Acyl Carrier Protein is Essential for Apicoplast Biogenesis in Malaria Parasites Independent of Fatty Acid Synthesis

Reviewer #1 (Public review):

This study provides evidence that the apicoplast-locaized isoform of acyl-carrier protein (ACP) has acquired important non-enzymatic functions in the malaria parasite. Previous studies have shown that the apicoplast-located FASII-dependent pathway of fatty acid synthesis is not essential in Plasmodium blood stages. In contrast, genome-wide knockout studies suggested that ACP, a key protein in this pathway, is essential in these stages, indicating that it may have additional non-canonical functions. In this study, the authors confirm that ACP is essential in Pf blood stages (using both apicoplast IPP rescue and conditional knockdown); show that this essential function requires modification with 4-phosphopantetheine and use proximity biotinylation and complementary immunoprecipitation pull-down approaches to provide compelling evidence that ACP binds to and stabilizes the apicoplast-located isoform of pyruvate kinase II. Notably, these interactions appear to differ from those associated with the binding of mitochondrial isoforms of ACP to proteins involved in Fe-S biosynthesis. Loss of ACP was shown to lead to a decrease in PKII levels and apicoplast DNA/RNA synthesis, consistent with loss of NTP synthesis in this organelle. The data are clear and very well described, and the findings represent a significant advance in our understanding of metabolic regulatory mechanisms in apicomplexan apicoplast studies.

Strengths:

The study uses a variety of complementary genetic approaches to demonstrate the essentiality of ACP and the enzyme involved in its activation with 4-PP in Pf blood stages, demonstrating that the ascribed non-enzymatic function is mediated by holo-ACP. Similarly, a number of complementary biochemical approaches, including proximity biotinylation, immunoprecipitation, and co-expression of PfACP and PK-II in a heterologous bacterial expression system, are used to confirm the physiological significance of the PfACP and PK-II interaction. The study also reports additional findings, such as the independence of P. faciparum blood stages on exogenous (media) fatty acids, indicating that intracellular stages can salvage all of their requirements from the red blood cell.

Weaknesses:

Overall, this is a very strong study. While questions remain around the function of other apicoplast ACP-interacting proteins detected in this study, I don't have any suggestions for significant improvements.

Reviewer #2 (Public review):

This study focuses on revealing the essential divergent function of the Acyl Carrier protein (ACP) in the deadliest human malaria parasite, Plasmodium falciparum. More precisely, using inducible KO, cellular and biochemical approaches, the authors determined that instead of a canonical role for ACP allowing the de novo synthesis of fatty acids in the apicoplast (essential relict plastid) of the parasite, the enzyme couples with pyruvate kinase II to generate nucleoside triphosphate to maintain parasite survival during blood stages. The study is novel, well-designed, providing interesting new data on Plasmodium and apicomplexa biology. The results convincingly support the major claim of the study. However, it is currently incomplete to support some claims on the essentiality of some apicoplast pathways.

In this study, Geher et al. focused on deciphering the role of the Acyl Carrier Protein (ACP) present in the relict non-photosynthetic plastid, i.e. the apicoplast of the most lethal human malaria parasite, Plasmodium falciparum. More particularly, they determined an essential function of ACP independent of its usual/typical function as the central protein for the normal function of the apicoplast Type II fatty acid synthesis (FASII) pathway. Rather, the protein seems to associate with the apicoplast Pyruvate Kinase II, together generating an essential nucleoside triphosphate (NTPs) source to fuel the apicoplast and parasite survival instead.

By generating a TetR-DOZY-based inducible KD line for ACP, they confirmed that the protein is indeed essential to maintain apicoplast integrity and parasite survival during asexual blood stages, as previously predicted and experimentally shown. They showed that ACP requires a biochemical modification, typically activating the protein for its function in the FASII pathway, i.e. binding of the 4-PP group by holoACP synthase. Then, they showed that the other enzymes of the FASII pathway are likely dispensable during the blood stage, as they were able to generate a KO line of the first enzyme of the pathway, FabD (which was predicted to be essential in P. falciparum). Based on a cell culture approach in a controlled culture medium, they further claimed that, unlike current evidence-based hypotheses, the FASII pathway (and thus a potentially FASII-linked ACP) has no role/activity during blood stages. Using a proximity biotinylation approach, they determined that ACP associates with the apicoplast pyruvate Kinase II (PKII), previously shown to generate NTPs in the apicoplast for energy and DNA/RNA maintenance (Xia et al. 2019), and not to fuel the FASII pathway as its main function in blood stages. Finally, they showed that the disruption of ACP induces the reduction of the presence/content in PKII in the parasite, as well as the drastic reduction of the apicoplast DNA and RNA content. Together, they concluded that the main function of ACP is indeed the NTP formation via its association with PKII, rather than its canonical role for the generation of fatty acids in the apicoplast.

This study is novel and focuses on a topic of particular interest in malaria biology, but also for most of the apicomplexa-related diseases, and beyond for plastid bearing orgnaisms and this unusual role for ACP. The study is well thought out with proper biochemical approaches that convincingly point to this association of ACP with PKII for NTP synthesis as a major function during P. falciparum blood stages. However, there are currently some important experimental issues/flaws, missing experiments that induced wrong interpretations and thus do not support some important claims of the study, notably for the role of FASII and the interaction between ACP and PKII.

