All living organisms breakdown food molecules to generate energy for processes, such as growing, reproducing and movement. The series of chemical reactions that breakdown sugars into smaller molecules – known as glycolysis – is so important that it occurs in all life forms, from bacteria to humans.

In higher organisms, such as fungi and animals, these reactions take place in the cytosol, the space surrounding the cell’s various compartments. A transport protein then shuttles the end-product of glycolysis – pyruvate – into specialised compartments, known as the mitochondria, where most energy is produced.

However, recently it was discovered that a group of living organisms, called the stramenopiles, have a branched glycolysis in which the enzymes involved in the second half of this process are located in both the cytosol and mitochondrial matrix. But it was not known how the intermediate molecules produced after the first half of glycolysis enter the mitochondria.

To answer this question, Pyrihová et al. searched for transport protein(s) that could link the two halves of the glycolysis pathway. Computational analyses, comparing the genetic sequences of many transport proteins from several different species, revealed a new group found only in stramenopiles. Pyrihová et al. then used microscopy to visualise these new transport proteins – called GIC-1 and GIC-2 – in the parasite Blastocystis, which infects the human gut, and observed that they localise to mitochondria.

Further biochemical experiments showed that GIC-1 and GIC-2 can physically bind these intermediate molecules, but only GIC-2 can transport them across membranes. Taken together, these observations suggest that GIC-2 links the two halves of glycolysis in Blastocystis.

Further analyses could reveal corresponding transport proteins in other stramenopiles, many of which have devastating effects on agriculture, such as Phytophthora, which causes potato blight, or Saprolegnia, which causes skin infections in farmed salmon. Since human cells do not have equivalent transporters, they could be new drug targets not only for Blastocystis, but for these harmful pathogens as well.

Glycolysis is a crucial metabolic pathway that most organisms use to obtain energy from the oxidation of sugars, which consists of 10 enzymatic reactions in total, divided into two phases. The first ‘preparatory’ phase consists of five reactions, the products of which are the triose phosphates dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The second ‘pay-off’ phase leads to the production of ATP, NADH, and pyruvate, which is transported into the mitochondrial matrix by the mitochondrial pyruvate carrier (Bricker et al., 2012; Herzig et al., 2012). In most eukaryotes, glycolysis is a cytosolic process with the exception of trypanosomes, where it is localized in the glycosomes (Opperdoes and Borst, 1977). However, it was recently discovered that a large group of eukaryotes, the stramenopiles, have a branched glycolytic pathway in which the pay-off phase is duplicated and also localized in mitochondria (Abrahamian et al., 2017; Río Bártulos et al., 2018). The stramenopiles are an extremely diverse eukaryotic group consisting mainly of protist algae, which are among the most important primary oxygen producers in the oceans (Burki et al., 2020). The group also includes multicellular algae (kelps) and the economically devastating oomycetes, such as Phytophthora infestans (Jiang and Tyler, 2012), which causes potato blight, one of the major contributing factors of the Great Famine in Ireland in the late 19th century.

The human gut protozoan Blastocystis also belongs to the stramenopiles. Despite more than 1 billion infected individuals worldwide (Andersen and Stensvold, 2016), the public health significance of Blastocystis is debated. Blastocystis is often linked to patients with diarrhoea, irritable bowel syndrome (Poirier et al., 2012), or inflammatory bowel disease (Dogruman-Al et al., 2009). However, it is also frequently found in healthy individuals, and a few microbiome studies associated Blastocystis with higher microbial diversity (Andersen et al., 2015; Audebert et al., 2016; Stensvold et al., 2022). The controversy may arise from the extreme genetic diversity of Blastocystis, which is classified into over 40 subtypes (Andersen et al., 2015; Stensvold et al., 2023), and thus its influence on gut microbiota is likely to be subtype specific (Tito et al., 2019). Nevertheless, Blastocystis ST7-B, which was used in this study, is most frequently associated with pathogenic phenotypes (Ajjampur et al., 2016; Deng et al., 2023; Stensvold et al., 2009; Wu et al., 2014; Yason et al., 2019) and was recently linked to a decreased microbial diversity in diarrheal patients (Deng et al., 2022). The treatment of choice is metronidazole (Stenzel and Boreham, 1996), but there have been reports of resistance (Haresh et al., 1999; Mirza et al., 2011; Moghaddam et al., 2005), highlighting the need for new therapeutic interventions.

Unlike other stramenopiles, Blastocystis has lost the cytosolic pay-off phase of glycolysis, consistent with the absence of a gene encoding a mitochondrial pyruvate carrier (Río Bártulos et al., 2018). The only exception is the last enzyme of the pathway, pyruvate kinase, which was also identified as a cytosolic protein (Lantsman et al., 2008). Blastocystis is an anaerobe (Zierdt, 1991) with highly divergent mitochondria (Stechmann et al., 2008). It has respiratory complexes I and II, but lacks the rest of the electron transport chain, as well as ATP synthase and mitochondrial ADP/ATP carriers (Gentekaki et al., 2017; Stechmann et al., 2008; Wawrzyniak et al., 2008). Therefore, Blastocystis relies mainly on glycolysis and fermentation for its metabolic energy requirements (Müller et al., 2012). The transport of glycolytic intermediates across the mitochondrial inner membrane is necessary for the completion of glycolysis and is critical for the survival of the parasite. Glycolytic enzymes are powerful drug targets in parasites that are strictly dependent on glycolysis for ATP production. The structural differences between host and parasitic glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase in Plasmodium falciparum have been studied with the aim of inhibiting glycolysis (Bruno et al., 2014; Penna-Coutinho et al., 2011; Ravindra and Balaram, 2005). Similarly, inhibitors against phosphofructokinase in Trypanosoma brucei have a rapid effect on the parasite viability without inhibiting the host enzyme (McNae et al., 2021). Understanding the unique aspects of parasite metabolism is crucial for the discovery of new drug targets.

