Biology subjects

Shoue, D.

Publications and source records attributed to Shoue, D..

6 recordsLinked to original sources

bioRxiv2026

A Multidimensional Analysis of the Bimodal Piperaquine Response in Plasmodium falciparum

Malaria remains a pressing global health challenge, with the continued emergence of resistance threatening the long-term efficacy of artemisinin-based combination therapies (ACTs). Piperaquine (PPQ), an important partner drug in artemisinin-based combination therapies exhibits a unique bimodal dose-response phenotype associated with reduced susceptibility, yet the biological mechanism underlying this phenotype remains unknown. This phenotype is strongly associated with mutations in pfcrt and copy number amplification of plasmepsin II/III (pm II/III). Given that plasmepsins play a central role in hemoglobin degradation within the blood stage parasite digestive vacuole, and that PPQ accumulates within this compartment and perturbs heme detoxification, this phenotype likely reflects alterations in fundamental biological processes alongside drug-specific effects. We used isogenic PPQ-resistant parasite clones differing only in pm II/III copy number to integrate phenotypes with metabolic changes, and transcriptional responses to ascertain the impact of genotype combinations on parasite response to PPQ. Across increasing PPQ concentrations, parasites with elevated pm II/III copy number exhibited distinct metabolic responses compared to single-copy parasites, specifically, an altered abundance of peptides derived from hemoglobin degradation, directly implicating a core biological pathway long associated with plasmepsin function. The combination of metabolic and transcriptional data with phenotypic measurements supports a model in which increased plasmepsin expression enhances the parasites capacity to sustain hemoglobin digestion and associated metabolic activity under high PPQ concentrations. This points to a mechanistic basis for continued parasite survival, indicating that changes in hemoglobin processing within the digestive vacuole contribute to the bimodal response to PPQ. Molecular dynamics simulations further support a direct interaction between PPQ and PM II/III, as a mechanism by which these proteins impact PPQ response dynamics through both modulation of hemoglobin digestion and protein-drug interactions within the digestive vacuole.

molecular biology↗

bioRxiv2026

Functional validation of the Plasmodium falciparum K13 C580Y mutation in recently collected Ethiopian isolates

Recent genomic investigation in Ethiopia identified the first detection of the Plasmodium falciparum kelch13 (K13) C580Y substitution in the Horn of Africa. To assess its functional impact, we introduced C580Y into two recently collected Ethiopian clinical isolates using CRISPR-Cas9 genome editing. Ring-stage survival assays showed significantly elevated in vitro dihydroartemisinin (DHA) survival in edited parasites relative to isogenic controls, demonstrating that C580Y confers artemisinin tolerance in contemporary Ethiopian parasite genetic backgrounds.

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↗

bioRxiv2023

Using a P. falciparum genetic cross to dissect the relative contributions of pfcrt and plasmepsin II/III to piperaquine response-related traits

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance. ImportanceResistance to PPQ used in combination with DHA has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention, and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and have the distinct advantage of assaying the effects of naturally evolved genetic variation. Our study was significantly strengthened because the full a range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multi-gene model suggests an important role for both loci in the evolution of this ACT resistant parasite lineage.

genomics↗

bioRxiv2019

The extended recovery ring stage survival assay provides superior prediction of patient clearance half life and increases throughput

BackgroundTracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. MethodsP. falciparum cultures were synchronized with single layer Percoll during the schizont stage of the erythrocytic cycle. Cultures were left to reinvade to early ring-stage and parasitemia was quantified using flow cytometry. Cultures were diluted to 2% hematocrit and 0.5% parasitemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700nM of dihydroartemisinin or an equivalent amount of dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. ConclusionseRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

microbiology↗

bioRxiv2019

Phlebotomus papatasi sand fly salivary protein diversity and immune response potential in Egypt and Jordan populations

Phlebotomus papatasi sand flies inject their hosts with a myriad of pharmacologically active salivary proteins to assist with blood feeding and to modulate host defenses. These salivary proteins have been studied for their role in cutaneous leishmaniasis disease outcome with different salivary proteins attenuating or exacerbating lesion size. Studies have shown that while co-administered sand fly saliva exacerbates Leishmania major infections in naive mice, animals pre-exposed to saliva are protected, with the infection attenuated via a delayed-type hypersensitivity immune reaction. These studies highlight the potential of the salivary components to be used as a vaccine. One protein in particular, P. papatasi salivary protein 15 (PpSP15) has been intensively studied because of its ability to protect mice against Le. major challenge. The number of antigenic molecules included in vaccines is restricted thus emphasizing the role of population genetics to identify molecules, like PpSP15, that are functionally significant, conserved across populations and do not experience selection. Three distinct ecotope study sites, one in Egypt (Aswan) and two in Jordan (Swaimeh and Malka), were chosen based on their elevation, rainfall, vegetation, differing reservoir species, and the presence or absence of Le. major. The objective of this work was to analyze the genetic variability of nine of the most abundantly expressed salivary proteins including PpSP12, PpSP14, PpSP28, PpSP29, PpSP30, PpSP32, PpSP36, PpSP42, and PpSP44 and to predict their ability to elicit an immune response. Two proteins, PpSP12 and PpSP14, demonstrated low genetic variability across the three sand fly populations represented in this study, with multiple predicted MHCII epitope binding sites, identified by alleles present in the human populations from the study sites. The other seven salivary proteins revealed greater allelic variation across the same sand fly populations indicating that their use as vaccine targets may prove to be challenging.

genetics↗