Mitochondria were derived from α-proteobacteria in an endosymbiotic event, and were present in the last common ancestor of all eukaryotes (Gray, 2012; Sagan, 1967). Mitochondria and related organelles are present in almost all extant eukaryotes. They function in the biosynthesis of molecules such as iron-sulfur clusters, lipids, coenzyme Q, heme and amino acids, the catabolism of molecules such as fatty acids, amino acids and monocarboxylates, the storage of ions and signalling molecules such as Ca²⁺, the regulation of apoptosis, and the production of energy-carrying molecules such as ATP and NADH (McBride et al., 2006; van Dooren et al., 2006; Zíková et al., 2016). Notably, the function of mitochondria and related organelles vary substantially between different eukaryotic lineages, reflective of the >1500 M years that separate these lineages (Chernikova et al., 2011; Zíková et al., 2016).

Apicomplexans are a phylum of intracellular parasites that impose a significant medical and economic burden on human populations around the world. Apicomplexans include the causative agents of malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.) and toxoplasmosis (Toxoplasma gondii). T. gondii is an opportunistic parasite that chronically infects approximately one-third of the world’s adult population. Symptoms of T. gondii infection in healthy individuals are typically mild. In immunocompromised individuals and unborn fetuses, however, T. gondii infection can cause severe neurological and developmental impairment, and, without treatment, can lead to death of the infected individual (Montoya and Liesenfeld, 2004). The tractable genetics of T. gondii make it a versatile model for studying conserved aspects of apicomplexan biology.

Mitochondria are critical for the survival of apicomplexans, and several important drugs target proteins that function in this organelle, including atovaquone and endochin-like quinolones, both of which target cytochrome c reductase (complex III) of the electron transport chain, and DSM1, which targets dihydroorotate dehydrogenase, a central enzyme in pyrimidine biosynthesis (Doggett et al., 2012; Phillips et al., 2008; Srivastava et al., 1999). The functions of apicomplexan mitochondria are largely implied through comparative genomic approaches that have identified homologs of genes encoding known mitochondrial proteins from other eukaryotes (Seeber et al., 2008; van Dooren et al., 2006). The genome of apicomplexans such as T. gondii and Plasmodium spp. encode homologs of proteins involved in core mitochondrial processes such as the electron transport chain, the tricarboxylic acid (TCA) cycle, and the synthesis of molecules such as iron-sulfur clusters, heme and coenzyme Q (Seeber et al., 2008; van Dooren et al., 2006). The mitosome of Cryptosporidium parvum is highly reduced compared to that of other apicomplexans, lacking a TCA cycle, and harboring a minimal electron transport chain, but retaining the capacity for iron-sulfur cluster synthesis (Mogi and Kita, 2009).

Apicomplexans belong to a group of eukaryotes known as the myzozoans, that also include dinoflagellates and chromerids (Cavalier-Smith and Chao, 2004). Chromerids are a phylum of free-living, photosynthetic eukaryotes, and are thought to be the closest extant relatives of apicomplexans (Moore et al., 2008). All known myzozoans retain a mitochondrion or mitochondrion-derived organelle, the functions of which have numerous differences from well-studied eukaryotes such as yeast and animals (Danne et al., 2013; Jacot et al., 2016). In addition to a limited gene content, the mitochondrial genomes of these organisms contain unusually fragmented ribosomal RNAs (Feagin et al., 2012). Myzozoan mitochondria lack pyruvate dehydrogenase, the enzyme complex that decarboxylates pyruvate to form acetyl-CoA (Danne et al., 2013; Foth et al., 2005). Instead, these organisms have repurposed a branched-chain α-ketoacid dehydrogenase (BCKDH) to catalyse this reaction (Oppenheim et al., 2014). Myzozoans have a functional TCA cycle, but some enzymes of this pathway are phylogenetically distinct from equivalent enzymes in yeast and humans (Danne et al., 2013; Ke et al., 2015; MacRae et al., 2013; MacRae et al., 2012; van Dooren et al., 2006). These organisms also contain a mitochondrial ATP synthase that lacks clear homologs to many of the proteins that comprise the membrane-bound F₀ component of the complex (Balabaskaran Nina et al., 2011; Sturm et al., 2015).

