PREreview of Mitochondrial ATP synthesis is essential for efficient gametogenesis inPlasmodium falciparum

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.