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Organisms Without Mitochondria, How It
May Happen?
Vladimír Hampl
Contents
1 Mitochondria in Anaerobes Are Reduced but Typically Not Lost .. . . . . . . . . . . . . . . .. . . . . . . . 310
2 Oxymonads: Protists Without Mitochondria ................................................ 311
3 Prerequisites and Consequences of Mitochondrial Loss .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . 313
4 Why Should We Be Interested in Amitochondriate Protists? .............................. 314
References .. . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . 316
Abstract Decades of investigations have clearly shown that protists living in
low-oxygen environments possess mitochondria despite their textbook function,
oxidative phosphorylation, is usually absent. The presence of these, in some cases,
very rudimental mitochondria has been ascribed to their irreplaceable role in the
synthesis of FeS clusters, prosthetic groups of several essential proteins. The deep
investigation of the oxymonad Monocercomonoides exilis (Preaxostyla,
Metamonada) revealed that this organism very likely represents a notable exception,
in which the synthesis of FeS clusters runs in the cytosol and mitochondrion is
absent. Investigation of a broader spectrum of oxymonads and their relatives pro-
vided evidence that the profound reorganisation of FeS cluster synthesis was initi-
ated by a HGT of the bacterial pathway SUF and a loss of the mitochondrial pathway
ISC already before the last common ancestor of this clade. This innovation was very
likely a preadaptation for (and not a consequence of) the mitochondrial loss, which
happened much later and only in the oxymonad lineage. M. exilis and other
oxymonads are being further studied because they represent valuable examples
relevant to our understanding of the reductive evolution of organelles and to the
origin of the eukaryotic cell.
V. Hampl (*)
Faculty of Science, Department of Parasitology, Charles University, BIOCEV, Vestec,
Czech Republic
e-mail: vlada@natur.cuni.cz
©Springer Nature Switzerland AG 2019
J. Tachezy (ed.), Hydrogenosomes and Mitosomes: Mitochondria
of Anaerobic Eukaryotes, Microbiology Monographs 9,
https://doi.org/10.1007/978-3-030-17941-0_13
309
1 Mitochondria in Anaerobes Are Reduced but Typically
Not Lost
Endosymbiosis greatly contributed to the formation of eukaryotic complexity,
because it is the only known mechanism how membrane-bound and genome-
containing cell compartments evolve (Sagan 1967; Lane 2011). Its most important
products, mitochondria and plastids, had changed the biosphere completely and
arguably are behind the evolutionary success of eukaryotes. At the same time,
while sampling the diverse eukaryotic lineages, it has become apparent that these
organelles are not being dispensed at occasions, when their most pronounced
functions, oxidative phosphorylation in the case of mitochondria and photosynthesis
in the case of plastids, are not needed or are disabled by environmental conditions.
So then, vast majority of secondary non-photosynthetic algae and plants still contain
colourless plastids (Hadariová et al. 2017), and eukaryotes inhabiting environments
with low-oxygen concentrations still contain mitochondria (Roger et al. 2017), the
examples of which have been in details described in this book.
Careful examinations provided raison d’etre of these sometimes very rudimental
mitochondria in the synthesis of FeS clusters (Williams et al. 2002). FeS clusters
serve as prosthetic groups of some proteins and are essential in every cell, and their
synthesis is initiated in the mitochondrion by the iron-sulphur cluster assembly
pathway of alpha-proteobacterial origin and continued in the cytosol by the cytosolic
iron-sulphur protein assembly (CIA) pathway (Kispal et al. 1999; Lill et al. 2015;
Braymer and Lill 2017). Interesting exception is represented by Archamoebae
(Entamoeba,Mastigamoeba), which lack ISC pathway, and their FeS clusters are
apparently synthesised by the combination of nitrogen fixation (NIF) and CIA
pathway; the former was acquired by horizontal gene transfer (HGT) from
Epsilonproteobacteria. In the human pathogen Entamoeba histolytica, the NIF
pathway is probably not localised in its mitochondrion, although controversies
remain (Mi-ichi et al. 2009; Maralikova et al. 2010; Nývltová et al. 2013), and so
it may be the first known mitochondrion not involved in the iron-sulphur cluster
assembly. If true, the case of E. histolytica demonstrates that under some circum-
stances, like acquisition of new upstream biosynthetic pathway via horizontal gene
transfer, the process of FeS cluster assembly can be performed entirely in the
cytosol. Also, it seems that FeS cluster assembly is not the reason why
E. histolytica keeps its mitosomes and another essential process, sulphate activation,
was proposed to localise into this organelle (Mi-ichi et al. 2009).
