Malaria Drug Resistance and Disease Virulence

National Institute Of Allergy And Infectious Diseases

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Abstract

FY2025 Research Project Accomplishments: 1. Progressive heterogeneity of enlarged and irregularly shaped apicoplasts in Plasmodium falciparum persister blood stages after drug treatment In this study, we investigated the morphological and spatial dynamics of the apicoplast, an essential chloroplast-like organelle of malaria parasites, in relation to features of the mitochondrion and nucleus in actively replicating and dormant (“persister”) forms of Plasmodium falciparum in human erythrocytes. Our research aimed to characterize cellular adaptations involved in parasite dormancy and recrudescence following antimalarial drug exposure. Using super-resolution Airyscan microscopy and highly synchronized cultures of parasites expressing a green fluorescent protein-labelled apicoplast, we quantitatively compared organelle volumes and distances in parasites treated with dihydroartemisinin followed by sorbitol (DHA/sorbitol), sorbitol alone, or the drug vehicle (dimethyl sulfoxide) as a control. Three-dimensional analyses revealed that mitochondria from both DHA/sorbitol- and sorbitol-induced persister cells were consistently enlarged and closely associated with the nucleus, whereas apicoplasts displayed progressive heterogeneity over time: some persisters maintained compact, oblate apicoplasts typical of ring stages, while others developed swollen, irregularly shaped apicoplasts that became more prevalent as dormancy advanced. Upon recrudescence of actively growing parasites, all organelles reverted to their normal morphology and organization. Analogies to stress responses in plants and other eukaryotes suggest that altered organelle morphologies and increased proximity to the nucleus may facilitate survival signaling and stress adaptation during dormancy. The swelling and aberrant morphology of the apicoplast – reminiscent of chloroplast autophagy (‘chlorophagy’) in plants – are consistent with organelle deterioration and reduced viability in persister forms. The established localization and function of autophagy-related protein 8 (ATG8) on the apicoplast, and the known role of this protein in organelle maintenance and stress responses in other cellular systems, support the possibility that ATG-dependent autophagy may participate in apicoplast remodeling and determine parasite viability during dormancy and recrudescence – a hypothesis that requires future investigation. Our findings underscore the remarkable adaptability of malaria parasite organelles under drug stress and suggest that understanding the molecular mechanisms underlying these adaptations may identify new targets to prevent parasite recrudescence and improve antimalarial treatment efficacy. 2. Isolation and characterization of Plasmodium falciparum blood-stage persisters by improved selection protocols using dihydroartemisinin alone This work developed improved protocols to efficiently isolate and characterize dormant forms (“persister forms”) of the Plasmodium falciparum malaria parasites after their exposure to artemisinin-based drugs. By treating mixed-stage parasite cultures with three or four consecutive daily 6-hour pulses of dihydroartemisinin at clinically relevant concentrations, we reliably induced the formation of persister populations, eliminating the need for any complex synchronization or purification steps. Microscopy imaging, flow cytometry, and fluorescence-activated cell sorting enabled reliable enrichment and objective characterization of persisters, which were found to display distinct morphological types. Recrudescence of actively replicating populations to 2% parasitemia was typically observed 17–22 days after drug exposure. Quantitative RT-PCR analysis of purified RNA showed dynamic changes in the abundance of gene transcripts associated with fatty acid synthesis (acetyl-CoA carboxylase, acc) and protein export (skeleton binding protein 1, sbp1) throughout dormancy and reactivation, consistent with the transitions of metabolic quiescence that support persister survival. Evidence for autophagy-like processes, including observation of cells with disorganized structures and “faded” cytoplasm, suggests that autophagy may support cellular remodeling and provide metabolic energy for processes of dormancy, possibly including the formation of persisters and their later reactivation to cause recrudescent infections. These protocols and findings provide a robust foundation for dissecting dormancy pathways and may accelerate the discovery of targeted therapeutic strategies designed to eliminate persisters and prevent malaria relapse. 