It is clear that the branched stramenopile glycolysis needs to be linked by a specific transporter in the mitochondrial inner membrane, as the solute exchange between the cytosol and mitochondrial matrix is tightly controlled (Kunji et al., 2020; Ruprecht and Kunji, 2020). The mitochondrial carrier family (SLC25) is the largest solute transporter family in humans (Kunji et al., 2020). Its members facilitate the transport of nucleotides, amino acids, inorganic ions, keto acids, vitamins, and other nutrients across the mitochondrial inner membrane. All SLC25 members have a threefold pseudo-symmetric structure, evident at the sequence level by the presence of three conserved homologous repeats (Saraste and Walker, 1982) and specific motifs important for their transport mechanism (Ruprecht et al., 2019; Ruprecht and Kunji, 2020; Ruprecht and Kunji, 2021).

Here, we report a stramenopile-specific group of transport proteins closely related to mitochondrial carboxylate carriers of the SLC25 family. We characterized one of these unique Blastocystis carriers, which has lost its ability to transport the canonical substrates malate and oxoglutarate. Instead, this carrier can transport several glycolytic intermediates, including dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate (PEP), thus providing a previously undiscovered transport link between cytosolic and mitochondrial glycolysis. Since Blastocystis relies solely on mitochondrial glycolysis, the inhibition of this carrier might represent a promising way to treat this parasite.

Glycolysis is a textbook example of a well-conserved cytosolic pathway. However, the discovery that glycolysis is branched and also mitochondrial in stramenopiles (Abrahamian et al., 2017; Río Bártulos et al., 2018; Nakayama et al., 2012) raises questions about how glycolytic intermediates are transported across the tightly controlled mitochondrial inner membrane. Here, we report the discovery and characterization of a mitochondrial carrier protein unique to stramenopiles that transports the glycolytic intermediates dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, and PEP, providing the heretofore missing transport link.

Since mitochondrial glycolysis is found exclusively in stramenopiles, we searched for a transporter that is ubiquitous in these species, but not in other eukaryotes. Phylogenetic analyses identified a new class of mitochondrial carboxylate carriers in stramenopiles that are most closely related to plant DTC (Figure 1). The plant carriers catalyse the transport of tricarboxylates (citrate, isocitrate, and aconitate) in exchange for dicarboxylates (oxoglutarate, oxaloacetate, malate, maleate, succinate, and malonate) (Picault et al., 2002) and are also present in Alveolata, Rhizaria, and Haptophyta (Toleco et al., 2020), which share a common evolutionary ancestor with stramenopiles (Burki et al., 2020). Therefore, the GIC of stramenopiles is probably derived from a common ancestor with DTC and does not exist in any other eukaryote outside of the stramenopile clade.

Blastocystis was used in this study as a stramenopile model organism, as it relies solely on glycolysis for its energy demands and does not have a second branch of glycolysis in the cytosol. We characterized human and Blastocystis carboxylate carriers and demonstrated that the stramenopile GIC proteins have a unique substrate specificity that is distinct from the human carriers (Figure 6). We also characterized the substrate specificity of Blastocystis OGC, which has the same specificity as human OGC (SLC25A11), and we demonstrated that it is unable to transport glycolytic intermediates and thus could not be the missing transport step in the branched glycolytic pathway.

