Virulence factors associated with amoebic gill disease from Neoparamoeba perurans

Abstract

Determining the virulence factors associated with pathogenic organisms is imperative in comprehending disease pathogenesis and ultimately paves the way for methods of treatment and disease management. Neoparamoeba perurans is an ectoparasitic amoeba responsible for the hyperplastic gill infection of marine cultured finfish referred to as amoebic gill disease (AGD). Neoparamoeba perurans is suspected to have a repertoire of putative virulence factors that enable the attachment and colonisation of the host, as well as resistance to host immune response. The primary aim of this study was to identify the virulence factors of N. perurans (both amoebic and bacterial) using a proteomic approach, and to increase understanding of the parasite’s mechanisms as an infectious agent in AGD of farmed fish. This thesis is divided into five chapters comprising: an introduction to AGD, N. perurans and virulence factors, three experimental chapters written as manuscripts for publication and finally, a general discussion that reviews the findings and future outcomes of this research. The first experimental chapter (chapter two) confirmed the reported loss of N. perurans virulence in long-term culture, as determined by an in vivo challenge trial. This work validated AGD infection in the virulent cohort and validated the lack of AGD infection in the attenuated cohort by gill scoring, histopathology, and qPCR of gill tissue. The microbiome of the virulent-1 and attenuated N. perurans were characterised by 16S rRNA Illumina MiSeq sequencing for informing the bacterial database protein search. Two-dimensional gel electrophoresis (2D gel) and LC-MS/MS analysis revealed that hydrophilic virulence proteins are differentially expressed between a virulent-1, virulent-2 and attenuated culture. Differentially expressed proteins were actin-associated proteins, lipoxygenase, an ABC membrane transporter and profilin. Overall, these proteins suggest that the virulent-2 culture of N. perurans maintains elevated levels of proteins involved in cytoskeletal, oxidative and immunomodulatory roles. The second experimental chapter (chapter three) assessed the extracellular secretions, or exoproteome, of a virulent-3 and attenuated parasite for virulence factors. Using label–free LC-MS/MS, proteins that were differentially expressed were found to be elevated in the attenuated parasite. Additionally, the extracellular secretions of the attenuated parasite had a greater cytotoxic effect on the salmonid RTgill W1 cell line compared with that of the virulent-3 isolate. This effect was believed to be associated with the increased growth of bacteria in the attenuated culture of N. perurans. Antibiotics were employed to reduce the presence of bacteria in the culture, which lessened the cytotoxicity observed in RTgill W1. Neoparamoeba perurans specific proteins were found in both exoproteomes that have serine protease activity which may facilitate AGD infection, but are not the main drivers of cytotoxicity. Additionally unknown and hypothetical proteins of N. perurans were identified that warrant further investigation. The third experimental chapter (chapter four) investigated the cell-surface and membrane proteome of a virulent-3 and attenuated N. perurans. Proteins involved in potential cell-mediated adhesion roles such as GAPDH and GTPase signalling proteins were found and may represent putative targets of treatment. Overall, the proteins identified reveal moonlighting roles for common proteins such as proteins involved in glycolysis that may aid host ligand attachment. The outcome of this work is expected to facilitate progress in the design of N. perurans targeted therapies for the treatment of AGD in farmed fish. Developing treatments that target and disrupt N. perurans will improve gill health in salmonids and will promote aquaculture sustainability and productivity.