Mitochondrial ATP synthesis is essential for efficient gametogenesis in Plasmodium falciparum

  1. Penny C Sparkes
  2. Mufuliat Toyin Famodimu
  3. Eduardo Alves
  4. Eric Springer
  5. Jude Przyborski
  6. Michael J Delves

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Interrupting parasite transmission from humans to mosquitoes is vital for malaria elimination and eradication. Plasmodium male and female gametocytes are the gatekeepers of human to mosquito transmission. Whilst dormant in the human host, their divergent roles during transmission become visually apparent soon after ingestion by the mosquito after rapid transformation into gametes – the males forming eight motile sperm-like cells that each aim to fertilise a single female gamete. Here we report that antibodies raised against PfLDH2 allow accurate identification of male gametocytes. Using this novel tool and functional mitochondrial labelling, we show that the male gametocyte mitochondrion is less active than that of female gametocytes. Rather than being a vestigial organelle discarded during male gametogenesis, we demonstrate that mitochondrial ATP synthesis is essential for male gametocytes to complete gametogenesis and inhibition leads to early arrest. Additionally, using a genetically encoded ratiometric sensor of ATP, we show that gametocytes can maintain cytoplasmic ATP homeostasis in the absence of mitochondrial respiration, indicating the essentiality of the gametocyte mitochondrion for transmission alone. Together, this reveals how gametocytes balance the conflicting energy demands of a dormant and active lifestyle and highlights the mitochondria as a rich source of transmission-blocking targets for future drug development.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/12768661.

    Summary

    In this manuscript from Sparkes et al., the authors investigated the role of mitochondria in Plasmodium falciparum gametogenesis. The authors have developed a useful tool for the malaria community to study male gametocytes, and shown that male gametogenesis is reliant on a functional mitochondrial ATP synthase. Previous work had demonstrated that although gametocytes are largely dormant stages in the parasite life cycle, both male and female gametocytes develop extensive mitochondrial networks with cristae. To investigate the mitochondria's role in these parasites and identify any sex-specific functional differences, the authors raised antibodies to a putative P. falciparum lactate dehydrogenase enzyme (LDH2). They showed that LDH2 is highly and specifically expressed in stage IV and V male gametocytes and male gametes, and antibodies raised against LDH2 are almost entirely nonreactive toward stage IV and V female gametocytes and female gametes. Using this marker, the authors were able to rigorously interrogate the role of the mitochondria in gametocytes and gametogenesis. They found that male gametocyte mitochondria appear to have lower membrane potential as measured by MitoTracker with no discernible differences in mitochondria shape, suggesting lower activity relative to mature female gametocytes. Both male and female gametocyte mitochondria could be inhibited with small molecules targeting the mitochondrial cytochrome bc1 complex, and male exflagellation could be inhibited altogether in a dose dependent manner with both mitochondrial cytochrome bc1 and ATP synthase inhibitors. By titrating serum glucose conditions and using the ATP synthase inhibitor oligomycin A, the authors were able to demonstrate that male gametogenesis is dependent on both glucose availability and a functional ATP synthase, suggesting that both glycolysis and mitochondrial oxidative phosphorylation contribute to functional male gamete formation. This work both informs our understanding of the metabolic underpinnings of an understudied stage of the Plasmodium life cycle, and highlights the potential of mitochondrial targets for malaria transmission blocking strategies.

    Major points:

    • The authors robustly demonstrate that glucose concentration impacts exflagellation, but then proceed to only use the optimized glucose and serum conditions to more thoroughly test the effect of oligomycin A ATP synthase inhibition. The authors should consider validating this result with other inhibitors, such as atovaquone and ELQ-300 which were used in Figure 4, to determine whether inhibition of mitochondrial electron transport chain activity broadly leads to the same exflagellation defect. The authors could also test the impact of glycolysis inhibitors such as 2-DG, rather than just removing glucose, to further support their finding of a specific requirement of glycolysis in exflagellation.

    Minor points:

    • In the introduction, the authors note that 6/8 TCA cycle members are dispensable for asexual development, but Rajaram et al. recently successfully knocked out the remaining two TCA cycle genes (FH and MQO) in P. falciparum (https://doi.org/10.1016/j.jbc.2022.101897). The authors should update their introduction to reflect this literature.

    • When introducing the thresholding algorithm in the text, rather than referencing (Fig. 2A + Fig. 3A) together, the authors could consider altering the text to state "An automated thresholding algorithm was then used to automatically draw ROIs around the nucleus (Fig. 2A) and mitochondria (Fig. 3A) in each cell" for clarity.