The presence of some form of mitochondrion in every studied eukaryote and the
fact that even the most reduced mitosomes harbour essential pathways strengthened
the paradigm about the ubiquity of mitochondria (Roger and Silberman 2002;
Embley et al. 2003; Hackstein et al. 2006). On the other hand, the example of
E. histolytica has shown that the seeming essentiality of mitochondria is not pro-
vided by the same function in all eukaryotes, and so the question remained, whether
it is possible for a mitochondrion to dispense all functions and disappear completely.
310 V. Hampl
Investigation of additional eukaryotic lineages has been instrumental, as many times
before, and this question was answered when it came to oxymonads.
2 Oxymonads: Protists Without Mitochondria
Oxymonads are relatively small collection of ~140 described species (Hampl 2016),
which live in the intestine of some insect and mammalian hosts, and together with
the genera Trimastix and Paratrimastix, they represent the third principal lineage of
Metamonada (Hampl et al. 2009; Adl et al. 2019). In wood-eating insects, certain
termites and cockroaches, oxymonads seem to be beneficial part of the intestinal
microflora; however, no solid data on their metabolic involvement is available.
Majority of studies involving oxymonads focused on description of new species,
phylogeny and ultrastructure (e.g. Nie 1950; Radek 1994; Brugerolle et al. 1997;
Heiss and Keeling 2006) and rarely on their other features. Exceptions are few
studies investigating the movement of axostyle, pronounced microtubular part of
their cytoskeleton (McIntosh 1973), relationships with ectosymbiotic bacteria (Lean-
der and Keeling 2004; Noda et al. 2006; Utami et al. 2018), modifications of
glycolysis (Slamovits and Keeling 2006a; Liapounova et al. 2006), introns
(Slamovits and Keeling 2006b) and noncanonical genetic codes (Keeling and
Leander 2003). Importantly for this text, ultrastructural investigations have not
brought clear evidence for the presence of neither mitochondrion nor Golgi body.
Notable is the study of Carpenter et al. (2008), in which was reported the presence of
relatively large electron dense and potentially double-membrane-bounded vesicles
more than 1 μm in diameter, putative mitochondria or peroxisomes. Similar vesicles
are visible also in earlier TEM images of (McIntosh 1973) and might be homologous
to cytoplasmic granules reported from light microscopy by Cleveland in
Saccinobaculus lata (1950). They were not investigated further.
In 2016 we have published a study (Karnkowska et al. 2016) in which the traces
of mitochondrion were carefully investigated in the genomic and transcriptomic
datasets of an oxymonad now known as Monocercomonoides exilis (Treitli et al.
2018; Fig. 1). The generated draft genome is ~75 MB in size and contains 16,629
predicted protein-coding loci, and according to the available measure, it is reason-
ably complete albeit divergent. Core Eukaryotic Genes Mapping Approach
(CEGMA) recovered 63.3% of core eukaryotic genes, and after manual curation of
gene models, and not considering mitochondrion-related genes in the CEGMA
dataset, the conserved gene recovery increased to 90%. The predicted proteins as
well as the six-frame translation of the genome have been searched for mitochondrial
hallmark proteins (e.g. components of TIM/TOM complexes, mitochondrial chap-
erones and ISC pathway enzymes); furthermore, the data were exhaustively searched
using homology-based and targeting signal-based approaches [summarised in Fig. 2
in Karnkowska et al. (2016)]. No approach has resulted in recovering a reliable
candidate(s) for mitochondrial proteins, and this led to the conclusion that the
mitochondrion is not present. This hypothesis still holds, while further investigation
Organisms Without Mitochondria, How It May Happen? 311
of M. exilis genome and genomic and transcriptomic data of other oxymonads is in
progress (Karnkowska et al. under review).
If we admit the absence of mitochondrion in M. exilis, we should provide an
explanation how the cell substitutes the essential functions typically provided by this
compartment. As in the mitosome-containing anaerobes, the cellular ATP require-
ments must be met by the cytosolic ATP production associated with the catabolism
of organic compounds. Indeed, M. exilis contains full set of glycolytic enzymes
including ATP saving analogues PPi-dependent phosphofructokinase (PFK) and
pyruvate-orthophosphate dikinase (PPDK), which are known to be present in other
eukaryotic anaerobes (Liapounova et al. 2006; Karnkowska et al. 2016). The
breakdown of one molecule of glucose to two molecules of pyruvate should theo-
retically provide the M. exilis cell with three ATPs, and because two of these ATP
molecules were formed from AMP and PPi, M. exilis gains five new high-energy
phosphate bonds on ATP molecules. Enzymes of extended glycolysis, pyruvate-
ferredoxin oxidoreductase (PFOR), [FeFe]hydrogenase and acetyl-CoA synthetase
(ADP forming) are also present, suggesting the yield of additional one ATP per
pyruvate molecule. Enzymes for the arginine dihydrolase pathway, known as sig-
nificant contributor to ATP pool in Giardia intestinalis and Trichomonas vaginalis
(Schofield et al. 1992; Yarlett et al. 1996), have been detected in M. exilis
genome (Novák et al. 2016), and other ATP-producing pathways were and will be
Fig. 1 Monocercomonoides exilis and Paratrimastix pyriformis. DIC images of (a)
Monocercomonoides exilis and (b)Paratrimastix pyriformis from cultures. Cells are shown in the
same scale. (c) TEM micrograph of Monocercomonoides exilis (photo by Naoji Yubuki), Ax
axostyle, ER endoplasmic reticulum, Fl flagellum, FV food vacuoles, Gly glycogen granules, Nu
nucleus, Pax preaxostyle
312 V. Hampl
revealed by the detailed analysis of the M. exilis genome (Karnkowska et al. under
review) and by follow-up metabolic studies.
