Genetic validation of PfFKBP35 as an antimalarial drug target

  1. Basil T Thommen
  2. Jerzy M Dziekan
  3. Fiona Achcar
  4. Seth Tjia
  5. Armin Passecker
  6. Katarzyna Buczak
  7. Christin Gumpp
  8. Alexander Schmidt
  9. Matthias Rottmann
  10. Christof Grüring
  11. Matthias Marti
  12. Zbynek Bozdech
  13. Nicolas MB Brancucci

  • Curated by eLife

    eLife logo

    eLife assessment

    FKBP35 is the only FK506-binding protein present in the malaria-causing parasite Plasmodium falciparum, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506, an immunosuppressant with antiplasmodial activity. This study demonstrates the essentiality of FKBP35 in parasite growth, based on compelling genetic evidence. The data also suggest that FK506 may exert its antimalarial activity through FKBP35-independent mechanisms that have not yet been characterised. This important study will be of interest to scientists working on the parasite biology and antimalarial drug development.

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Plasmodium falciparum accounts for the majority of over 600,000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein Pf FKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting Pf FKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting Pf FKBP35 levels are lethal to P. falciparum and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its antiproliferative activity in a Pf FKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond Pf FKBP35. In addition to revealing first insights into the function of Pf FKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action – with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.

Article activity feed

  1. Version published to 10.7554/elife.86975.4 on eLife
  2. Version published to 10.7554/elife.86975 on eLife
  3. Version published to 10.7554/elife.86975.3 on eLife
  4. eLife

    Author Response

    The following is the authors’ response to the previous reviews

    We thank the Reviewers and Editors for the evaluation of our revised manuscript.

    We especially value the careful assessment of Reviewer 1; at the same time, we clearly disagree with the reviewer’s statement that the revised manuscript “is essentially unchanged”. As appreciated by the other Reviewers, we performed a key experiment (in our opinion the only conclusive experiment) to further solidify that FK506-treatment kills parasites in a FK506-independent manner. Of note, however, Reviewer 1 made us aware of an error in the legend of Figure 4F, which likely contributed to the confusion regarding the antiplasmodial effect of FK506: Unfortunately, we missed updating this legend to appropriately imbed the new experiment. We therefore incorrectly stated that parasites were exposed to FK506 for 48 hours after FK506 treatment at 4-10 hpi and 36-42 hpi in G1. In contrast to the experiments described in the initial submission, parasite survival was not measured 48 h later, but in G2 ring stage parasites, i.e. at a time point during which parasitemia is not affected by the knockout of PfFKBP35. We have now corrected this. As pointed out correctly by Reviewer 1, it would otherwise not be possible to disentangle the effects of the gene knockout and the drug. The setup we now present in Figure 4F, however, is clearly able to do so.

    We apologize for the inaccuracy and hope this resolves the ambiguities regarding the FKBP35-independent antimalarial effect of FK506. In line with the comments of Reviewers 2 and 3, we believe that our findings on FK506 activity are of particular importance for the malaria research community. We therefore hope that the final eLife assessment will reflect this.

  5. eLife

    eLife assessment

    FKBP35 is the only FK506-binding protein present in the malaria-causing parasite Plasmodium falciparum, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506, an immunosuppressant with antiplasmodial activity. This study demonstrates the essentiality of FKBP35 in parasite growth, based on compelling genetic evidence. The data also suggest that FK506 may exert its antimalarial activity through FKBP35-independent mechanisms that have not yet been characterised. This important study will be of interest to scientists working on the parasite biology and antimalarial drug development.

  6. eLife

    Joint Public Review:

    In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript addresses the biological function of PfFKBP35 and the antimalarial activity of FK506.

    The authors combine conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide conclusive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death-like phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors clearly demonstrate that FKBP35 is essential for parasite growth and that ribosome biogenesis is disrupted, but further insights into the pathway itself would be more convincing that this is a direct consequence rather than a secondary feature of parasite death.

    The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death-like phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

    The authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35, based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, indicating that FK506 activity is independent of FKBP35 levels. Using cellular thermal shift assays, the authors confirm the interaction between FK506 and FKBP35, and further identify candidate proteins bound by the compound, albeit at lower affinity. Further work is needed to validate whether these putative targets contribute to the FKBP35-independent antimalarial activity of FK506.

  7. Version published to 10.1101/2022.12.09.519720v4 on bioRxiv
  8. Version published to 10.7554/elife.86975.2 on eLife
  9. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    eLife assessment

    This important study addresses both the native role of the Plasmodium falciparum protein PfFKBP35 and whether this protein is the target of FK506, an immunosuppressant with antiplasmodial activity. The genetic evidence for the essentiality of FKBP35 in parasite growth is compelling. However, the conclusion that the role of FKBP35 is to secure ribosome homeostasis and the claim that FK506 exerts its antimalarial activity independently of FKBP35 rely on incomplete evidence.

    We thank the Reviewers and Editors for their careful evaluation of our manuscript and the constructive criticism. We realized that some of our conclusions may be regarded/misunderstood as overstatements. This was by no means our intention and we apologize for the unnecessary inconvenience. The phenotype of FKBP35 knock-out parasites clearly centers on failing ribosomes and protein synthesis, which in our opinion, provides an important leap towards understanding the role of this drug target in P. falciparum biology. It is however correct that, at this point, we can only make evidence-based hypotheses about direct interaction partners and we will emphasize this more clearly in a revised version of the manuscript. In order to prevent misinterpretation of our work, and as detailed in the point-by-point responses to the reviewer comments, we propose changing the manuscript title to “Genetic validation of PfFKBP35 as an antimalarial drug target”. To address the criticism regarding the effects of FK506, we will perform specific additional experiments. We are convinced that this new data set will resolve any remaining ambiguities and allows for a conclusive assessment of FK506 drug activity in P. falciparum.

    Reviewer #1 (Public Review):

    In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase (PPIase) and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript is structured in two parts addressing the biological function of PfFKBP35 and the antimalarial activity of FK506, respectively.

    The first part combines conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide definitive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors propose that FKBP35 regulates ribosome homeostasis but an alternative explanation could be that changes in the ribosome proteome are downstream consequences of the abrogation of FKBP35 essential activities as chaperone and/or PPIase. It is unclear whether FKBP35 has a specific function in P. falciparum as compared to other organisms. The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

    We thank the Reviewer for the compliments regarding our experimental setup and the clarity of our manuscript. We agree that the link between FKBP35 knock-out and ribosome homeostasis is indirect and we now emphasize this more clearly in the revised manuscript. To prevent a general misinterpretation of our manuscript, we will adapt the title accordingly.

    We would still like to reiterate that the phenotype of FKBP35 knock-out parasites is best described by their defects in maintaining functional ribosomes. It is for several reasons that we believe the links between FKBP35 and ribosome function are purely evidence driven: First, pre-ribosomal and nucleolar factors are the first proteins (in generation 1 schizonts) to be affected upon knock-out of fkbp35 (Figure 2A, Table S1). We realized that Figure 2A falls short in showing this observation, which is why will update the figure accordingly. Second, the dysregulation of ribosomal factors and the general stall in protein synthesis is dominating the phenotype of FKBP35 knock-out parasites in generation 2. We thus believe it is appropriate to say that knock-out cells are most likely killed in response to defective ribosome maintenance – which is a consequence of reduced FKBP35 levels. We are aware that our experiments (and possibly any other reverse genetics approach) cannot rule out that FKBP35 affects ribosomal factors indirectly. Clearly, more work is required to disentangle this question in more detail in the future.