Elucidating the proteomes of mitochondria is key to understanding their functions. Organellar proteomics in intracellular parasites such as T. gondii is limited by available material and a lack of established organellar purification techniques. To overcome these obstacles, we adopted two complementary spatially-restricted biotin tagging approaches to define the proteome of the mitochondrial matrix of T. gondii. We identified over 400 putative mitochondrial matrix proteins, many of which have no ascribed function and no clear homology to proteins found in well-characterised eukaryotes such as yeast, and most of which are important for parasite growth and survival. We functionally characterise one protein that had no previously ascribed function, and demonstrate that this is a critical component of the cytochrome c oxidase (COX) complex of the mitochondrial electron transport chain in the parasite. We subsequently identify numerous apicomplexan-specific components of COX. These data reveal considerable divergence in the COX complex, and in mitochondria more generally, of T. gondii and related apicomplexans, compared to the animals they infect.

In this study, we utilised two spatially-restricted biotin tagging approaches to elucidate the proteome of the mitochondrial matrix of T. gondii. These complementary approaches identified approximately 400 putative mitochondrial proteins. This number is slightly lower than the 495 proteins identified in the mtAPEX-derived proteome of mammalian cells (Rhee et al., 2013), and is less than the 750 – 900 proteins identified in yeast studies using highly purified mitochondria (Morgenstern et al., 2017; Sickmann et al., 2003). Our proteome identified most of the proteins ‘expected’ to localize to the T. gondii mitochondrion (Figure 2D; Supplementary file 3), suggesting a high level of coverage. The lowered numbers of proteins in the T. gondii mitochondrial proteome compared to the yeast mitochondrial proteome may represent reduced functions in the parasite organelle compared to that in a metabolically flexible organism such as yeast, although our subsequent analyses (discussed below) are consistent with the presence of a high degree of divergent biology in apicomplexan mitochondria.

We experimentally localized 27 previously uncharacterised proteins from the T. gondii mitochondrial proteome, finding that 22 of these localised to the mitochondrion (Figure 3). From this, we estimate that ~80% of the ‘uncharacterized’ proteins from the proteome localize to the mitochondrion. Our findings suggest a low false positive rate in the proteins identified from the APEX proteome alone (~5%), but a higher false positive rate in the mtBirA* proteome (~20%). Based on these analyses, we consider the 213 proteins from the mtAPEX and shared mtAPEX/mtBirA* proteomes to be ‘likely’ mitochondrial proteins, and the 208 proteins found in the mtBirA* proteome alone to be ‘possible’ mitochondrial proteins.

To our knowledge, our study is the first time the APEX and BirA* restricted biotinylation approaches have been directly compared in organellar proteomic approaches. Bioinformatic and localization analyses indicate a high level of overlap between the two approaches, but also indicate the presence of unique proteins in both data sets. This suggests that the two approaches provide complementary information that expands the organellar proteome, while at the same time providing confidence in the shared set of proteins that were identified. The mtBirA*-derived proteome identified greater numbers of proteins, while also featuring greater numbers of false positives.

The mtAPEX labelling was performed over a short timeframe in extracellular parasites, whereas the mtBirA* labelling was performed across 24 hr in intracellular parasites. The differences we see in these proteomes may, in part, result from differences in the labelling conditions. In this regard, it is notable that of the 20 ‘expected’ mitochondrial ribosomal protein and ribosome assembly factors, 17 were only present in the (intracellular) mtBirA* proteome (Supplementary file 3). Future work could examine whether there are differences in mitochondrial ribosome assembly and abundance between intra- and extra-cellular parasites. Similarly, mitochondrial carrier proteins, which mediate the transport of solutes into and out of the mitochondria, were enriched in the APEX proteome (5 of the six carrier proteins identified were present in the ‘APEX only’ proteome, and the other in both proteomes; Supplementary file 1). This may reflect either differences in the abundance of these proteins in intra- vs extra-cellular parasites, or in different labelling affinities for these integral membrane proteins between biotin-phenol and BirA*-catalysed biotinylation.

