Guetens, P. | Search bioRxiv

bioRxiv2025

Elevated Plasmodium falciparum sexual conversion in HbAC and HbAS red blood cells without altered IgG/IgM levels to trophozoite and stage I gametocyte-specific antigens

Malaria transmission relies on the differentiation of asexual parasites into gametocytes, a process initiated by sexual conversion (SC). Mutations in the host hemoglobin beta (HBB) gene are known to influence parasite growth and disease outcome, yet their impact on SC remains unclear. We investigated the effect of HBB mutant genotypes on Plasmodium falciparum SC and humoral immunity in individuals from Nanoro, Burkina Faso. To measure SC rates in natural human malaria infections, we developed a new ex vivo SC assay (evSCA). We found that in human natural P. falciparum infections, SC rates were higher in individuals with HbAS or HbAC than in individuals with wild-type HBB (HbAA). Consistently, using an in vitro SC assay (ivSCA) based on the NF54-gexp02-Tom reporter line we found that cultures grown in HbAS red blood cells (RBCs) had higher SC rates than those grown in HbAA RBCs. Furthermore, IgG and IgM responses against trophozoite- and stage I gametocyte-infected RBC antigens, quantified by flow cytometry, did not differ between plasma from individuals with different HBB genotypes. These results demonstrate that exposure to RBCs with HBB mutations enhances SC, highlighting a host-genetic factor that may influence malaria transmission potential. AUTHOR SUMMARYTransmission of Plasmodium falciparum -a malaria causing parasite- depends on the parasites ability to produce gametocytes, the stage that infects mosquitoes. In many African regions where malaria is common, hemoglobin (Hb) variants such as hemoglobin S (the sickle-cell trait) and C are highly prevalent reflecting their role in protecting against severe disease. However, it remains unclear whether these variants also influence the parasites early shift toward gametocyte development (i.e., sexual conversion [SC]). In this study, we combined ex vivo and in vitro SC assays to measure SC in red blood cells with different hemoglobin genotypes. We found that SC was higher in parasites growing in red blood cells carrying hemoglobin AC and AS genotypes compared to normal AA hemoglobin. These findings provide experimental support for earlier epidemiological observations reporting that individuals with these hemoglobin variants often carry more gametocytes and are more infectious to mosquitoes. We also measured antibody levels in individuals with mutant RBCs and found no differences in immunity that could explain the variation in SC. Our results show that host genetic background can influence gametocyte development and may shape the infectious reservoir in high-burden settings. This knowledge is important for designing effective strategies for malaria elimination.

microbiology↗

bioRxiv2024

Novel single-cell preservation and RNA sequencing technology unlocks field studies for Plasmodium natural infections

Single-cell RNA sequencing (scRNA-seq) is a powerful technology used to investigate cellular heterogeneity. When applied to unicellular eukaryotes such as Plasmodium parasites, scRNA-seq provides a single-cell resolution particularly valuable to study complex infections which are often comprised of mixed life stages and clones. Until now, the application of scRNA-seq has been mainly limited to in vitro and animal malaria models, despite known transcriptional differences as compared to circulating parasite populations. This is primarily due to the challenges of working with Plasmodium natural infections in endemic settings. We validated sample preparation methods and a novel single-cell RNA sequencing technology for the first time in P. knowlesi parasites which can be effectively implemented to analyze natural infections in low-resource settings. We recovered 22,345 P. knowlesi single-cell transcriptomes containing all asexual blood stages from 6 in vitro culture samples, with conditions mimicking natural infections, and generated the most extensive P. knowlesi single-cell dataset to date. All 6 samples produced reproducible circular UMAP projections with consistent cluster localization and high gene expression correlation, regardless of the sample preparation methods used. Biomarker expression and life stage annotation using the Malaria Cell Atlas P. knowlesi reference dataset further confirmed these results. In conclusion, the combination of adaptable sample preparation methods with novel preservation and scRNA-seq technology has the potential to fundamentally transform the study of natural infections. This approach unlocks the use of scRNA-seq in field studies which will lead to new insights into Plasmodium parasite biology. ImportanceSequencing unicellular organisms, such as malaria parasites, at the single-cell level is important to understand the diversity present in cell populations. Until now, single-cell sequencing of malaria has been primarily limited to laboratory models. While these models are key to understanding biological processes, there are known differences between lab models and parasite populations circulating in natural human infections. This study presents sample preparation methods and a new single-cell RNA sequencing technology that enables sample collection from natural infections in low-resource settings. Using a mock natural infection, we validated this new single-cell RNA sequencing technology using marker genes with known expression patterns and a reference dataset from the Malaria Cell Atlas. We demonstrate that high-quality single-cell transcriptomes with consistent expression patterns can be recovered using various sample preparation methods, thereby unlocking single-cell sequencing for field studies and leading to additional insights into parasite biology in the future.

microbiology↗