A new fluorescent chytrid infection model allows researchers to follow pathogen invasion in real-time, a key step toward developing treatments
At least 90 amphibian species have been driven to extinction as the frog-infecting chytrid fungus has spread across the world.
Caused by Batrachochytrium dendrobatidis (Bd), the severe impact of the fungus is partly due to its remarkably large host range, being able to infect over 700 amphibian species.
Understanding how Bd can infect and kill such a wide range of amphibians is key to mitigating this pathogen and preventing further loss of biodiversity.
Despite decades of study into this important pathogen, researchers previously lacked the tools to precisely investigate the factors involved in infecting the amphibian host and evading its immune system.
Now, a team from the University of Melbourne and the University of Rochester has developed stable transformation protocols to produce a fluorescent chytrid infection model, making it possible to track the pathogen in real time and help tease out the factors that make the chytrid fungus so deadly by watching as it infects and grows inside the cells.
This is the first step in developing novel chytridiomycosis treatment options and hopefully, slowing down the amphibian decline.
The research team included Dr Rebecca Webb, Professor Lee Skerratt and Associate Professor Lee Berger from the University of Melbourne and Dr Andrea L Vu, Mr Francisco De Jesús Andino and Professor Jacques Robert from the University of Rochester.
Cycle of chytrid infection
Bd produces zoospores (mobile fungal spores) which infect amphibian skin via germ tube, before developing into zoosporangia within epidermal (skin) cells. Zoosporangia then produce a new generation of infectious zoospores that are then released into the amphibian’s bloodstream.
High levels of zoospore infection result in the disease chytridiomycosis, characterised by abnormal skin, including increased sloughing, ulcers and erythema. The skin disruption can lead to electrolyte depletion in amphibians, resulting in cardiac failure and death.
So understanding aspects of Bd virulence like zoosporangia maturation, detection and evasion of Bd by the amphibian immune system will hopefully inform targeted interventions.
Developing the new tool
Genetic transformation is an incredible tool for testing gene function. This is especially useful for pathogens because it can be used to pinpoint exactly how they infect and kill the host.
Stable genetic transformation of an organism involves the integration of a transgene into its genome, creating a stable line displaying the desired traits. Inserting or removing different genes in Bd will help researchers understand their role in the infection process.
As a proof of concept, researchers started by transforming the chytrid fungus so it would express a red fluorescent protein. Using the fluorescent chytrid in a cell-line infection assay, the team could then easily watch the fungus as it infects and grows inside the cells.
For many years, in vitro studies of Bd have used a tryptone-based growth media that is not representative of the host environment, limiting the ability to fully understand virulence factors.
These improvements in the cell-line assay have created an essential tool for investigating the pathogen's virulence factors, host resistance traits and potential treatments while helping to reduce the use of live animals in experiments.
Next steps
The team has already had requests from researchers around the world, keen to use the fluorescent chytrid to track infection patterns and evasion of the host's immune system.
Funding
This work was supported by a 2024 Early Career Researcher Grant, ECR Global Collaboration Award and Australian Research Council grants DP220101361 and FT190100462
Publication
Webb RJ, Vu AL, Skerratt LF, Berger L, De Jesús Andino F, Robert J (2024) Stable in vitro fluorescence for enhanced live imaging of infection models for Batrachochytrium dendrobatidis. PLoS ONE 19(8): e0309192. https://doi.org/10.1371/journal.pone.0309192
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First published on 3 September 2024.
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