    We agree with the Reviewer that it is not possible to tell if the delayed death-like phenotype is due to a “delayed protein depletion”. We would however like to note that the DiCre/loxP approach allows for an immediate knock-out at the genome level and is thus as precise as possible. Further, in addition to the substantial depletion of FKBP35 in knock-out cells during the phenotypically silent generation, knocking out of fkbp35 at earlier time points (TPs 24-30 and 34-40 hpi in the preceding generation) resulted in the very same phenotype cycle (Figure 1). Here, parasite death was delayed substantially longer, i.e. more than one complete cycle. Together with the dysregulation of early ribosome maturation in generation 1, these findings point towards a delayed death phenotype. It is of course still possible to explain the delayed death-like phenotype by remnant activity of proteins synthetized prior to the genomic knock-out. We address this possibility and describe the two scenarios mentioned by the Reviewer in lines 141-144. Disentangling the two possibilities in future experiments will be difficult, not only with regards to FKBP35, but regarding “delayed death” phenotypes in general.

    In the second part, the authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35. This conclusion is based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, suggesting that FK506 activity is independent of FKBP35 levels, and on the fact that FK506 kills the parasite rapidly whereas inducible gene knockout results in delayed death phenotype. However, there are alternative explanations for these observations. As mentioned above, the delayed death phenotype could be due to delayed depletion of the protein upon induction of gene knockout. FK506 could have a similar activity on WT and mutant parasites when added before sufficient depletion of FKBP35 protein. In some experiments, the authors exposed KO parasites to FK506 later, presumably when the KO is effective, and obtained similar results. However, in these conditions, the death induced by the knockout could be a confounding factor when measuring the effects of the drug. Furthermore, the authors show that FK506 binds to FKBP35, and propose that the FK506-FKBP35 complex interferes with ribosome maturation, which would point towards a role of FKBP35 in FK506 action. In summary, the study does not provide sufficient evidence to rule out that FK506 exerts its effects via FKBP35.

    Noteworthy, we were also very much surprised by data indicating that the antimalarial activity of FK506 is independent of FKBP35. It is for this reason that we conducted a comprehensive set of experiments to disprove our initial observations, but couldn`t find any evidence for an FKBP35-dependent mode of action of FK506:

    We were not able to see altered FK506 sensitivity in (i) inducible knock-down parasites, (ii) inducible overexpression parasites and (iii) inducible knock-out parasites. Parasites with altered FKBP35 levels (as assessed by Western blot and quantitative proteomics at 36-42 hpi, respectively) were equally sensitive to FK506. Importantly, at no sub-lethal FK506 concentration did lower FKBP35 levels lead to an altered response of FKBP35KO compared to the wild-type control population. Furthermore, (iv) induction of the knock-out in the cycle preceding FK506 exposure also had no effect on parasite sensitivity. As mentioned by the Reviewer, we also exposed the parasites to FK506 at 30-36 hpi and (v) did not see any effect, even though we measured a 19-fold difference in FKBP35 protein levels between the parasite populations at 36-42 hpi. At this point, parasite death induced by the knock-out cannot be a confounding factor (as it was mentioned by the Reviewer), because the FKBP35 knock-out has no effect on parasite survival in generation 1 in the absence of FK506 (Figure 1F). This demonstrates that the observed effect is only due to drug-mediated killing and not due to the FKBP35 knock-out.

    To account for a scenario in which the drop in FKBP35 levels only occurs after 36 hpi, we will perform an additional set of experiments, in which we induce the knock-out at 0-6 hpi and treat the parasites at 36-42 hpi (i.e. the time point at which the 19-fold difference in protein levels was measured by quantitative proteomics). This setup will allow determining whether or not the parasite killing activity of FK506 depends on FKBP35 levels.

    So far, our experiments cannot support any scenario in which FK506 kills P. falciparum parasites via inhibiting the essential role of FKBP35 and we would therefore want to insist that this statement is based on highly solid evidence. In this context, it is important to note that our conclusion includes two scenarios: “This indicates that either the binding of FK506 does not interfere with the essential role of PfFKBP35, or that PfFKBP35 is inhibited only at high FK506 concentrations that also inhibit other essential factors.” While this phrase is already present in our initial submission, we will emphasize this point more clearly in the revised manuscript. We are convinced that this information is of high importance for ongoing and future drug development.

    Reviewer #2 (Public Review):

    The manuscript by Thomen et al. FKBP secures ribosome homeostasis in Plasmodium falciparum and focuses on the importance of PfKBP35 protein, its interaction with the FK506 compound, and the role of PfKBP35 in ribosome biogenesis. The authors showed the interaction of the PfKBP54 with FK506, but the part of the FK506 and PfKBP54 in ribosome biogenesis based on the data is unclear.

    The introduction is plotted with two parallel stories about PfKBP35 and FK506, with ribosome biogenesis as the central question at the end. In its current form, the manuscript suffers from two stories that are not entirely interconnected, unfinished, and somewhat confusing. Both stories need additional experiments to make the manuscript(s) more complete. The results from PfFBP35 need more evidence for the proposed ribosome biogenesis pathway control. On the other hand, the results from the drug FK506 point to different targets with lower EC50, and other follow-up experiments are needed to substantiate the authors' claims.

    The strengths of the manuscript are the figures and experimental design. The combination of omics methods is informative and gives an opportunity for follow-up experiments.

    We thank the Reviewer for the evaluation of the manuscript. We apologize for the fact that the Reviewer found the manuscript to be inaccessible. We will use the comments as an incentive to restructure the manuscript and do our best to clarify the presentation, interpretation and conclusion of the presented data in the revised version. We believe that the FKBP35 data are strongly interlinked with the findings on FK506. We will emphasize these links more clearly and are convinced that the complementary nature of the datasets are a particular strength of the presented work.

    Reviewer #3 (Public Review):

    The study by Thommen et al. sought to identify the native role of the Plasmodium falciparum FKBP35 protein, which has been identified as a potential drug target due to the antiplasmodial activity of the immunosuppressant FK506. This compound has multiple binding proteins in many organisms; however, only one FKBP exists in P. falciparum (FKBP35). Using genetically-modified parasites and mass spectrometry-based cellular thermal shift assays (CETSA), the authors suggest that this protein is in involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is separate from its activity on the FKBP35 protein. The authors first created a conditional knockdown using the destruction domain/shield system, which demonstrated no change in asexual blood stage parasites. A conditional knockout was then generated using the DiCre system. FKBP35KO parasites survived the first generation but died in the second generation. The authors called this "a delayed death phenotype", although it was not secondary to drug treatment, so this may be a misnomer. This slow death was unrelated to apicoplast dysfunction, as demonstrated by lack of alterations in sensitivity to apicoplast inhibitors. Quantitative proteomics on the FKBP35KO vs FKBP35WT parasites demonstrated enrichment of proteins involved in pre-ribosome development and the nucleolus. Interestingly, the KO parasites were not more susceptible to cycloheximide, a translation inhibitor, in the first generation (G1), suggesting that mature ribosomes still exist at this point. The SunSET technique, which incorporates puromycin into nascent peptide chains, also showed that in G1 the FKBP35KO parasites were still able to synthesize proteins. But in the second generation (G2), there was a significant decrease in protein synthesis. Transcriptomics were also performed at multiple time points. The effects of knockout of FKBP35 were transcriptionally silent in G1, and the parasites then slowed their cell cycles as compared to the FKBP35WT parasites.

    The authors next sought to evaluate whether killing by FK506 was dependent upon the inhibition of PfKBP35. Interestingly, both FKBP35KO and FKBP35WT parasites were equally susceptible to FK506. This suggested that the antiplasmodial activity of FK506 was related to activity targeting essential functions in the parasite separate from binding to FKBP35. To identify these potential targets, the authors used MS-CETSA on lysates to test for thermal stabilization of proteins after exposure to drug, which suggests drug-protein interactions. As expected, FK506 bound FKBP35 at low nM concentrations. However, given that the parasite IC50 of this compound is in the uM range, the authors searched for proteins stabilized at these concentrations as putative secondary targets. Using live cell MS-CETSA, FK506 bound FKBP35 at low nM concentrations; however, in these experiments over 50 ribosomal proteins were stabilized by the drug at higher concentrations. Of note, there was also an increase in soluble ribosomal factors in the absence of denaturing conditions. The authors suggested that the drug itself led to these smaller factors disengaging from a larger ribosomal complex, leading to an increase in soluble factors. Ultimately, the authors conclude that the native function of FKBP35 is involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is not related to the binding of FKBP35, but instead results from inhibition of essential functions of secondary targets.

