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Combining Liver Stage Culture System with Backcross Genetics to Discover Antimalarial Drug Resistance Loci

Award Number: R21AI144591
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/ABSTRACT Major advances in understanding the molecular basis of antimalarial drug resistance have been achieved by using the power of genetics. Since sexual combination occurs in the mosquito vector, gametocytes of cloned parent lines with different genetic backgrounds and drug resistance phenotypes can be mixed, fed to mosquitos, and the resulting haploid progeny in erythrocytes can be cloned and used to identify genetic loci associated with the desired phenotype. Genetic crosses with Plasmodium falciparum have been limited due to limitations of using non-human primates and or costs of mice engrafted with human hepatocytes. Herein we propose a novel approach of using a highly efficient, facile in vitro culture system to produce liver stage P. falciparum for genetic crosses. Backcross breeding is a forward genetics tool that can be used to introduce a specific genetic trait from one line into a second line. Most often this has been used in agriculture to introduce a desired trait (e.g., disease resistance) into an elite breeding line. Herein we propose the first phenotype-assisted backcross with P. falciparum to identify genetic loci that either confer or enhance K13 mediated resistance to artemisinin. In Aim 1 we will conduct backcross experiments with drug susceptible (Nf54) and an artemisinin-resistant clone of P. falciparum that possesses K13 E252Q mutation and a resistant phenotype in the ring stage survival assay (RSA). Gametocytes of the parent lines will be mixed, fed to mosquitos, and sporozoites used to inoculate 384 well cultures of human hepatocytes. F1 progeny will be collected and subjected to drug selection in a modified RSA to enrich for resistant progeny and then backcrossed with the drug susceptible parent line (Nf54). We will repeat this phenotype-enhanced backcross three more times and clone progeny from backcross generation 4 (BC4) for deep sequencing. In Aim 2 we will conduct phenotype and genotype analysis of cloned BC4 progeny. Artemisinin resistance phenotypes will be assessed by using a plate-based ring stage survival assay (RSA) in cloned progeny from BC4. QTL analysis will be performed to identify loci associated with artemisinin resistance and the role of K13 E252Q mutation will be assessed. The results from this study could significantly enrich the toolbox for experimental genetics of the human malaria parasite and uncover essential background mutations that either confer or enhance E252Q K13 mutations in artemisinin resistance.


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: 2023 ( Subtotal = -$54 )
2023	2020	UNIVERSITY OF GEORGIA RESEARCH FOUNDATION, INC.	310 E CAMPUS RD RM 409	ATHENS	GA	30602	CLARKE	USA	Allergy and Infectious Diseases Research	000	2	9/14/2023	NON-COMPETING CONTINUATION	-$54
														Subtotal = -$54

Issue Date FY: 2022 ( Subtotal = $0 )
2022	2020	UNIVERSITY OF GEORGIA RESEARCH FOUNDATION, INC.	310 EAST CAMPUS RD TUCKER HALL ROOM 409	ATHENS	GA	30602	CLARKE	USA	Allergy and Infectious Diseases Research	000	2	3/4/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2020 ( Subtotal = $188,750 )
2020	2020	UNIVERSITY OF GEORGIA RESEARCH FOUNDATION, INC.	310 EAST CAMPUS RD TUCKER HALL ROOM 409	ATHENS	GA	30602	CLARKE	USA	Allergy and Infectious Diseases Research	000	2	2/18/2020	NON-COMPETING CONTINUATION	$188,750
														Subtotal = $188,750

Issue Date FY: 2019 ( Subtotal = $226,000 )
2019	2019	UNIVERSITY OF GEORGIA RESEARCH FOUNDATION, INC.	310 EAST CAMPUS RD TUCKER HALL ROOM 409	ATHENS	GA	30602	CLARKE	USA	Allergy and Infectious Diseases Research	000	1	3/11/2019	NEW	$226,000
														Subtotal = $226,000

Grand Total All Awards = $414,696

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Combining Liver Stage Culture System with Backcross Genetics to Discover Antimalarial Drug Resistance Loci

Award Number: R21AI144591

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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