Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors

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.

Figure 1

(A) Schematic representation of the structure of the AOX gene of P. dicentrarchi, indicating the position of exons (squares) and introns (lines) in the nucleotide sequence, as well as their translation to mRNA. Linear scale = 100 nucleotides (B) Philasterides dicentrarchi AOX mRNA, complete cds (GenBank: MH427340.1), together with its corresponding translation into amino acids (GenBank: QAR17767.1), including the mitochondrial targeting sequences (MTS), the diiron binding motifs [ion binding site] (DBM), and predictions for O-ß-GlcNAc attachment sites. (C) Diagram of the bioinformatic prediction of the cellular location of the amino acid sequences of the AOX provided by the LocTree 3 program (Rostlab, Technical University of Munich, Germany; URL: https://rostlab.org/services/loctree3/).

Figure 2

Structural modelling and active sites of P. dicentrarchi AOX. (A) Model of spatial structure for AOX homodimeric complex showing regions where the active sites containing the diiron binding motifs in the two polypeptide chains (1 and 2) of the enzyme are located (circles). (B) Diiron active site in chain 1, showing diiron and hydroxo atoms as well as the interactions of the diiron complex with the glutamate and histidine residues. (C) Diiron active site in chain 2, showing diiron and hydroxyl atoms as well as the interactions of the diiron complex with the glutamate and histidine residues. In both cases, the diiron site (iron moieties in orange, hydroxyl in red) is contained within a four alpha-helix bundle. The images of build model were generated by the Swiss-Model program (BIOZENTRUM, The Centre for Molecular Life Sci, University of Basel, Switzerland; URL: https://swissmodel.expasy.org/).

Figure 3

Phylogenetic analysis of amino acid sequences of P. dicentrarchi AOX inferred by Maximum Likelihood method (ML) and Bayesian inference (BI) methods, respectively. The analysis includes sequences of AOXs from representative species of Metazoa, Viridiplantae, Fungi and SAR. The GenBank access number is included after the name of the species. The tree with the highest log likelihood (− 8,244.09) is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown beside the branches and represent the bootstrap value for ML analysis (black) and posterior probability value of BI analysis (red). Dashes (–) indicate disagreement between the ML and BI analysis. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Analysis involved 28 amino acid sequences. All positions containing gaps and missing data were eliminated using the trimAl program. In total, 226 positions were included in the final dataset. Evolutionary analyses were conducted with the MEGAX and MrBayes programs. The scale bar corresponds to 20 substitutions per 100 aa positions.

Figure 4

Biochemical characterization and cellular localization of the AOX of P. dicentrarchi. (A) Western blot analysis with a polyclonal anti-recombinant AOX (anti-rAOX) on a total ciliate lysate (CL, lanes 1 and 2) from trophonts of P. dicentrarchi maintained for 24 h in hypoxia and on the rAOX (lines 3 and 4). The proteins were separated by SDS-PAGE under non-reducing (− DTT, lanes 1 and 3) and reducing (+ DTT, lanes 2 and 4) conditions. (B) SDS-PAGE profile of rAOX stained with Coomassie blue under non-reducing (lane 1) and reducing conditions (lane 2). Arrows indicate the presence of recognition bands. MW molecular weight markers. (C) Representative photomicrograph of an immunofluorescence assay performed on a trophont of P. dicentrarchi and using a polyclonal anti-rAOX antibody in which intense fluorescent mitochondrial labelling is observed (arrows). (D) TEM microphotograph of a trophont of P. dicentrarchi showing the location of the mitochondria below the plasma membrane (arrows).

Figure 5

Analysis of the expression of the P. dicentrarchi AOX gene. (A) Ciliate growth kinetics under conditions of normoxia and hypoxia. (B) AOX gene transcription levels in trophonts maintained under normoxia for 6 days. (C) Western blot analysis using a polyclonal anti-rAOX antibody and ciliated lysates obtained on different days from a normoxic culture. (D) mRNA levels in trophonts obtained from in vitro cultures under normoxia (control) conditions, and in the same ciliates after intraperitoneal injection in turbot and subsequent extraction at 24 h post-infection (24 h). (E) AOX transcript amount in trophonts maintained under normoxia and hypoxia for 6 days (Control, C) and treated with 1 mM of KCN or salicylhydroxamic acid (SHAM). (F) Western blot analysis with a polyclonal anti-rAOX antibody in ciliate lysate of trophonts maintained in the same conditions as in (E). The results presented in the graphs correspond to the mean values ± the standard error (SE; n = 5). Statistically significant difference between groups are indicated by different letters (P < 0.05).