them in our storage servers.

One aim of this exhibition was to dispel the image of fungi being predominantly plant-like forest organisms and project the deadly nature of these pathogens in humans. This collection shows these pathogens in niche host environments rarely seen at such high resolution. I was keen to get across the talent at both sides of the coverslip (sample prep and image capture). Our team have developed novel therapeutic fungal-targeting systems, multi-species infection models, and multicolour staining of the brain, to name a few techniques… before even approaching a microscope! It has been especially satisfying to see microscopy novices become exceptional in medical MYCOscopy, which is a testament to how accessible these technologies are to all stages of careers, whether they are experienced or never used a microscope before. It’s a real thrill to support these scientists in our MYCOscopy lab.”

Here the researchers talk about the images they exhibited and how they captured them.

Dr Seána Duggan, Rachel Etherington, Orlando Ross, Dr Darren Thomson, Dr Vanessa Francis, Dr Iana Kalinina, Dr Dora Corzo Leon, Dr Tina Bedekovic

The Fungal Zoo

A range of fungal hyphae and yeast with cell wall, immune antigen and organelle dyes. Image credit (L to R): Dr Dora Corzo Leon, Dr Darren Thomson, Dr Tina Bedekovic, Dr Mariano Malamud (not pictured), Dr Alyssa Hudson (not pictured).

“In making this collage, we wanted to highlight the diversity and beauty of these killer fungi. They’re not just round or filamentous; they can shape shift based on their environment. The multi colour aspect of these images are really rich, which pays tribute to the skill at the bench in being able to label these structures so carefully. Each pathogen requires a different protocol to stain, so the team effort here was important to achieve this collage.

A lot of work was done at the PC to get the images aligned and straight looking for the final piece. I’m very happy with the pathogen diversity, symmetry and scale of each panel. This image underpins and inspires scientists at the CMM to study infectious disease pan-pathogen, across species in a higher throughput manner. I’m certain we’re going to learn a lot when we compare the infection biology between these pathogens.”

This image shows a collection of diverse killer fungi which cause fatal infections in humans. Fungal spores are dispersed by the structures called conidiophores, which act as fruiting bodies and eject spores into the air. Yeast cells are round and grow by rapidly budding to cause infection, whereas some fungi only grow by forming long filaments (called hyphae), which invade human tissues in search of nutrients. Fungi enter our bodies in different ways; some live within us quite happily without causing harm (C. albicans), whereas fungi like Aspergillus live in the environment and are inhaled. Our immune system usually prevents fungi from causing disease using neutrophils. Using fluorescence microscopy techniques, we can see the internal workings of the cell which helps our researchers understand how they live and cause disease. We are also developing novel antibodies which can ‘tag’ deadly fungi, paving the way for improved diagnosis and treatment of these infections.

Better (or Worse) Together

Candida glabrata (pink) and Staphylococcus aureus (gold) dual-species biofilm. Credit: Orlando Ross & Dr. Seána Duggan, University of Exeter

Image description:

Orlando Ross is a PhD student in Professor William Horsnell’s group, researching host responses to Candida albicans in the intestine, with a focus on the intestinal epithelial cells and immunological consequences of colonisation.

Dr Seána Duggan is an Early Career Fellow at the MRC Centre for Medical Mycology, researching Candida albicans – Staphylococcus aureus co-infections, looking at fungal-bacterial-host interactions.

The image was taken during Orlando’s MRes project with Seána. The project looked at C. glabrata and S. aureus biofilms, which are communities of microbes that stick to surfaces such as human cells or intravenous catheters and grow together as a dense clump of cells. Biofilms produce a protective substance (coloured green in the above image), which adds additional protection from antimicrobial medicines.

“As part of my training,” says Orlando, “I really wanted to use a scanning electron microscope (SEM), and this project was perfect for that, as SEM allows for super-high resolution imaging of the surface of your object (in this case the two species and their secreted matrix). SEM is a challenging technique, because you can easily lose definition and focus if the electron beam is not set up correctly. The sample preparation can be difficult, as SEM requires complete dehydration of the cells, which can result in the biofilm cracking or turning into dust!”

This image was pseudo coloured to bring out the different components in the biofilm, such as the fungi (magenta), bacteria (yellow) and the biofilm’s protective glucan layer (green). This was carefully done at the PC after the microscope and required some artistry!

“Having our image selected to be part of the exhibition gave us the opportunity to share our science with a new audience and explore just how beautiful and artistic our data can be.” Added Orlando. “I am already itching to get back into the Bioimaging facility with my current research!”