Blastocystis GIC-1 did not transport any of the tested substrates (Figure 5A, C, E), but it is expressed and localized to mitochondria (Figure 2A), demonstrating that it is not a pseudogene. We did not test radiolabelled glycolytic intermediates directly in transport, as PEP and dihydroxyacetone phosphate are not available, and radiolabelled glyceraldehyde-3-phosphate is prohibitively expensive. We cannot, therefore, exclude the possibility that GIC-1 catalyses glycolytic intermediate exchange, but not in hetero-exchange reactions with malate, phosphate, and sulphate. It is more likely that bGIC-1 has lost its transport activity and could possibly fulfil a different role. For example, in Tetrahymena thermophila, a non-functional OGC-like carrier is incorporated into a respiratory supercomplex (Zhou et al., 2022). In contrast to bGIC-1, bGIC-2 transports several inorganic ions and glycolytic intermediates, including dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (Figure 6D), but not the canonical substrates of Blastocystis OGC, such as malate, maleate, malonate, and succinate. Most mitochondrial carriers operate as strict antiporters, where the chemical gradient of one substrate drives the uptake of the counter-substrate (Ruprecht and Kunji, 2020). A mitochondrial phosphate carrier homologue was identified in the phylogenetic analysis of Blastocystis (Figure 1—figure supplement 1), which is driven by the proton motive force to maintain high concentrations of phosphate in the mitochondrial matrix, which in turn could drive the uptake of glycolytic intermediates. Blastocystis lacks many of the conserved carrier proteins found in the mitochondria of aerobes. In total, we have only identified 18 mitochondrial carriers in Blastocystis (Figure 1—figure supplement 1). However, we show that bGIC-2 seems to be able to compensate for the loss of some mitochondrial carriers, including the transport of inorganic ions in the absence of a DIC homologue. Additionally, the distantly related citrate carrier, which transports dicarboxylates, tricarboxylates, and PEP, was also lost in Blastocystis, possibly due to an incomplete tricarboxylic acid (TCA) cycle devoid of citrate synthase, aconitase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase (Stechmann et al., 2008; Zierdt et al., 1988). In Blastocystis, PEP can be synthesized from mitochondrial pyruvate via oxaloacetate (Gentekaki et al., 2017; Stechmann et al., 2008), and here we show that PEP transport is catalysed by bGIC-2. When PEP is exported by bGIC-2, it could be converted into pyruvate and lactate by cytosolic pyruvate kinase and lactate dehydrogenase, respectively (Lantsman et al., 2008), which would lead to the synthesis of ATP and the conversion of NADH to NAD⁺ in the cytosol. This reaction may play a significant role in cytosolic ATP generation, as there is no mitochondrial ADP/ATP carrier in Blastocystis for adenine nucleotide exchange (Figure 1—figure supplement 1). Blastocystis also contains a glycerol-3-phosphate dehydrogenase (Gentekaki et al., 2017), which oxidizes NADH to convert glycerol-3-phosphate to dihydroxyacetone phosphate, which in turn can be transported into mitochondria by bGIC-2 to generate ATP and reducing equivalents. In most other eukaryotes, cytosolic NADH is oxidized by the malate–aspartate shuttle, but this pathway requires an orthologue of the mitochondrial aspartate/glutamate carrier, which could not be identified in Blastocystis, even though it might have a mitochondrial glutamate carrier (Figure 1—figure supplement 1).

What selective advantage could there be to have the pay-off phase of glycolysis in the mitochondrial matrix? Blastocystis is well adapted to an anoxic environment with its anaerobic metabolism but has a limited ability to detoxify oxygen. In its natural environment, Blastocystis likely uses protons as terminal electron acceptors by using pyruvate:ferredoxin oxidoreductase, which would result in the production of hydrogen and a range of fermentative products, such as lactate, acetate, and propionate (Gentekaki et al., 2017; Lantsman et al., 2008; Stechmann et al., 2008). Under aerobic stress, such as in a dysbiotic gut (Stensvold and van der Giezen, 2018), it possibly uses oxygen as the terminal electron acceptor by using alternative oxidase, which produces water (Tsaousis et al., 2018). However, as a likely adaptation to the anoxic environment, Blastocystis has lost some of the canonical components of oxidative phosphorylation, including complexes III and IV of the respiratory chain, ATP-synthase and mitochondrial ADP/ATP carriers (Stechmann et al., 2008). Consequently, it has lost its ability to synthesize mitochondrial ATP via this route. This role was taken over by transfer of the pay-off phase of glycolysis to the mitochondrial matrix in Blastocystis, which has several advantages. The degradation of one glucose molecule in the cytosol requires one ATP and one pyrophosphate molecule, but will generate four ATP molecules in the mitochondrial matrix, which can be used for ATP-requiring processes, such as protein import, mitochondrial DNA replication and transcription, and mitochondrial protein synthesis (Denoeud et al., 2011; Gentekaki et al., 2017). In addition, two NAD⁺ molecules are reduced to NADH, which can be used by complex I to generate a proton motive force to drive the import of proteins and the uptake of ions and molecules into mitochondria, such as phosphate. Cytosolic ATP can be supplied either through the export of PEP by bGIC-2, as explained, or via a mitochondrial carrier that shares properties with the mitochondrial Mg-ATP/Pi carrier, which is generally used to equilibrate adenine nucleotide pools between the matrix and cytosol.

Our results show that the canonical metabolic pathways studied in common model organisms can be altered in non-model eukaryotic species (Figure 7). Moreover, the newly discovered glycolytic intermediates carrier demonstrates the plasticity of mitochondrial carriers in terms of their substrate specificity. Through the discovery of this carrier, we have linked the two halves of glycolysis and provided a potential target for specific treatment not only for Blastocystis but also for other stramenopile pathogens that threaten global food security.

Figure 7

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All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for Figures 2–6.

1. 1. Abrahamian M
   2. Kagda M
   3. Ah-Fong AMV
   4. Judelson HS

   (2017) Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria

   BMC Evolutionary Biology 17:241.

   https://doi.org/10.1186/s12862-017-1087-8

   • PubMed
   • Google Scholar
2. 1. Ajjampur SSR
   2. Png CW
   3. Chia WN
   4. Zhang Y
   5. Tan KSW

   (2016) Ex Vivo and In Vivo Mice Models to Study Blastocystis spp. Adhesion, Colonization and Pathology: Closer to Proving Koch’s Postulates

   PLOS ONE 11:e0160458.

   https://doi.org/10.1371/journal.pone.0160458

   • PubMed
   • Google Scholar
3. 1. Alexander CG
   2. Wanner R
   3. Johnson CM
   4. Breitsprecher D
   5. Winter G
   6. Duhr S
   7. Baaske P
   8. Ferguson N

   (2014) Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

   Biochimica et Biophysica Acta 1844:2241–2250.

   https://doi.org/10.1016/j.bbapap.2014.09.016

   • PubMed
   • Google Scholar
4. 1. Andersen LO
   2. Bonde I
   3. Nielsen HB
   4. Stensvold CR

   (2015) A retrospective metagenomics approach to studying Blastocystis

   FEMS Microbiology Ecology 91:fiv072.

   https://doi.org/10.1093/femsec/fiv072

   • PubMed
   • Google Scholar
5. 1. Andersen LO
   2. Stensvold CR

   (2016) Blastocystis in health and disease: are we moving from a clinical to a public health perspective?

