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Principal Investigator: PHILLIP A YATES
Organization: OREGON HEALTH & SCIENCE UNIVERSITY
Fiscal Year: 2024
Award: $199,812
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as
one million people per year. At least 20,000 are killed each year by the deadly visceral form of the disease,
which is caused by L. donovani. Genetic tools for understanding important questions in Leishmania biology are
currently quite limited. Most Leishmania species lack the capacity for RNA interference (RNAi), which
precludes the implementation of genome-scale RNAi library screens like those that have revolutionized
molecular genetics in the related kinetoplastid T. brucei. As is the case for knocking down protein expression
via RNAi, expressing proteins at higher levels than normal (i.e., overexpression) can confer resistance to drugs
or otherwise enhance survival (Gain-of-Fitness = GnFt), or cause deleterious effects on cell fitness (Loss-of-
Fitness = LsFt) that can reveal important pathways and protein functions. Genome-scale overexpression
libraries have proven valuable for a wide range of genetic screens in multiple species, including T. brucei. The
goal of this proposal is to generate and validate a novel, genome-scale, inducible overexpression library in
Leishmania that will serve as a versatile and broadly applicable new genetic tool for the field.
We propose to generate a library encoding the majority of L. donovani proteins (~7500 Open Reading
Frames, or LdORFs). PCR amplified LdORFs will be directionally cloned into Gateway Entry vectors to
facilitate transfer of the library to any Gateway compatible vector (Aim 1), resulting in a LdORFeome plasmid
library. The LdORFeome will be transferred into novel inducible Leishmania expression vectors developed in
our lab (Aim 1). The resulting library will be stably transfected into Leishmania to generate an inducible
Leishmania donovani ORFeome overexpression library (the LdOX library). Because the overall goal is to
make the LdOX library available as a resource for the community, we will validate the library by performing
proof-of-principle overexpression screens to identify drug resistance genes (GnFt) and pathways required for
normal growth (LsFt) (Aim2).
The LdOX library will provide an innovative and much-needed new tool for high throughput genetic
screens in Leishmania. We anticipate that screens with the LdOX library will have a sustained impact on the
field by revealing mechanisms of drug action and resistance, and uncovering proteins and pathways required
for surviving any growth condition or lifecycle stage.
Terms: <Acceleration><American trypanosome><Bar Codes><Biology><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Communities><Complex><DFMO><DL-alpha-Difluoromethylornithine><DL-α-Difluoromethylornithine><Defect><Disease><Disorder><Drug Targeting><Drug Therapy><Drug resistance><Ensure><Expression Library><Gene Action Regulation><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Generalized Growth><Generations><Genes><Genetic><Genetic Screening><Genetic Technics><Genetic Techniques><Genome Library><Genomic Library><Glean><Goals><Growth><Hyperactivity><Incidence><Individual><Intracellular Communication and Signaling><Kala-Azar><L donovani><L-Ornithine carboxy-lyase><L. donovani><Laboratories><Leishmania><Leishmania (Leishmania) donovani><Leishmania donovani><Libraries><Life Cycle><Life Cycle Stages><Melarsoprol><Methods><Molecular Genetics><ORFs><Open Reading Frames><Ornithine Carboxy-lyase><Ornithine Decarboxylase><Ornithine Decarboxylase Inhibitor><Parasites><Pathway interactions><Persons><Pharmacotherapy><Phenotype><Plasmids><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Protein Coding Region><Proteins><RNA Interference><RNA Silencing><RNA interference screen><RNAi><RNAi screen><RNAi-based screen><Regulatory Protein><Reproducibility><Research Resources><Resistance><Resources><Ribosomal RNA><Sequence-Specific Posttranscriptional Gene Silencing><Signal Transduction><Signal Transduction Systems><Signaling><Stress><System><T brucei><T cruzi><T. brucei><T. cruzi><Time><Tissue Growth><Transfection><Trypanosoma brucei><Trypanosoma brucei brucei><Trypanosoma cruzi><Validation><Visceral><Visceral Leishmaniasis><Yeasts><barcode><biological signal transduction><difluoromethylornithine><dosage><drug action><drug resistant><drug treatment><experience><experiment><experimental research><experimental study><experiments><expression vector><fitness><genetic regulatory protein><genome scale><genome-wide><genomewide><human disease><inducible expression><inducible gene expression><innovate><innovation><innovative><knock-down><knockdown><life course><loss of function><neglected tropical diseases><new drug target><new drug treatments><new druggable target><new drugs><new pharmacological therapeutic><new pharmacotherapy target><new therapeutic target><new therapeutics><new therapy><new therapy target><next generation therapeutics><novel><novel drug target><novel drug treatments><novel druggable target><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel pharmacotherapy target><novel therapeutic target><novel therapeutics><novel therapy><novel therapy target><ontogeny><overexpress><overexpression><pathway><protein expression><protein function><rRNA><regulatory gene product><resistance gene><resistance locus><resistance mechanism><resistance to Drug><resistant><resistant gene><resistant mechanism><resistant to Drug><tool><validations><vector>