Biology subjects

Boucher, M. J.

Publications and source records attributed to Boucher, M. J..

6 recordsLinked to original sources

bioRxiv2026

Therapeutic vulnerabilities of the drug-resistant fungal meningitis pathogen Cryptococcus neoformans

Treatment options are limited for the priority fungal pathogen Cryptococcus neoformans. We describe CRISPR-insert, a scalable gene inactivation method for global interrogation of drug targets. Genome-wide screens using this methodology combined with machine learning predict 1,297 essential genes under liberal criteria. Unexpectedly absent from this list is FKS1, encoding the target of echinocandin drugs to which C. neoformans is resistant. Contrary to longstanding literature, targeted deletion shows that FKS1 is not essential. Solid-state NMR reveals that fks1{triangleup} mutants lack {beta}-1,3-glucan, arguing against compensation by another synthase. We experimentally test essentiality predictions for genes lacking human orthologs, identifying high-priority drug target candidates. To uncover targets for combination therapies, we establish genome-scale quantitative genetic interaction mapping. We use this method to uncover synthetic lethal dependencies of echinocandin resistance determinants, including the druggable calcineurin and Cdc50-Apt1 pathways. CRISPR-insert illuminates therapeutic vulnerabilities of an invasive fungal pathogen.

microbiology↗

bioRxiv2021

Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging

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 KN99 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.

microbiology↗

bioRxiv2019

The antimalarial natural product salinipostin A identifies essential α/β serine hydrolases involved in lipid metabolism in P. falciparum parasites

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 /{beta} 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.

cell biology↗

bioRxiv2019

Structure-function analysis reveals a DNA polymerization-independent role for mitochondrial DNA polymerase IC in African trypanosomes.

The 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 the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.\n\nSummary statementMitochondrial DNA segregation in African trypanosomes is supported by a dual-functioning DNA polymerase.

microbiology↗

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

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↗