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Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or “CnoCAS9”) reliably enabled genome editing in the widely-used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

Trypanosoma brucei has a unique catenated mitochondrial DNA (mtDNA) network called kinetoplast DNA (kDNA). Replication of kDNA occurs once per cell cycle in near synchrony with nuclear S phase and requires the coordination of many proteins. Among these are three essential DNA polymerases (TbPOLIB, IC, and ID). Localization dynamics of these proteins with respect to kDNA replication stages and how they coordinate their functions during replication are not well understood. We previously demonstrated that TbPOLID undergoes dynamic localization changes that are coupled to kDNA replication events. Here, we report the localization of TbPOLIC, a second essential DNA polymerase, and demonstrate the accumulation of TbPOLIC foci at active kDNA replication sites (antipodal sites) during stage II of the kDNA duplication cycle. While TbPOLIC was undetectable by immunofluorescence during other cell cycle stages, steady-state protein levels measured by Western blot remained constant. TbPOLIC foci colocalized with the fraction of TbPOLID that localized to the antipodal sites. However, the partial colocalization of the two essential DNA polymerases suggests a highly dynamic environment at the antipodal sites to coordinate the trafficking of replication proteins during kDNA synthesis. These data indicate that cell cycle–dependent localization is a major regulatory mechanism for essential mtDNA polymerases during kDNA replication.

SUMMARYSalinipostin A (Sal A) is a potent antimalarial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in thePlasmodium falciparumparasite. All of the identified proteins contain α/β serine hydrolase domains, and several are essential for parasite growth. One of the essential targets displays high homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism and produces disorganized and stalled schizonts similar to Sal A. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a protein linked to drug resistance inToxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Summary Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles, called kinetoplast DNA (kDNA). Replication of a single nucleoid requires at least three DNA polymerase I-like proteins (i.e. POLIB, POLIC and POLID), each showing discrete localizations near the kDNA during S phase. POLIB and POLID have roles in minicircle replication but the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNA interference (RNAi)-complementation system to dissect the functions of two distinct POLIC regions, i.e. the conserved family A DNA polymerase (POLA) domain and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization of kDNA, active site point mutations in the POLA domain impaired minicircle replication similar to that of POLIB and POLID depletions. Complementation with POLA domain alone abolished the formation of POLIC foci and partially rescued the RNAi phenotype. Furthermore, we provide evidence that the UCR is crucial in cell cycle-dependent protein localization and facilitates proper distribution of progeny networks. This is the first report of a DNA polymerase that impacts on mitochondrial nucleoid distribution. This article has an associated First Person interview with the first author of the paper.

Salinipostin A (Sal A) is a potent antimalarial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays high homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism and produces disorganized and stalled schizonts similar to Sal A. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Malaria 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. Unfortunately, some drugs targeting apicoplast pathways exhibit a delayed-death phenotype, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. Identification of druggable apicoplast biogenesis factors that will avoid the delayed-death phenotype is an important priority. Here, we find that chemical stabilization of an apicoplast-targeted mDHFR domain disrupts apicoplast biogenesis and inhibits parasite growth after a single lytic cycle, suggesting a non-delayed-death target. Our finding indicates that further interrogation of the mechanism-of-action of this exogenous fusion protein may reveal novel therapeutic avenues., 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. The biogenesis pathways responsible for apicoplast growth, division, and inheritance are of key interest as potential drug targets. Unfortunately, several known apicoplast biogenesis inhibitors are of limited clinical utility because they cause a peculiar “delayed-death” phenotype in which parasites do not stop replicating until the second lytic cycle posttreatment. Identifying apicoplast biogenesis pathways that avoid the delayed-death phenomenon is a priority. Here, we generated parasites targeting a murine dihydrofolate reductase (mDHFR) domain, which can be conditionally stabilized with the compound WR99210, to the apicoplast. Surprisingly, chemical stabilization of this exogenous fusion protein disrupted parasite growth in an apicoplast-specific manner after a single lytic cycle. WR99210-treated parasites exhibited an apicoplast biogenesis defect beginning within the same lytic cycle as drug treatment, indicating that stabilized mDHFR perturbs a non-delayed-death biogenesis pathway. While the precise mechanism-of-action of the stabilized fusion is still unclear, we hypothesize that it inhibits apicoplast protein import by stalling within and blocking translocons in the apicoplast membranes. IMPORTANCE Malaria 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. Unfortunately, some drugs targeting apicoplast pathways exhibit a delayed-death phenotype, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. Identification of druggable apicoplast biogenesis factors that will avoid the delayed-death phenotype is an important priority. Here, we find that chemical stabilization of an apicoplast-targeted mDHFR domain disrupts apicoplast biogenesis and inhibits parasite growth after a single lytic cycle, suggesting a non-delayed-death target. Our finding indicates that further interrogation of the mechanism-of-action of this exogenous fusion protein may reveal novel therapeutic avenues.

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.ImportanceMalaria 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.

Malaria parasites (Plasmodium spp.) and related apicomplexan pathogens contain a nonphotosynthetic 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., Author summary Plasmodium parasites, which cause malaria, and related pathogens belonging to the phylum Apicomplexa contain a relict chloroplast called the apicoplast. During evolution, the apicoplast lost photosynthetic functions but retained critical metabolic pathways that are required for host cell infection. Because of its importance for parasite survival and its unusual evolutionary origin, the apicoplast is of major interest for its unique biology and its potential to yield new antimalarial drug targets. However, an accurate inventory of proteins that localize to the apicoplast is lacking, hindering our ability to study the most novel and biologically interesting aspects of apicoplast biology. To address this limitation, we combine proximity biotinylation-based proteomics and a new machine learning algorithm to identify apicoplast proteins in an accurate and unbiased manner. We identify 346 candidate apicoplast proteins with high confidence and find that many of these proteins are new and expected to be important for parasite survival. This proteomic resource will therefore be a valuable tool for elucidating the unique cell biology of the apicoplast and for identifying new antimalarial drug targets.