The Xenopus tropicalis response to infection by Batrachochytrium dendrobatidis

The individual-level response of cold-blooded vertebrates (ectotherms) to infectious disease is a neglected area of research. However, the realized and potential economic costs of recently emerged diseases of ectotherms are substantial. The chytrid fungal pathogen, Batrachochytrium dendrobatidis, was recently described in 1997 as a pathogen of captive amphibians, and chytridiomycosis is presently devastating commercial amphibian farms, research colonies and natural populations on a global basis. The current state of knowledge regarding this fungal infection is manifestly inadequate; major gaps in our understanding are how amphibians respond to this chytrid and how this chytrid causes the death of its host. We lack this information due to the absence of a reliable amphibian model system that can be used to identify the biological mechanisms underpinning the host-pathogen interaction. Our project will rectify this imbalance by investigating the feasibility of using the African clawed frog Xenopus tropicalis as a model system to research chytridiomycosis. X. topicalis, unlike its sister taxa X. laevis, is highly susceptible to chytridiomycosis. The fully annotated genome sequence for X. tropicalis is now complete which facilitates the analysis of amphibian responses to infection as measured by changes in key infection-related genes. Our initial experiments will focus on characterising the X. tropicalis-response to infection under (i) different dosages of the pathogen and (ii) different temperatures. Using confocal microscopy, we will visualise the Xenopus-pathogen interaction by staining against key antigens that are associated with the humoral and cell-mediated arms of the Xenopus immune-response. Subsequently, we will then analyse the changes in gene-expression between uninfected, non-lethally infected and lethally infected animals. We will do this by designing robust quantitative PCR assays that monitor gene-expression over the time-course of an infection for classes of genes that are involved in (i) the Th1 (cell-mediated), (ii) the Th2 (antibody-based) and (iii) innate immune response. We will focus our analyses on genes that are known to be important in controlling infection in other vertebrate species, and that we have data-mined from the X. tropicalis genome by our use of comparative-genomic techniques. These analyses will reveal, for the first time, the type and magnitude of the amphibian adaptive and innate response(s) to infection. Understanding these processes will allow us to further develop X. tropicalis as a model species within which we can investigate host-pathogen biology within a tractable system that has well-developed immunological and genetic-toolboxes. This will allow us to apply our research to vaccine development against B. dendrobatidis, an urgent goal given the global economic cost of this disease. Finally, determining the type of host response that is associated with infectious disease in amphibians will have a broad relevance for understanding host responses in other, thus far neglected, cold-blooded species.

This research project will undertake a focused 1-year pilot study to assess the applicability of Xenopus tropicalis as model system within which to investigate the mechanisms that underpin the host response to infection by B. dendrobatidis. Using X. tropicalis, we will perform challenge experiments within defined inbred lines and will monitor the intensity of infection in experimental animals by using quantitative realtime PCR. We will use confocal microscopy and FACS against a range of immune-associated antigens (specifically, B-cells, IgM, IgY, IgX and T-cell receptors) to visualise the response that X. tropicalis mounts against B. dendrobatidis, and will quantify the degree of involvement, and type of response, within different tissues. We will subsequently use our model to assess the time-dependent expression profiles of candidate infection-related genes. We will do this by developing robust quantitative PCR assays to measure expression of key components of the X. topicalis adaptive and innate immune-response. These candidate infection-related genes have been data-mined from the X. tropicalis genome using gene-ontology (GO) analyses. These data will be used to investigate the hypothesis that successful response to infection in epidermal pathogens requires the activation of a Th1-type inflammatory reaction. To this end, we will specifically focus our attentions on discriminating the differential upregulation of cytokine subsets that are associated with Th1 and Th2 responses. We will also focus our attention on genes involved in innate immunity, such as scavenger and Toll-like receptors, as well as genes involved in the production of antimicrobial peptides (Magainin, Xenopsin, PYLa).