   Journal of Clinical Microbiology 54:524–528.

   https://doi.org/10.1128/JCM.02520-15

   • PubMed
   • Google Scholar
6. 1. Audebert C
   2. Even G
   3. Cian A
   4. Blastocystis Investigation Group
   5. Loywick A
   6. Merlin S
   7. Viscogliosi E
   8. Chabé M

   (2016) Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota

   Scientific Reports 6:25255.

   https://doi.org/10.1038/srep25255

   • PubMed
   • Google Scholar
7. 1. Bisaccia F
   2. Indiveri C
   3. Palmieri F

   (1988) Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier

   Biochimica et Biophysica Acta 933:229–240.

   https://doi.org/10.1016/0005-2728(88)90030-8

   • PubMed
   • Google Scholar
8. 1. Bricker DK
   2. Taylor EB
   3. Schell JC
   4. Orsak T
   5. Boutron A
   6. Chen Y-C
   7. Cox JE
   8. Cardon CM
   9. Van Vranken JG
   10. Dephoure N
   11. Redin C
   12. Boudina S
   13. Gygi SP
   14. Brivet M
   15. Thummel CS
   16. Rutter J

   (2012) A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans

   Science 337:96–100.

   https://doi.org/10.1126/science.1218099

   • PubMed
   • Google Scholar
9. 1. Bruno S
   2. Pinto A
   3. Paredi G
   4. Tamborini L
   5. De Micheli C
   6. La Pietra V
   7. Marinelli L
   8. Novellino E
   9. Conti P
   10. Mozzarelli A

   (2014) Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria

   Journal of Medicinal Chemistry 57:7465–7471.

   https://doi.org/10.1021/jm500747h

   • PubMed
   • Google Scholar
10. 1. Burki F
    2. Roger AJ
    3. Brown MW
    4. Simpson AGB

    (2020) The new tree of eukaryotes

    Trends in Ecology & Evolution 35:43–55.

    https://doi.org/10.1016/j.tree.2019.08.008

    • PubMed
    • Google Scholar
11. 1. Castegna A
    2. Scarcia P
    3. Agrimi G
    4. Palmieri L
    5. Rottensteiner H
    6. Spera I
    7. Germinario L
    8. Palmieri F

    (2010) Identification and functional characterization of a novel mitochondrial carrier for citrate and oxoglutarate in Saccharomyces cerevisiae

    The Journal of Biological Chemistry 285:17359–17370.

    https://doi.org/10.1074/jbc.M109.097188

    • PubMed
    • Google Scholar
12. 1. Crichton PG
    2. Lee Y
    3. Ruprecht JJ
    4. Cerson E
    5. Thangaratnarajah C
    6. King MS
    7. Kunji ERS

    (2015) Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers

    The Journal of Biological Chemistry 290:8206–8217.

    https://doi.org/10.1074/jbc.M114.616607

    • PubMed
    • Google Scholar
13. 1. Crompton M
    2. Palmieri F
    3. Capano M
    4. Quagliariello E

    (1974) The transport of thiosulphate in rat liver mitochondria

    FEBS Letters 46:247–250.

    https://doi.org/10.1016/0014-5793(74)80379-0

    • PubMed
    • Google Scholar
14. 1. Deng L
    2. Lee JWJ
    3. Tan KSW

    (2022) Infection with pathogenic Blastocystis ST7 is associated with decreased bacterial diversity and altered gut microbiome profiles in diarrheal patients

    Parasites & Vectors 15:312.

    https://doi.org/10.1186/s13071-022-05435-z

    • PubMed
    • Google Scholar
15. 1. Deng L
    2. Wojciech L
    3. Png CW
    4. Kioh DYQ
    5. Gu Y
    6. Aung TT
    7. Malleret B
    8. Chan ECY
    9. Peng G
    10. Zhang Y
    11. Gascoigne NRJ
    12. Tan KSW

    (2023) Colonization with two different Blastocystis subtypes in DSS-induced colitis mice is associated with strikingly different microbiome and pathological features

    Theranostics 13:1165–1179.

    https://doi.org/10.7150/thno.81583

    • PubMed
    • Google Scholar
16. 1. Denoeud F
    2. Roussel M
    3. Noel B
    4. Wawrzyniak I
    5. Da Silva C
    6. Diogon M
    7. Viscogliosi E
    8. Brochier-Armanet C
    9. Couloux A
    10. Poulain J
    11. Segurens B
    12. Anthouard V
    13. Texier C
    14. Blot N
    15. Poirier P
    16. Ng GC
    17. Tan KSW
    18. Artiguenave F
    19. Jaillon O
    20. Aury J-M
    21. Delbac F
    22. Wincker P
    23. Vivarès CP
    24. El Alaoui H

    (2011) Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite

    Genome Biology 12:R29.

    https://doi.org/10.1186/gb-2011-12-3-r29

    • PubMed
    • Google Scholar
17. 1. Dogruman-Al F
    2. Kustimur S
    3. Yoshikawa H
    4. Tuncer C
    5. Simsek Z
    6. Tanyuksel M
    7. Araz E
    8. Boorom K