Fungal Meadow

Candida albicans biofilm (blue) formed from a population of Goliath cells (yellow), visualised in 3D. Image credit: Dr Iana Kalinina, Wilson Lab, University of Exeter

Dr Iana Kalinina is a postdoctoral researcher in Dr Duncan Wilson’s group at the MRC Centre for Medical Mycology. Specialising in biofilms of Candida albicans, Iana shared her experience from her recent imaging work:

“It was crucial to capture images to understand the behaviour of Goliath cells, a novel morphological type of Candida albicans, which are the yellow seed cells for the blue biofilm we imaged” explained Iana. “These Goliath cells exhibit increased adherence to plastic, impacting biofilm formation, which can cause disease in patients.”

“Capturing images of biofilms posed challenges due to light scattering within the thick biofilm layers,” Iana detailed. “Adjustment of the refractive index for light penetration deep in the thick biofilm, while developing new staining approaches, was not straight forward. Staining Goliath cells during biofilm seeding allowed us to visualize their role in the subsequently fully formed filamentous biofilms. These images would not have been possible without the hard work at the bench and image processing on the PC for optimal visualisation. The actual image capture took less than 5 minutes to generate almost 100 gigabytes of data, but the image processing and visualisation can take hours and hours to get right. I’m very happy with the ability to resolve individual cells and hyphae within the ‘meadow’ of biofilm!”

A Deadly Combination

Rhizopus delemar (blue) and Klebsiella pneumoniae (yellow colour, but pink if inside the fungus.) Credit: Dr Dora Corzo Leon, Ballou Lab, University of Exeter

Image description: Rhizopus delemar, the most common fungus causing mucormycosis worldwide, and Klebsiella pneumoniae, a common bacteria and hospital pathogen, can be found together in clinical settings. Mucormycosis is a life-threatening fungal infection which can affect people with weakened immune systems. In humans, infections involving this microbial pairing, pose a huge challenge for the human immune system to kill the pathogens and clear the infection.

This image shows how bacteria interact with fungi in two ways: first, by making direct contact with the fungus and secondly, by forming an interdependent relationship (endosymbiosis). Here, it is demonstrated for the first time that Mucorales cells can accommodate pathogenic bacteria.

“To look at how these bugs interact, we needed to be able to stain and co-locate them, in 3D. A lot of work went into getting the correct concentration of bug:bug ratio to induce this endosymbiosis. We learnt a lot by capturing these on the microscope and it has led us to much more questions than answers, which is what we love as scientists!”

Dora Corzo Leon

The Fungal Superhighway

Cryptococcus neoformans (blue) in mouse nose tissue (pink). Credit: Dr Vanessa Francis, Coelho Lab, University of Exeter

This image was captured by post-doctoral researcher Dr Vanessa Francis, who researches how fungi invade organs.

Image description: In this image, the airways in the nose are full of deadly C. neoformans (blue), as it travels to the lungs. To capture this image, the tissue underwent a complex chemical treatment to make it transparent, which allows us to see deeper within the infected airways. This project forms part of a portfolio of wider interdisciplinary research involving neuroscientists, chemists, bioinformaticians, and mathematicians from the University of Exeter and across the world, all working to improve our understanding of this deadly pathogen. This is the first-time researchers have seen Cryptococcus in this formation in the airways.

Vanessa used a very fast spinning disk confocal microscope to capture this stunning image, which is part of a recently published piece of work that investigated how the fungus Cryptococcus neoformans invades the brain. The MYCOscopy lab enabled Vanessa to look at large areas of tissue and identify rare infection events in the brain.

“This whole project was a complete learning curve for me,” said Vanessa. “Before I started this project, I had never used a microscope past the basic light microscopy I had done in my undergraduate degree. I had to learn how to use multiple different microscopes, which all have their own quirks and software to also learn. I then had to learn how to process the images I took again using different advanced software.”

“The microscopes we used to capture large number of images rapidly, and in detail, really allowed us to do what was previously impractical to do, at scale. However, this meant 100’s of gigabytes of data had to be dealt with and a lot of time was spent processing this data, which thankfully is embedded in the MYCOscopy lab. Large data management was not a skill I was anticipating learning, but it was crucial for this project. “

“The resolution and clarity of the different textures on the surface of the fungal cells was unexpected. Some look like they have spots on them and although it was not the focus of this project it would be interesting for other labs who are working on the cell wall of Cryptococcus to follow this up.”

Vanassa continued “I really enjoyed this project and I think that it produced some really powerful images. Being able to visualise infections and interactions in this way opens the research to a wider audience, as you can see what is happening. We’ve had great feedback from the exhibition which is fantastic to disseminate the message of killer fungi.”

