Characterisation of Galleria mellonella innate immune response to Mycobacterium tuberculosis infection

The Greater wax moth (Galleria mellonella, GM) is an insect pest of honeybee hives. However, it has been found that the caterpillar of GM can be infected in the laboratory by human bacterial pathogens. Such GM "infection models" can be used to understand how pathogens cause diseases in humans, helped by the many similarities between the insect and human immune system. GM are now being increasingly being used as an alternative to animal infection models. However, a major limitation to the more widespread use of GM-based infection models is the lack of reagents that can be used to characterise the GM immune system. Availability of such GM reagents would lead to a greater uptake of GM-based infection models and will further reduce the use of animals in research.

In this exciting Fellowship, I will generate reagents, which for the first time, can distinguish the six sub-types of haemocytes (considered equivalent to white blood cells populations in humans) found in GM. These reagents will be used to characterise the immune response in a GM-Mycobacterium tuberculosis (MTB) infection model. MTB is the cause of human tuberculosis (TB) which, until the covid pandemic, was the leading worldwide cause of death due to infection. To meet the WHO goal of ending the TB epidemic by 2035, alternative infection models - like GM are urgently needed. In the work leading up to this Fellowship, I have established a GM-MTB infection model. Unlike the most widely used mouse model, granulomas - the hallmark of human TB - are produced in GM. We know that haemocytes are required for their formation. However, we do not know if only a select few or all six sub-types of GM haemocytes are needed. Knowing which sub-types are involved will enable a greater understanding of insect immunity and, in particular, the haemocyte sub-types that are involved in the response of GM to MTB. In addition, I will analyse which genes are expressed in GM caterpillars infected with MTB, allowing direct comparison with human studies. The outcome of this project will be the generation of reagents and methods that will allow a greater understanding of GM immunity, in particular, the response of larvae to MTB infection, and how close it is to the human immune response to MTB. All reagents, methods and results will be made publicly available. A successful conclusion to this project will result in a greater uptake of not only the GM-MTB infection model, but also in other GM infection models (of which there are over 65) that are currently in use.

I have previously established and characterised the use of Galleria mellonella (GM) as an infection model for Mycobacterium tuberculosis (MTB). The GM-MTB model develops granuloma-like structures, a hallmark of human tuberculosis. Preliminary data has shown that MTB induces GM haemocytes to form such structures, but it is unknown which of the six sub-types are required for their formation or maintenance. I hypothesise that a select sub-types are required for both. However, as a model in its infancy, further structural and functional innate immune analysis of GM is hindered by the lack of reagents. Transcriptomes from naïve and MTB-infected GM haemocytes will also be generated in this Fellowship, from which genes whose expression distinguishes each of the six GM haemocyte sub-types will be identified. I plan to synthesis RNA probes for genes of interest and through fluorescence in situ hybridisation (FISH) determine, for the first time, the cellular composition of the granuloma-like structures both in in vivo and ex vivo GM-MTB infection models. The generated transcriptomes will also be used to comprehensively compare GM and human MTB transcriptomes, which is urgently needed to encourage further uptake of the GM-MTB model. The transcriptome data will be used to identify innate immune effector inhibitory targets for functional gene knockdown assays using RNA interference. This will enable genes of interest, important for granuloma formation and maintenance, to be identified and validated. Reagents (RNA probes and inhibitors) and resources (GM haemocyte transcriptomes) generated will be of significant value to the wider GM community, and will be made publicly available, facilitating uptake of not only GM-MTB but also other GM-based infection models.