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Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum

Felix Evers, Alfredo Cabrera-Orefice, Dei M. Elurbe, Mariska Kea-te Lindert, Sylwia D. Boltryk, Till S. Voss, Martijn A. Huynen, Ulrich Brandt, Taco W.A. Kooij

Preprint posted on October 05, 2020 https://www.biorxiv.org/content/10.1101/2020.10.05.326496v1

Peeking into Plasmodium mitochondria

Selected by Mariana De Niz

Categories: molecular biology

Background

Malaria parasites harbour only a single, indispensable mitochondrion with a minimalistic mitochondrial DNA encoding three proteins, COX1, COX3 and CYTB – the latter of which is the target of the antimalarial atovaquone. Although this unusual mitochondrion in Plasmodium parasites is a validated drug target, its function remains poorly understood. Due to its high sequence diversity and poor mitochondrial targeting predictions, the Plasmodium mitochondrial genome remains poorly explored. Function annotations, co-expression patterns and homology data have been integrated in silico to predict possible protein interactions, however, this approach is limited by a lack of annotated orthologues for many proteins, limited temporal resolution of expression data, and imperfect correlation between transcription and translation timing in Plasmodium species. Recent advances in methodology, including complexome profiling, has the potential of providing an inventory of protein complexes in a single experiment. It has allowed finding components of OXPHOS complexes and assembly intermediates and interactions in the mitochondria of various organisms. In their work, Evers et al (1) applied complexome profiling to map the inventory of protein complexes across the Plasmodium asexual blood and transmissible stages, enriched for mitochondria.

Figure 1. Electron micrographs of Plasmodium falciparum blood stages show
ultrastructural differences between the mitochondrion (red arrow) in asexual blood stage parasites and gametocytes. (From Ref 1.)

Key findings and developments

The authors began by demonstrating the existence of stage-specific mitochondrial ultrastructure in P. falciparum, by performing transmission electron microscopy (TEM) of the three developmental stages of asexual blood stage parasites (namely, rings, trophozoites and schizonts), and stage V gametocytes. From the obtained data, the authors point out to specific differences in cristae inside the mitochondria across the various stages. Particularly, in gametocytes, mitochondria appear more electron-dense and cover larger distinct areas, suggesting an increase in size and level of branching. The clear differences between all asexual blood stages on one side, and the gametocytes on the other, led the authors to further explore how these ultrastructural changes are reflected at the protein level. For this, the authors performed complexome profiling of mixed asexual stages and stage V gametocytes, using different enrichment methods based on syringe lysis with saponin, syringe lysis without saponin, and nitrogen cavitation, and the use of different detergents. All enrichment methods were found to be consistent, and digitonin solubilization was the chosen method for gametocyte samples. Large numbers of gametocytes were obtained by induction by conditional overexpression of GDV1 in a gametocyte producer line. Across all asexual blood stage and gametocyte samples, 1759 unique proteins were identified.

Validation of complexome profiling

In order to validate the approach, the authors verified whether other well-known, previously identified complexes could be correctly identified and whether differences existed if different isolation methods were used:

  1. The protein folding/degradation-involved endoplasmic reticulum membrane complex (EMC) subunits were identified (with one exception).
  2. The proteasome complex subunits were identified. Saponin treatment was shown to deplete all proteasome-associated assemblies, while membrane proteins were not affected.
  3. The rhoptry protein complex (RhopH) was identified, and a new component was described, which the authors term RhopH associated protein 1 (RhopA1).
  4. Six LCCL domain-containing proteins form a complex in the crystalloid (an organelle unique to Plasmodium insect stages) were identified. The authors found that in stage V gametocytes, LAP1-3 and LAP4-5 formed two distinct subcomplexes, and identified a possible LAP2-3 assembly intermediate.
  5. The Plasmodium translocon of exported proteins (PTEX) is a complex essential for the export of parasite proteins into the host erythrocyte. The authors identified the various components (EXP2, PTEX150, HSP101, PTEX88 and TRX2 – the latter 2 as monomers without clear co-migration with the complex being evident), as well as noting that EXP2 might be present independent from the PTEX.

