Biology subjects

Florentin, A.

Publications and source records attributed to Florentin, A..

8 recordsLinked to original sources

bioRxiv2026

Mechanistic Constraints on ClpM Expression Underlie Apicoplast Genome Retention in Malaria Parasites

The apicoplast of malaria parasites retains a reduced genome encoding a small set of genes with unknown functions. Among these genes is a putative ClpM chaperone, which unlike other apicoplast Clp-family members is not nuclear but plastid-encoded. In this study, we used ClpM as a model case to investigate evolutionary and molecular basis for plastid genome retention. Phylogenetic analyses across plastid-containing eukaryotes revealed that ClpM orthologues are broadly conserved and consistently plastid-encoded in all organisms with a red alga-derived plastid, irrespective of parasitism, photosynthesis or physiology. This broad phenomenon suggested gene-specific evolutionary constraints that were subsequently tested experimentally. To test whether clpM can be functionally expressed from the nucleus, we generated transgenic parasites carrying a nuclear ClpM copy fused to an apicoplast-targeting transit peptide. Unexpectedly, standard transgenesis resulted in transcriptional silencing, and we therefore forced transcription using integration into an endogenous essential locus. This led to robust clpM mRNA, however no detectable ClpM protein was observed. Multiple analyses ruled out apicoplast-dependent instability, ER-associated degradation, misfolding or membrane sequestration. Attempts to express clpM or other plastid-derived genes using endogenous sequences were found to be toxic, suggesting nucleotide-sequence incompatibility. In contrast, a transgene carrying a second copy of the nuclear ClpC ortholog was readily expressed. Comparative analysis of ClpM and ClpC domain architecture showed that their ATPase domains form distinct evolutionary clusters, suggesting conserved but functionally divergent roles. Subsequently, domain-swap experiments between ClpC and ClpM rescued partial expression and identified specific domains as contributors to the nuclear-expression barrier. Together, these findings demonstrate that clpM retention in the apicoplast genome is enforced by multilayered constraints involving evolutionary conservation, nucleotide-sequence incompatibility, transcriptional block and protein-intrinsic translational barriers. This work provides experimental evidence for mechanisms that restrict organelle-to-nucleus gene transfer and contribute to organelle genome retention.

microbiology↗

bioRxiv2025

Organelle Development and Inheritance are Driven by Independent Nuclear and Organellar Mechanisms in Malaria Parasites

The apicoplast organelle of Plasmodium falciparum is essential for parasites replication, however the details of its biogenesis, inheritance and regulation throughout the cell cycle are unknown. Here, we report the development of a dynamic imaging platform coupled with an analytical pipeline that enables us to follow and measure subcellular structures throughout the 48-hour cell cycle of live parasites. We reveal a predetermined sequence of four discrete morphological steps in organelle development, which are tightly correlated with nuclear replication. We show that one of these steps, which we term the Crown morphology, is required for nucleus-apicoplast attachment. During Crown, apicoplast is stretched over multiple nuclei, fastened by centriolar tubulin. A complementary molecular approach was used to discover the basic ploidy of apicoplast and mitochondrial genomes, their replication rates and association with nuclear DNA replication. We inhibited nuclear DNA replication and found that it completely blocks apicoplast biogenesis in its most initial stages, demonstrating dependency on S-phase initiation. Conversely, specific inhibition of apicoplast genome replication resulted in an almost-undisturbed organelle development and division. However, it affected the Crown step, preventing association to tubulin-nuclear structures, leading to failure in accurate organelle sorting into daughter cells. Collectively, these experiments reveal a central cellular pathway linking apicoplast development to the parasites cell cycle, and a second independent organellar mechanism responsible for segregation into daughter cells.

