Why did we fund this project?
This award aims to expand the use of Galleria mellonella as a replacement for mice in bacterial and fungal immune challenge studies by developing transgenic larvae with fluorescent phagocytic cells (hemocytes).
One of the barriers to the uptake of Galleria as a model organism has been the inability to genetically modify the larva. Galleria possess a number of advantages over other invertebrates, cell lines and murine models including being able to infect and maintain the larvae at 37oC. This allows infection studies to be performed at the same temperature as humans, which is important for cellular kinetics and temperature-sensitive protein production. However, wider uptake of Galleria is dependent on methods being developed to create transgenic Galleria to study interactions between pathogens and components of the immune system. Studies are currently limited to qualitative analysis of infection progression, typically by either assessing the melanisation of the larvae (a response initiated by the immune system to capture pathogens at the wound site), or by assessing the larvae survival rate. In a proof-of-concept study, Dr James Wakefield and colleagues have developed a DNA injection protocol and created a transgenic ubiquitously fluorescent Galleria larva for the first time.
With NC3Rs funding, James and colleagues will expand on their successful pilot study to fluorescently label Galleria hemocytes. The protocols will be optimised and validated in a functional study with Candida albicans to build confidence in the methodology. A collaboration is in place with BioSystems Technology for the distribution of the transgenic Galleria to promote the dissemination and uptake of this model.
Growing evidence supports the use of the larvae of the waxmoth, Galleria Mellonella, as an in vivo animal partial replacement model, particularly in the related fields of infection, immunity and inflammation. Although they are insects, their immune system is very similar to humans. There is huge potential impact on the number of rodents used in scientific research, if Galleria can be optimised as a model organism. However, currently, the health of larvae after injection with bacteria or fungal pathogens is monitored only by a crude assessment of whether/when they turn black and die.
In the first part of this project, we will make transgenic larvae that glow (fluoresce) differentially under stress, infection, immune challenge or inflammatory status. We will light up their macrophages, the cells in the immune system that wander around, looking for foreign pathogens and "eating them". This will allow us to measure the movement of this important type of cell before and after infection, revolutionising the amount and type of information we can obtain about the way animals, and we, respond to infection.
The second aim of the project is to use these fluorescent Galleria larvae to understand how the most common cause of human fungal disease in the world, a pathogen called
Candida albicans, hides itself from our immune system. We know that Candida has something on their cell surface that the immune system normally recognises but that, in some cases, their environment triggers a change that ends up masking this signal. We will expose Candida to many of these sorts of environments and ask how effective the glowing Galleria macrophages are at recognising the fungus.
Our work will therefore not only figure out the key molecular signals that mask Candida from the immune system, potentially identifying candidate molecules for new anti-fungal agents, but also has the potential to revolutionise the use of Galleria as mouse replacement model, saving many thousands of mice from being used in scientific research in the future.