Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease

Abstract

The causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance. In this study, the impact of prolonged culture on N. perurans virulence and its proteome was investigated. Two isolates, attenuated and virulent, had their virulence assessed in an experimental trial using Atlantic salmon smolts and their bacterial community composition was evaluated by 16S rRNA Illumina MiSeq sequencing. Soluble proteins were isolated from three isolates: a newly acquired, virulent and attenuated N. perurans culture. Proteins were analysed using two-dimensional electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS). The challenge trial using naïve smolts confirmed a loss in virulence in the attenuated N. perurans culture. A greater diversity of bacterial communities was found in the microbiome of the virulent isolate in contrast to a reduction in microbial community richness in the attenuated microbiome. A collated proteome database of N. perurans, Amoebozoa and four bacterial genera resulted in 24 proteins differentially expressed between the three cultures. The present LC–MS/MS results indicate protein synthesis, oxidative stress and immunomodulation are upregulated in a newly acquired N. perurans culture and future studies may exploit these protein identifications for therapeutic purposes in infected farmed fish.

Differential Exoproteome and Biochemical Characterisation of Neoparamoeba perurans

“…However, N. perurans specific proteins were identified, many of which had endopeptidase activity which correlates with cytotoxicity, such as Cn-Zn superoxide dismutase (SOD) and physarolisin, (Table 1). SOD expression has previously been found to be significantly higher in the cytoplasmic fraction of a wild type isolate of N. perurans [20]. Although SOD is reported to defend parasites against the host response, its presence here may be part of the attenuated isolate's defence mechanism in response to excess production of endogenous oxidative stressors from its own commensal microbiota.…”

“…Encompassing the complexity of N. perurans and its microbiota, protein databases of several key genera were included from a recent N. perurans microbiome study utilising these two amoeba isolates [20]. Furthermore, to facilitate the identification of N. perurans proteins the Amoebozoa reference proteome from UniProt was included in the analysis, in conjunction with the N. perurans database (20,887 proteins (v2, 07/08/2019, CSIRO) [20]). Overall, the LC-MS/MS results revealed a stark contrast between exoproteomes of attenuated and virulent cultures and most remarkably, that the attenuated culture significantly secretes more proteins than the virulent.…”

“…The inclusion of proteins from a broad range of amoebae in the Amoebozoa dataset was to both facilitate and validate the identification of amoeba-specific proteins. Co-culturing bacteria of N. perurans contributed to the protein identifications in the cytoplasmic protein investigation of N. perurans [20], therefore, it is imperative to distinguish between N. perurans specific and bacterial specific proteins. The microbial communities of the attenuated and virulent cultures were previously evaluated using 16S rRNA gene sequencing [20].…”

“…Co-culturing bacteria of N. perurans contributed to the protein identifications in the cytoplasmic protein investigation of N. perurans [20], therefore, it is imperative to distinguish between N. perurans specific and bacterial specific proteins. The microbial communities of the attenuated and virulent cultures were previously evaluated using 16S rRNA gene sequencing [20]. The key genera identified as part of this analysis were used to inform the creation of a bacteria protein database consisting of 148,582 proteins.…”

“…The naïve smolts were issued an AGD gill score [24] of 1. An attenuated culture of N. perurans, previously shown to have lost virulence [20] was used as the attenuated isolate (4 years in culture at the time of protein extraction). To acquire a virulent isolate, fresh trophozoites were collected by swabbing AGD affected gills and placing swabs in 0.2 µm filtered sterile seawater for 4 h to release trophozoites from the swab into the seawater.…”