    • As the analyses performed in figures 2 and 3 are from the same images, the authors could combine these into a single figure describing both the changes in nuclei and mitochondria composition.

    • The susceptibility of male gametocyte mitochondria to atovaquone does not appear to follow a clean dose response, with the 1 uM dose showing similar inhibition to DMSO. The authors should repeat these experiments or comment on possible explanations for this result. For example, the error bars look wider in this group. Was there substantial replicate variability?

    • The data in Figure 5 may not warrant an entire main text figure on its own, as it is primarily optimizing the media conditions for experiments in Figure 6. These two figures could be combined.

    • To support their qualitative observations in Figure 6B of small, focal spots of DAPI in the no glucose and glucose + 5 uM OA conditions, the authors could consider performing similar quantitative nuclear analyses to those performed in Figure 2.

    • Authors could consider adding a schematic of the FRET sensor parasite line developed to aid in reader understanding, either as an additional panel in figure 7 or as a supplemental figure.

    • Authors should make all of the image datasets analyzed in this paper available in a public repository, in addition to the representative images currently shown in figures.

    Competing interests

    The authors declare that they have no competing interests.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/12775137.

    In the manuscript by Sparkes et al they investigate the role of mitochondrial ATP synthesis for gametogenesis in Plasmodium falciparum parasite. Due to biological differences between asexual stage and gametocyte stage parasites, mainly antimalarials are ineffective against gametocyte parasites leaving them free to continue their life-cycle within the mosquito.  Thus, there is a pressing need to further examine this critical life-stage for development of novel transmission blocking tools. Here, the authors first identify by transcriptional analysis a male-specific stage marker LDH2 for late stage IV gametocytes and subsequently validate the marker through immunofluorescence assays. They next contrasted the differences between nuclear and mitochondrial organization between male and female gametocytes, finding that male gametocytes have larger nuclear morphology while females have higher mitochondrial activity. They then used different mitochondrial inhibitors to determine the impact on the male/females during gametogenesis. As males show large inhibition upon compound treatment, they investigated the necessity of active mitochondrial ATP synthesis during male gametogenesis for exflagellation using FRET-based assay. Overall, the manuscript is well written. The introduction section contains enough background information for a new reader to understand P. falciparum gametocyte biology and states the gap in the field that is to be addressed. The methods (use of a transgenic parasite lines for the promoter experiments, Mitotracker for the impact on mitochondrial activity, and Dual Gamete Formation Assay for male and female gametogenesis) are correctly used in the study. The results section follows a logical progression into each experiment, using RNA-seq data to identify male specific promoters, using that information to test different mitochondrial inhibitors against male and females gametocytes, and then finally the impact of drug treatment under different media conditions (with glucose and glucose repleted media). Collectively, the identification of LDH2 as a molecular marker for male gametocytes and the defined role of ATP-synthesis required for male gametogenesis is a significant finding and supports the authors major conclusions in this study. The discussion section starts by summarizing the manuscript's main findings and then leads into a discussion on the different potential possibilities to address the complex question on the necessity of ATP generation for gametogenesis despite the absence of a mitochondrion in male gametes. The final section than details a path forward with future project goals in male/female gametocyte biology.

    Major Points:

    The results for Figure 5 examining the optimal media conditions going forward for the contribution of ATP from glycolysis or oxidative phosphorylation are interesting. Could this question not also be addressed by using chemical inhibitors of glycolysis? It would be insightful (and a much simpler way to test their hypothesis) It would be beneficial to have an additional compound (atovaquone or another ATP-synthase inhibitor) used in the male gametogenesis in Figure 6 to further support their findings. Although oligomycin A is an inhibitor of the ATP-synthase, which is what the authors would like to test, the Figure 4 results for the mitotracker and EC50 curves for the female gametocytes may indicate additional

    Minor Points:

    A few sentences are required to explain the methodology behind the readout for the Dual Gamete Formation Assay (i.e. what is Pfs25) for readers who are not familiar with the previous publication. Similarly, a schematic model of the design of the FRET system would be a great addition to Figure 7. The model shown in Figure 8 is helpful for summarizing the main findings of the manuscript for the reader, but it is confusing the three small circles representing the arrested male gametocytes all look the same. I believe the authors are trying to state the different nutrient conditions cause the arrest to occur at different points in development (the male gametocytes in the glucose-repleted sample with the mitochondrial ATP synthesis inhibitor seems to still develop 1-2 flagellum, while the other conditions in no glucose arrest much earlier); but this is lost when the representations all look similar in the figure. 

    Competing interests

    The author declares that they have no competing interests.

  3. Version published to 10.1101/2024.04.23.590695v1 on bioRxiv