The authors conclude that complexome profiling helps to distinguish the presence of proteins in different sub-assemblies, highlighting it as an advantageous tool to investigate interactions of promiscuous components or assess assembly pathways.

Investigation of mitochondrial complexes

Respiratory chain complex III (CIII)

Following the above validation, the authors next focused on the OXPHOS complexes. They found that all but one of the canonical components of cytochrome bc1 (CIII) with obvious Plasmodium orthologues, comigrated. MPPα and MPPβ, as well as 4 newly identified proteins (PF3D7_0306000, and respiratory chain complex 3 associated proteins 1-3 (C3AP1-3)) comigrated consistently with CIII subunits. The complexome profiles for CIII suggested abundant differences between asexual blood stage parasites and stage V gametocytes, with enrichment-related intensity values being 9-fold higher in gametocytes.

Respiratory chain complex IV (CIV)

So far, only 5 canonical subunits of Plasmodium cytochrome c oxidase (CIV) have been identified (COX1, COX2, COX3, COX5b and COX6b). Fragments COX2a and 2b were both identified in the complexome profiles.  Recent research showed a highly divergent composition of CIV in T. gondii, containing 11 subunits specific to Apicomplexa. In their work, the authors identified orthologues for all these subunits in P. falciparum. In addition, they also identified 5 previously uncharacterized myzozan-specific proteins that consistently comigrated with the complex (which they termed respiratory chain complex 4 associated proteins 1-5 (C4AP1-5). The complexome profiles for CIV suggested abundant differences between asexual blood stage parasites and stage V gametocytes, with enrichment-related intensity values being 20-fold higher in gametocytes.

Composition of respiratory chain complexes III and IV in an evolutionary context

The authors began by mapping the gains and losses of the respective subunits along the evolutionary tree. They suggest that the three novel CIII proteins (C3AP1-3) appear to be relatively recent appearances in Apicomplexans and their close relatives. Conversely, there are proteins present in CIII from fungi and Metazoa, but absent in P. falciparum, with some being lost specifically in the Apicomplexans. Regarding the CIV complex, most of the novel proteins identified in this study appear to have myzozan origin. In addition to presence of orthologues in other species, the authors also examined addition/loss of protein domains in conserved complex membranes, and found important differences within direct evolutionary relationships.

Complex V

Classical mitochondrial function includes harnessing energy in the chemical bonds of ATP, and this process is predominantly executed by complex V (CV). The authors lysed large amounts of asexual blood stage parasites or gametocytes through nitrogen cavitation, without saponin – as this proved to be the most suitable method for this specific point. They found 14 proteins associated with CV.

Complex II

Succinate dehydrogenase couples succinate oxidation as part of the citric acid cycle to the reduction of ubiquinone in the OXPHOS pathway. CII is generally composed of at least 4 different subunits: SDHA and SDHB, catalysing succinate oxidation, and SDHC and SDHD anchoring the complex in the inner mitochondrial membrane and providing the binding pocket for haem and ubiquinone. As for CV, only the method involving nitrogen cavitation worked successfully. Although some previously suggested candidates were not found, the authors identified five putative subunits sharing a common dominant band. They assigned one of them as putative PfSDHC, and named the other 4 components, respiratory chain complex 2 associated proteins 1-4 (C2AP1-4), one of which is myzozoan-specific and plays an important role in ookinete mitochondria in P. berghei.