    Strengths:

    This study has many strengths. It addresses an important gap in parasite biology and drug development, by addressing the native role of the potential antiplasmodial drug target FKBP35 and whether the compound FK506 works through inhibition of that putative target. The knockout data provide compelling evidence that the KBP35 protein is essential for asexual parasite growth after one growth cycle. Analysis of the FKBP35KO line also provides evidence that the effects of FK506 are likely not solely due to inhibition of that protein, but instead must have secondary targets whose function is essential. These data are important in the field of drug development as they may guide development away from structure-based FK506 analogs that bind more specifically to the FKBP35 protein.

    Weaknesses:

    There are also a few notable weaknesses in the evidence that call into question the conclusion in the article title that FKBP35 is definitely involved in ribosomal homeostasis. While the proteomics supports alterations in ribosome biogenesis factors, it is unclear whether this is a direct role of the loss of the FKBP35 protein or is more related to non-specific downstream effects of knocking down the protein. The CETSA data clearly demonstrate that FK506 binds PfKB35 at low nM concentrations, which is different than the IC50 noted in the parasite; however, the evidence that the proteins stabilized by uM concentrations of drug are actual targets is not completely convincing. Especially, given the high uM amounts of drug required to stabilize these proteins. This section of the manuscript would benefit from validation of a least one or two of the putative candidates noted in the text. In the live cell CETSA, it is noted that >50 ribosomal components are stabilized in drug treated but not lysate controls. Similarly, the authors suggest that the -soluble fraction of ribosomal components increases in drug-exposed parasites even at 37{degree sign}C and suggests that this is likely from smaller ribosomal proteins disengaging from larger ribosomal complexes. While the evidence is convincing that this protein may play a role in ribosome homeostasis in some capacity, it is not sure that the title of the paper "FKBP secures ribosome homeostasis" holds true given the lack of mechanistic data. A minor weakness, but one that should nonetheless be addressed, is the use of the term "delayed death phenotype" with regards to the knockout parasite killing. This term is most frequently used in a very specific setting of apicoplast drugs that inhibit apicoplast ribosomes, so the term is misleading. It is also possible that the parasites are able to go through a normal cycle because of the kinetics of the knockout and that the time needed for protein clearance in the parasite to a level that is lethal.

    Overall, the authors set out to identify the native role of FKB35 in the P. falciparum parasites and to identify whether this is, in fact, the target of FK506. The data clearly demonstrate that FKBP35 is essential for parasite growth and provide evidence that alterations in its levels have proteomic but not transcriptional changes. However, the conclusion that FKBP35 actually stabilizes ribosomal complexes remains intermediate. The data are also very compelling that FK506 has secondary targets in the parasite aside from FKBP35; however, the high uM concentrations of the drug needed to attain results and the lack of biological validation of the CETSA hits makes it difficult to know whether any of these are actually the target of the compound or instead are nonspecific downstream consequences of treatment.

    We appreciate the detailed and valuable suggestions to improve the manuscript. We agree that CETSA could only identify potential targets of FK506 in the micromolar range, while FK506 showed a high affinity for FKBP35, consistent with earlier reports (2). We would however like to point out that FK506 kills P. falciparum at exactly these relatively high concentrations and not at those presumed from the high affinity interactions between FK506 and FKBP35. The relatively high FK506 concentration required to stabilize potential off target proteins is therefore not a concerning observation, but rather corroborates our conclusion that FK506 fails to inhibit the essential function of FKBP35 at concentrations that leave off targets unaffected. As mentioned in response to Reviewer 1, we will describe and discuss these data more clearly in the revised manuscript. We thank the Reviewer for pointing out the potential issues regarding the use of the term “delayed death phenotype”. We now refer to the FKBP35 phenotype as “delayed death-like” in the revised manuscript.

    We believe that follow-up work on specific FK506 CETSA hits is out of scope of the current and already quite complex manuscript.

    As mentioned in the response to Reviewer 1, we realize that the short title of the manuscript can be regarded as an overstatement. Again, this was clearly not our intention and we apologize that the Reviewers had to indicate this issue. While we believe that the message of the title holds true (see response to Reviewer 1), we recognize the misconception that might arise from it, which is why we propose the new title: “Genetic validation of PfFKBP35 as an antimalarial drug target”.

    Reviewer #1 (Recommendations For The Authors):

    1. Documentation of FKBP35 protein levels over time in knockout, knockdown and overexpressing parasites is missing here. Since the knockdown of PfFKBP35 has no phenotypic consequence, very low amounts of FKBP35 are probably sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays. In particular, the delayed death phenotype could simply reflect delayed protein depletion, contrasting with the immediate inhibition of FKBP35 by FK506. The quantification by mass spectrometry does indicate substantial depletion but provides no information on the kinetics and levels. What is 19 fold compared to the knockdown condition? Also, expression of FKBP35 in overexpressing parasites should be compared side by side with the iKD (in the presence of Shield).

    We agree with the Reviewer that low FKBP35 levels are likely sufficient for parasite survival. This is addressed in the manuscript (lines 141-143). Assessing protein levels in the transgenic parasites side by side in time course experiments would be interesting. However, our conclusions are independent of the outcome of such experiments because the relative difference in FKBP35 levels resulting from conditional expression systems did not change the parasites` susceptibility to FK506. We believe that comparing isogenic populations is much more informative than comparing independent cell lines with each other.

    1. The authors claim that FK506 fails at inhibiting the essential function of PfFKBP35 (line 103), however this is not directly supported by data. FK506 kills the parasite and so inhibits essential functions. The data indicate that FK506 antimalarial activity does not seem to be influenced by FKBP35 levels, which would support the authors claim. However, as mentioned above, it is important to better define experimentally FKBP35 expression levels. Also, in experiments where FK506 is added late after rapamycin treatment, the authors need to clarify how they could distinguish drug killing and death due to the knockout.

    In the experiment described by the Reviewer, the FKBP35 knock-out was induced in young ring stages (0-6 hpi) and FK506 was added at 30-36 hpi, we measured the parasite survival from G1 to G2 (see figure 5A). In the absence of FK506, the FKBP35 knock-out has no effect on parasite survival (Figure 1), demonstrating that the observed effect is only due to drug killing and not due to the KO.

    To address the concerns regarding delayed depletion of FKBP35, we have performed an additional set of experiments. This data corroborates that the effect of FK506 is independent of FKBP35 levels. We discuss this topic in more detail in the Public Review. In brief, the additional experiment included exposing knock-out parasites (KO induced 0-6 hpi) with FK506 at 36-42 hpi, i.e. at a time point when FKBP35 protein levels are reduced by more than 90% (19-fold difference compared to the control parasites based on quadruplicate quantitative mass spectrometry data). However, despite the clear difference, the IC50 of FK506 remained the same as determined before (see new figure 4F).

    1. Since FK506 is known to inhibit FKBP35 PPIase activity, it could be relevant to compare the effects of FK506 versus KO on ribosomes and translation. This could provide further evidence supporting a FKBP35-independent activity of FK506.

    We agree with the Reviewer that this would be very informative. However, it would be difficult to disentangle indirect downstream processes on translation caused by both the FK506 drug treatment and the FKBP35 knock-out in a cellular assay. Establishing a biochemical in vitro assay to study the role of PPIase activity in translation is out of scope of this manuscript.

    Minor points

    -The title is rather vague, which reflects the fact that the function of PfFKBP35 is not precisely defined in the study.

    We thank the Reviewer for this assessment, which is in agreement with Reviewer 3. Based on these concerns, and in order to prevent misinterpretation of our manuscript, we propose changing the title to “Genetic validation of PfFKBP35 as an antimalarial drug target” (see public response above).