microbiology↗

bioRxiv2024

A Chaperonin Complex Regulates Organelle Proteostasis in Malaria Parasites

The apicoplast of Plasmodium parasites serves as a metabolic hub that synthesize essential biomolecules. Like other endosymbiotic organelles, 90% of the apicoplast proteome is encoded by the cell nucleus and transported to the organelle. Evidence suggests that the apicoplast has minimal control over the synthesis of its proteome and therefore it is unclear how organelle proteostasis is regulated. Here, we identified and investigated a large and conserved chaperonin (CPN) complex with a previously unknown function. Using genetic tools, we demonstrated that ablation of the apicoplast CPN60 subunit leads to parasite death due to organellar damage, immediately within its first replication cycle, deviating from the delayed death phenotype commonly observed for apicoplast translation inhibitors. Unlike its close orthologues in other prokaryotic and eukaryotic cells, CPN60 is not upregulated during heat shock (HS) and does not affect HS response in the parasite. Instead, we found that it is directly involved in proteostasis through interaction with the Clp (caseinolytic protease) proteolytic complex. We showed that CPN60 physically binds both the active and inactive forms of the Clp complex, and manipulates its stability. A computational structural model of a possible interaction between these two large complexes suggests a stable interface. Finally, we screened a panel of inhibitors for the bacterial CPN60 orthologue GroEL, to test the potential of chaperonin inhibition as antimalarial. These inhibitors demonstrated an anti-Plasmodium activity that was not restricted to apicoplast function, with additional targets outsides of this organelle. Taken together, this work reveals how balanced activities of proteolysis and refolding safeguard the apicoplast proteome, and is essential for organelle biogenesis. Author SummaryThe cell of the human malaria parasite Plasmodium falciparum has a unique organelle called the apicoplast that produces essential metabolites, but it is unclear how it maintains a stable proteome. Here, we address the question of organelle proteostasis by investigating the function of a large chaperonin complex and its main subunit CPN60. We show that CPN60 mutants die due to organellar damage immediately within the first replication cycle, avoiding the typical apicoplast-delayed cell death. We demonstrate that it binds and stabilizes another large proteolytic complex and use computational predicting tools to demonstrate how a stable interface is attained. We use bacterial inhibitors to explore their potential as an antimalarial drug target. This study reveals how balanced refolding and proteolysis safeguard the apicoplast proteome and opens a new avenue for antimalarial drug discovery.

microbiology↗

bioRxiv2019

The Clp proteolytic system acts as an essential nexus for Plasmodium plastid biogenesis

The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most of apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrated that the Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9 based system to express catalytically-dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and matured via trans-autocatalysis. The expression of a Clp chaperone (PfClpC) mutant led to the discovery of a functional chaperone-protease interaction essential for plastid function. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

microbiology↗

bioRxiv2019

Metabolomic profiling reveals developmentally regulated biosynthesis of polyprenols and dolichols in the malaria parasite

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Polyprenols have been hardly detectable in eukaryotic cells under normal conditions with the exception of plants and sporulating yeast. Our metabolomics studies revealed that cis-polyisoprenoids are more prevalent and diverse in the parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was also observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasites development. In addition, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite. Isotopic labeling and metabolomic analyses of a conditional mutant of PfCPT or PfPPRD suggest that polyprenols may be able to substitute dolichols in their biological functions when dolichol synthesis is impaired in Plasmodium.

biochemistry↗

bioRxiv2018

The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2 kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

cell biology↗

bioRxiv2018

CRISPR/Cas9 gene editing to make conditional mutants of the human malaria parasite Plasmodium falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of better drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology, but historically, the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. The method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

cell biology↗

bioRxiv2017

The exported chaperone PfHsp70x is dispensable for the Plasmodium falciparum intraerythrocytic lifecycle

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual lifecycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 co-chaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, PfHsp70x, is exported with them.\n\nPfHsp70x is absent from most Plasmodium species and is found only in P. falciparum and closely-related species that infect Apes. Herein, we have utilized CRISPR/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the DHFR-based Destabilization Domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic lifecycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host RBC, including the critical virulence factor PfEMP1, was tested and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum.\n\nImportanceHalf of the worlds population lives at risk for malaria. The intraerythrocytic lifecycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasites ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the human-disease causing Plasmodium species, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (1). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors nor is it essential for parasite survival inside of the RBC.

microbiology↗