Abstract
Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host.
Highlights
Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus)
Analysis of the topology of the aa sequence using the Phobius bioinformatic program revealed that this protein has two transmembrane regions associated with the mitochondrial inner membrane between aa 125 and and 188–211, a region located between aa 1–125 and between aa 212–305 that is located inside the mitochondrial matrix, and a region located between aa and 187 that is located towards the intermembrane space of the mitochondria
The program for predicting transmembrane helices in proteins (TMHMM-ver. 2.0) only identified a single transmembrane region associated with the mitochondrial inner membrane between aa 188–211, a region between aa 1–188 that is located inside the mitochondrial matrix, and a region located between aa 212–305 directed towards the mitochondrial intermembrane space
Summary
Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). AOX participates in important biological processes, such as survival and adaptation to stress factors in biotic and abiotic environments, resistance to oxidative stress and maintenance of mitochondrial and cellular energy h omeostasis[18,19] In parasitic protists, it is associated with metabolic adaptation to the host and pathogenicity[20,21,22]. We have demonstrated the presence of a cyanide-resistant alternative respiratory pathway (AP) in mitochondria of the scuticociliate parasite P. dicentrarchi, in addition to the conventional cytochrome pathway (CP)[13] This provided physiological evidence for the presence of a cyanide-insensitive oxidase, which was recognised by antibodies raised against a synthetic peptide derived from a fully conserved C-terminal consensus motif from plant AOX isoforms[13]. We investigated the transcriptional modulation of this enzyme in vitro, in response to experimentally generated hypoxia, as well as in response to the presence of inhibitors of CP and AP mitochondrial respiration
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