    -The transcriptomics data (Fig 3) provide little information on the function of FKBP35 and could be included as supplemental material. On the contrary, data in FigS5 convey important information and should be moved to the main figures.

    We believe that the transcriptomics data are important to characterize the effect of limiting FKBP35 levels in G2, as they show that, unlike certain homologs of other organisms (3), FKBP35 has no role in transcriptional control and its knock-out does not have any downstream consequences on the transcriptional level (except for the death-related stall in cell cycle progression). We would therefore would like to keep this dataset represented in the main figures. The updated Figure 4F now includes more information about the effect of adding FK506 at different time points, which was only addressed in Figure S5 in the previous version of the manuscript. We believe that the key message of Figure S5 is now covered in Figure 4.

    -Line 30: "action" rather than "role"

    We corrected this.

    Reviewer #2 (Recommendations For The Authors):

    I have no comments on data, code, or other issues.

    General comments:

    The introduction is plotted with two parallel stories about PfKBP35 and FK506, with ribosome biogenesis as the central question at the end. In its current form, the manuscript suffers from two stories that are not entirely interconnected, unfinished, and somewhat confusing. I recommend focusing only on one story - either characterizing PfBP35 and its role in Plasmodium falciparum biology - future investigation of PfBP35 control of cellular processes or focusing on the actual targets of the FK506 drug (identified in figure 4). Both stories need additional experiments to make the manuscript(s) more complete and ready for publication. The results from PfFBP35 need more evidence for the proposed ribosome biogenesis pathway control. On the other hand, the results from the drug FK506 point to different targets with lower EC50, and other follow-up experiments are needed to substantiate the authors' claims.

    The strengths of the manuscript are the figures and experimental design. The combination of omics methods is informative and gives an opportunity for follow-up experiments.

    Detailed points and suggestions for authors:

    Line 99

    There is no such thing as "protein translation"; it is mRNA translation or protein synthesis, which needs to be updated throughout the manuscript.

    We thank Reviewer 2 for pointing out this error that we now corrected.

    Line 174

    The statement needs a reference(s).

    We added an appropriate review reference.

    Lines 229-235

    While transcriptomics and proteomics data can argue that FKBP35 maybe acts at the post-transcriptional level, its function, as well as presented data, could point to post-translational mechanisms as well, cell cycle checkpoint misregulation, and multiple other pathways that control cell size, cell proliferation, translation, and ribosome biogenesis. More solid and direct evidence on ribosome biogenesis (rRNA processing, polysome profiles, or similar experiments) would be needed to show the function of FKBP35 in this cellular process.

    We have changed the term “post-transcriptional processes” to “transcription-independent processes”. As detailed in the Public Review, we agree with the Reviewer and lowered our statements regarding the function of FKBP35 throughout the manuscript.

    Lines 237-313

    The authors showed again the interaction of PfFKBP35 with the FK506 drug, but the phenotype differs from the protein deletion. Moreover, EC50s for multiple other proteins (i.e., PF3D7_1138700 or PF3D7_1325900, among others) are lower than for PfFKBP35 but are never further tested. This would be necessary to characterize FK506 drug targets, and it would be a different study.

    We believe that characterizing putative targets of FK506 is out of the scope of this already complex study and should be addressed, as suggested by the Reviewer, in a future and independent efforts.

    Lines 293 - 301

    The point of lower EC50 for PfFKBP35 and FK506 in in vitro cell lysate experiment compared to in vivo IC50 data is not surprising, given that drug delivery is not an issue in a lysate experiment. It is unclear why the authors pick some proteins and not others for further characterization of FK506 binding. There is no explanation for this selection. They did not follow up on the best targets of FK406 drugs from Fig 4 (above comment).

    As mentioned above, validation of FK506 targets is out of scope of this study.

    Lines 313 -352

    An alternative scenario for the FK506 drug data in CETSA experiments is that they bind directly to ribosomes interacting with rRNA, as many macrolides do. One should note that these are not ribosomal factors (line 334) but ribosomal proteins mentioned in Fig.4 F, mainly associated with large ribosomal subunit.

    We agree with Reviewer 2 that FK506 could bind indirectly to ribosomal proteins. This scenario is already described in the initial version of the manuscript (see lines 285-287: “Of note, these ribosomal proteins were stabilized at virtually identical FK506 concentrations (Figs. 4D, and S7), indicating that the drug – directly or indirectly – interacts with ribosomal complexes.”).

    We thank the Reviewer for pointing out that we are indeed talking about “ribosomal proteins” rather than “ribosomal factors”. We now corrected this.

    Reviewer #3 (Recommendations For The Authors):

    Please see Public review for suggestions about experimental validation of the link to ribosome homeostasis.

    We would like to thank Reviewer 3 for the detailed suggestions.

    References

    1. Kennedy K, Cobbold SA, Hanssen E, Birnbaum J, Spillman NJ, McHugh E, et al. Delayed death in the malaria parasite Plasmodium falciparum is caused by disruption of prenylation-dependent intracellular trafficking. PLoS Biol. 2019;17(7):e3000376.

    2. Kotaka M, Ye H, Alag R, Hu G, Bozdech Z, Preiser PR, et al. Crystal structure of the FK506 binding domain of Plasmodium falciparum FKBP35 in complex with FK506. Biochemistry. 2008;47(22):5951-61.

    3. Kasahara K, Nakayama R, Shiwa Y, Kanesaki Y, Ishige T, Yoshikawa H, et al. Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genes cooperatively with Hmo1 in Saccharomyces cerevisiae. PLoS Genet. 2020;16(6):e1008865.

  10. eLife

    eLife assessment

    This important study addresses both the native role of the Plasmodium falciparum protein PfFKBP35 and whether this protein is the target of FK506, an immunosuppressant with antiplasmodial activity. The demonstration of the essentiality of FKBP35 in parasite growth relies on compelling genetic evidence. However, it remains unclear whether FK506 exerts its antimalarial activity through an FKBP35-independent mechanism.

  11. eLife

    Reviewer #1 (Public Review):

    In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase (PPIase) and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript is structured in two parts addressing the biological function of PfFKBP35 and the antimalarial activity of FK506, respectively.

    The first part combines conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide definitive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors propose that FKBP35 regulates ribosome homeostasis but an alternative explanation could be that changes in the ribosome proteome are downstream consequences of the abrogation of FKBP35 essential activities as chaperone and/or PPIase. It is unclear whether FKBP35 has a specific function in P. falciparum as compared to other organisms. The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

    In the second part, the authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35. This conclusion is based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, suggesting that FK506 activity is independent of FKBP35 levels, and on the fact that FK506 kills the parasite rapidly whereas inducible gene knockout results in delayed death phenotype. However, there are alternative explanations for these observations. As mentioned above, the delayed death phenotype could be due to delayed depletion of the protein upon induction of gene knockout. FK506 could have a similar activity on WT and mutant parasites when added before sufficient depletion of FKBP35 protein. In some experiments, the authors exposed KO parasites to FK506 later, presumably when the KO is effective, and obtained similar results. However, in these conditions, the death induced by the knockout could be a confounding factor when measuring the effects of the drug. Furthermore, the authors show that FK506 binds to FKBP35, and propose that the FK506-FKBP35 complex interferes with ribosome maturation, which would point towards a role of FKBP35 in FK506 action. In summary, the study does not provide sufficient evidence to rule out that FK506 exerts its effects via FKBP35.

  12. eLife

    Reviewer #2 (Public Review):

    The introduction is plotted with two parallel stories about PfKBP35 and FK506, with ribosome biogenesis as the central question at the end. In its current form, the manuscript suffers from two stories that are not entirely interconnected, unfinished, and somewhat confusing. I recommend focusing only on one story - either characterizing PfBP35 and its role in Plasmodium falciparum biology - future investigation of PfBP35 control of cellular processes or focusing on the actual targets of the FK506 drug (identified in figure 4). Both stories need additional experiments to make the manuscript(s) more complete. The results from PfFBP35 need more evidence for the proposed ribosome biogenesis pathway control. On the other hand, the results from the drug FK506 point to different targets with lower EC50, and other follow-up experiments are needed to substantiate the authors' claims. The strengths of the manuscript are the figures and experimental design. The combination of omics methods is informative and gives an opportunity for follow-up experiments.

