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

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

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.

Technical Summary

Studying innate immunity at mucosal surfaces in mammals is technically difficult because these surfaces are located internally, and adaptive immunity complicates the picture. Here, we will use the Xenopus tropicalis tadpole as a model system to explore the innate defence function of the mucus barrier that overlays the tadpole skin. Importantly, this model will allow us to study innate defence mechanisms in the absence of adaptive immunity, which does not develop until metamorphosis. We have shown that the tadpole skin secretes a mucin, Otogelin, which forms the molecular framework of a mucus barrier; similar to mucus that protects the mucosal surfaces in mammals. We have identified other mucus components (IgG Fc gamma binding protein (FCGBP), Intelectin and Wap) that have been implicated in mucosal innate defence. These molecules have homologues in mammalian mucus and yet their innate defence roles remain unclear.
We will use the X. tropicalis model system to define roles for these molecules in the innate defence of the tadpole against infection and provide insight to their roles in other organisms, including man. We will first knock down the individual molecules and assess the effectiveness of the mucus barrier. We will challenge tadpoles with Aeromonas hydrophila, which we have shown causes infection of tadpoles at high dose. Depletion of these molecules should render tadpoles more sensitive to infection. We will investigate the structural properties of the mucus with and without these molecules. We will also establish their antimicrobial activity, in protecting the tadpole from infection and use recombinant proteins to test their potency. Mucus contains other proteins implicated in innate defence. We will use this system to identify and define their roles.
Uncovering fundamental mechanisms of innate defence may lead to novel strategies and targets to treat disease, and discovery of novel antimicrobial proteins will be useful in the fight against infection.

Planned Impact

Studying the innate defence mechanisms of X. tropicalis tadpoles will have impact beyond its academic significance. Understanding how the tadpole defends itself against infection will uncover fundamental principles in the innate defence of this organism. A potential impact of our research concerns global amphibian decline. This is a phenomenon driven by a number of factors and chief among them is disease and the impact of global warming. Using an amphibian model such as X. tropicalis to study natural innate defence mechanisms may give new insight into how disease can be tackled and the effect of climate change on innate defences to infection. Amphibian conservation may benefit from our research through generating publicity and awareness of the problems that amphibian's face, whilst our own research may benefit from such a partnership.
These studies will also generate new discoveries about the biology of mucosal surfaces that are relevant to animal health. We have identified a number of molecules secreted from the tadpole skin that we expect to be involved in innate defence and furthermore a number of these molecules, in humans, show altered expression or mutation in disease. Investigating their functional roles may result in new clinical targets for the development of therapeutics. This would ultimately benefit patients suffering from debilitating mucosal diseases such as inflammatory bowel disease, gastric ulcers and asthma, but may also benefit the pharmaceutical and biotechnology companies that will have new drug targets; better understanding barrier organisation may aid with improved drug delivery across mucosal surfaces. One of the molecules we have identified is potentially a novel antimicrobial protein and we will be testing its activity in this regard. If these activities were proven then there would be potential for commercial exploitation, which would benefit the University of Manchester in terms of its goal to develop impact from research and also economically. Also, there is a pressing societal need to develop new types of antimicrobial molecules to combat increasing antibiotic resistance, which is a global issue. Identifying natural antimicrobials is one strategy and our research could help in this regard.
The findings of our research may also be important in terms of the development of the model as a platform to study disease and to screen novel therapeutics. The tadpole skin is a mucosal surface and contains motile multi-ciliated cells, so we envisage that it could be used to mimic both mucosal disease and diseases of cilia motility (ciliopathies). Such a system may be useful for screening new molecules for efficacy and therefore our research may benefit the pharmaceutical industry and clinicians interested in developing therapies for these diseases. The researcher Co-I, Eamon Dubaissi, has a keen interest in the translational impact of research and has attended many courses and workshops in this field. He intends to continue to develop his interest in this area, which may be of economic and societal benefit in future, either within this research area or elsewhere.
During the project, we expect to have societal impact, particularly in the local area, with active participation in public engagement events. All the applicants have taken part in public engagement events in the recent past and are keen to do so in future. Likely beneficiaries include the local community and also young students who will have the opportunity to interact with scientists and gain an insight into a research career and why research is important for everyone in society. Public enthusiasm for, and understanding of, science, is of benefit to all.

Publications

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Dubaissi E (2018) Functional characterization of the mucus barrier on the skin surface. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have comprehensively characterised (at a biochemical and biophysical level) and defined the host protective role of the major structural component (a mucin glycoprotein polymer) of the Xenopus tropicalis tadpole skin surface mucus barrier. We have shown this mucin (which we have named MucXS) bears a strong similarity in sequence, domain organisation, O-glycosylation and structural properties to mammalian gel-forming mucins that underpin mucus barriers that line the internal mucosal surfaces (eg. lung and gut). We demonstrated that this mucin forms the structural basis of a surface mucus barrier (~ 6 microns thick). Depletion of MucXS using genetic approaches markedly reduces the thickness of the mucus barrier (~ 900nm) and results in susceptibility to infection by an opportunistic bacterial pathogen (Aeromonas hydrophila). This work has identified the Xenopus tropicalis tadpole skin as a powerful model system to define mucus barrier function and host-microbe interactions in mammals.

We have also undertaken work to characterise other potential components of the mucus barrier (FCGBP, Wap, intelectin). Using the same genetic approaches successfully employed to study MucXS, we depleted each of these molecules. We discovered that alongside their predicted roles in innate immunity they had unexpected roles in tadpole development. While this closed off avenues for using this approach to investigate their roles in innate defence, it has highlighted alternative functions in development of the mucosal surface which warrants further study in the future. Importantly, we have generated reagents (antibodies and RNA probes) that can be used in future work and made available to the research community.

Finally, we undertook a proteomic approach to identify other molecules in the mucus barrier with predicted innate defence function in mammals. However, this approach did not identify any obvious candidates. We therefore took an alternative approach and profiled gene expression changes in the early stages of bacterial infection. This led to the identification of a number of genes related to immune defence at mucosal surfaces (e.g. Interleukin 17c, Chemokines and the poly immunoglobulin receptor, pIgR). This may form the basis of a system to explore the role of these molecules in innate defence in mammals.
Exploitation Route We have shown that the tadpole skin surface mucus has similar protective barrier properties to mammalian mucus barriers. Moreover, the ciliated tadpole skin surface provides a novel model to study mucociliary epithelia as found lining the lungs of mammals. Therefore, this model system could provide a powerful in vivo tool to study host-pathogen interactions at mucosal surfaces and could be used as an initial screening tool for drugs and other therapeutic modalities that are delivered across mucosal surfaces.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description A live model to study mucociliary clearance in health and disease
Amount £494,136 (GBP)
Funding ID NC/S001034/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 08/2018 
End 07/2021
 
Description Science uncovered (Manchester Museum) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Manchester Museum - 'Science Uncovered' part of the Europe-wide event, European Researchers' Night' (2015 & 2016). Showcased our research on the mucus barrier - discussed our research with members of the general public and increased awareness and interest in the role of the mucus barrier in the protection of mucosal surfaces.
Year(s) Of Engagement Activity 2015,2016