Protein dynamics are in line with a significant metabolic shift in P. falciparum gametocytes

Metabolomics approaches have indicated a shift in carbon metabolism in gametocytes from anaerobic glycolysis towards increased TCA cycle utilization and increased respiration. This is reflected in a general increase of mitochondrial proteins and specifically of TCA proteins in gametocytes. A further indication is the de novo appearance of cristae in gametocytes, as they typically serve as hubs for respiration. The authors investigated whether this mitochondrial phenotype would be reflected in the abundance of OXPHOS complexes. Overall, the authors found that proteins associated with CIII and CIV were significantly more abundant in gametocytes than asexual blood stage parasites. However, they also reported some variability across gametocyte samples. The authors validated their findings using complexome profiling, by using mass spectrometry, and confirmed significantly higher abundance levels of OXPHOS complex components (CII, CIII, CIV and CV) in gametocytes than in asexual blood stages. The authors also identified a few outliers (one per complex), which showed higher abundance in asexual blood stage parasites. For determining whether this trend was indicative of larger metabolic shift in gametocytes, the authors also investigated abundance dynamics of other proteins involved in central energy metabolism. They found that enzymes involved in glycolysis were more abundant in asexual blood stage parasites, while other proteins seem to be gametocyte-specific. Altogether, their data support previous metabolomic-based suggestions of a switch towards respiration and away from anaerobic glycolysis in P. falciparum gametocytes.

 

What I like about this preprint

I chose this preprint because it touched on a very interesting topic (i.e. mitochondrial biology) which is poorly understood in Plasmodium. As the authors emphasize in their discussion, this is surprising given that mitochondrial components are in fact antimalarial targets. I found also very interesting that they chose to compare mature gametocytes and asexual blood stages, and I like that they present an interesting method which seems to offer various advantages over others previously used. I think it will hopefully close important gaps in our knowledge of Plasmodium biology.

References

  1. Evers et al, Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum, bioRxiv, 2020.

 

Posted on: 22nd December 2020 , updated on: 23rd December 2020

doi: https://doi.org/10.1242/prelights.26595

Read preprint (1 votes)




Author's response

Taco Kooij and Felix Evers shared

Open questions 

1. This work is very exciting. Throughout your work, you divided the findings into stage V gametocytes, and asexual blood stages. Are there important differences in terms of mitochondrial complexes and their function, between rings/trophozoites/schizonts, and between the different stages of gametocyte maturation (I-V)?

As we did not perform the corresponding experiments on synchronized asexual parasites or younger gametocytes – these are subject of our ongoing efforts – we can only speculate on differences. In asexual blood stages, transcriptional and functional data suggests higher OXPHOS abundance in schizonts compared to trophozoites or especially rings. This is in line with demand for pyrimidine biosynthesis, which requires ubiquinone cycling by cytochrome bc1 complex that is expected for schizonts due to the high rate of DNA biosynthesis. In gametocytes, it would be interesting to see whether the increase in respiratory chain components is a preadaptation to facilitate survival in the insect host, or whether OXPHOS is crucial for gametocytes to meet energy requirements in the human host. In the latter case, one would expect early emergence of cristae and increases in OXPHOS complex abundance while in the former changes might only occur relatively close to maturity of the gametocytes.    

2. A more general question is that you studied the mitochondrial complexes from parasites grown in culture. Do you think the in vivo environment within mammalian hosts influences the function of mitochondria in any way? For instance, the locations for schizont sequestration, or the bone marrow for gametocyte development?

As is the case for any model, our current in vitro system is not capable of faithfully replicating conditions a parasite would encounter in the human body. Obvious deviations such as absence of an immune system, difference in haematocrit, nutrient availability, and many other host factors have obvious implications. However, it has been shown in P. falciparum that the transcriptome, and presumably the proteome, is relatively hard-wired and unresponsive to environmental challenges, which would suggest our proteome-level observations to be a good proxy for the in vivo situation. This is further supported by the fact that NF54, the underlying strain for this IGP2 line, is perfectly capable of infecting mosquitoes and establishing infection in human volunteers. Regarding sequestration of schizonts and gametocytes: neither of those microenvironments have obvious deviations in nutrient availability and oxygen availability from standard circulation, so we would not expect sequestration to significantly alter viability of different bioenergetic approaches or mitochondrial makeup but it can of course not be excluded.

3. You found throughout your work significantly higher abundance levels of OXPHOS complex components (CII, CIII, CIV and CV) in gametocytes than in asexual blood stages. Can you expand further on the implications of this finding? Why is this the case for gametocytes?

