Establishment of the thermotolerant Arabian killifish as a model for infection studies
Lead Research Organisation:
UNIVERSITY OF EXETER
Department Name: Biosciences
Abstract
Zebrafish have been widely used in recent years to study the interaction of the immune system with a range of microbes that cause human disease - yet none of these reflects the importance of temperature on the expression of virulence traits. We have therefore specifically chosen the Arabian killifish (Aphanius dispar) for this proposal as it is naturally acclimated to high temperature environments (37-40'C) - allowing us to investigate the pathobiology of microbial infections, and the behaviour of pathogens at temperatures consistent with our own. The proposal builds upon work we have already undertaken in a doctoral project (Kudoh, Ramsdale & Hamied) to develop the model for fungal (Candida albicans) infections. So far our model has shown all of the benefits of the zebrafish model, including excellent live cell fluorescence and brightfield imaging, antifungal screens, and (as we have shown) the ability to create transgenic and CRISPR/Cas9 mutant lines. In the project we are proposing, we now have the following scientific aims:
(1) Generate transgenic fish lines that facilitate live cell imaging of key immune cell populations - replacing rodent models.
(2) Generate mutant fish lines with defects in genes linked to human disease that are predisposing factors for infection - replace rodent models for mechanistic infection studies.
(3) Establish a publicly available, searchable Arabian killifish genome database /portal based on our existing sequence data - to allow the community to design experiments in our system. Use RNAseq data from different stages of embryo development and infection at different temperatures (30'C and 37'C) to map out key markers of the immunome that can be made available as a track on the killifish genome viewer so that users of the model can assess stage specific pathobiology related events.
(4) Disseminate our tools e.g. (fish lines), data (immunome) and methods (imaging and infection) to the wider infection biology research community and provide opportunities to researchers in zebrafish, mice and other models to use the Arabian killifish as an alternative model.
In relation to NC3Rs our model has the following advantages over rodents and zebrafish:
Replacement (Partial): Our Arabian killifish embryo model could replace many mice that are used for studies of interactions with immune cells and tissues, initial virulence testing and antifungal / antibacterial drug screens. TOTAL in local facilities, with 25% uptake within the MRC Centre for Medical Mycology at the University of Exeter over 5 years = 1,700 mice.
Reduction: In the Arabian killifish data can be collected from different stages of infection in a single live animal, greatly reducing the unnecessary culling of mice and associated high husbandry costs. Assuming 5% take-up over five years a reduction in use of 45,687 mice for infection studies could be achieved.
Refinement: Standard screens of human pathogens in zebrafish embryos are undertaken at 32-33'C , imposing unnecessary thermal stress. Scientifically the model has greater validity as human pathogens need to grow at 37'C in order to express their full suite of virulence traits. There is also a reduced need for tricaine anaesthesia due to less developmental twitching. A TOTAL of 74,820 zebrafish are used p.a (based on literature analysis) so with a 5% take up over five years 18,750 zebrafish embryo studies could be refined.
As core members of the MRC Centre for Medical Mycology (CMM - Ramsdale and Farrer) and the Aquatic Resource Centre (ARC - Kudoh) at the University of Exeter we have state-of-the-art expertise in fish biology, fungal molecular and cell biology and cell biology, immunology and bioinformatics to support this work. Exeter therefore provides a vibrant, exciting and scientifically excellent research environment, and is the perfect place for these studies to be executed.
(1) Generate transgenic fish lines that facilitate live cell imaging of key immune cell populations - replacing rodent models.
(2) Generate mutant fish lines with defects in genes linked to human disease that are predisposing factors for infection - replace rodent models for mechanistic infection studies.
(3) Establish a publicly available, searchable Arabian killifish genome database /portal based on our existing sequence data - to allow the community to design experiments in our system. Use RNAseq data from different stages of embryo development and infection at different temperatures (30'C and 37'C) to map out key markers of the immunome that can be made available as a track on the killifish genome viewer so that users of the model can assess stage specific pathobiology related events.
(4) Disseminate our tools e.g. (fish lines), data (immunome) and methods (imaging and infection) to the wider infection biology research community and provide opportunities to researchers in zebrafish, mice and other models to use the Arabian killifish as an alternative model.
In relation to NC3Rs our model has the following advantages over rodents and zebrafish:
Replacement (Partial): Our Arabian killifish embryo model could replace many mice that are used for studies of interactions with immune cells and tissues, initial virulence testing and antifungal / antibacterial drug screens. TOTAL in local facilities, with 25% uptake within the MRC Centre for Medical Mycology at the University of Exeter over 5 years = 1,700 mice.