Planetary Craters

Candida albicans mother cells showing bud scars falsely coloured in blue. Image credit: Dr Tina Bedekovic, Brand Lab, University of Exeter

Image description: Candida albicans lives within our bodies, for example in the gut, mouth, throat and vagina. They usually live as part of our microbiome, without causing any problems. However, if conditions in their environment change, C. albicans can multiply and cause inflammation, which can be uncomfortable and debilitating for those affected. This image shows bud scars as bright circles (giving the impression of planetary craters) on the surface of C. albicans, which are circular spots on the cell wall that mark the site where a daughter cell has emerged and separated from a mother cell.

Dr Bedekovic focusses on the invasive hyphal growth of the human fungal pathogen, Candida albicans. Her research investigated how this growth led to tissue damage and disease progression. Recently, she captured an image that vividly illustrated her findings, which was featured in an exhibition blending science and art.

The image was the result of a collaborative effort. Tina meticulously prepared samples and used a laser scanning confocal microscope to obtain high-resolution images. The 3D image of the cells aimed to reinforce their data from other experiments, showing that modifying cell growth mechanisms, like the positioning of where cells bud off from yeast, called a bud scar, affected the pathogen’s ability to invade host tissue and cause damage. The process was challenging, especially in immobilizing cells to capture intricate details accurately, but the result exceeded their expectations, revealing novel cellular behaviours.

“Having previously mastered live-cell imaging of timelapse microscopy, it was great to have a chance to sit down at these cells and really image them deeply at super resolution without having to worry about the next time point.” Tina explained how the super-resolved structures were invaluable as they continued to explore the molecular mechanisms of Candida albicans pathogenesis. Microscopy remains a key tool for her research. Tina expressed her enthusiasm, saying, “I am thrilled that our image was chosen for the exhibition, allowing our science to reach a broader audience.” Presenting their work through art engaged and inspired diverse viewers, fostering a greater appreciation for the complexities of microbial life and the beauty of scientific discovery. In summary, capturing this image was a journey of collaboration, learning, and discovery. It advanced their understanding of Candida albicans and highlighted the intersection of science and art.

Immune Cells Fighting Back

Aspergillus fumigatus hyphae (blue) surrounded by human neutrophils stained for their intracellular and extracellular trap DNA (NETs; red). Credit: Dr Mariano Malamud Guillan, Brown Lab, and Dr Darren Thomson, University of Exeter

Image description: Aspergillus fumigatus is a mould fungus, which is found in the air we breathe. Most people breathe it in every day, without becoming unwell. However, people with weakened immune systems or lung disease are at risk of developing health problems due to Aspergillus, and infections can be life-threatening.

This image shows the fungus forming hyphae (elongated invasive filaments which grow through the host tissue). Neutrophils, a type of white blood cell, are forming a ‘net’ (coloured red) around the hyphae to stop their growth. By understanding the role of NETs in the immune response to A. fumigatus we can find new therapeutic targets to treat the disease.

“As someone who predominantly uses flow cytometry to count cell types from infected organs, microscopy always seemed too specialised for me and beyond my grasp. But then the MYCOscopy lab opened and the coincidental need for spatial understanding in my project of host-pathogen cell interactions called for its use. We work with immune cells which form these DNA nets to trap and kill pathogenic fungi. We needed to know where and when these nets were forming, so we managed to stain our cells for microscopy and asked Darren to capture this process in very high resolution. The data from these experiments opened new ways of thinking about the spatio-temporal dynamics of this important defence mechanism against killer fungi. We still look at the data now and ponder some questions for follow up on these beautiful neutrophil nets.”

Mariano Malamud

Target Locked

Rhizopus arrhizus. Cells from in vitro culture at different stages of germination and hyphal growth. Blue indicates chitin cell wall. Orange indicates the presence of a protein that can be recognised by the diagnostic antibody TG11 (developed by the University of Exeter spin-out company ISCA Diagnostics Ltd.) along the growing hyphae.

Credit: Dr Alyssa Hudson, Ballou Lab, University of Exeter. This work was undertaken as part of the Noah’s Pink Balloon Fellowship awarded to Dr Alyssa Hudson, funded by Noah’s Pink Balloon Leukaemia Fund.

Image description: Noah’s Pink Balloon Leukaemia Fund was founded in July 2021, by the parents of Noah Tesselaar, who was diagnosed with a rare form of leukaemia when he was 5 years old. Despite a courageous fight during treatment, Noah’s immune system was too weak to fight off infections and he died of mucormycosis in June 2020. Noah’s Pink Balloon Leukaemia Fund focuses on funding neglected areas of leukaemia research, including the early diagnosis of deadly infections, to give hope to other children and their families.

