Biology subjects

Yeh, E.

Publications and source records attributed to Yeh, E..

7 recordsLinked to original sources

bioRxiv2018

Evidence that disruption of apicoplast protein import in malaria parasites evades delayed-death growth inhibition

Malaria parasites (Plasmodium spp.) contain a nonphotosynthetic plastid organelle called the apicoplast, which houses essential metabolic pathways and is required throughout the parasite life cycle. Hundreds of proteins are imported across 4 membranes into the apicoplast to support its function and biogenesis. The machinery that mediates this import process is distinct from proteins in the human host and may serve as ideal drug targets. However, a significant concern is whether inhibition of apicoplast protein import will result in a \"delayed-death\" phenotype that limits clinical use, as observed for inhibitors of apicoplast housekeeping pathways. To assess the growth inhibition kinetics of disrupting apicoplast protein import, we targeted a murine dihydrofolate reductase (mDHFR) domain, which is stabilized by the compound WR99210, to the apicoplast to enable inducible blocking of apicoplast-localized protein translocons. We show that stabilization of this apicoplast-targeted mDHFR disrupts parasite growth within a single lytic cycle in an apicoplast-specific manner. Consistent with inhibition of apicoplast protein import, stabilization of this fusion protein disrupted transit peptide processing of endogenous apicoplast proteins and caused defects in apicoplast biogenesis. These results indicate that disruption of apicoplast protein import avoids delayed-death growth inhibition and that target-based approaches to develop inhibitors of import machinery may yield viable next-generation antimalarials.\n\nImportanceMalaria is a major cause of global childhood mortality. To sustain progress in disease control made in the last decade, new antimalarial therapies are needed to combat emerging drug resistance. Malaria parasites contain a relict chloroplast called the apicoplast, which harbors new targets for drug discovery, including import machinery that transports hundreds of critical proteins into the apicoplast. Unfortunately, some drugs targeting apicoplast pathways show delayed growth inhibition, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. We used a chemical biology approach to disrupt apicoplast protein import and showed that chemical disruption of this pathway avoids delayed growth inhibition. Our finding indicates that prioritization of proteins involved in apicoplast protein import for target-based drug discovery efforts may aid in the development of novel fast-acting antimalarials.

microbiology↗

bioRxiv2018

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid, the apicoplast, which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in P. falciparum. Apicoplast-minus mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a TIM-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly-identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

cell biology↗

bioRxiv2018

Host cell metabolism contributes to delayed-death kinetics of apicoplast inhibitors in Toxoplasma gondii

Toxoplasma gondii and related human parasites contain an essential plastid organelle called the apicoplast. Clinically-used antibiotics and other inhibitors that disrupt apicoplast biogenesis cause a mysterious \"delayed-death\" phenotype, in which parasite growth is unaffected during the first lytic cycle of inhibitor treatment but is severely inhibited in the second lytic cycle even after drug removal. Critical to understanding the complex downstream cellular effects of these drug classes is the timing of apicoplast loss during inhibitor treatment and how it relates to this peculiar growth phenotype. Here we show that, upon treatment with diverse classes of apicoplast inhibitors, newly-replicated T. gondii parasites in the first lytic cycle initially form apicoplasts with defects in protein import or genome replication and eventually fail to inherit the apicoplast altogether. Despite the accumulation of parasites with defective or missing apicoplasts, growth is unaffected during the first lytic cycle, as previously observed. Strikingly, concomitant inhibition of host cell isoprenoid biosynthesis results in growth inhibition in the first lytic cycle and unmasks the apicoplast defects. These results suggest that defects in and even complete loss of the apicoplast in T. gondii are partially rescued by scavenging of host cell metabolites leading to death that is delayed. Our findings uncover host cell interactions that can alleviate apicoplast inhibition and highlight key differences in \"delayed-death\" inhibitors between T. gondii and Plasmodium falciparum.

microbiology↗

bioRxiv2018

The proteome of the malaria plastid organelle, a key anti-parasitic target

Malaria parasites (Plasmodium spp.) and related apicomplexan pathogens contain a non-photosynthetic plastid called the apicoplast. Derived from an unusual secondary eukaryote-eukaryote endosymbiosis, the apicoplast is a fascinating organelle whose function and biogenesis rely on a complex amalgamation of bacterial and algal pathways. Because these pathways are distinct from the human host, the apicoplast is an excellent source of novel antimalarial targets. Despite its biomedical importance and evolutionary significance, the absence of a reliable apicoplast proteome has limited most studies to the handful of pathways identified by homology to bacteria or primary chloroplasts, precluding our ability to study the most novel apicoplast pathways. Here we combine proximity biotinylation-based proteomics (BioID) and a new machine learning algorithm to generate a high-confidence apicoplast proteome consisting of 346 proteins. Critically, the high accuracy of this proteome significantly outperforms previous prediction-based methods and extends beyond other BioID studies of unique parasite compartments. Half of identified proteins have unknown function, and 77% are predicted to be important for normal blood-stage growth. We validate the apicoplast localization of a subset of novel proteins and show that an ATP-binding cassette protein ABCF1 is essential for blood-stage survival and plays a previously unknown role in apicoplast biogenesis. These findings indicate critical organellar functions for newly discovered apicoplast proteins. The apicoplast proteome will be an important resource for elucidating unique pathways derived from secondary endosymbiosis and prioritizing antimalarial drug targets.