  13. eLife

    Reviewer #3 (Public Review):

    The study by Thommen et al. sought to identify the native role of the Plasmodium falciparum FKBP35 protein, which has been identified as a potential drug target due to the antiplasmodial activity of the immunosuppressant FK506. This compound has multiple binding proteins in many organisms; however, only one FKBP exists in P. falciparum (FKBP35). Using genetically-modified parasites and mass spectrometry-based cellular thermal shift assays (CETSA), the authors suggest that this protein is in involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is separate from its activity on the FKBP35 protein. The authors first created a conditional knockdown using the destruction domain/shield system, which demonstrated no change in asexual blood stage parasites. A conditional knockout was then generated using the DiCre system. FKBP35KO parasites survived the first generation but died in the second generation. The authors called this "a delayed death phenotype", although it was not secondary to drug treatment, so this may be a misnomer. This slow death was unrelated to apicoplast dysfunction, as demonstrated by lack of alterations in sensitivity to apicoplast inhibitors. Quantitative proteomics on the FKBP35KO vs FKBP35WT parasites demonstrated enrichment of proteins involved in pre-ribosome development and the nucleolus. Interestingly, the KO parasites were not more susceptible to cycloheximide, a translation inhibitor, in the first generation (G1), suggesting that mature ribosomes still exist at this point. The SunSET technique, which incorporates puromycin into nascent peptide chains, also showed that in G1 the FKBP35KO parasites were still able to synthesize proteins. But in the second generation (G2), there was a significant decrease in protein synthesis. Transcriptomics were also performed at multiple time points. The effects of knockout of FKBP35 were transcriptionally silent in G1, and the parasites then slowed their cell cycles as compared to the FKBP35WT parasites.

    The authors next sought to evaluate whether killing by FK506 was dependent upon the inhibition of PfKBP35. Interestingly, both FKBP35KO and FKBP35WT parasites were equally susceptible to FK506. This suggested that the antiplasmodial activity of FK506 was related to activity targeting essential functions in the parasite separate from binding to FKBP35. To identify these potential targets, the authors used MS-CETSA on lysates to test for thermal stabilization of proteins after exposure to drug, which suggests drug-protein interactions. As expected, FK506 bound FKBP35 at low nM concentrations. However, given that the parasite IC50 of this compound is in the uM range, the authors searched for proteins stabilized at these concentrations as putative secondary targets. Using live cell MS-CETSA, FK506 bound FKBP35 at low nM concentrations; however, in these experiments over 50 ribosomal proteins were stabilized by the drug at higher concentrations. Of note, there was also an increase in soluble ribosomal factors in the absence of denaturing conditions. The authors suggested that the drug itself led to these smaller factors disengaging from a larger ribosomal complex, leading to an increase in soluble factors. Ultimately, the authors conclude that the native function of FKBP35 is involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is not related to the binding of FKBP35, but instead results from inhibition of essential functions of secondary targets.

    Strengths
    This study has many strengths. It addresses an important gap in parasite biology and drug development, by addressing the native role of the potential antiplasmodial drug target FKBP35 and whether the compound FK506 works through inhibition of that putative target. The knockout data provide compelling evidence that the KBP35 protein is essential for asexual parasite growth after one growth cycle. Analysis of the FKBP35KO line also provides evidence that the effects of FK506 are likely not solely due to inhibition of that protein, but instead must have secondary targets whose function is essential. These data are important in the field of drug development as they may guide development away from structure-based FK506 analogs that bind more specifically to the FKBP35 protein.

    Weaknesses:
    There are also a few notable weaknesses in the evidence that call into question the conclusion in the article title that FKBP35 is definitely involved in ribosomal homeostasis. While the proteomics supports alterations in ribosome biogenesis factors, it is unclear whether this is a direct role of the loss of the FKBP35 protein or is more related to non-specific downstream effects of knocking down the protein. The CETSA data clearly demonstrate that FK506 binds PfKB35 at low nM concentrations, which is different than the IC50 noted in the parasite; however, the evidence that the proteins stabilized by uM concentrations of drug are actual targets is not completely convincing. Especially, given the high uM amounts of drug required to stabilize these proteins. This section of the manuscript would benefit from validation of a least one or two of the putative candidates noted in the text. In the live cell CETSA, it is noted that >50 ribosomal components are stabilized in drug treated but not lysate controls. Similarly, the authors suggest that the -soluble fraction of ribosomal components increases in drug-exposed parasites even at 37{degree sign}C and suggests that this is likely from smaller ribosomal proteins disengaging from larger ribosomal complexes. While the evidence is convincing that this protein may play a role in ribosome homeostasis in some capacity, it is not sure that the title of the paper "FKBP secures ribosome homeostasis" holds true given the lack of mechanistic data. A minor weakness, but one that should nonetheless be addressed, is the use of the term "delayed death phenotype" with regards to the knockout parasite killing. This term is most frequently used in a very specific setting of apicoplast drugs that inhibit apicoplast ribosomes, so the term is misleading. It is also possible that the parasites are able to go through a normal cycle because of the kinetics of the knockout and that the time needed for protein clearance in the parasite to a level that is lethal.

    Overall, the authors set out to identify the native role of FKB35 in the P. falciparum parasites and to identify whether this is, in fact, the target of FK506. The data clearly demonstrate that FKBP35 is essential for parasite growth and provide evidence that alterations in its levels have proteomic but not transcriptional changes. However, the conclusion that FKBP35 actually stabilizes ribosomal complexes remains intermediate. The data are also very compelling that FK506 has secondary targets in the parasite aside from FKBP35; however, the high uM concentrations of the drug needed to attain results and the lack of biological validation of the CETSA hits makes it difficult to know whether any of these are actually the target of the compound or instead are nonspecific downstream consequences of treatment.

  14. Version published to 10.1101/2022.12.09.519720v3 on bioRxiv
  15. Version published to 10.7554/elife.86975.1 on eLife
  16. eLife

    eLife assessment

    This important study addresses both the native role of the Plasmodium falciparum protein PfFKBP35 and whether this protein is the target of FK506, an immunosuppressant with antiplasmodial activity. The genetic evidence for the essentiality of FKBP35 in parasite growth is compelling. However, the conclusion that the role of FKBP35 is to secure ribosome homeostasis and the claim that FK506 exerts its antimalarial activity independently of FKBP35 rely on incomplete evidence.

  17. eLife

    Reviewer #1 (Public Review):

    In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase (PPIase) and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript is structured in two parts addressing the biological function of PfFKBP35 and the antimalarial activity of FK506, respectively.

    The first part combines conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide definitive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors propose that FKBP35 regulates ribosome homeostasis but an alternative explanation could be that changes in the ribosome proteome are downstream consequences of the abrogation of FKBP35 essential activities as chaperone and/or PPIase. It is unclear whether FKBP35 has a specific function in P. falciparum as compared to other organisms. The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

    In the second part, the authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35. This conclusion is based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, suggesting that FK506 activity is independent of FKBP35 levels, and on the fact that FK506 kills the parasite rapidly whereas inducible gene knockout results in delayed death phenotype. However, there are alternative explanations for these observations. As mentioned above, the delayed death phenotype could be due to delayed depletion of the protein upon induction of gene knockout. FK506 could have a similar activity on WT and mutant parasites when added before sufficient depletion of FKBP35 protein. In some experiments, the authors exposed KO parasites to FK506 later, presumably when the KO is effective, and obtained similar results. However, in these conditions, the death induced by the knockout could be a confounding factor when measuring the effects of the drug. Furthermore, the authors show that FK506 binds to FKBP35, and propose that the FK506-FKBP35 complex interferes with ribosome maturation, which would point towards a role of FKBP35 in FK506 action. In summary, the study does not provide sufficient evidence to rule out that FK506 exerts its effects via FKBP35.

