The Plasmodium falciparum apicoplast cysteine desulfurase provides sulfur for both iron-sulfur cluster assembly and tRNA modification

  1. Russell P Swift
  2. Rubayet Elahi
  3. Krithika Rajaram
  4. Hans B Liu
  5. Sean T Prigge

  • Curated by eLife

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    This study provides important new insights into iron sulfur biosynthesis in the human malaria parasite Plasmodium falciparum. The work is based on elegant and robust genetic approaches, and not only confirms the essentiality of the plastid-hosted Suf iron-sulfur cluster synthesis pathway, but also highlights an important additional role for the cysteine desulfurase SufS in apicoplast maintenance via tRNA modification. The work provides compelling evidence for a dual function of parasite SufS, although impact on tRNA has not been established directly. These findings reveal a potential new target for metabolic intervention, and will be of interest to researchers studying apicomplexan parasites, and more broadly, in the field of plastid biology.

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Abstract

Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum . Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

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  1. Version published to 10.7554/elife.84491 on eLife
  2. eLife

    eLife assessment

    This study provides important new insights into iron sulfur biosynthesis in the human malaria parasite Plasmodium falciparum. The work is based on elegant and robust genetic approaches, and not only confirms the essentiality of the plastid-hosted Suf iron-sulfur cluster synthesis pathway, but also highlights an important additional role for the cysteine desulfurase SufS in apicoplast maintenance via tRNA modification. The work provides compelling evidence for a dual function of parasite SufS, although impact on tRNA has not been established directly. These findings reveal a potential new target for metabolic intervention, and will be of interest to researchers studying apicomplexan parasites, and more broadly, in the field of plastid biology.

  3. eLife

    Reviewer #1 (Public Review):

    Malaria parasites contain a relict plastid organelle, called apicoplast, which harbors essential metabolic pathways such as iron-sulfur cluster and isoprenoid precursor biosynthetic pathways. In this paper, the authors investigated the apicoplast iron-sulfur (FeS) pathway in P. falciparum. Using an elegant chemical bypass genetic method, they deleted four nuclear genes encoding apicoplast FeS pathway proteins involved in sulfur acquisition or FeS cluster assembly (SufS, SufE, SufC and SufD), and demonstrated that all four are essential for parasite survival. Interestingly, an additional phenotype characterized by disruption of the apicoplast was observed with sufS (but not other mutants). The authors hypothesized that the loss of the apicoplast in the absence of SufS could be due to an additional function of SufS in tRNA thiolation, a pathway that relies on sulfur transfer. Based on sequence homology they identified a putative apicoplast tRNA thiolation enzyme, PfMnmA, and confirmed by genetic tagging that PfMnmA localizes to the parasite apicoplast. Using the chemical bypass system, they further show through knockdown or knockout strategies that PfMnmA is required for parasite survival and for apicoplast maintenance, similar to SufS.

    The authors then used a series of genetic complementation with bacterial enzymes, and show that SufS and MnmA can be replaced by two enzymes from Bacillus subtilis, the cysteine desulfurase BsYrvO and the tRNA thio-uridylase BsMnmA, respectively. In B. subtilis, YrvO mediates the direct transfer of sulfur to MnmA, which mediates tRNA thiolation. Based on the genetic complementation results, the authors infer that SufS has a dual function in P. falciparum, in FeS biosynthesis (together with other Suf proteins), and in apicoplast maintenance via tRNA thiolation. The work is very well performed and the manuscript is well written. The evidence for a dual role of SufS is compelling. However, the claimed role of PfSufS/PfMnmA in tRNA modification is not directly addressed, which would make this exciting story more complete. The identification of new essential metabolic pathways is of great interest as the apicoplast is a potential target for antimalarial therapies.