Several recent studies have used BirA* to identify novel proteins in apicomplexan organelles (Boucher et al., 2018; Chen et al., 2015; Kehrer et al., 2016; Nadipuram et al., 2016). Our study is the first time APEX has been used for proteomic applications in apicomplexan parasites, and we suggest that this approach will prove useful in elucidating the proteomes of other apicomplexan organelles (e.g. the apicoplast) or organellar sub-compartments (e.g. the mitochondrial intermembrane space), either by itself or in combination with BirA* approaches. Our study suggests that APEX and BirA* are powerful tools in determining organellar proteomes in apicomplexans.

Our phylogenetic comparisons suggest that a large number of proteins in the T. gondii mitochondrion have homologs in P. falciparum (Figure 4A; Supplementary file 1). This is consistent with the presence of similar mitochondrial processes and biochemical pathways in these organisms (Seeber et al., 2008). The lower value in C. parvum is consistent with the reduced function of the mitosome of this organism (Mogi and Kita, 2010). The shared mitochondrial/mitosomal proteome of T. gondii and C. parvum likely consists of proteins involved in pathways shared between these organisms, such as Fe-S cluster synthesis. Notably, we identified homologs to 83% of T. gondii mitochondrial proteins in the chromerid V. brassicaformis (Figure 4A; Supplementary file 1). This mirrors recent findings that several ‘derived’ features of apicomplexan mitochondria, including the repurposing of the BCKDH, the loss of Complex I, and the acquisition of novel TCA cycle enzymes, were already present in the free-living, autotrophic common ancestor of myzozoans (dinoflagellates, chromerids and apicomplexans; Danne et al., 2013; Jacot et al., 2016). Notably, most of the ApiCox proteins we identified in the T. gondii COX complex had homologs in chromerids (Table 1), indicating that even conserved mitochondrial processes in this group of organisms have a considerable degree of novelty. Our findings mirror those of two recent studies that examined the mitochondrial ATP synthase complex of T. gondii, which also contains many subunits that are conserved between apicomplexans and their nearest free-living relatives (Huet et al., 2018; Salunke et al., 2018). Together, these data indicate that much of the mitochondrial biology in T. gondii was present in the free-living ancestor that they share with chromerids.

Many (~40 – 50%) mitochondrial proteins in T. gondii lack apparent orthologs in animals and other eukaryotes (Figure 4B–C). Surprisingly, proteins found only in coccidians, or restricted to apicomplexans and chromerids, were just as likely to be important for parasite growth as proteins conserved across eukaryotic evolution (Figure 4D). This suggests that many derived or unique features of the T. gondii mitochondrion (and apicomplexan mitochondria more generally) are critical for parasite survival. This is in contrast to the general proteome of T. gondii, where proteins with a restricted phylogenetic distribution are typically less important for parasite survival (Sidik et al., 2016).

To understand the functions of apicomplexan-specific mitochondrial proteins that are important for parasite growth, we have commenced a project to characterize these proteins. In this study, we describe the characterisation of TgApiCox25, demonstrating that this is a component of the T. gondii COX complex. TgApiCox25 is important for parasite growth and mitochondrial oxygen consumption (Figure 5). Knockdown of TgApiCox25 also leads to defects in the integrity of the COX complex. In particular, TgApiCox25 knockdown leads to a depletion of TgCox2a abundance, and also results in the appearance of a smaller,~400 kDa TgCox2a-containing complex (Figure 7E–F). These data imply an important role for TgApiCox25 in the assembly and/or stability of the COX complex. It remains unclear whether loss of TgApiCox25 leads to loss of a ~ 200 kDa module from the complex, or whether TgApiCox25 knockdown leads to defects in COX assembly, with the ~400 kDa complex representing an assembly intermediate. Regardless, loss of TgApiCox25 results in defects in the abundance and integrity of the parasite COX complex, which likely explains the defects in mOCR observed upon TgApiCox25 knockdown. Future studies will examine the functional role of TgApiCox25 in the COX complex of the parasite.