  18. eLife

    Reviewer #2 (Public Review):

    The manuscript by Thomen et al. FKBP secures ribosome homeostasis in Plasmodium falciparum and focuses on the importance of PfKBP35 protein, its interaction with the FK506 compound, and the role of PfKBP35 in ribosome biogenesis. The authors showed the interaction of the PfKBP54 with FK506, but the part of the FK506 and PfKBP54 in ribosome biogenesis based on the data is unclear.

    The introduction is plotted with two parallel stories about PfKBP35 and FK506, with ribosome biogenesis as the central question at the end. In its current form, the manuscript suffers from two stories that are not entirely interconnected, unfinished, and somewhat confusing. Both stories need additional experiments to make the manuscript(s) more complete. The results from PfFBP35 need more evidence for the proposed ribosome biogenesis pathway control. On the other hand, the results from the drug FK506 point to different targets with lower EC50, and other follow-up experiments are needed to substantiate the authors' claims.

    The strengths of the manuscript are the figures and experimental design. The combination of omics methods is informative and gives an opportunity for follow-up experiments.

  19. eLife

    Reviewer #3 (Public Review):

    The study by Thommen et al. sought to identify the native role of the Plasmodium falciparum FKBP35 protein, which has been identified as a potential drug target due to the antiplasmodial activity of the immunosuppressant FK506. This compound has multiple binding proteins in many organisms; however, only one FKBP exists in P. falciparum (FKBP35). Using genetically-modified parasites and mass spectrometry-based cellular thermal shift assays (CETSA), the authors suggest that this protein is in involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is separate from its activity on the FKBP35 protein. The authors first created a conditional knockdown using the destruction domain/shield system, which demonstrated no change in asexual blood stage parasites. A conditional knockout was then generated using the DiCre system. FKBP35KO parasites survived the first generation but died in the second generation. The authors called this "a delayed death phenotype", although it was not secondary to drug treatment, so this may be a misnomer. This slow death was unrelated to apicoplast dysfunction, as demonstrated by lack of alterations in sensitivity to apicoplast inhibitors. Quantitative proteomics on the FKBP35KO vs FKBP35WT parasites demonstrated enrichment of proteins involved in pre-ribosome development and the nucleolus. Interestingly, the KO parasites were not more susceptible to cycloheximide, a translation inhibitor, in the first generation (G1), suggesting that mature ribosomes still exist at this point. The SunSET technique, which incorporates puromycin into nascent peptide chains, also showed that in G1 the FKBP35KO parasites were still able to synthesize proteins. But in the second generation (G2), there was a significant decrease in protein synthesis. Transcriptomics were also performed at multiple time points. The effects of knockout of FKBP35 were transcriptionally silent in G1, and the parasites then slowed their cell cycles as compared to the FKBP35WT parasites.

    The authors next sought to evaluate whether killing by FK506 was dependent upon the inhibition of PfKBP35. Interestingly, both FKBP35KO and FKBP35WT parasites were equally susceptible to FK506. This suggested that the antiplasmodial activity of FK506 was related to activity targeting essential functions in the parasite separate from binding to FKBP35. To identify these potential targets, the authors used MS-CETSA on lysates to test for thermal stabilization of proteins after exposure to drug, which suggests drug-protein interactions. As expected, FK506 bound FKBP35 at low nM concentrations. However, given that the parasite IC50 of this compound is in the uM range, the authors searched for proteins stabilized at these concentrations as putative secondary targets. Using live cell MS-CETSA, FK506 bound FKBP35 at low nM concentrations; however, in these experiments over 50 ribosomal proteins were stabilized by the drug at higher concentrations. Of note, there was also an increase in soluble ribosomal factors in the absence of denaturing conditions. The authors suggested that the drug itself led to these smaller factors disengaging from a larger ribosomal complex, leading to an increase in soluble factors. Ultimately, the authors conclude that the native function of FKBP35 is involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is not related to the binding of FKBP35, but instead results from inhibition of essential functions of secondary targets.

    Strengths:

    This study has many strengths. It addresses an important gap in parasite biology and drug development, by addressing the native role of the potential antiplasmodial drug target FKBP35 and whether the compound FK506 works through inhibition of that putative target. The knockout data provide compelling evidence that the KBP35 protein is essential for asexual parasite growth after one growth cycle. Analysis of the FKBP35KO line also provides evidence that the effects of FK506 are likely not solely due to inhibition of that protein, but instead must have secondary targets whose function is essential. These data are important in the field of drug development as they may guide development away from structure-based FK506 analogs that bind more specifically to the FKBP35 protein.

    Weaknesses:

    There are also a few notable weaknesses in the evidence that call into question the conclusion in the article title that FKBP35 is definitely involved in ribosomal homeostasis. While the proteomics supports alterations in ribosome biogenesis factors, it is unclear whether this is a direct role of the loss of the FKBP35 protein or is more related to non-specific downstream effects of knocking down the protein. The CETSA data clearly demonstrate that FK506 binds PfKB35 at low nM concentrations, which is different than the IC50 noted in the parasite; however, the evidence that the proteins stabilized by uM concentrations of drug are actual targets is not completely convincing. Especially, given the high uM amounts of drug required to stabilize these proteins. This section of the manuscript would benefit from validation of a least one or two of the putative candidates noted in the text. In the live cell CETSA, it is noted that >50 ribosomal components are stabilized in drug treated but not lysate controls. Similarly, the authors suggest that the -soluble fraction of ribosomal components increases in drug-exposed parasites even at 37{degree sign}C and suggests that this is likely from smaller ribosomal proteins disengaging from larger ribosomal complexes. While the evidence is convincing that this protein may play a role in ribosome homeostasis in some capacity, it is not sure that the title of the paper "FKBP secures ribosome homeostasis" holds true given the lack of mechanistic data. A minor weakness, but one that should nonetheless be addressed, is the use of the term "delayed death phenotype" with regards to the knockout parasite killing. This term is most frequently used in a very specific setting of apicoplast drugs that inhibit apicoplast ribosomes, so the term is misleading. It is also possible that the parasites are able to go through a normal cycle because of the kinetics of the knockout and that the time needed for protein clearance in the parasite to a level that is lethal.

    Overall, the authors set out to identify the native role of FKB35 in the P. falciparum parasites and to identify whether this is, in fact, the target of FK506. The data clearly demonstrate that FKBP35 is essential for parasite growth and provide evidence that alterations in its levels have proteomic but not transcriptional changes. However, the conclusion that FKBP35 actually stabilizes ribosomal complexes remains intermediate. The data are also very compelling that FK506 has secondary targets in the parasite aside from FKBP35; however, the high uM concentrations of the drug needed to attain results and the lack of biological validation of the CETSA hits makes it difficult to know whether any of these are actually the target of the compound or instead are nonspecific downstream consequences of treatment.

  20. eLife

    Author Response

    eLife assessment

    This important study addresses both the native role of the Plasmodium falciparum protein PfFKBP35 and whether this protein is the target of FK506, an immunosuppressant with antiplasmodial activity. The genetic evidence for the essentiality of FKBP35 in parasite growth is compelling. However, the conclusion that the role of FKBP35 is to secure ribosome homeostasis and the claim that FK506 exerts its antimalarial activity independently of FKBP35 rely on incomplete evidence.

    We thank the Reviewers and Editors for their careful evaluation of our manuscript and the constructive criticism. We realized that some of our conclusions may be regarded/misunderstood as overstatements. This was by no means our intention and we apologize for the unnecessary inconvenience. The phenotype of FKBP35 knock-out parasites clearly centers on failing ribosomes and protein synthesis, which in our opinion, provides an important leap towards understanding the role of this drug target in P. falciparum biology. It is however correct that, at this point, we can only make evidence-based hypotheses about direct interaction partners and we will emphasize this more clearly in a revised version of the manuscript. In order to prevent misinterpretation of our work, and as detailed in the point-by-point responses to the reviewer comments, we propose changing the manuscript title to “Genetic validation of _Pf_FKBP35 as an antimalarial drug target”. To address the criticism regarding the effects of FK506, we will perform specific additional experiments. We are convinced that this new data set will resolve any remaining ambiguities and allows for a conclusive assessment of FK506 drug activity in P. falciparum.