  4. eLife

    Reviewer #2 (Public Review):

    This manuscript explores the importance of the plastid-hosted SUF iron-sulfur cluster synthesis pathway for plastid maintenance and for the viability of blood stages of the human parasite Plasmodium falciparum. The authors convincingly demonstrate that while most of the proteins of the SUF pathway are essential to P. falciparum survival only one, the cysteine desulfurase SufS, also leads to the loss of the plastid. The authors then explore the possibility that SufS may be providing sulfur to a plastid-localised putative tRNA modifying enzyme, MnmA. They demonstrate that, like when SufS is depleted, specific depletion of MnmA impairs parasite viability and causes plastid loss. They also elegantly complement this phenotype with bacterial MnmA expressed together with a bacterial cysteine desulfurase or even alone, suggesting that SufS from the parasite is able to directly transfer sulfur to the bacterial MnmA.

    Overall, this is a well-conducted and well-controlled study, for which I do not have any major criticism, although tRNA purification, identification, and quantification in the SufS and MnmA mutants would bring more compelling evidence that tRNA thiolation is affected in these mutants.

  5. eLife

    Reviewer #3 (Public Review):

    In recent work, Prigge and collaborators reported the essential function of the apicoplast in the synthesis of isoprenoids which serves as a precursor of several biochemical processes. The pathway involving the synthesis of IPP includes Fe-S enzymes IspH/IspG. Thus, the inactivation of the gene products promoting the assembly of Fe-S clusters for these enzymes in the apicoplast indirectly affects IPP formation and makes the function of these genes likewise essential. Recently, the authors established that the essential requirement of IspH/IscG can be bypassed if an alternate IPP pathway is provided. The mevalonate (MEV) pathway does not require the involvement of Fe-S enzymes and allows for the mevalonate-dependent organism's survival even after disruption of IspH/IspG or ferredoxin (involved in Fe-S cluster formation). The MEV bypass genetic construct provides a valuable experimental handle to expand the analysis of additional functions essential to the apicoplast. Using this genetic tool, this report provides experimental evidence demonstrating the essentiality of sufS, sufE, sufC, sufD, and sufB in IPP synthesis and supporting their previously proposed roles in Fe-S cluster biosynthesis. Although the results from these experiments were anticipated, the novel finding of this study is that phenotypes associated with sufS inactivation differ from the phenotypes associated with the inactivation of other components of the Fe-S cluster biosynthetic apparatus pointing to additional function(s) of this enzyme.

    Cysteine desulfurases are enzymes involved in sulfur mobilization for the synthesis of Fe-S cluster and other sulfur-containing cofactors. Thus, the inactivation of sufS would likely lead to the depletion of additional sulfur-containing biomolecules in the apicoplast. Using the MEV bypass, the authors showed sufS inactivation led to the loss of the apicoplast genome, indicating the involvement of SufS in additional essential functions in this organelle. Based on this premise, the authors tested the hypothesis that tRNA thiolation was also an essential process in this organelle. Experimental validation supporting this hypothesis included 1) genetic evidence that the putative tRNA 2-thiouridylase MnmA is also essential and that mnmA inactivation leads to phenotypes that mirror those of sufS inactivation in the MEV bypass genetic background, 2) B. subtilis MnmA or MnmA-YrvO fusion complements the PfMnmA inactivation, and 3) B. subtilis MnmA-YrvO fusion is able to complement PfsufS inactivation in an MEV bypass. Collectively, these results support a model in which SufS is involved in two essential functions Fe-S cluster formation and tRNA thiolation. Interestingly, genetic analysis suggests that SufS but not SufE are involved in tRNA thiolation, indicating the occurrence of a direct SufS-MnmA sulfur transfer reaction, a mechanistically distinct feature from other characterized SufS-like enzymes that require a dedicated E-like sulfur transferase. Thus the absence of SufS-like sequences in the host cells combined with the essentiality of this enzyme for the parasite life cycle offers an attractive target for metabolic intervention.

  6. Version published to 10.1101/2022.10.31.514511v1 on bioRxiv