Less usual explanation was proposed for the way how FeS clusters are
synthetized without the mitochondrial ISC system. Like in the case of
E. histolytica, HGT apparently played the key role. In the genome and transcriptome
of M. exilis, a set of genes for sulphur mobilisation system (SUF) was found. It
consists of a fusion gene SufDSU, and genes for SufB and SufC, which branch from
within eubacteria and have no close bacterial sister lineage (Karnkowska et al. 2016).
SUF pathway is frequently present in prokaryotes including E. coli, in all plastids,
which inherited the pathway from cyanobacteria, and it rarely occurs also in eukary-
otic anaerobes (Blastocystis,Pygsuia and Stygiella) (Tsaousis et al. 2012; Roche
et al. 2013; Stairs et al. 2014; Leger et al. 2016). The SUF pathway of M. exilis is
simple but theoretically functional with SufDSU extracting sulphur from cysteine
and SufB and SufC creating a scaffold for the cluster formation. Besides the SUF
pathway, M. exilis contains basic inventory of genes for the CIA pathway, namely,
Nbp35, Nar1, Cia1, Cia2a and Cia2b. As there are no functional data regarding the
SUF and CIA pathways, it is unclear whether and how this unique combination of
pathway cooperates during the FeS cluster formation.
3 Prerequisites and Consequences of Mitochondrial Loss
With such an “extreme”product of mitochondrial reduction in hand, it would be
interesting to learn more about the mitochondria of M. exilis ancestors. Luckily, the
mitochondrion-bearing relatives of oxymonads, genera Trimastix and Paratrimastix,
split from two points of the lineage leading to M. exilis, and so they represent
independent descendants of two such intermediate stages (Zhang et al. 2015). Bits
of knowledge on the function of the mitochondrion in Trimastix marina acquired
thanks to transcriptomic project on this species suggest that this organelle possesses
[FeFe]hydrogenase and a complete glycine cleavage system (GCS) (Leger et al.
2017). GCS is a complex of four enzymes with strictly mitochondrial localisation
catabolising glycine to CO
2
and NH
3
with concomitant production of cofactors
NADH and 5,10-methylene-H
4
folate. ATP is probably not produced in the
T. marina mitochondrion, and pyruvate is oxidatively decarboxylated in the cytosol
by PFO and ATP is produced by substrate-level phosphorylation by acetyl-CoA
synthetase (ADP forming). The mitochondrion of Paratrimastix (formerly
Trimastix)pyriformis has been scrutinised more thoroughly supporting the picture
sketched for T. marina (Hampl et al. 2008; Zubáčová et al. 2013). GCS has been
proven to be localised in the organelle by specific antibodies, and the ongoing
genomic project on this organism promises to provide more complete picture of its
metabolism. Importantly, the mitochondrial ISC pathway is absent in P. pyriformis,
and it is substituted by the closely related set of the three SUF pathway enzymes
orthologous to those in M. exilis. The localisation of these enzymes is unknown, and
Organisms Without Mitochondria, How It May Happen? 313
it is of interest for understanding the functioning of P. pyriformis mitochondrion, as
well as the loss of mitochondrion in M. exilis.
Taxon-wide investigation of the presence/absence of FeS cluster assembly path-
ways using genomic and transcriptomic datasets of seven species of oxymonads and
three species of (para)trimastigids has confirmed the observation from P. pyriformis
and M. exilis that in this whole clade, the ISC was lost and substituted by SUF
composed of the same three enzymes found in M. exilis (Vacek et al. 2018). This
indicates that the switch from ISC to SUF happened relatively early in the common
ancestor of the Preaxostyla clade, but the loss of mitochondrion, which is well-
documented for M. exilis but expected to be common for all oxymonads, happened
much later. So, ISC to SUF transition was likely a preadaptation for the mitochon-
drial loss, but it was definitely not the direct and immediate cause of it, as these
protists have undergone millions of years of evolution with mitochondrion along
with the SUF system before losing the organelle in oxymonads and (para)
trimastigids have preserved this status until present. Dating the protist evolution is
a tricky task, but in this particular case, we have a solid hint from 100-MYA-old
amber fossils of termites, which demonstrate that at this time, oxymonads in their
hindguts were already present and diversified (Poinar 2009). This leads to conclu-
sion that the loss of mitochondria is at least 100-MYA-old event and the substitution
of ISC by SUF is an event considerably older.
