Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor

eLife, Год журнала: 2025, Номер 13

Опубликована: Фев. 24, 2025

The ability to analyze the function of all genes in a genome is highly desirable, yet challenging Leishmania due repetitive genome, limited DNA repair mechanisms, and lack RNA interference most species. While our introduction cytosine base editor (CBE) demonstrated potential overcome these limitations (Engstler Beneke, 2023), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, competition between deleterious non-deleterious mutations. Here, we present an optimized approach addressing issues. We identified T7 RNAP promoter variant ensuring high species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed prioritize STOP codon generation. Additionally, triple-expression construct created for stable integration sgRNA expression cassettes into safe harbor locus using AsCas12a ultra-mediated double-strand breaks, increasing efficiency by ~400-fold 1 transfectant per 70 transfected cells. Using this improved small-scale proof-of-principle pooled screen, successfully confirmed essential fitness-associated functions CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140, RAB5A mexicana , demonstrating significant improvement over previous method. Lastly, show utility co-expressing ultra, RNAP, hybrid CRISPR gene replacement within same cell line. Overall, improvements will broaden range possible applications enable variety loss-of-function screens near future.

Язык: Английский

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Evolutionary adaptations of doublet microtubules in trypanosomatid parasites

Science, Год журнала: 2025, Номер 387(6739)

Опубликована: Март 13, 2025

The movement and pathogenicity of trypanosomatid species, the causative agents trypanosomiasis leishmaniasis, are dependent on a flagellum that contains an axoneme dynein-bound doublet microtubules (DMTs). In this work, we present cryo–electron microscopy structures DMTs from two Leishmania tarentolae Crithidia fasciculata , at resolutions up to 2.7 angstrom. revealed 27 trypanosomatid-specific microtubule inner proteins, specialized dynein-docking complex, presence paralogous proteins enable higher-order periodicities or proximal-distal patterning. Leveraging genetic tractability quantified location contribution each structure-identified protein swimming behavior. Our study shows proper B-tubule closure is critical for flagellar motility, exemplifying how integrating structural identification with systematic gene deletion can dissect individual contributions motility.

Язык: Английский

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Converting Blastocrithidia Nonstop, a Trypanosomatid With Non‐Canonical Genetic Code, Into a Genetically‐Tractable Model

Molecular Microbiology, Год журнала: 2025, Номер unknown

Опубликована: Апрель 9, 2025

ABSTRACT Blastocrithidia nonstop is a protist with highly unusual nuclear genetic code, in which all three standard stop codons are reassigned to encode amino acids, UAA also serving as sole termination codon. In this study, we demonstrate that parasitic flagellate amenable manipulation, enabling gene ablation and protein tagging. Using preassembled Cas9 ribonucleoprotein complexes, successfully disrupted tagged the non‐essential encoding catalase. These advances establish single‐celled eukaryote model organism for investigating malleability evolution of code eukaryotes.

Язык: Английский

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Characterization of a novel Leishmania antigen containing a repetitive domain and its potential use as a prophylactic and therapeutic vaccine

mSphere, Год журнала: 2025, Номер unknown

Опубликована: Апрель 22, 2025

ABSTRACT Human visceral leishmaniasis (HVL) is the second most lethal tropical parasitic disease. Currently, no prophylactic or therapeutic vaccines exist for HVL. Thus, development of an efficacious vaccine still needed. We previously performed immunoproteomics analysis on Leishmania amazonensis parasite extracts to identify immunodominant antigens recognized by sera vaccinated and protected mice. Among identified antigens, we discovered a novel, unstudied repetitive protein, initially annotated in genomes as kinetoplast-associated protein-like protein from infantum (LinKAP), containing conserved domains (trichohyalin-plectin-homology [TPH] TolA) that are associated with other mitochondrial proteins. LinKAP sequences across trypanosomatids, including Endotrypanum, Leishmania, Trypanosoma species. Using differential centrifugation subcellular structures, showed was enriched fractions colocalizing mNeonGreen labeling at endogenous locus using CRISPR-Cas9 confocal microscopy confirmed mitochondrial-associated but not specifically colocalized kDNA. cloned expressed truncated version (rLinKAP), part (15) several amino acid repeats, demonstrating over 85% homology L. infantum, amazonensis, braziliensis, mexicana An adjuvanted formulation Poly ICLC, polyinosinic-polycytidylic (Poly I:C) stabilized carboxymethylcellulose polylysine, used vaccinate mice hamsters leishmaniasis. Animals immunized rLinKAP potent cellular humoral response significant decrease tissue parasitism when challenged . also tested Following vaccination, antibody responses were enhanced, became apparent. Our treatment protocol inhibited splenic burden 75% treated In conclusion, our antigen discovery strategy observed protective effect highlight promising candidate IMPORTANCE A previous reverse vaccinology study (LinKAP) potential new target, this promastigotes. Interestingly, trichohyalin-plectin-homology (TPH) TolA protein. further characterized highly among trypanosomatids. validated (rLinKAP) both vaccine, immunize animals against (VL). This disease, caused parasite, affects populations globally lacks effective vaccines. Identifying its preliminary characterization provides perspectives studying role parasite's biology.

