Amoebic gill disease (AGD), caused by the opportunistic amoebozoan parasite Neoparamoeba perurans, is a major disease in salmonid aquaculture [1-3] and there is an urgent need to mitigate the impact parasitic infections have on both fish welfare and sustainable growth in the sector. Development of vaccine technology over the last 20 years has dramatically reduced losses associated with viral and bacterial disease. However, the industry remains beset by the biological threat posed by parasitic infections, including caligid sea lice as well as AGD, which are causing intolerable losses (c.20% annually). AGD is a primary driver of proliferative gill inflammation which impairs the respiratory capacity of fish leading to asphyxia, respiratory acidosis, acute hypertension, circulatory collapse and death . Further, gill inflammation driven by exposure to micro-cnidarian blooms, for example, increases N. perurans gill colonisation and the prevalence of AGD at farm sites .
Treatment options for AGD are currently extremely limited. However, it may be possible to exploit the unique biology of N. perurans in future drug development. Strikingly, within each N. perurans cell is an endosymbiotic kinetoplastid, named Perkinsela, on which it relies for essential metabolic processes [6, 7]. This is a secondary endosymbiosis that is unique among eukaryotes because it does not involve an originally photosynthetic symbiont. Genomic data indicate that the basic physiology of Perkinsela shares many biochemical features with other kinetoplastid pathogens of man and livestock, although due to its small genome size (9.5 Mb, <5,500 genes), it has reduced metabolic capacity . Furthermore, while molecular data indicates that Perkinsela retains some key features of kinetoplastid biology, including polycistronic transcription, trans-splicing and RNA editing, it has lost the genes required for flagellum biogenesis and is therefore aflagellate [6, 8].
Despite its importance, the life cycle of N. perurans is not well understood, and little is known of how the amoeba or its endosymbiont Perkinsela replicate and divide throughout the life cycle. This project seeks to define the life and cell cycles of N. perurans and Perkinsela in detail using light, fluorescence and electron microscopy. In particular, whether the replication of N. perurans and Perkinsela are linked and/or interdependent will be investigated. Further, their genomes will be bioinformatically mined for orthologues of cell cycle protein kinases and other regulators to catalogue the cell cycle machinery and permit functional characterisation. Cell cycle inhibitors may be employed to investigate cell cycle checkpoints, and molecular approaches (e.g. fluorescent tagging, RNAi, CRISPR/Cas9 etc) will be developed to allow functional characterisation of individual cell cycle proteins. Key cell cycle kinases of interest will also be expressed recombinantly to allow activity assays to be developed, which could facilitate future screening of small molecule inhibitor libraries for compounds that exert antiparasitic effects either directly on N. perurans or indirectly by targeting Perkinsela.
It is envisaged that these studies will not only lead to a greater understanding of the intriguing cell biology of the N. perurans-Perkinsela symbiosis, but will identify potential novel drug targets and provide context to investigations of the mode of action of other experimental drugs being developed to treat AGD.