Phru, C. S. | Search bioRxiv

bioRxiv2026

Experimental evolution and functional genetics identify KIC1 as a determinant of reduced artemisinin susceptibility in Bangladeshi Plasmodium falciparum

Bangladesh has greatly reduced malaria transmission, but persistent Plasmodium falciparum transmission in the Chittagong Hill Tracts (CHT), bordering Myanmar, remains a concern for elimination. Emerging artemisinin resistance could threaten remaining control efforts, making it important to identify determinants of reduced artemisinin susceptibility in CHT parasites. We combined long-term in vitro dihydroartemisinin (DHA) selection, whole-genome sequencing, functional genetics, and analysis of contemporaneous clinical isolates to define artemisinin-response variation in CHT P. falciparum. Starting with a 2018 patient-isolated, artemisinin-sensitive CHT clone, three independent cultures were exposed over 18 months to 27 cycles of stepwise DHA pressure up to 1600 nM. This generated CHT-R lines with elevated RSA survival relative to parental/sibling controls (4.0-6.5% versus <1%; 7-11-fold increase) and faster post-DHA recovery than CHT-S-sib (5.2 {+/-} 0.3 versus 10.1 {+/-} 0.57 days). Whole-genome sequencing identified convergent disruption of PF3D7_0606000, encoding KIC1 (Kelch13 Interacting Candidate1), a protein linked to the Kelch13-associated endocytic compartment; no pfkelch13 mutations emerged. Independent DHA-selected lines acquired distinct stop-gain or frameshift mutations predicted to truncate KIC1, and targeted pfkic1 disruption in parental CHT-S increased RSA survival and accelerated recovery, functionally validating KIC1 as a contributor to reduced artemisinin susceptibility. To link the in vitro selection findings to naturally circulating parasites, we tested whether pfkic1 variation was associated with artemisinin response in an independent set of CHT isolates. We integrated newly generated RSA measurements and whole-genome variation from lab adapted field isolates all carrying wild-type pfkelch13, with patient clearance values from our CHT artemether-lumefantrine efficacy study. Among 22 isolates, RSA survival ranged from 0.00 to 6.44%, with 12 exceeding the 1% in vitro ART-R threshold and was associated with PC50, the time required to clear 50% of the initial parasite density, but not parasite clearance half-life (PCt1/2). In this field-isolate dataset, targeted pfkic1 gene-score analysis and elastic-net modeling provided supportive evidence that natural variation at the pfkic1 locus is associated with PC50 and RSA survival; an exploratory genome-wide gene-score scan nominated additional candidate loci for future study. Together, these findings identify KIC1 as a functionally validated determinant of reduced artemisinin susceptibility in a Bangladeshi parasite background and suggest that artemisinin-response variation in CHT parasites may involve perturbation of the Kelch13-associated endocytic pathway beyond canonical pfkelch13 mutations.

microbiology↗

bioRxiv2025

Globally prevalent Kelch13 mutations increase partial artemisinin resistance and fitness in Bangladeshi Plasmodium falciparum parasites

Artemisinin partial resistance (ArtR), mediated by Kelch13 (K13) gene mutations in Plasmodium falciparum, has caused delayed parasite clearance and, together with partner drug resistance, treatment failures in the Greater Mekong Subregion (GMS). The Chittagong Hill Tracts (CHTs), located in southeastern Bangladesh and bordering Myanmar and India, regions with prevalent K13 mutations, account for approximately 90% of the countrys malaria infections but have not yet reported ArtR-causing K13 mutations. Importantly, however, some isolates from the CHTs have demonstrated moderate in vitro ArtR in the absence of K13 mutations. To assess the potential threat of K13-mediated ArtR in Bangladesh, we proactively evaluated the impact of three prevalent neighboring K13 mutations (F446I, R561H, and C580Y) on ArtR and parasite fitness in parasites isolated from the CHTs. We edited these mutations into two distinct genetic backgrounds: an artemisinin-sensitive strain (CHT-S) and a K13-independent, moderately resistant strain (CHT-R). In these edited lines, we then evaluated ArtR levels and fitness using ring-stage survival assays (RSA), post-drug treatment recovery assays, and competitive fitness assays against isogenic control strains. Prior to genome editing, both isolates were characterized for baseline drug susceptibility, resistance-associated mutations, and population structure. We found that C580Y and R561H mutations, but not F446I, confer increased in vitro ArtR in the CHT-R background already exhibiting K13-independent moderate resistance. All three mutations incurred minimal or no fitness costs in both genetic backgrounds. Notably, R561H, the dominant allele at the Thai-Myanmar border and currently expanding in Rwanda, mediates extreme resistance in the CHT-R background, with mean RSA survival rates of 30.9{+/-}1.9%, an unprecedented resistance level among K13-engineered lines. R561H also showed fitness advantages (0.5% per generation) and the highest growth recovery post-treatment. This represents the first experimental study modeling this K13 mediated ArtR risk in Bangladeshi parasites. In conclusion, we found that indigenous P. falciparum isolates in the CHTs possess inherent genetic potential to sustain high-level ArtR and fitness advantages if K13 mutations emerge, raising urgent concerns for containment and surveillance strategies during Bangladeshs malaria elimination phase.

microbiology↗