Язык: Английский

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Bar-seq strategies for the LeishGEdit toolbox

Molecular and Biochemical Parasitology, Год журнала: 2020, Номер 239, С. 111295 - 111295

Опубликована: Июль 10, 2020

Язык: Английский

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Intraflagellar transport speed is sensitive to genetic and mechanical perturbations to flagellar beating

The Journal of Cell Biology, Год журнала: 2024, Номер 223(9)

Опубликована: Май 14, 2024

Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins dyneins carry IFT trains bidirectionally along axoneme. Despite assembling cilia flagella, train speeds have only previously been quantified in immobilized flagella—mechanical immobilization or genetic paralysis. This has limited investigation interaction between flagellar beating. Here, uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy visualize movement beating flagella. We discovered that adhesion flagella a microscope slide is detrimental, reducing speed increasing stalling. In free move, not strongly dependent on beat type; however, permanent disruption by deletion genes necessary formation regulation showed an inverse correlation frequency speed.

Язык: Английский

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Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1

PLoS Pathogens, Год журнала: 2024, Номер 20(7), С. e1012382 - e1012382

Опубликована: Июль 11, 2024

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease poverty. The mechanism action (AmB) thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption integrity key functions plasma membrane. Emergence clinically refractory isolates Leishmania donovani L . infantum ongoing issue knowledge potential resistance mechanisms can help alleviate this problem. Here we report characterisation four independently selected clones that are resistant AmB. Whole genome sequencing revealed three moderately clones, was due solely deletion gene encoding C24-sterol methyltransferase ( SMT1 ). fourth, hyper-resistant clone (>60-fold) found have 24 bp both alleles putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid (0.2% versus 18% total sterols wild-type) had marked accumulation 14-methylfecosterol (75% 0.1% 14-alpha methylcholestanes. These substrates sterol demethylase CYP51 ) suggesting enzyme may be bona fide P450R specifically involved electron transfer from NADPH during catalysis. Deletion P450R1 wild-type cells phenocopied metabolic changes observed our AmB as well nulls. Likewise, addition wild type restored profiles type. Our studies indicate essential amastigote viability, thus loss unlikely driver clinical resistance. Nevertheless, investigating underpinning provided insights refine understanding biosynthetic pathway.

Язык: Английский

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CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence in Leishmania major parasites

Medical Microbiology and Immunology, Год журнала: 2025, Номер 214(1)

Опубликована: Март 25, 2025

Язык: Английский

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Construction and characterization of a gE/gI/TK-gene-deleted recombinant pseudorabies virus variant expressing the GP5 of the Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (HP-PRRSV) and NADC30-like PRRSV

Microbial Pathogenesis, Год журнала: 2025, Номер unknown, С. 107522 - 107522

Опубликована: Апрель 1, 2025

Язык: Английский

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The RNA export factor TbMex67 connects transcription and RNA export in Trypanosoma brucei and sets boundaries for RNA polymerase I

Nucleic Acids Research, Год журнала: 2023, Номер 51(10), С. 5177 - 5192

Опубликована: Март 25, 2023

Abstract TbMex67 is the major mRNA export factor known to date in trypanosomes, forming part of docking platform within nuclear pore. To explore its role co-transcriptional export, recently reported Trypanosoma brucei, pulse labelling nascent RNAs with 5-ethynyl uridine (5-EU) was performed cells depleted and complemented a dominant-negative mutant (TbMex67-DN). RNA polymerase (Pol) II transcription unaffected, but procyclin loci, which encode mRNAs transcribed by Pol I from internal sites on chromosomes 6 10, showed increased levels 5-EU incorporation. This due readthrough transcription, proceeded beyond procyclin-associated genes up start site opposite strand. Complementation TbMex67-DN also I-dependent formation R-loops γ-histone 2A foci. The DN exhibited reduced localisation binding chromatin compared wild-type TbMex67. Together interaction remodelling TbRRM1 II, transcription-dependent association nucleoporins, our findings support for connecting T. brucei. In addition, stalls specific contexts, thereby limiting R-loop replication stress.

Язык: Английский

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