microbiology↗

bioRxiv2017

Atg8 is essential specifically for an autophagy-independent function in apicoplast biogenesis in blood-stage malaria parasites

Plasmodium parasites and related pathogens contain an essential non-photosynthetic plastid organelle, the apicoplast, derived from secondary endosymbiosis. Intriguingly, a highly conserved eukaryotic protein, autophagy-related protein 8 (Atg8), has an autophagy-independent function in the apicoplast. Little is known about the novel apicoplast function of Atg8 and its importance in blood-stage P. falciparum. Using a P. falciparum strain in which Atg8 expression was conditionally regulated, we showed that PfAtg8 is essential for parasite replication. Significantly, growth inhibition caused by the loss of PfAtg8 was reversed by addition of isopentenyl pyrophosphate (IPP), which was previously shown to rescue apicoplast defects in P. falciparum. Parasites deficient in PfAtg8, but growth rescued by IPP, had lost their apicoplast. We designed a suite of functional assays, including a new fluorescence in situ hybridization (FISH) method for detection of the low-copy apicoplast genome, to interrogate specific steps in apicoplast biogenesis and detect apicoplast defects which preceded the block in parasite replication. Though protein import and membrane expansion of the apicoplast were unaffected, the apicoplast was not inherited by daughter parasites. Our findings demonstrate that, though multiple autophagy-dependent and independent functions have been proposed for PfAtg8, only its role in apicoplast biogenesis is essential. We propose that PfAtg8 is required for fission or segregation of the apicoplast during parasite replication.

cell biology↗

bioRxiv2017

A Specific Non-Bisphosphonate Inhibitor Of The Bifunctional Farnesyl/Geranylgeranyl Diphosphate Synthase In Malaria Parasites

Isoprenoid biosynthesis is essential for Plasmodium falciparum (malaria) parasites and contains multiple validated antimalarial drug targets, including a bifunctional farnesyl and geranylgeranyl diphosphate synthase (FPPS/GGPPS). We identified MMV019313 as an inhibitor of PfFPPS/GGPPS. Though PfFPPS/GGPPS is also inhibited by a class of bisphosphonate drugs, MMV019313 has significant advantages for antimalarial drug development. MMV019313 has superior physicochemical properties compared to charged bisphosphonates that have poor bioavailability and strong bone affinity. We also show that it is highly selective for PfFPPS/GGPPS and showed no activity against human FPPS or GGPPS. Inhibition of PfFPPS/GGPPS by MMV019313, but not bisphosphonates, was disrupted in an S228T variant, demonstrating that MMV019313 and bisphosphonates have distinct modes-of-inhibition against PfFPPS/GGPPS. Altogether MMV019313 is the first specific, non-bisphosphonate inhibitor of PfFPPS/GGPPS. Our findings uncover a new small molecule binding site in this important antimalarial drug target and provide a promising starting point for development of Plasmodium-specific FPPS/GGPPS inhibitors.

microbiology↗

bioRxiv2017

A first-in-class inhibitor of parasite FtsH disrupts plastid biogenesis in human pathogens

The malaria parasite Plasmodium falciparum and related apicomplexan pathogens contain an essential plastid organelle, the apicoplast, which is a key anti-parasitic target. Derived from secondary endosymbiosis, the apicoplast depends on novel, but largely cryptic, mechanisms for protein/lipid import and organelle inheritance during parasite replication. These critical biogenesis pathways present untapped opportunities to discover new parasite-specific drug targets. We used an innovative screen to identify actinonin as having a novel mechanism-of-action inhibiting apicoplast biogenesis. Resistant mutation, chemical-genetic interaction, and biochemical inhibition demonstrate that the unexpected target of actinonin in P. falciparum and Toxoplasma gondii is FtsH1, a homolog of a bacterial membrane AAA+ metalloprotease. Pf FtsH1 is the first novel factor required for apicoplast biogenesis identified in a phenotypic screen. Our findings demonstrate that FtsH1 is a novel and, importantly, druggable antimalarial target. Development of FtsH1 inhibitors will have significant advantages with improved drug kinetics and multistage efficacy against multiple human parasites.

microbiology↗