    (2009) Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey

    Memorias Do Instituto Oswaldo Cruz 104:724–727.

    https://doi.org/10.1590/s0074-02762009000500011

    • PubMed
    • Google Scholar
18. 1. Fiermonte G
    2. Walker JE
    3. Palmieri F

    (1993) Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria

    The Biochemical Journal 294:293–299.

    https://doi.org/10.1042/bj2940293

    • PubMed
    • Google Scholar
19. 1. Fiermonte G
    2. Dolce V
    3. Palmieri L
    4. Ventura M
    5. Runswick MJ
    6. Palmieri F
    7. Walker JE

    (2001) Identification of the human mitochondrial oxodicarboxylate carrier: bacterial expression, reconstitution, functional characterization, tissue distribution, and chromosomal location

    The Journal of Biological Chemistry 276:8225–8230.

    https://doi.org/10.1074/jbc.M009607200

    • PubMed
    • Google Scholar
20. 1. Gentekaki E
    2. Curtis BA
    3. Stairs CW
    4. Klimeš V
    5. Eliáš M
    6. Salas-Leiva DE
    7. Herman EK
    8. Eme L
    9. Arias MC
    10. Henrissat B
    11. Hilliou F
    12. Klute MJ
    13. Suga H
    14. Malik S-B
    15. Pightling AW
    16. Kolisko M
    17. Rachubinski RA
    18. Schlacht A
    19. Soanes DM
    20. Tsaousis AD
    21. Archibald JM
    22. Ball SG
    23. Dacks JB
    24. Clark CG
    25. van der Giezen M
    26. Roger AJ

    (2017) Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis

    PLOS Biology 15:e2003769.

    https://doi.org/10.1371/journal.pbio.2003769

    • PubMed
    • Google Scholar
21. 1. Haresh K
    2. Suresh K
    3. Khairul Anus A
    4. Saminathan S

    (1999) Isolate resistance of Blastocystis hominis to metronidazole

    Tropical Medicine & International Health 4:274–277.

    https://doi.org/10.1046/j.1365-3156.1999.00398.x

    • PubMed
    • Google Scholar
22. 1. Herzig S
    2. Raemy E
    3. Montessuit S
    4. Veuthey J-L
    5. Zamboni N
    6. Westermann B
    7. Kunji ERS
    8. Martinou J-C

    (2012) Identification and functional expression of the mitochondrial pyruvate carrier

    Science 337:93–96.

    https://doi.org/10.1126/science.1218530

    • PubMed
    • Google Scholar
23. 1. Hoang DT
    2. Chernomor O
    3. von Haeseler A
    4. Minh BQ
    5. Vinh LS

    (2018) UFBoot2: improving the ultrafast bootstrap approximation

    Molecular Biology and Evolution 35:518–522.

    https://doi.org/10.1093/molbev/msx281

    • PubMed
    • Google Scholar
24. 1. Jaiquel Baron S
    2. King MS
    3. Kunji ERS
    4. Schirris TJJ

    (2021) Characterization of drug-induced human mitochondrial ADP/ATP carrier inhibition

    Theranostics 11:5077–5091.

    https://doi.org/10.7150/thno.54936

    • PubMed
    • Google Scholar
25. 1. Jiang RHY
    2. Tyler BM

    (2012) Mechanisms and evolution of virulence in oomycetes

    Annual Review of Phytopathology 50:295–318.

    https://doi.org/10.1146/annurev-phyto-081211-172912

    • PubMed
    • Google Scholar
26. 1. Jones P
    2. Binns D
    3. Chang H-Y
    4. Fraser M
    5. Li W
    6. McAnulla C
    7. McWilliam H
    8. Maslen J
    9. Mitchell A
    10. Nuka G
    11. Pesseat S
    12. Quinn AF
    13. Sangrador-Vegas A
    14. Scheremetjew M
    15. Yong S-Y
    16. Lopez R
    17. Hunter S

    (2014) InterProScan 5: genome-scale protein function classification

    Bioinformatics 30:1236–1240.

    https://doi.org/10.1093/bioinformatics/btu031

    • PubMed
    • Google Scholar
27. 1. King MS
    2. Kunji ERS

    (2020) Expression and purification of membrane proteins in Saccharomyces cerevisiae

    Methods in Molecular Biology 2127:47–61.

    https://doi.org/10.1007/978-1-0716-0373-4_4

    • PubMed
    • Google Scholar
28. 1. Kunji ERS
    2. Robinson AJ

    (2006) The conserved substrate binding site of mitochondrial carriers

    Biochimica et Biophysica Acta 1757:1237–1248.

    https://doi.org/10.1016/j.bbabio.2006.03.021

    • PubMed
    • Google Scholar
29. 1. Kunji ERS
    2. King MS
    3. Ruprecht JJ
    4. Thangaratnarajah C

    (2020) The SLC25 carrier family: important transport proteins in mitochondrial physiology and pathology

    Physiology 35:302–327.

    https://doi.org/10.1152/physiol.00009.2020

    • PubMed
    • Google Scholar
30. 1. Lantsman Y
    2. Tan KSW
    3. Morada M
    4. Yarlett N

    (2008) Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7

    Microbiology 154:2757–2766.

