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A CRISPR-Based Mutagenesis Strategy for Examining CLAG3 Helix 44 Contribution to Malaria Parasite Nutrient Uptake Channels

Year: 2026

Type: article

Abstract: Background: Malaria parasites import essential nutrients from plasma into their host erythrocytes through the plasmodial surface anion channel (PSAC), a conserved ion and nutrient channel on the infected cell surface. A parasite-encoded ternary complex consisting of CLAG3, RhopH2, and RhopH3 determines PSAC activity, but the precise contributions of each member to formation of the nutrient uptake pore remains uncertain. Methods: Here, we devised a two-step CRIPSR transfection strategy to examine an amphipathic CLAG3 helix, termed α-helix 44 (α-H44), as a candidate pore-lining domain. Results: A CLAG3 truncation protein without α-H44 phenocopies a CLAG3 knockout line, suggesting a critical role of α-H44 in formation of the nutrient channel; CLAG3 restoration using a recodonized α-H44 restores PSAC activity fully. A saturation mutagenesis library that splits the helix into four sequential segments was devised and implemented. Two engineered mutants exhibit distinct PSAC phenotypes; their cultures failed to expand in a modified medium that approximates in vivo nutrient availability. Conclusions: These studies support a α-H44 role in channel permeation and block by a strain-specific inhibitor. Our strategy will enable saturation mutagenesis to determine how PSAC achieves its unique ion and nutrient selectivity and should help guide drug discovery against this antimalarial target.

Source: Genes

DOI: 10.3390/genes17040462

Authors: Zabdi Gonzalez-Chavez, Mansoor A. Siddiqui, Sundar Ganesan, Sanjay A. Desai

Institutions: National Institute of Allergy and Infectious Diseases, National Institutes of Health

Language: English

Topic: Malaria Research and Control

Subfield: Public Health, Environmental and Occupational Health

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,137