    Reviewer #1 (Public Review):

    In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase (PPIase) and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript is structured in two parts addressing the biological function of PfFKBP35 and the antimalarial activity of FK506, respectively.

    The first part combines conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide definitive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors propose that FKBP35 regulates ribosome homeostasis but an alternative explanation could be that changes in the ribosome proteome are downstream consequences of the abrogation of FKBP35 essential activities as chaperone and/or PPIase. It is unclear whether FKBP35 has a specific function in P. falciparum as compared to other organisms. The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

    We thank the Reviewer for the compliments regarding our experimental setup and the clarity of our manuscript. We agree that the link between FKBP35 knock-out and ribosome homeostasis is indirect and we now emphasize this more clearly in the revised manuscript. To prevent a general misinterpretation of our manuscript, we will adapt the title accordingly.

    We would still like to reiterate that the phenotype of FKBP35 knock-out parasites is best described by their defects in maintaining functional ribosomes. It is for several reasons that we believe the links between FKBP35 and ribosome function are purely evidence driven: First, pre-ribosomal and nucleolar factors are the first proteins (in generation 1 schizonts) to be affected upon knock-out of fkbp35 (Figure 2A, Table S1). We realized that Figure 2A falls short in showing this observation, which is why will update the figure accordingly. Second, the dysregulation of ribosomal factors and the general stall in protein synthesis is dominating the phenotype of FKBP35 knock-out parasites in generation 2. We thus believe it is appropriate to say that knock-out cells are most likely killed in response to defective ribosome maintenance – which is a consequence of reduced FKBP35 levels. We are aware that our experiments (and possibly any other reverse genetics approach) cannot rule out that FKBP35 affects ribosomal factors indirectly. Clearly, more work is required to disentangle this question in more detail in the future.

    We agree with the Reviewer that it is not possible to tell if the delayed death-like phenotype is due to a “delayed protein depletion”. We would however like to note that the DiCre/loxP approach allows for an immediate knock-out at the genome level and is thus as precise as possible. Further, in addition to the substantial depletion of FKBP35 in knock-out cells during the phenotypically silent generation, knocking out of fkbp35 at earlier time points (TPs 24-30 and 34-40 hpi in the preceding generation) resulted in the very same phenotype cycle (Figure 1). Here, parasite death was delayed substantially longer, i.e. more than one complete cycle. Together with the dysregulation of early ribosome maturation in generation 1, these findings point towards a delayed death phenotype. It is of course still possible to explain the delayed death-like phenotype by remnant activity of proteins synthetized prior to the genomic knock-out. We address this possibility and describe the two scenarios mentioned by the Reviewer in lines 141-144. Disentangling the two possibilities in future experiments will be difficult, not only with regards to FKBP35, but regarding “delayed death” phenotypes in general.

    In the second part, the authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35. This conclusion is based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, suggesting that FK506 activity is independent of FKBP35 levels, and on the fact that FK506 kills the parasite rapidly whereas inducible gene knockout results in delayed death phenotype. However, there are alternative explanations for these observations. As mentioned above, the delayed death phenotype could be due to delayed depletion of the protein upon induction of gene knockout. FK506 could have a similar activity on WT and mutant parasites when added before sufficient depletion of FKBP35 protein. In some experiments, the authors exposed KO parasites to FK506 later, presumably when the KO is effective, and obtained similar results. However, in these conditions, the death induced by the knockout could be a confounding factor when measuring the effects of the drug. Furthermore, the authors show that FK506 binds to FKBP35, and propose that the FK506-FKBP35 complex interferes with ribosome maturation, which would point towards a role of FKBP35 in FK506 action. In summary, the study does not provide sufficient evidence to rule out that FK506 exerts its effects via FKBP35.

    Noteworthy, we were also very much surprised by data indicating that the antimalarial activity of FK506 is independent of FKBP35. It is for this reason that we conducted a comprehensive set of experiments to disprove our initial observations, but couldn`t find any evidence for an FKBP35-dependent mode of action of FK506:

    We were not able to see altered FK506 sensitivity in (i) inducible knock-down parasites, (ii) inducible overexpression parasites and (iii) inducible knock-out parasites. Parasites with altered FKBP35 levels (as assessed by Western blot and quantitative proteomics at 36-42 hpi, respectively) were equally sensitive to FK506. Importantly, at no sub-lethal FK506 concentration did lower FKBP35 levels lead to an altered response of FKBP35KO compared to the wild-type control population. Furthermore, (iv) induction of the knock-out in the cycle preceding FK506 exposure also had no effect on parasite sensitivity. As mentioned by the Reviewer, we also exposed the parasites to FK506 at 30-36 hpi and (v) did not see any effect, even though we measured a 19-fold difference in FKBP35 protein levels between the parasite populations at 36-42 hpi. At this point, parasite death induced by the knock-out cannot be a confounding factor (as it was mentioned by the Reviewer), because the FKBP35 knock-out has no effect on parasite survival in generation 1 in the absence of FK506 (Figure 1F). This demonstrates that the observed effect is only due to drug-mediated killing and not due to the FKBP35 knock-out.

    To account for a scenario in which the drop in FKBP35 levels only occurs after 36 hpi, we will perform an additional set of experiments, in which we induce the knock-out at 0-6 hpi and treat the parasites at 36-42 hpi (i.e. the time point at which the 19-fold difference in protein levels was measured by quantitative proteomics). This setup will allow determining whether or not the parasite killing activity of FK506 depends on FKBP35 levels.

    So far, our experiments cannot support any scenario in which FK506 kills P. falciparum parasites via inhibiting the essential role of FKBP35 and we would therefore want to insist that this statement is based on highly solid evidence. In this context, it is important to note that our conclusion includes two scenarios: “This indicates that either the binding of FK506 does not interfere with the essential role of _Pf_FKBP35, or that _Pf_FKBP35 is inhibited only at high FK506 concentrations that also inhibit other essential factors.” While this phrase is already present in our initial submission, we will emphasize this point more clearly in the revised manuscript. We are convinced that this information is of high importance for ongoing and future drug development.

    Reviewer #2 (Public Review):

    The manuscript by Thomen et al. FKBP secures ribosome homeostasis in Plasmodium falciparum and focuses on the importance of PfKBP35 protein, its interaction with the FK506 compound, and the role of PfKBP35 in ribosome biogenesis. The authors showed the interaction of the PfKBP54 with FK506, but the part of the FK506 and PfKBP54 in ribosome biogenesis based on the data is unclear.

    The introduction is plotted with two parallel stories about PfKBP35 and FK506, with ribosome biogenesis as the central question at the end. In its current form, the manuscript suffers from two stories that are not entirely interconnected, unfinished, and somewhat confusing. Both stories need additional experiments to make the manuscript(s) more complete. The results from PfFBP35 need more evidence for the proposed ribosome biogenesis pathway control. On the other hand, the results from the drug FK506 point to different targets with lower EC50, and other follow-up experiments are needed to substantiate the authors' claims.

    The strengths of the manuscript are the figures and experimental design. The combination of omics methods is informative and gives an opportunity for follow-up experiments.

    We thank the Reviewer for the evaluation of the manuscript. We apologize for the fact that the Reviewer found the manuscript to be inaccessible. We will use the comments as an incentive to restructure the manuscript and do our best to clarify the presentation, interpretation and conclusion of the presented data in the revised version. We believe that the FKBP35 data are strongly interlinked with the findings on FK506. We will emphasize these links more clearly and are convinced that the complementary nature of the datasets are a particular strength of the presented work.