    https://doi.org/10.1099/mic.0.2008/017897-0

    • PubMed
    • Google Scholar
31. 1. Leonardi SS
    2. Li F-J
    3. Chee MS-J
    4. Yason JA
    5. Tay HY
    6. Chen JY-S
    7. Koh EY
    8. He CY-X
    9. Tan KS-W

    (2021) Characterisation of novel functionality within the Blastocystis tryptophanase gene

    PLOS Neglected Tropical Diseases 15:e0009730.

    https://doi.org/10.1371/journal.pntd.0009730

    • PubMed
    • Google Scholar
32. 1. Majd H
    2. King MS
    3. Palmer SM
    4. Smith AC
    5. Elbourne LD
    6. Paulsen IT
    7. Sharples D
    8. Henderson PJ
    9. Kunji ER

    (2018) Screening of candidate substrates and coupling ions of transporters by thermostability shift assays

    eLife 7:e38821.

    https://doi.org/10.7554/eLife.38821

    • PubMed
    • Google Scholar
33. 1. Mavridou V
    2. King MS
    3. Tavoulari S
    4. Ruprecht JJ
    5. Palmer SM
    6. Kunji ERS

    (2022) Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle

    Nature Communications 13:3585.

    https://doi.org/10.1038/s41467-022-31366-5

    • PubMed
    • Google Scholar
34. 1. McNae IW
    2. Kinkead J
    3. Malik D
    4. Yen L-H
    5. Walker MK
    6. Swain C
    7. Webster SP
    8. Gray N
    9. Fernandes PM
    10. Myburgh E
    11. Blackburn EA
    12. Ritchie R
    13. Austin C
    14. Wear MA
    15. Highton AJ
    16. Keats AJ
    17. Vong A
    18. Dornan J
    19. Mottram JC
    20. Michels PAM
    21. Pettit S
    22. Walkinshaw MD

    (2021) Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice

    Nature Communications 12:1052.

    https://doi.org/10.1038/s41467-021-21273-6

    • PubMed
    • Google Scholar
35. 1. Minh BQ
    2. Schmidt HA
    3. Chernomor O
    4. Schrempf D
    5. Woodhams MD
    6. von Haeseler A
    7. Lanfear R

    (2020) IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era

    Molecular Biology and Evolution 37:1530–1534.

    https://doi.org/10.1093/molbev/msaa015

    • PubMed
    • Google Scholar
36. 1. Mirza H
    2. Teo JDW
    3. Upcroft J
    4. Tan KSW

    (2011) A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities

    Antimicrobial Agents and Chemotherapy 55:637–648.

    https://doi.org/10.1128/AAC.00900-10

    • PubMed
    • Google Scholar
37. 1. Moghaddam DD
    2. Ghadirian E
    3. Azami M

    (2005) Blastocystis hominis and the evaluation of efficacy of metronidazole and trimethoprim/sulfamethoxazole

    Parasitology Research 96:273–275.

    https://doi.org/10.1007/s00436-005-1363-1

    • PubMed
    • Google Scholar
38. 1. Müller M
    2. Mentel M
    3. van Hellemond JJ
    4. Henze K
    5. Woehle C
    6. Gould SB
    7. Yu R-Y
    8. van der Giezen M
    9. Tielens AGM
    10. Martin WF

    (2012) Biochemistry and evolution of anaerobic energy metabolism in eukaryotes

    Microbiology and Molecular Biology Reviews 76:444–495.

    https://doi.org/10.1128/MMBR.05024-11

    • PubMed
    • Google Scholar
39. 1. Nakayama T
    2. Ishida K
    3. Archibald JM

    (2012) Broad distribution of TPI-GAPDH fusion proteins among eukaryotes: evidence for glycolytic reactions in the mitochondrion?

    PLOS ONE 7:e52340.

    https://doi.org/10.1371/journal.pone.0052340

    • PubMed
    • Google Scholar
40. 1. Opperdoes FR
    2. Borst P

    (1977) Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome

    FEBS Letters 80:360–364.

    https://doi.org/10.1016/0014-5793(77)80476-6

    • PubMed
    • Google Scholar
41. 1. Palmieri F
    2. Quagliariello E
    3. Klingenberger M

    (1972) Kinetics and specificity of the oxoglutarate carrier in rat-liver mitochondria

    European Journal of Biochemistry 29:408–416.

    https://doi.org/10.1111/j.1432-1033.1972.tb02003.x

    • PubMed
    • Google Scholar
42. 1. Penna-Coutinho J
    2. Cortopassi WA
    3. Oliveira AA
    4. França TCC
    5. Krettli AU

    (2011) Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies

    PLOS ONE 6:e21237.

    https://doi.org/10.1371/journal.pone.0021237

    • PubMed
    • Google Scholar
43. 1. Picault N
    2. Palmieri L
    3. Pisano I
    4. Hodges M
    5. Palmieri F

    (2002) Identification of a novel transporter for dicarboxylates and tricarboxylates in plant mitochondria: bacterial expression, reconstitution, functional characterization, and tissue distribution

    The Journal of Biological Chemistry 277:24204–24211.

    https://doi.org/10.1074/jbc.M202702200

    • PubMed
    • Google Scholar
44. 1. Poirier P
    2. Wawrzyniak I
    3. Vivarès CP
    4. Delbac F
    5. El Alaoui H

    (2012) New insights into Blastocystis spp.: a potential link with irritable bowel syndrome

    PLOS Pathogens 8:e1002545.