Our analysis of the parasite COX complex revealed that it is approximately 600 kDa in mass (Figure 6A), which is larger than the equivalent complex in yeast (200 kDa), mammals (200 kDa) and plants (220/300 kDa) (Eubel et al., 2003; Lenaz and Genova, 2010; Maréchal et al., 2012; Schägger and Pfeiffer, 2000; Tsukihara et al., 1996). It is conceivable that the T. gondii COX complex exists in a multimeric form (e.g. a homodimer or homotrimer), and/or that it contains subunits not present in the complex of these other organisms that inflate its mass. Another possibility is that T. gondii COX exists in a ‘super-complex’ with other respiratory complexes, as has been observed in other systems (Eubel et al., 2003; Schägger and Pfeiffer, 2000). Although we cannot rule out that respiratory chain supercomplexes exist in T. gondii, the 600 kDa complex is probably not a supercomplex, since (i) we do not observe enrichment of canonical components of other respiratory chain proteins in our proteomic analysis of the complex, (ii) we solubilised the cytochrome c oxidase complex with Triton X-100 detergent, rather than digitonin (which is typically used to solubilize supercomplexes in other organisms; Schägger and Pfeiffer, 2000), and (iii) the molecular masses of other respiratory chain complexes in T. gondii do not correspond in size to COX (GvD, unpublished observations).

We identified 15 proteins in the T. gondii COX complex. In addition to these, it is likely that the complex contains TgCoxI, a protein encoded on the mitochondrial genome of the parasite. 16 COX proteins in T. gondii is comparable in number to those found in the mammalian (13 proteins) and plant (10 or 12 proteins) complexes (Eubel et al., 2003; Tsukihara et al., 1996). Surprisingly, only a few T. gondii COX proteins have obvious homologs in eukaryotes outside the myzozoan lineage. Of note, 10 of the 11 ApiCox proteins we identified have homologs in chromerids (Table 1), while only TgApiCox13 has a clear homolog in ciliates (http://orthomcl.org), the eukaryotic lineage that is the sister taxon to myzozoans. This suggests either a high degree of novelty in the proteins that comprise the T. gondii/myzozoan COX complex, or that the sequences of ApiCox proteins have diverged to the extent that they are no longer easily recognisable by sequence comparisons. Notably, similarity searches that incorporate secondary structure information suggest that ApiCox25 and ApiCox23 may have homology to Cox6a and Cox4, respectively, from animals. A priority in the field is to establish the structure of the COX complex in myzozoans, which will reveal whether ApiCox proteins have structural equivalents in other eukaryotes. Regardless of their degree of novelty, our observations are consistent with other observations that suggest a considerable divergence in the mitochondrial biology of organisms from the myzozoan lineage compared to other eukaryotes (Danne et al., 2013; Huet et al., 2018; Jacot et al., 2016; Salunke et al., 2018).

Our study highlights the divergence of mitochondrial proteomes across the eukaryotic domain of life. Future studies will be aimed at elucidating the function of other T. gondii- and myzozoan-specific mitochondrial proteins, since these proteins will provide insights into unique functions of the organelle. Given their importance to parasite growth, and their absence (or divergence) from the host organisms that they infect, these T. gondii and apicomplexan-specific proteins are prime drug targets.

Mitochondrial proteomics data are available in on the ToxoDB website (http://toxodb.org).

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Author details

1. Azadeh Seidi

   Research School of Biology, Australian National University, Canberra, Australia

   Present address

   Millennium Science, Victoria, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Linden S Muellner-Wong

   Competing interests

   No competing interests declared

2. Linden S Muellner-Wong

   Research School of Biology, Australian National University, Canberra, Australia

   Present address

   Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Azadeh Seidi

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0348-6408

3. Esther Rajendran

   Research School of Biology, Australian National University, Canberra, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

4. Edwin T Tjhin

   Research School of Biology, Australian National University, Canberra, Australia

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

5. Laura F Dagley

   1. The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia
   2. Department of Medical Biology, University of Melbourne, Victoria, Australia

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

6. Vincent YT Aw

   Research School of Biology, Australian National University, Canberra, Australia

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

7. Pierre Faou

   Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Victoria, Australia

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

8. Andrew I Webb

   1. The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia
   2. Department of Medical Biology, University of Melbourne, Victoria, Australia

   Contribution

   Resources, Supervision, Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

9. Christopher J Tonkin

   1. The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia
   2. Department of Medical Biology, University of Melbourne, Victoria, Australia

   Contribution

   Resources, Supervision, Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

10. Giel G van Dooren

    Research School of Biology, Australian National University, Canberra, Australia

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    giel.vandooren@anu.edu.au

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2455-9821

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