Therefore, at this point, the study is only partial and would require major additions and/or important text edits/revisions before being considered for acceptance.

Major points:

From the graph of P. falciparum growth, we can see that in the lipid-rich condition, where both FabH KO and ACP KO can survive, the addition of mevalonate was essential for the growth of ACP KO. Along with the other evidence (PKII association, DNA levels...), we therefore agree that PfACP is involved in the mevalonate pathway. The authors claim that the FASII pathway is inactive/not essential in the P. falciparum blood stage. However, the authors have not shown any evidence on whether ACP is or not involved in the FASII pathway during the asexual blood stage. As currently designed, the experiments presented cannot conclude on that point for several reasons. Indeed, it was previously shown that (i) the expression of the protein from the FASII pathway are all present in blood stages and are significantly upregulated in patients that are under under "nutrient starvation" (Daily et al. Nature 2007), (ii) that, growing parasites under similar low lipid conditions in vitro induces an activation/upregulation of FASII, which can be measured by stable isotope precursor labelling and lipidomics (Botté et al. 2013), (iii) that growing the PfFabI KO line under deprived lipid conditions leads to parasite death (Amiar et al. 2020), indicating that the FASII pathway can become critical, if not essential, depending on the host nutritionnal content together correlating patients' data and metabolic adaptation for the same reasons in the related parastie Toxoplasma gondii (Amiar et al. 2020, Krishnan et al. 2020, Liang et al. 2020, Primo et al. 2021, Charital et al. 2024, Dass et al. 2024, Bitew et al. 2025).

Here, the authors are expecting to show that FabH (and thus the FASII pathway) is not essential in an experiment that is not designed to be in low lipid conditions but rather in lipid rich conditions: Such high lipid conditions of culture in this study is granted by daily feedings with high fatty acid supplement (30-90 uM palmitic acid and 30-60 uM oleic acid). These fatty acid concentrations were used previously by Mitamura et al. (2005) and Mi-ichi et al.(2007) to replace non-determined supplements such as Serum or Albumax supplement to grant similar growth by a completely controlled culture medium.

This means the concentrations above do not represent limited fatty acid concentrations, especially not with daily feeding (representing an excess supplied amount of lipids, unlike regular 48h feedings) that allowed the authors to easily reach very high non-physiological parasitaemia of more than 20%!! Amiar et al. previously showed essentiality of FabI in P. falciparum in the limited fatty acid culture at a lower concentration (<30uM 16:0, <45um 18:1), than the Mi-Ichi et al. controlled medium with regular 48 h culture feeding. Therefore, with the current experimental settings, the FAH KO is placed in high lipid conditions, thus preventing any conclusion on its essentiality under low lipid conditions.

Furthermore, it is too uncertain to conclude that ACP is only essential for the mevalonate pathway. This would be a similar discussion to the Yeh et al. 2011 and the Swift et al., where induced Apicoplast knockout caused parasites to require IPP to survive, but there were always remnant apicoplast vesicles and thus the putative presence of an active FASII in the parasite, where de novo fatty acid synthesis could be maintained. Amiar et al. (2020) and Krishnan et al. (2020) showed that disruption of FASII and absence of de novo FA synthesis in T. gondii could be compensated by the exogenous supplementation of myristic acid, C14:0. Here, high fatty acid supplementation using commercially available fatty acids may include unexpected fatty acid species such as myristic acid in palmitic acid or oleic acid, since all commercially available fatty acids guarantee only >99% but not 100%. If P. falciparum requires a very, very low amount of myristic acid to survive, the amount of possible contamination, like 1 nM, may be sufficient to maintain their survival. Thus, ACP and FabH might be very important to generate de novo fatty acids within parasites, but this was not shown by the authors.

Therefore, the manuscript currently contains incorrect conclusions on the potential essentiality/use of FASII, against current experimental evidence.

Author response:

We thank the editor and reviewers for the positive comments and critical feedback on our manuscript. We are currently preparing revisions to address the critiques provided by reviewer 2, which focused primarily on growth experiments performed with ∆ACP and ∆FabD P. falciparum parasites in minimal lipid conditions. We note that the major conclusions of our manuscript regarding an essential, FASII-independent function for ACP in apicoplast biogenesis do not require or rely on these experiments in minimal lipid conditions.

Nevertheless, we believe that these observations have value and agree that they contrast with similar experiments reported in the Amiar et al. 2020 study referenced by the reviewer. We note that this prior study (and others cited by the reviewer) primarily focused on the related apicomplexan parasite, Toxoplasma gondii. We fully agree that available evidence in these and other papers supports a key, fitness-conferring role for FASII activity in growth of T. gondii parasites, including possible expanded functions for ACP that may differ from P. falciparum. We will revise our manuscript to clarify that our results only apply to P. falciparum. We note that our minimal lipid growth experiments with P. falciparum utilized culture conditions and concentrations that appear identical to those reported in the Amiar et al. 2020 study. Nevertheless, we agree with the reviewer that additional experiments will be required to fully test and understand FASII functions in asexual blood-stage malaria parasites, including possible functions in low-lipid conditions. We plan to revise our manuscript to clarify this and other points, and we will include expanded responses to the reviewer critiques.