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

Kerrie Ní Dhufaigh,Ian O’Connor,Orla Slattery,Natasha Botwright,Eugene Dillon,Eugene Maccarthy,Victor Birlanga,Anita Talbot

doi:10.1038/s41598-021-85988-8

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.

Highlights

The causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance
The control cohort remained negative for N. perurans for the duration of the trial, as confirmed by gill scoring, quantitative polymerase chain reaction (qPCR) and histology
One fish from the attenuated cohort and all fish from the virulent cohort sampled at 7 dpi were qPCR positive for N. perurans

Summary

Introduction

The causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance. The microbiome of an attenuated and virulent isolate of N. perurans, both maintained at 16 °C, was characterised by 16S rRNA Illumina MiSeq sequencing to identify similarities and divergences between isolates. This collated information was used to inform the ’bacterial protein database’ that was incorporated with an amoebozoa database and N. perurans specific database, into the Andromeda search engine of M axQuant[31] for protein identification from 2D PAGE. These phenotypes were compared by characterising their soluble proteome using 2D PAGE, alongside a newly acquired culture of N. perurans, providing a proteomic timeline of differential virulence

Methods

Results

Conclusion