TransLeish: Identification of membrane transporters essential for survival of intracellular Leishmania parasites in a systematic gene deletion screen

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 2, 2025

Abstract For the protozoan parasite Leishmania , completion of its life cycle requires sequential adaptation cellular physiology and nutrient scavenging mechanisms to different environments a sand fly alimentary tract acidic mammalian host cell phagolysosome. Transmembrane transporters are gatekeepers intracellular environments, controlling flux solutes ions across membranes. To discover which vital for survival as amastigote forms, we carried out systematic loss-of-function screen L. mexicana transportome. A total 312 protein components small molecule carriers, ion channels pumps were identified targeted in CRISPR-Cas9 gene deletion promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss fitness macrophage mouse infections. striking example is Vacuolar H + ATPase (V-ATPase), which, unexpectedly, was dispensable growth vitro but essential disease-causing amastigotes.

Language: Английский

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DNDI-6148: A Novel Benzoxaborole Preclinical Candidate for the Treatment of Visceral Leishmaniasis

Journal of Medicinal Chemistry, Journal Year: 2021, Volume and Issue: 64(21), P. 16159 - 16176

Published: Oct. 29, 2021

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and fatal if untreated. Current therapies are unsuitable, there an urgent need for safe, short-course, low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates treatment other diseases. Here, we describe development optimization series, leading identification compounds with potent in vitro vivo antileishmanial activity. lead compound (DNDI-6148) combines impressive efficacy (>98% reduction parasite burden) pharmaceutical properties suitable onward acceptable safety profile. Detailed mode action studies confirm that DNDI-6148 acts principally through inhibition Leishmania cleavage polyadenylation specificity factor (CPSF3) endonuclease. As result these its promising profile, been declared preclinical candidate VL.

Language: Английский

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Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

eLife, Journal Year: 2023, Volume and Issue: 12

Published: May 24, 2023

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania , the causative agent of leishmaniasis. As lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection drug resistance-associated edits or time-consuming isolation clones. Genome-wide screens across different conditions and multiple species are therefore unfeasible at present. Here, we report cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs to introduce STOP codons by converting into thymine created http://www.leishbaseedit.net/ for CBE primer design kinetoplastids. Through reporter assays targeting single- multi-copy genes L. mexicana major donovani infantum demonstrate how this tool can efficiently generate expressing just one single-guide RNA, reaching up 100% rate non-clonal populations. then generated -optimised successfully targeted an essential plasmid library delivered screen . Since our method does not require double-strand breaks, homologous recombination, clones, believe enables first time genetic via delivery libraries.

Language: Английский

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Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics

eLife, Journal Year: 2023, Volume and Issue: 12

Published: May 11, 2023

African trypanosomes proliferate as bloodstream forms (BSFs) and procyclic in the mammal tsetse fly midgut, respectively. This allows them to colonise host environment upon infection ensure life cycle progression. Yet, understanding of mechanisms that regulate drive cell replication these is limited. Using single-cell transcriptomics on unsynchronised populations, we have obtained high resolution regulated (CCR) transcriptomes both slender BSF Trypanosoma brucei without prior sorting or synchronisation. Additionally, describe an efficient freeze–thawing protocol transcriptomic analysis cryopreserved T. . Computational reconstruction using periodic pseudotime inference allowed dynamic expression patterns cycling genes be profiled for forms. Comparative analyses identify a core transcriptome highly conserved between forms, well several where transcript levels dynamics are form specific. Comparing with protein abundance revealed majority exhibit relative delay peak expression. work reveals novel detail CCR which available further interrogation via interactive webtool.

Language: Английский

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Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification

Cell Reports, Journal Year: 2024, Volume and Issue: 43(5), P. 114203 - 114203

Published: May 1, 2024

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions individuals worldwide. Pseudouridine (Ψ), most abundant modification on rRNA, changes during parasite life cycle. Alterations in level a specific Ψ helix 69 (H69) affected ribosome function. To decipher molecular mechanism this phenotype, we determine structure ribosomes lacking single its parental strain at ∼2.4–3 Å resolution using cryo-EM. Our findings demonstrate significance H69 to importance for interactions with 44 tRNAs. study suggests that rRNA affects translation mRNAs carrying codon bias due selective accommodation tRNAs by ribosome. Based high-resolution structures, propose explaining how selects

Language: Английский

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Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach

ACS Infectious Diseases, Journal Year: 2024, Volume and Issue: 10(8), P. 2913 - 2928

Published: July 18, 2024

The lack of effective vaccines and the development resistance to current treatments highlight urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source Leishmania-specific targets these lipids are key structural components eukaryotic plasma membrane involved in distinct cellular events. Inositol phosphorylceramide (IPC) is primary sphingolipid Leishmania species product reaction mediated by IPC synthase (IPCS). antihistamine clemastine fumarate identified an inhibitor IPCS L. major potent anti-leishmanial vivo. Here we sought further examine target this compound more tractable mexicana, using approach combining genomic, proteomic, metabolomic lipidomic technologies, with molecular biochemical studies. While data demonstrated that response was largely conserved, unexpected disturbances beyond were identified. Furthermore, while deletion gene encoding LmxIPCS had little impact vitro, it did influence efficacy and, importantly, vivo pathogenicity. Together, demonstrate does inhibit cause associated metabolic disturbances, but its may lie elsewhere.

Language: Английский

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Impact of Genetic Diversity and Genome Plasticity of Leishmania spp. in Treatment and the Search for Novel Chemotherapeutic Targets

Frontiers in Cellular and Infection Microbiology, Journal Year: 2022, Volume and Issue: 12

Published: Jan. 24, 2022

Leishmaniasis is one of the major public health concerns in Latin America, Africa, Asia, and Europe. The absence vaccines for human use lack effective vector control programs make chemotherapy main strategy to all forms disease. However, high toxicity available drugs, limited choice therapeutic agents, occurrence drug-resistant parasite strains are challenges related chemotherapy. Currently, only a small number drugs leishmaniasis treatment, including pentavalent antimonials (Sb V ), amphotericin B its formulations, miltefosine, paromomycin sulphate, pentamidine isethionate. In addition drug toxicity, failure serious concern. parasites causes closely diversity this genus. Owing enormous plasticity genome, resistance can occur by altering different metabolic pathways, demonstrating that mechanisms multifactorial extremely complex. Genetic variability genome cause not have limitations, but also search new challenging. Here, we examined biological characteristics hinder discovery.

Language: Английский

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A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Nov. 20, 2023

Abstract Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts the causative agent of sleeping sickness. Here, we describe changes pseudouridine (Ψ) modification on rRNA in two life stages parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs all snoRNAs guiding Ψ helix 69 (H69) large subunit were lethal. A single knock-out a snoRNA Ψ530 H69 altered composition 80S monosome. These specifically affected translation only subset proteins. This study correlates site with ribosomal protein stoichiometry, supported by high-resolution cryo-EM structure. We propose alteration modifications could generate ribosomes preferentially translating state-beneficial

Language: Английский

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Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

PLoS Pathogens, Journal Year: 2020, Volume and Issue: 16(10), P. e1008784 - e1008784

Published: Oct. 27, 2020

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur Leishmania parasite differentiates between its main morphological forms, promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in mexicana genome but, currently, little is known about role of E1, enzymes this parasite. Bar-seq analysis 23 HECT/RBR null mutants generated promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes to amastigote differentiation mammalian infection. The E2s UBC1/CDC34, UBC2 UEV1 HECT HECT2 required successful transformation from UBA1b, UBC9, UBC14, HECT7 HECT11 normal proliferation during mouse Of all enzyme examined screen, Δ ubc2 uev1 exhibited most extreme differentiation. Null could not UBA1a or UBC3, UBC7, UBC12 UBC13, suggesting these essential promastigotes. X-ray crystal structure UEV1, orthologues human UBE2N UBE2V1/UBE2V2 respectively, reveal a heterodimer with highly conserved interface. Furthermore, recombinant L . load onto UBC2, allowing UBC2-UEV1 form K63-linked di-ubiquitin chains vitro Notably, cooperate E3s RNF8 BIRC2 non-K63-linked polyubiquitin chains, showing facilitate independent but association inhibits ability. Our study demonstrates dual essentiality intracellular survival shows interaction two proteins crucial regulation their activity function.

Language: Английский

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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

Published: Feb. 11, 2025

The ability to analyse 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 one 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 L. mexicana , demonstrating significant improvement over previous method. Lastly, show utility co-expressing ultra, hybrid CRISPR gene replacement within same cell line.Overall, improvements will broaden range possible applications enable variety loss-of-function screens near future.

Language: Английский

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