Biology subjects

Wellems, T.

Publications and source records attributed to Wellems, T..

5 recordsLinked to original sources

bioRxiv2024

Progressive heterogeneity of enlarged and irregularly shaped apicoplasts in P. falciparum persister blood stages after drug treatment

Morphological modifications and shifts in organelle relationships are hallmarks of dormancy in eukaryotic cells. Communications between altered mitochondria and nuclei are associated with metabolic quiescence of cancer cells that can survive chemotherapy. In plants, changes in the pathways between nuclei, mitochondria, and chloroplasts are associated with cold stress and bud dormancy. Plasmodium falciparum parasites, the deadliest agent of malaria in humans, contain a chloroplast-like organelle (apicoplast) derived from an ancient photosynthetic symbiont. Antimalarial treatments can fail because a small fraction of the blood stage parasites enter dormancy and recrudesce after drug exposure. Altered mitochondrial-nuclear interactions in these persisters have been described for P. falciparum, but interactions of the apicoplast remained to be characterized. In the present study, we examined the apicoplasts of persisters obtained after exposure to dihydroartemisinin (a first-line antimalarial drug) followed by sorbitol treatment, or after exposure to sorbitol treatment alone. As previously observed, the mitochondrion of persisters was consistently enlarged and in close association with the nucleus. In contrast, the apicoplast varied from compact and oblate, like those of active ring stage parasites, to enlarged and irregularly shaped. Enlarged apicoplasts became more prevalent later in dormancy, but regular size apicoplasts subsequently predominated in actively replicating recrudescent parasites. All three organelles, nucleus, mitochondrion, and apicoplast, became closer during dormancy. Understanding their relationships in erythrocytic-stage persisters may lead to new strategies to prevent recrudescences and protect the future of malaria chemotherapy. Significance StatementDormancy of blood-stage malaria parasites (as persister forms) frequently undermines treatment and may facilitate the evolution of drug resistance. Here, we examine changes that occur in dormancy with two P. falciparum organelles relative to the nucleus: the mitochondrion and the plastid-like apicoplast. As previously reported, the mitochondrion of persisters is consistently enlarged, irregularly shaped, and shifted into close apposition with the nucleus. However, apicoplasts exhibit a greater variety of shapes, volumes, and relative positioning during dormancy: some persisters maintain a regular appearing apicoplast, while others show dramatically altered apicoplasts, reminiscent of the chloroplast swelling and degradation that occurs with death from reactive oxygen species in various plant cells. Improved understanding of these processes will support new approaches in antimalarial chemotherapy.

microbiology↗

bioRxiv2022

Long read single cell RNA sequencing reveals the isoform diversity of Plasmodium vivax transcripts

Plasmodium infections often consist of heterogenous populations of parasites at different developmental stages and with distinct transcriptional profiles, which complicates gene expression analyses. The advent of single cell RNA sequencing (scRNA-seq) enabled disentangling this complexity and has provided robust and stage-specific characterization of Plasmodium gene expression. However, scRNA-seq information is typically derived from the end of each mRNA molecule (usually the 3-end) and therefore fails to capture the diversity in transcript isoforms documented in bulk RNA-seq data. Here, we describe the sequencing of scRNA-seq libraries using Pacific Biosciences (PacBio) chemistry to characterize full-length Plasmodium vivax transcripts from single cell parasites. Our results show that many P. vivax genes are transcribed into multiple isoforms, primarily through variations in untranslated region (UTR) length or splicing, and that the expression of these isoforms is often developmentally regulated. Our findings demonstrate that long read sequencing can be used to characterize mRNA molecules at the single cell level and provides an additional resource to better understand the regulation of gene expression throughout the Plasmodium life cycle. Author SummarySingle cell RNA-sequencing is a valuable tool for identifying cell specific differences in heterogenous populations. However, scRNA-seq has limitations in assigning reads to genes of organisims with poorly annotated UTRs, due to the poly-A caputre utilized by some scRNA-seq technologies, this technical limitation also makes identifying transcript specific differences, like alternative splicing, difficult. Despite its importance in human disease the P. vivax genome annotation is still relatively sparce, especially in the UTRs, and very little is known about transcript differece in the different life stages of the parasite life cycle. Here, we utilize a modified version of 10X scRNA-seq technology to capture full length transcripts via PacBio sequencing from both sporozoite and blood stages of P. vivax. This allowed us to predict full length stage specific transcripts for P. vivax as well as identify important variation in the previously poorly annotated UTRs. These findings will aide in futhering our understanding of P. vivax transcript regulation across the life cycle stages.

genomics↗

bioRxiv2021

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle

BackgroundPlasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. MethodsIn vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated P. vivax or Plasmodium falciparum 5 UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. ResultsLuciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, antimalarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. ConclusionAll three P. vivax promoters tested in this study functioned in P. knowlesi whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new antimalarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

microbiology↗

bioRxiv2020

Modulation of triple artemisinin-based combination therapy pharmacodynamics by Plasmodium falciparum genotype

The first-line treatments for uncomplicated Plasmodium falciparum malaria are artemisinin-based combination therapies (ACTs), consisting of an artemisinin derivative combined with a longer acting partner drug. However, the spread of P. falciparum with decreased susceptibility to artemisinin and partner drugs presents a significant challenge to malaria control efforts. To stem the spread of drug resistant parasites, novel chemotherapeutic strategies are being evaluated, including the implementation of triple artemisinin-based combination therapies (TACTs). Currently, there is limited knowledge on the pharmacodynamics and pharmacogenetic interactions of proposed TACT drug combinations. To evaluate these interactions, we established an in vitro high-throughput process for measuring the drug dose-response to three distinct antimalarial drugs present in a TACT. Sixteen different TACT combinations were screened against fifteen parasite lines from Cambodia, with a focus on parasites with differential susceptibilities to piperaquine and artemisinins. Analysis revealed drug-drug interactions unique to specific genetic backgrounds, including antagonism between piperaquine and pyronaridine associated with gene amplification of plasmepsin II/III, two aspartic proteases that localize to the parasite digestive vacuole. From this initial study, we identified parasite genotypes with decreased susceptibility to specific TACTs, as well as potential TACTs that display antagonism in a genotype-dependent manner. Our assay and analysis platform can be further leveraged to inform drug implementation decisions and evaluate next-generation TACTs.One Sentence Summary In vitro process to evaluate triple-drug combinations for prioritizing antimalarial combinations for in vivo evaluation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2020

Isomerization of antimalarial drug WR99210 explains its inactivity in a commercial stock

ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing human dhfr have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of 1 and 2; however, the compounds displayed differences by thin layer chromatography, reverse phase high performance liquid chromatography, and ultraviolet-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed 1 as WR99210 and 2 as an isomeric dihydrotriazine. Induced fit, computational docking models showed that 1 binds tightly and specifically in the P. falciparum DHFR active site whereas 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of isomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that can promote isomerization. Absorption spectroscopy may serve for assays of the unrearranged and rearranged triazines.Competing Interest StatementOne author, Gavin Heffernan, is an employee of Jacobus Pharmaceutical Company, Inc. The rest of the authors have no competing interest.View Full Text

biochemistry↗