“During my clinical research fellow post at the MRC Centre for Medical Mycology I worked with two diagnostic antibodies that were developed to diagnose mucormycosis. I wanted to visualise where these antibodies bind to their target fungal killers. I spent months optimising the experiment and imaging different fungi with fluorescently labelled antibody. I was amazed when I first visualised the TG11 diagnostic antibody in my experiments and captured incredible images like this one. These images capture the power of this diagnostic antibody, as it locks onto its fungal target, shown brightly in orange. We have since been able to describe where and when the antibody binds to the fungal pathogen using 4D timelapse microscopy, and have also used it to detect the fungus in an infection model. As a medical doctor, learning 3D advanced fluorescence microscopy in the MYCOscopy lab, and capturing this striking image of fungal diagnostics in action, has been so rewarding.”

Alyssa Hudson

Alyssa has completed her project with Noah’s Pink Balloon Leukaemia Fund and has now returned to work as a hospital microbiologist, but still continues to present this important work at conferences.

Caught in a Thunderstorm

 

Cryptococcus neoformans (orange) in a mouse brain showing immune cells (microglia, blue and pink) and blood vessels (yellow).

Credit: Dr Vanessa Francis, Coelho Lab, University of Exeter

 

Image description: Cryptococcus neoformans lives in the environment, and we breathe it in daily. Infections are rare among people with healthy immune systems, however C. neoformans is a major cause of illness in people living with HIV/AIDS. There are an estimated 152,000 cases of cryptococcal meningitis occurring worldwide each year, resulting in approximately 112,000 deaths.

In this image, a cluster of C. neoformans (orange) can be seen in the mouse brain surrounded by specialised immune cells called microglia (blue and pink). Microglia form part of the immune system that protects the brain from infection. To capture this image, the tissue underwent a complex chemical treatment, which allows us to see deeper within brain tissue. It is incredibly challenging to find C. neoformans in the brain during early infection, so large areas of tissue were imaged to find these rare clusters of cells. This project forms part of a portfolio of wider interdisciplinary research involving neuroscientists, chemists, bioinformaticians, and mathematicians from the University of Exeter and across the world, all working to improve our understanding of this deadly pathogen. These images have shown that microglia rapidly interact with C. neoformans during early infection in the brain.

A recent study provided unprecedented insights into brain invasion by the fungus Cryptococcus neoformans. The research visualized the fungus in situ, allowing scientists to examine large tissue areas and identify rare brain infection events. The investigation also extended to the nasal cavity, revealing a diverse population of fungal cells, including large Titan cells crucial for infection.

“This was the fastest rate at which these cells have been show to develop in vivo” notes Vanessa. “The project marked a steep learning curve, having to very quickly get to grips with the anatomy of the brain and how to label the multiple cells of interest simultaneously, such as microglia and blood vessels.”

This image has been really engaging for fellow scientists and the public alike. We played with lots of different colours during image processing, but felt the blue thunderstorm element really chimed. It’s especially apt that the pathogen cluster in the middle of the image is on it’s own, given that it’s rare to find this event in the brain. These events are akin to a needle-in-a-haystack and sometimes takes many biological repeats and hours sitting at the microscope hunting them down… which makes their discovery all the more exciting!”

Despite the challenges in microscopy and sample preparation, the project yielded powerful images that enhance the understanding of fungal infections in the brain. Vanessa has since moved on to studying Aspergillus fumigatus, focusing on lung invasion and protein localization, aiming to leverage and expand her microscopy skills. “Visualizing infections in this way opens up the research to a wider audience”.

Art Meets Medical Mycology

  1. Comic book A Brick in the Wall. Credit: Peony Gent.
  2. Pharmakon statue, Streatham campus outside the MRC CMM. Sculpture design: Still/Moving.
  3. Image of Dr Liliane Mukaremera (University of Exeter) from the Researching Resistance photography exhibition. Credit: Simon Ryder.
  4. Performing the Mycobiome by Carolyn Deby / sirenscrossing. An MRC CMM supported research project which fed into site-specific performances of becoming fungi, becoming forest: performing the mycelial city, commissioned by 2021 Coventry UK City of Culture and Coventry Biennial of Contemporary Art. Photo: Adele Mary Reed. Performer pictured: Jia-Yu Chang-Corti
  5. Still from the movie Our Body is a Planet. Credit: Leonie Hampton, Still/Moving.

 

This image shows a selection of projects by artists employing various expression forms (illustration, sculpture, photography, performance and film). These artists have collaborated with the MRC CMM to develop, co-create and deliver new ways of communicating our science in uplifting, innovative and inspirational ways. These projects enable artists to come into the Centre and immerse themselves in its research. There is a two-way exchange between the artist and the researchers, enabling them to work closely together and share knowledge, understanding and build new skills. The picture also features MRC CMM’s Public Engagement and Communication Manager, Rachel Etherington.

Rachel commented “It has been a real pleasure to work with the scientists to produce these eye-catching images. I now have a much deeper appreciation of the beauty and danger in these microscopic organisms, and I am proud of the fact that we can share our work with different audiences”.