Reduction: In the Arabian killifish data can be collected from different stages of infection in a single live animal, greatly reducing the unnecessary culling of mice and associated high husbandry costs. Assuming 5% take-up over five years a reduction in use of 45,687 mice for infection studies could be achieved.
Refinement: Standard screens of human pathogens in zebrafish embryos are undertaken at 32-33'C , imposing unnecessary thermal stress. Scientifically the model has greater validity as human pathogens need to grow at 37'C in order to express their full suite of virulence traits. There is also a reduced need for tricaine anaesthesia due to less developmental twitching. A TOTAL of 74,820 zebrafish are used p.a (based on literature analysis) so with a 5% take up over five years 18,750 zebrafish embryo studies could be refined.
As core members of the MRC Centre for Medical Mycology (CMM - Ramsdale and Farrer) and the Aquatic Resource Centre (ARC - Kudoh) at the University of Exeter we have state-of-the-art expertise in fish biology, fungal molecular and cell biology and cell biology, immunology and bioinformatics to support this work. Exeter therefore provides a vibrant, exciting and scientifically excellent research environment, and is the perfect place for these studies to be executed.
Technical Summary
To establish the Arabian killifish as a novel model for infection we will:
(1) Generate transgenic fish lines in Arabian Killifish to allow 4D-imaging of the interactions between the pathogen (tagged with complementary CFP/GFP/RFP markers) and the host fish embryo cells (tagged with mpx:GFP, a neutrophil specific green fluorescent line; mpeg:mCherry, a macrophage specific red fluorescent line; lck:GFP, a T cell specific fluorescent line and IgM1:mCherry, an activated B cell specific fluorescent line). We have already established a method for generating transgenic Arabian killifish lines (eg EF1a:Kaede and bAct:mCherry) with high success rates (only 40 eggs were injected and several TG founder adult fish were identified).
(2) Create CRSIPR/Cas9 mediated mutant fish lines that promote candidiasis focussing on the cftr gene (cystic fibrosis), card9 (familial candidiasis-2), il17rc (familial candidiasis-9) and nox2 (chronic granulomatous disease). We have already successfully used CRISPR/Cas9 in the Arabian killifish to generate a mutant (Hamied et al. 2020). The success rate of generating homozygous mutations by injecting CRISPR RNA was 100% (about 40 eggs were injected and all founder fish became homozygous mutants for gch and tyr genes, CFS Fig.2).
(3) Undertake transcript profiling (RNAseq) of Arabian Killifish embryos with and without Candida albicans at two temperatures (30'C comparable to human skin temperature and 37'C for human body temperature) to better understand Candida virulence, the killifish innate and acquired immune response, and to support gene models for ab initio prediction of genes from our existing Nanopore Promethion genome assembly.
(4) We will establish an Arabian killifish database including a genome browser for the Arabian killifish, which will serve as a powerful community resource, allowing detailed searches for genes and transcripts that will allow infectious disease biologists to plan their own experiments.
(1) Generate transgenic fish lines in Arabian Killifish to allow 4D-imaging of the interactions between the pathogen (tagged with complementary CFP/GFP/RFP markers) and the host fish embryo cells (tagged with mpx:GFP, a neutrophil specific green fluorescent line; mpeg:mCherry, a macrophage specific red fluorescent line; lck:GFP, a T cell specific fluorescent line and IgM1:mCherry, an activated B cell specific fluorescent line). We have already established a method for generating transgenic Arabian killifish lines (eg EF1a:Kaede and bAct:mCherry) with high success rates (only 40 eggs were injected and several TG founder adult fish were identified).
(2) Create CRSIPR/Cas9 mediated mutant fish lines that promote candidiasis focussing on the cftr gene (cystic fibrosis), card9 (familial candidiasis-2), il17rc (familial candidiasis-9) and nox2 (chronic granulomatous disease). We have already successfully used CRISPR/Cas9 in the Arabian killifish to generate a mutant (Hamied et al. 2020). The success rate of generating homozygous mutations by injecting CRISPR RNA was 100% (about 40 eggs were injected and all founder fish became homozygous mutants for gch and tyr genes, CFS Fig.2).
(3) Undertake transcript profiling (RNAseq) of Arabian Killifish embryos with and without Candida albicans at two temperatures (30'C comparable to human skin temperature and 37'C for human body temperature) to better understand Candida virulence, the killifish innate and acquired immune response, and to support gene models for ab initio prediction of genes from our existing Nanopore Promethion genome assembly.
(4) We will establish an Arabian killifish database including a genome browser for the Arabian killifish, which will serve as a powerful community resource, allowing detailed searches for genes and transcripts that will allow infectious disease biologists to plan their own experiments.