Answering the “why” question is often quite challenging in an evolutionary context. It is safe to assume that the higher abundance of these complexes confers the ability to accommodate a higher rate of oxidative phosphorylation compared to asexual blood stages. One possible hypothesis is that gametocytes, have to preadapt to the comparatively nutrient deprived mosquito midgut, which necessitates the much more efficient oxidative phosphorylation instead of anaerobic glycolysis to meet their energy requirements. This is supported by the high number of OXPHOS related genes that have turned out to have minor effects on blood stages but are crucial for colonization of the insect host.  One could also argue from the other direction in that the rapid growth and high metabolic activity observed in asexual blood stages would generate too much oxidative stress to handle, while the comparatively slow gametocyte development does allow for more of the efficient but oxidatively taxing oxidative phosphorylation.

4. In your work, you enriched for gametocytes by conditional overexpression of GDV1. Would you expect that your findings might be affected in any way a) by gametocyte density and b) by the inducible nature of the line? What would you expect from samples taken from an infected person, or naturally-arising gametocytes from culture?

How well the results with the IGP2 line and corresponding culture conditions resemble a natural infection is a very valid concern. However, given the nature of the experiments and the vast amount of material required, this was the best available model. In the manuscript, we analyzed one sample of wild-type, non-inducible NF54 gametocytes for comparison. Though this does still not reflect a natural infection, at least these laboratory-adapted parasites retain the ability to infect mosquitoes and human volunteers. We find that at least on the proteome level, there are no striking differences in either abundance dynamics or complex composition between NF54 and IGP2, indicating that the induction of high gametocyte densities in vitro has no significant effect. This is further explored in the recently submitted preprintby our co-authors Till Voss and Sylwia Boltryk.

5. Going further into your study of composition and stage dynamics of mitochondrial complexes, is there anything known regarding the liver stages of Plasmodium? This might be an interesting target as well, given the complex nature of the interaction with a much more sophisticated host cell so to speak.

It would be fascinating to study the interplay of the liver cells and host cells on a protein complex level. One major challenge to achieve this would be to obtain liver stage material in sufficient quantities and also enrich it over the host cells to a degree to allow mapping peptides distinctly to parasite and not host mitochondria as well as finding parasite signal among the expected quantity of host cell noise. But once technically feasible this would add a lot to our understanding of the currently enigmatic liver stage.

6. As a continuation of question 4, what differences or similarities would you expect upon comparison of your findings in P. falciparum, with P. berghei or other rodent models which can be isolated from a mammalian host?

It has been shown previously that P. berghei shows the phenotype of acristate asexual blood stage and cristate gametocytes. In addition, much of the observations that mitochondrial function becomes more important during transmission stem from work in P. berghei. Finally, all newly identified complex components were well conserved within the genus. Therefore, it seems likely that much of our observations would represent conserved trades of all Plasmodium species. However, one may also anticipate some differences due to the in vivo conditions, the preference of P. berghei for young red blood cells (reticulocytes) and, not in the least, because of the dramatic difference in gametocyte maturation time observed between P. berghei – just over one day – and P. falciparum – approximately ten days. Unfortunately, it will not be trivial to harvest sufficient material to make the actual comparison.

7. You performed various optimizations for complexome profiling which you describe in depth in your work. You also compare the usefulness of this method with others that have been used in the past to try and understand mitochondrial biology. What would be some limitations still existing, which you think can still be further improved for future work in the parasitology field?

One limitation currently is that we are limited in size range (BN-PAGE used in this study resolves complexes from 20 kDa up ~ 5MDa) as well as stability of some protein complexes. The former may be addressed through usage of large pore gels, different acrylamide gradients, or alternative separation methods such as the use of size exclusion columns. The latter could be potentially addressed through incorporation of crosslinking to stabilize and fix transient interactions or interactions that are highly dependent on the exact cellular context. This would also be exciting for a potential nuclear complexome as many protein interactions there have been shown to heavily rely on the DNA context.

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