Defining mechanisms of mucosal innate defence using the X. tropicalis tadpole

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Pathogens are disease causing microorganisms and viruses, which represent a severe challenge to the health and survival of animals. When animals succumb to infectious disease, it is because pathogens have breached the body's natural defence mechanisms. Broadly, these defences fall into two types, innate and adaptive immune defences. Adaptive immunity is a specific defence, where antibodies generated against a particular pathogen are used to rapidly respond to infection. However, it is advantageous for animals if pathogens never have the opportunity to infect the body in the first place. Innate immunity (non-specific immunity) is the first line of defence against infection and includes the generation of physical barriers to entry and the production of molecules that actively destroy pathogens. One such physical barrier used by exposed tissues in the body (e.g. lungs and gut) is the production of a mucus layer. Mucus traps pathogens preventing them from accessing the underlying cells and also contains other innate defence molecules such as antibacterial agents. It is important to study mucus to understand how it functions and what happens when it becomes dysfunctional, and is breached. Severe diseases can arise due to lack of a properly functioning mucus barrier; these include inflammatory bowel disease, stomach ulcers, and asthma. Mammalian models, including mice, are often used to study mucus in the lungs and gut. However, simple non-mammalian organisms can also provide valuable insight into evolutionarily conserved mechanisms of mucus function. In this proposal, we introduce the frog, Xenopus tropicalis, and specifically the mucus on the surface of the tadpole stage, as a model to study innate defence.X. tropicalis is a model organism commonly used in developmental biology to understand basic concepts in how embryos develop and the molecular processes that occur. In recent years, we have been studying how the embryonic skin changes as the embryo develops into a tadpole, prior to becoming a frog. We have identified a number of cell types in the tadpole skin, including two types of secretory cells that together secrete molecules that form a mucus layer over the surface of the skin. We are proposing that this simple model can be used to study aspects of mucosal innate defence that will be applicable to other organisms, including humans. Indeed, we have identified a number of innate defence molecules in the tadpole mucus that have known homologues in humans and yet their functions are largely unexplored. In this grant proposal, we intend to interrogate their functions through a number of means. The X. tropicalis tadpole model has some distinct advantages in that the skin directly faces the environment and so conditions can be altered to observe the effects. This is not the case with mammalian mucus barriers, which are usually found on tissues within the body and are thus more difficult to access. In addition, X. tropicalis tadpoles have yet to acquire adaptive immunity (which happens later in their development) so any observed effects will be due to innate defence mechanisms, rather than a more complicated, combined adaptive response to infection. We intend to genetically alter the expression of the innate defence molecules in the tadpoles skin mucus layer and then challenge the tadpoles with a potential pathogen found in their native environment (the bacterium, Aeromonas hydrophila), in order to understand the importance of each molecule. We will look at their structural roles in the physical barrier and their potential functional roles as anti-microbial agents. We aim to advance the understanding of how the tadpole defends itself against infection and ultimately how evolutionarily conserved innate defence mechanisms function in mucus barriers. This could potentially lead to new targets for treating disease.