    https://doi.org/10.1371/journal.ppat.1002545

    • PubMed
    • Google Scholar
45. 1. Priyam A
    2. Woodcroft BJ
    3. Rai V
    4. Moghul I
    5. Munagala A
    6. Ter F
    7. Chowdhary H
    8. Pieniak I
    9. Maynard LJ
    10. Gibbins MA
    11. Moon H
    12. Davis-Richardson A
    13. Uludag M
    14. Watson-Haigh NS
    15. Challis R
    16. Nakamura H
    17. Favreau E
    18. Gómez EA
    19. Pluskal T
    20. Leonard G
    21. Rumpf W
    22. Wurm Y

    (2019) Sequenceserver: a modern graphical user interface for custom BLAST databases

    Molecular Biology and Evolution 36:2922–2924.

    https://doi.org/10.1093/molbev/msz185

    • PubMed
    • Google Scholar
46. 1. Ravindra G
    2. Balaram P

    (2005) Plasmodium falciparum triosephosphate isomerase: new insights into an old enzyme

    Pure and Applied Chemistry 77:281–289.

    https://doi.org/10.1351/pac200577010281

    • Google Scholar
47. 1. Río Bártulos C
    2. Rogers MB
    3. Williams TA
    4. Gentekaki E
    5. Brinkmann H
    6. Cerff R
    7. Liaud MF
    8. Hehl AB
    9. Yarlett NR
    10. Gruber A
    11. Kroth PG
    12. van der Giezen M

    (2018) Mitochondrial glycolysis in a major lineage of eukaryotes

    Genome Biology and Evolution 10:2310–2325.

    https://doi.org/10.1093/gbe/evy164

    • PubMed
    • Google Scholar
48. 1. Robinson AJ
    2. Kunji ERS

    (2006) Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

    PNAS 103:2617–2622.

    https://doi.org/10.1073/pnas.0509994103

    • PubMed
    • Google Scholar
49. 1. Ruprecht JJ
    2. King MS
    3. Zögg T
    4. Aleksandrova AA
    5. Pardon E
    6. Crichton PG
    7. Steyaert J
    8. Kunji ERS

    (2019) The molecular mechanism of transport by the mitochondrial ADP/ATP carrier

    Cell 176:435–447.

    https://doi.org/10.1016/j.cell.2018.11.025

    • PubMed
    • Google Scholar
50. 1. Ruprecht JJ
    2. Kunji ERS

    (2020) The SLC25 mitochondrial carrier family: structure and mechanism

    Trends in Biochemical Sciences 45:244–258.

    https://doi.org/10.1016/j.tibs.2019.11.001

    • PubMed
    • Google Scholar
51. 1. Ruprecht JJ
    2. Kunji ERS

    (2021) Structural mechanism of transport of mitochondrial carriers

    Annual Review of Biochemistry 90:535–558.

    https://doi.org/10.1146/annurev-biochem-072820-020508

    • PubMed
    • Google Scholar
52. 1. Saraste M
    2. Walker JE

    (1982) Internal sequence repeats and the path of polypeptide in mitochondrial ADP/ATP translocase

    FEBS Letters 144:250–254.

    https://doi.org/10.1016/0014-5793(82)80648-0

    • PubMed
    • Google Scholar
53. 1. Saris NEL

    (1980) Sulphate transport by H+ symport and by the dicarboxylate carrier in mitochondria

    The Biochemical Journal 192:911–917.

    https://doi.org/10.1042/bj1920911

    • PubMed
    • Google Scholar
54. 1. Stechmann A
    2. Hamblin K
    3. Pérez-Brocal V
    4. Gaston D
    5. Richmond GS
    6. van der Giezen M
    7. Clark CG
    8. Roger AJ

    (2008) Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

    Current Biology 18:580–585.

    https://doi.org/10.1016/j.cub.2008.03.037

    • PubMed
    • Google Scholar
55. 1. Stensvold CR
    2. Lewis HC
    3. Hammerum AM
    4. Porsbo LJ
    5. Nielsen SS
    6. Olsen KEP
    7. Arendrup MC
    8. Nielsen HV
    9. Mølbak K

    (2009) Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite

    Epidemiology and Infection 137:1655–1663.

    https://doi.org/10.1017/S0950268809002672

    • PubMed
    • Google Scholar
56. 1. Stensvold CR
    2. van der Giezen M

    (2018) Associations between gut microbiota and common luminal intestinal parasites

    Trends in Parasitology 34:369–377.

    https://doi.org/10.1016/j.pt.2018.02.004

    • PubMed
    • Google Scholar
57. 1. Stensvold CR
    2. Sørland BA
    3. Berg R
    4. Andersen LO
    5. van der Giezen M
    6. Bowtell JL
    7. El-Badry AA
    8. Belkessa S
    9. Kurt Ö
    10. Nielsen HV

    (2022) Stool microbiota diversity analysis of blastocystis-positive and blastocystis-negative individuals

    Microorganisms 10:326.

    https://doi.org/10.3390/microorganisms10020326

    • Google Scholar
58. 1. Stensvold CR
    2. Berg RPKD
    3. Maloney JG
    4. Molokin A
    5. Santin M

    (2023) Molecular characterization of Blastocystis and Entamoeba of muskoxen and sheep in Greenland

    International Journal for Parasitology 53:673–685.

    https://doi.org/10.1016/j.ijpara.2023.05.005

    • PubMed
    • Google Scholar
59. 1. Stenzel DJ
    2. Boreham PF

    (1996) Blastocystis hominis revisited

    Clinical Microbiology Reviews 9:563–584.