In the situation of having the organism that lives millions of years without such a
canonical part of the eukaryotic cell as the mitochondrion, it is fair to ask how the
other systems of such cell have been affected by this unique loss. With this question
in mind, we have performed careful analysis of the genomic and transcriptomic data
focusing on many cellular systems and the answer will hopefully be published soon
(Karnkowska et al. under review).
4 Why Should We Be Interested in Amitochondriate
Protists?
The case of M. exilis, which is given all currently available data a mitochondrion-free
eukaryote, provides example relevant to several ongoing debates related to evolution
of eukaryotes. Firstly, it indicates that mitochondrion, despite being essential and
evolutionarily very stable part of the cell, can be disposed under circumstances,
when the lineage modifies its FeS cluster synthetic pathway into a cytosolic one
(Karnkowska and Hampl 2016). This is apparently plausible but very improbable
process that has never been achieved with the ISC pathway, and in both known cases
(Monocercomonoides exilis and Entamoeba histolytica), it was initiated by a rare
event of HGT. From this perspective, the cellular setup of mitosome-containing
eukaryotes (Giardia, Entamoeba, microsporidia, Microcytos) is not in the “global
optimum”. Maintenance of mitosomes represents a non-zero cost, and it is also not
error-prone. Imagine synthesising costly lipids, setting up proteins and metabolite
314 V. Hampl
transporters, performing specific protein import, dividing vesicles in synchrony with
cell cycle, and this everything only for one or few pathways that are in principle
operable in the cytosol as the example of M. exilisdemonstrates. I expect that many if
not all mitosome-containing eukaryotes keep mitosomes not because the setup of
their cells without mitosomes is impossible but because the step-by-step evolution-
ary journey to a mitosome-free cell is so narrow and involves so improbable (but
possible) steps that the random walk of evolution has not made it through yet.
The second topic for which I propose M. exilis relevancy is the hotly debated
origin of eukaryotes; see Zachar and Szathmáry (2017) for the recent summary. In a
nutshell, two major schools of thought have formed in this debate, which disagree on
the role of mitochondria in the whole process. While some (Yutin et al. 2009;
Martijn and Ettema 2013; Cavalier-Smith 2014) consider mitochondrion as an
important innovation that was acquired during the formation of the eukaryotic cell
but not at the very beginning of this evolutionary transformation, others (Lane and
Martin 2010) argue that acquisition of mitochondrial symbiont was the starter of
eukaryogenesis. The latter claim that without mitochondrial ATP, evolution of many
other eukaryotic novelties would be energetically just implausible. Here comes the
example case of M. exilis but also other eukaryotic anaerobes that thrive without
mitochondrial ATP but maintain their eukaryotic features. Existence of these organ-
isms suggests, as we have recently argued in detail (Hampl et al. 2018), that life
forms of phagotrophic, nucleus-containing eukaryotes existing prior to the mito-
chondrial endosymbiosis are plausible. Hypotheses, which include amitochondriate
(Archezoa) stages, are therefore not logically incorrect or energetically unbearable,
which at the same time does not mean that evolution must have proceeded through
this path.
There are several reasons why Monocercomonoides exilis and other representa-
tives of Preaxostyla should be deeply explored. The main is to confirm or reject our
hypothesis that mitochondrion is absent, which still needs to be treated with caution
and critically scrutinised. Further studies of cell biology and biochemistry, currently
hindered by the absence of laboratory tools including axenic cultivation, should
follow to depict in more details how the amitochondriate cells function. The highest
priority remains to elucidate the process of the FeS cluster synthesis in such cells.
Finally, other oxymonads and Preaxostyla should be investigated for the presence of
mitochondria and, if present, the functions of these organelles should be understood
as much as possible, because their evolution has gone in such an interesting direction
within this group of protists.
Acknowledgement The salary of VH was funded from the European Research Council (ERC)
under the European Union’s Horizon 2020 research and innovation programme (grant agreement
No 771592), from the Centre for research of pathogenicity and virulence of parasites reg. nr.:
CZ.02.1.01/0.0/0.0/16_019/0000759, from the Ministry of Education, Youth and Sports of CR
within the National Sustainability Program II (Project BIOCEV-FAR) LQ1604 and from the project
‘BIOCEV’(CZ.1.05/1.1.00/02.0109).
Organisms Without Mitochondria, How It May Happen? 315
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