    Reviewer #3 (Public Review):

    The study by Thommen et al. sought to identify the native role of the Plasmodium falciparum FKBP35 protein, which has been identified as a potential drug target due to the antiplasmodial activity of the immunosuppressant FK506. This compound has multiple binding proteins in many organisms; however, only one FKBP exists in P. falciparum (FKBP35). Using genetically-modified parasites and mass spectrometry-based cellular thermal shift assays (CETSA), the authors suggest that this protein is in involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is separate from its activity on the FKBP35 protein. The authors first created a conditional knockdown using the destruction domain/shield system, which demonstrated no change in asexual blood stage parasites. A conditional knockout was then generated using the DiCre system. FKBP35KO parasites survived the first generation but died in the second generation. The authors called this "a delayed death phenotype", although it was not secondary to drug treatment, so this may be a misnomer. This slow death was unrelated to apicoplast dysfunction, as demonstrated by lack of alterations in sensitivity to apicoplast inhibitors. Quantitative proteomics on the FKBP35KO vs FKBP35WT parasites demonstrated enrichment of proteins involved in pre-ribosome development and the nucleolus. Interestingly, the KO parasites were not more susceptible to cycloheximide, a translation inhibitor, in the first generation (G1), suggesting that mature ribosomes still exist at this point. The SunSET technique, which incorporates puromycin into nascent peptide chains, also showed that in G1 the FKBP35KO parasites were still able to synthesize proteins. But in the second generation (G2), there was a significant decrease in protein synthesis. Transcriptomics were also performed at multiple time points. The effects of knockout of FKBP35 were transcriptionally silent in G1, and the parasites then slowed their cell cycles as compared to the FKBP35WT parasites.

    The authors next sought to evaluate whether killing by FK506 was dependent upon the inhibition of PfKBP35. Interestingly, both FKBP35KO and FKBP35WT parasites were equally susceptible to FK506. This suggested that the antiplasmodial activity of FK506 was related to activity targeting essential functions in the parasite separate from binding to FKBP35. To identify these potential targets, the authors used MS-CETSA on lysates to test for thermal stabilization of proteins after exposure to drug, which suggests drug-protein interactions. As expected, FK506 bound FKBP35 at low nM concentrations. However, given that the parasite IC50 of this compound is in the uM range, the authors searched for proteins stabilized at these concentrations as putative secondary targets. Using live cell MS-CETSA, FK506 bound FKBP35 at low nM concentrations; however, in these experiments over 50 ribosomal proteins were stabilized by the drug at higher concentrations. Of note, there was also an increase in soluble ribosomal factors in the absence of denaturing conditions. The authors suggested that the drug itself led to these smaller factors disengaging from a larger ribosomal complex, leading to an increase in soluble factors. Ultimately, the authors conclude that the native function of FKBP35 is involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is not related to the binding of FKBP35, but instead results from inhibition of essential functions of secondary targets.

    Strengths:

    This study has many strengths. It addresses an important gap in parasite biology and drug development, by addressing the native role of the potential antiplasmodial drug target FKBP35 and whether the compound FK506 works through inhibition of that putative target. The knockout data provide compelling evidence that the KBP35 protein is essential for asexual parasite growth after one growth cycle. Analysis of the FKBP35KO line also provides evidence that the effects of FK506 are likely not solely due to inhibition of that protein, but instead must have secondary targets whose function is essential. These data are important in the field of drug development as they may guide development away from structure-based FK506 analogs that bind more specifically to the FKBP35 protein.

    Weaknesses:

    There are also a few notable weaknesses in the evidence that call into question the conclusion in the article title that FKBP35 is definitely involved in ribosomal homeostasis. While the proteomics supports alterations in ribosome biogenesis factors, it is unclear whether this is a direct role of the loss of the FKBP35 protein or is more related to non-specific downstream effects of knocking down the protein. The CETSA data clearly demonstrate that FK506 binds PfKB35 at low nM concentrations, which is different than the IC50 noted in the parasite; however, the evidence that the proteins stabilized by uM concentrations of drug are actual targets is not completely convincing. Especially, given the high uM amounts of drug required to stabilize these proteins. This section of the manuscript would benefit from validation of a least one or two of the putative candidates noted in the text. In the live cell CETSA, it is noted that >50 ribosomal components are stabilized in drug treated but not lysate controls. Similarly, the authors suggest that the -soluble fraction of ribosomal components increases in drug-exposed parasites even at 37{degree sign}C and suggests that this is likely from smaller ribosomal proteins disengaging from larger ribosomal complexes. While the evidence is convincing that this protein may play a role in ribosome homeostasis in some capacity, it is not sure that the title of the paper "FKBP secures ribosome homeostasis" holds true given the lack of mechanistic data. A minor weakness, but one that should nonetheless be addressed, is the use of the term "delayed death phenotype" with regards to the knockout parasite killing. This term is most frequently used in a very specific setting of apicoplast drugs that inhibit apicoplast ribosomes, so the term is misleading. It is also possible that the parasites are able to go through a normal cycle because of the kinetics of the knockout and that the time needed for protein clearance in the parasite to a level that is lethal.

    Overall, the authors set out to identify the native role of FKB35 in the P. falciparum parasites and to identify whether this is, in fact, the target of FK506. The data clearly demonstrate that FKBP35 is essential for parasite growth and provide evidence that alterations in its levels have proteomic but not transcriptional changes. However, the conclusion that FKBP35 actually stabilizes ribosomal complexes remains intermediate. The data are also very compelling that FK506 has secondary targets in the parasite aside from FKBP35; however, the high uM concentrations of the drug needed to attain results and the lack of biological validation of the CETSA hits makes it difficult to know whether any of these are actually the target of the compound or instead are nonspecific downstream consequences of treatment.

    We appreciate the detailed and valuable suggestions to improve the manuscript. We agree that CETSA could only identify potential targets of FK506 in the micromolar range, while FK506 showed a high affinity for FKBP35, consistent with earlier reports (2). We would however like to point out that FK506 kills P. falciparum at exactly these relatively high concentrations and not at those presumed from the high affinity interactions between FK506 and FKBP35. The relatively high FK506 concentration required to stabilize potential off target proteins is therefore not a concerning observation, but rather corroborates our conclusion that FK506 fails to inhibit the essential function of FKBP35 at concentrations that leave off targets unaffected. As mentioned in response to Reviewer 1, we will describe and discuss these data more clearly in the revised manuscript.

    We thank the Reviewer for pointing out the potential issues regarding the use of the term “delayed death phenotype”. We now refer to the FKBP35 phenotype as “delayed death-like” in the revised manuscript.

    We believe that follow-up work on specific FK506 CETSA hits is out of scope of the current and already quite complex manuscript.

    As mentioned in the response to Reviewer 1, we realize that the short title of the manuscript can be regarded as an overstatement. Again, this was clearly not our intention and we apologize that the Reviewers had to indicate this issue. While we believe that the message of the title holds true (see response to Reviewer 1), we recognize the misconception that might arise from it, which is why we propose the new title: “Genetic validation of _Pf_FKBP35 as an antimalarial drug target”.

    1. Kennedy K, Cobbold SA, Hanssen E, Birnbaum J, Spillman NJ, McHugh E, et al. Delayed death in the malaria parasite Plasmodium falciparum is caused by disruption of prenylation-dependent intracellular trafficking. PLoS Biol. 2019;17(7):e3000376.
    2. Kotaka M, Ye H, Alag R, Hu G, Bozdech Z, Preiser PR, et al. Crystal structure of the FK506 binding domain of Plasmodium falciparum FKBP35 in complex with FK506. Biochemistry. 2008;47(22):5951-61.
    3. Kasahara K, Nakayama R, Shiwa Y, Kanesaki Y, Ishige T, Yoshikawa H, et al. Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genes cooperatively with Hmo1 in Saccharomyces cerevisiae. PLoS Genet. 2020;16(6):e1008865.
  21. Version published to 10.1101/2022.12.09.519720v2 on bioRxiv
  22. Version published to 10.1101/2022.12.09.519720v1 on bioRxiv