    https://doi.org/10.1128/CMR.9.4.563

    • PubMed
    • Google Scholar
60. 1. Tavoulari S
    2. Schirris TJJ
    3. Mavridou V
    4. Thangaratnarajah C
    5. King MS
    6. Jones DTD
    7. Ding S
    8. Fearnley IM
    9. Kunji ERS

    (2022) Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

    Molecular Metabolism 60:101469.

    https://doi.org/10.1016/j.molmet.2022.101469

    • PubMed
    • Google Scholar
61. 1. Tito RY
    2. Chaffron S
    3. Caenepeel C
    4. Lima-Mendez G
    5. Wang J
    6. Vieira-Silva S
    7. Falony G
    8. Hildebrand F
    9. Darzi Y
    10. Rymenans L
    11. Verspecht C
    12. Bork P
    13. Vermeire S
    14. Joossens M
    15. Raes J

    (2019) Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota

    Gut 68:1180–1189.

    https://doi.org/10.1136/gutjnl-2018-316106

    • PubMed
    • Google Scholar
62. 1. Toleco MR
    2. Naake T
    3. Zhang Y
    4. Heazlewood JL
    5. Fernie AR

    (2020) Plant mitochondrial carriers: molecular gatekeepers that help to regulate plant central carbon metabolism

    Plants 9:117.

    https://doi.org/10.3390/plants9010117

    • PubMed
    • Google Scholar
63. 1. Tsaousis AD
    2. Hamblin KA
    3. Elliott CR
    4. Young L
    5. Rosell-Hidalgo A
    6. Gourlay CW
    7. Moore AL
    8. van der Giezen M

    (2018) The human gut colonizer Blastocystis respires using complex ii and alternative oxidase to buffer transient oxygen fluctuations in the gut

    Frontiers in Cellular and Infection Microbiology 8:371.

    https://doi.org/10.3389/fcimb.2018.00371

    • PubMed
    • Google Scholar
64. 1. Wawrzyniak I
    2. Roussel M
    3. Diogon M
    4. Couloux A
    5. Texier C
    6. Tan KSW
    7. Vivarès CP
    8. Delbac F
    9. Wincker P
    10. El Alaoui H

    (2008) Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis

    International Journal for Parasitology 38:1377–1382.

    https://doi.org/10.1016/j.ijpara.2008.06.001

    • PubMed
    • Google Scholar
65. 1. Wu Z
    2. Mirza H
    3. Tan KSW

    (2014) Intra-subtype variation in enteroadhesion accounts for differences in epithelial barrier disruption and is associated with metronidazole resistance in Blastocystis subtype-7

    PLOS Neglected Tropical Diseases 8:e2885.

    https://doi.org/10.1371/journal.pntd.0002885

    • PubMed
    • Google Scholar
66. 1. Yason JA
    2. Liang YR
    3. Png CW
    4. Zhang Y
    5. Tan KSW

    (2019) Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies

    Microbiome 7:30.

    https://doi.org/10.1186/s40168-019-0644-3

    • PubMed
    • Google Scholar
67. 1. Zhou L
    2. Maldonado M
    3. Padavannil A
    4. Guo F
    5. Letts JA

    (2022) Structures of Tetrahymena’s respiratory chain reveal the diversity of eukaryotic core metabolism

    Science 376:831–839.

    https://doi.org/10.1126/science.abn7747

    • PubMed
    • Google Scholar
68. 1. Zierdt CH
    2. Donnolley CT
    3. Muller J
    4. Constantopoulos G

    (1988) Biochemical and ultrastructural study of blastocystis hominis

    Journal of Clinical Microbiology 26:965–970.

    https://doi.org/10.1128/jcm.26.5.965-970.1988

    • PubMed
    • Google Scholar
69. 1. Zierdt CH

    (1991) Blastocystis hominis--past and future

    Clinical Microbiology Reviews 4:61–79.

    https://doi.org/10.1128/CMR.4.1.61

    • PubMed
    • Google Scholar

Author details

1. Eva Pyrihová

   1. Medical Research Council Mitochondrial Biology Unit, The Keith Peters Building, Cambridge, United Kingdom
   2. University of Stavanger, Department of Chemistry, Bioscience, and Environmental Engineering, Stavanger, Norway

   Contribution

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

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5611-1234

2. Martin S King

   Medical Research Council Mitochondrial Biology Unit, The Keith Peters Building, Cambridge, United Kingdom

   Contribution

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

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6030-5154

3. Alannah C King

   Medical Research Council Mitochondrial Biology Unit, The Keith Peters Building, Cambridge, United Kingdom

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4100-3657

4. M Rey Toleco

   University of Stavanger, Department of Chemistry, Bioscience, and Environmental Engineering, Stavanger, Norway

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0002-7888-8479

5. Mark van der Giezen

   1. University of Stavanger, Department of Chemistry, Bioscience, and Environmental Engineering, Stavanger, Norway
   2. Research Department Stavanger University Hospital, Stavanger, Norway

   Contribution

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

   For correspondence

   mark.vandergiezen@uis.no

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1033-1335

6. Edmund RS Kunji

   Medical Research Council Mitochondrial Biology Unit, The Keith Peters Building, Cambridge, United Kingdom

   Contribution

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

   For correspondence

   ek@mrc-mbu.cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0610-4500

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