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Generation and validation of a novel inducible overexpression library for genome-scale genetic screens in Leishmania

Award Number: R21AI166002
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $214,865 )
2024	2024	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Allergy and Infectious Diseases Research	001	2	2/15/2024	SUPPLEMENT FOR EXPANSION	$35,035
2024	2024	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Allergy and Infectious Diseases Research	000	2	2/2/2024	NON-COMPETING CONTINUATION	$179,830
														Subtotal = $214,865

Issue Date FY: 2023 ( Subtotal = $277,753 )
2023	2023	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Allergy and Infectious Diseases Research	002	1	8/17/2023	SUPPLEMENT FOR EXPANSION	$39,655
2023	2023	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Allergy and Infectious Diseases Research	001	1	8/16/2023	NEW	$0
2023	2023	OREGON HEALTH & SCIENCE UNIVERSITY	3181 SW SAM JACKSON PARK RD	PORTLAND	OR	97239	MULTNOMAH	USA	Allergy and Infectious Diseases Research	000	1	2/13/2023	NEW	$238,098
														Subtotal = $277,753

Grand Total All Awards = $492,618

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Generation and validation of a novel inducible overexpression library for genome-scale genetic screens in Leishmania

Award Number: R21AI166002

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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