3. Epigenetically conferred ring-stage survival in Plasmodium falciparum against artemisinin treatment These studies investigated the epigenetic mechanisms underlying artemisinin resistance in the malaria parasite Plasmodium falciparum, with a focus on how certain parasites can survive and later recrudesce after short exposures to artemisinin and related drugs. The experiments systematically evaluated all putative histone acetyltransferase (HAT) genes in a large set of Plasmodium falciparum field and subcloned isolates to identify epigenetic regulators of the ring-stage survival (RS) phenotype—a key feature classified as partial artemisinin resistance. Using methods including CRISPR-Cas9 genetic modification, ribozyme-based gene knockdowns, chemical inhibition, single-cell RNA sequencing, and chromatin immunoprecipitation (ChIP) sequencing, we found that only the PfMYST HAT consistently correlated with RS rates, regardless of mutations in the Kelch domain protein PfK13. Genetic knockdown investigations demonstrated that reducing PfMYST expression led to increased ring-stage survival after artemisinin treatment, altered cell cycle progression, and produced widespread changes in parasite metabolism. Metabolomic profiling revealed that PfMYST knockdown and PfK13 mutation both lead to energy metabolic disruption, but through partially distinct pathways. Further, in studies of recrudescence from dormant parasites induced by dihydroartemisinin (DHA), we found that PfMYST knockdown parasites recrudesced earlier after DHA exposure than control parasites, indicating that epigenetic modulations by PfMYST can speed recovery from dormancy. Functional transfection experiments identified PfMYST-regulated genes that may contribute to RS. Multi-omics analysis suggested significant interplay between genetic (PfK13) and epigenetic (PfMYST) mechanisms in the RS artemisinin response. Single-cell transcriptomics further identified clusters of surviving cells and gene expression programs that may underlie PfMYST-mediated drug tolerance. Finally, PfMYST-mediated regulation also affected the survival of parasites exposed to certain other antimalarial drugs. Taken together, our findings suggest that epigenetic modulation, particularly via PfMYST-mediated histone acetylation, plays an important role in how Plasmodium falciparum responds to artemisinin exposure and that drug resistance can involve complex interactions between genetic and epigenetic mechanisms. 4. Single-cell analyses of polyclonal Plasmodium vivax infections and their consequences for parasite transmission In these investigations on factors that determine malaria virulence and transmission, we systematically examined the biology of infections that contain genetically distinct parasites (polyclonal infections) of Plasmodium vivax, a species that cannot be reliably cultivated in vitro. Using Saimiri monkeys as a malaria model, we investigated infections with two strains of Plasmodium vivax and analyzed more than 80,000 individual parasites using single-cell RNA sequencing (scRNA-seq). The experimental design enabled rigorous comparisons among mono-infections, consecutive infections, and simultaneous coinfections, including assessments of parasite transmission through mosquitoes and the subsequent establishment of blood infections after sporozoite inoculation. Findings showed that consecutive infections of different strains did not establish robust polyclonal infections, whereas simultaneous inoculation of the strains led to balanced polyclonality without significant changes in parasite gene regulation or sexual commitment. All parasite genotypes were transmitted to mosquitoes but, after inoculations of the monkeys with sporozoites from mosquitoes, only a subset of the inoculated parasites successfully progressed through the liver stage (an early developmental phase that must occur before parasites can invade red blood cells) to produce blood infections, thereby demonstrating a pronounced bottleneck during pre-erythrocytic (liver-stage) development. These findings underscore the importance of non-human primate models and single-cell transcriptomic approaches to elucidate the genetic and biological determinants of polyclonality and its consequences for malaria infection dynamics. 5. Pf8: an open dataset of Plasmodium falciparum genome variation in 33,325 worldwide samples This work provides the largest openly available dataset of genome variation in any eukaryotic species, comprising curated genomic data from 33,325 Plasmodium falciparum samples collected over 50 years from 34 countries and 122 locations. The dataset includes open access to raw sequencing data and genotypes at over 12 million genomic positions, as well as novel findings on copy number variations (CNVs) in key drug resistance genes (Pfcrt, Pfmdr1, PfPM2, PfPM3, and Pfgch1) and deletions of Pfhrp2 and Pfhrp3 which can cause failures of rapid diagnostic tests. Data were generated via high-throughput whole genome sequencing on Illumina platforms and were analyzed using advanced bioinformatics pipelines for variant calling and annotation. The analyses demonstrate geographic population structure, longitudinal trends in drug resistance including ongoing declines in chloroquine resistance in several African countries, and patterns of genetic diversity and CNV linked to selective pressures from treatment policies. Pf8 is accessible through interactive web applications, open analysis notebooks, and an updated Python software package to enable and support collaborative research in malaria genomic epidemiology and pathogen evolution. (A report on Pf8 is submitted to the Wellcome Open Research publishing platform, DOI 10.12688/wellcomeopenres.24031.1)