The role of DAF-16 in driving the evolution of immunity mechanisms in nematodes

Although adaptive immunity (the production of antibodies, for example) is unique to vertebrates (animals with backbones), some level of innate immunity is found in all invertebrates and indeed even in plants (R-genes, hypersensitive response) and in bacteria (restriction enzymes). Thus all living organisms have the ability to respond at the molecular level to infectious agents. It is clear that immune systems must change rapidly during evolution / pathogens are under pressure to evolve ever more effective mechanisms to infect their hosts, so the host must evolve ever more effective immune mechanisms to resist them. Given this, one might expect that species that are closely related may nonetheless show significant differences in their resistance to different pathogens. This is indeed true / HIV, for example, causes a fatal disease (AIDS) in humans but very mild, if any, illness in chimpanzees. We have shown that this also holds true for species of nematode (microscopic worms). For example, the fungus Cryptococcus kills the nematode Caenorhabditis elegans within four or five days, but takes three times as long to kill the related species Caenorhabditis remanei. Interestingly, this pattern is also seen when lifespan in the absence of infection is measured. If both animals are kept in disease-free conditions, C. remanei lives much longer, suggesting that immunity and longevity are linked at the molecular level. We hypothesise that this 'link' may result from the key role played in both immunity and longevity by one 'master regulator' gene, called daf-16, which makes the corresponding protein DAF-16. DAF-16 acts to help worms survive in times of stress; it triggers the production of detoxification proteins, immunity factors and proteins that help resist high temperatures ('heat-shock' proteins). We suspect that DAF-16, and the so-called 'downstream' genes that it controls, are prime candidates to be changed during evolution, either via manipulation of the whole pathway (for example, by changing the level or activity of DAF-16) or by changing individual components (the 'downstream' genes). We propose to test this by using a group of nematode species that all belong to the Caenorhabditis genus (i.e. they are 'sister' species). We already know that these species vary in their sensitivity to infectious pathogens and that they vary in their natural lifespan. We also know that they all have a daf-16 gene and, for one species, we know most of the genes that lie 'downstream' of it. Importantly, DNA-sequencing projects have provided the full ('whole genome') sequence for two species in the genus (C. elegans and C. briggsae) and will shortly provide whole genome sequences for three more (C. japonica, C. remanei and an as-yet unnamed species, PB2801). We are therefore in a unique position to be able to explore how evolution has shaped the genes that control ageing and immunity as these species have evolved from one another. By investigating this process, we hope to learn more both about how innate immune systems work, and how evolution shapes these immune systems.

Stress, immunity and ageing are intrinsically linked at the molecular level in many animal species, including humans. The nature of this 'link' is unknown, but it is nonetheless likely that the underlying molecular mechanism(s) are under intense selective pressure, since subtle changes in function will influence all three traits. We hypothesise that different selective pressures that occur during speciation (for example, in response to differing burdens of infection) will lead to diversification of the molecular components that regulate immunity, stress responses and ageing. In most animals this hypothesis is untestable / we know too little about the molecular basis of these traits and about how they vary. However, the nematode C. elegans is an exception / forty years of research has provided an unprecedented level of detail about the molecular biology of ageing, stress responses and, more recently, immunity in this organism. In addition, not only is the genome of C. elegans fully sequenced and annotated, but genome sequencing projects are either complete or near completion for several other species within the genus. Caenorhabditid nematodes thus represent a unique resource to examine the impact of evolution on immunity, ageing and stress responses. We know that all three responses in C. elegans are controlled by the activity of the forkhead transcripton factor DAF-16. This proposal is therefore aimed at asking how DAF-16 and its downstream genes have changed during the diversification of the Caenorhabditis genus. By a combination of expression analysis, gene knockdown, whole genome bioinformatics and inter-species transgenesis, we intend to provide a detailed analysis of the regulation and impact of the DAF-16 pathway in several species within the same genus. This will be the first time that such an analysis has been possible and will therefore provide evidence as to the molecular regulation of immunity during evolutionary diversification.