Plasmodium falciparum vacuolar pyrophosphatase 1 for ring stage development and its transition to trophozoite

It is widely accepted that glycolysis alone is sufficient to support the energy demand of intraerythrocytic malaria parasites when they grow inside RBCs. However, here we show that the metabolic by-product pyrophosphate (PPi) is a critical energy source for ring stage development and the transition from the ring to trophozoite stage. During early phases of the asexual lifecycle, the parasite utilizes PfVP1 (Plasmodium falciparum vacuolar pyrophosphatase 1), an ancient PPi-driven proton pump, to pump protons across the parasite plasma membrane to maintain the membrane potential and cytosolic pH. Conditional deletion of PfVP1 leads to delayed ring stage development and a complete blockage of the ring to trophozoite transition, which can be partially rescued by Arabidopsis thaliana vacuolar pyrophosphatase 1, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae. Proton-pumping pyrophosphatases are absent in humans and animals, which highlights the possibility of developing highly selective VP1 inhibitors against the malaria parasite.

5WBF: A low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates

Background: Whole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programs. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7-8 μm and exhibit some deformability, we hypothesized that cheap and commercially available 5 μm filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 μm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS. Methods: Whole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitemia range: 0.04-5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitemia range: 0.02-1.1%). These whole blood samples (50 to 400 μL) were diluted in RPMI 1640 medium or PBS 1X buffer and filtered with syringes connected to a 5 μm commercial filter. DNA was extracted from filtered and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, we also applied to the same unprocessed whole blood samples the selective whole-genome amplification (sWGA) method which does not rely on blood filtration. Results: After applying 5WBF, qPCR indicated an average of 2-fold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4,096- to 65,536-fold loss of human template DNA (human β actin gene). WGS analyses revealed that > 95% of the nuclear genome and the entire whole organellar genomes were covered at ≥ 10x depth for all samples tested. In sWGA counterparts, none of the organellar genomes were covered, and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10x depth depending on parasitemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5,460 genes; mean coverage: 91x; median coverage: 93x; 5th percentile: 70x; 95th percentile: 103x), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as we observed a much more heterogeneous distribution of reads among gene sequences (mean coverage: 80x; median coverage: 51x; 5th percentile: 7x; 95th percentile: 245x. Conclusions: The novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized, 5 μm filters which cost from 1.0 to 1.7€ per unit, depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum DNA from minute amounts of whole blood even with parasitemias as low as 0.02%.

Label-free imaging and classification of live P. falciparum enables high performance parasitemia quantification without fixation or staining

Manual microscopic inspection of fixed and stained blood smears has remained the gold standard for Plasmodium parasitemia analysis for over a century. Unfortunately, smear preparation consumes time and reagents, while manual microscopy is skill-dependent and labor-intensive. Here, we demonstrate that deep learning enables both life stage classification and accurate parasitemia quantification of ordinary brightfield microscopy images of live, unstained red blood cells. We tested our method using both a standard light microscope equipped with visible and near-ultraviolet (UV) illumination, and a custom-built microscope employing deep-UV illumination. While using deep-UV light achieved an overall four-category classification of Plasmodium falciparum blood stages of greater than 99% and a recall of 89.8% for ring-stage parasites, imaging with near-UV light on a standard microscope resulted in 96.8% overall accuracy and over 90% recall for ring-stage parasites. Both imaging systems were tested extrinsically by parasitemia titration, revealing superior performance over manually-scored Giemsa-stained smears, and a limit of detection below 0.1%. Our results establish that label-free parasitemia analysis of live cells is possible in a biomedical laboratory setting without the need for complex optical instrumentation. We anticipate future extensions of this work could enable label-free clinical diagnostic measurements, one day eliminating the need for conventional blood smear analysis.

Plasmodium falciparum Acetyl-CoA Synthetase is essential for parasite intraerythrocytic development and chromatin modification

The malaria parasite Plasmodium falciparum possesses a unique Acetyl-CoA Synthetase (PfACS) which provides acetyl moieties for different metabolic and regulatory cellular pathways. We characterized PfACS and studied its role focusing on epigenetic modifications using the var gene family as reporter genes. For this, mutant lines to modulate plasmodial ACS expression by degron-mediated protein degradation or ribozyme induced transcript decay were created. Additionally, an ACS inhibitor was tested for its effectiveness and specificity in interfering with PfACS. The knockdown of PfACS or its inhibition led to impaired parasite growth. Decreased levels of PfACS also led to differential histone acetylation patterns, altered variant gene expression and concomitantly decreased cytoadherence of infected red blood cells containing knocked-down parasites. Further, ChIP analysis revealed the presence of PfACS in many loci in ring stage parasites, underscoring its involvement in the regulation of chromatin. Due to its significant differences to human ACS, PfACS seems an interesting target for drug development.

Delay in primordial germ cell migration in adamts9 knockout zebrafish

Adamts9 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 9) is one of a few metalloproteinases structurally conserved from C. elegans to humans and is indispensable in germ cell migration in invertebrates. However, adamts9′s roles in germ cell migration in vertebrates has not been examined. In the present study, we found zygotic expression of adamts9 started around the germ ring stage and reached peak levels at 3 days post fertilization (dpf) in zebrafish. The migration of primordial germ cells (PGC) was completed within 24 hours (h) in wildtype siblings, while a delay in PGC migration was found at 15 and 24-h post-fertilization (hpf) in the Adamts9 knockout (KO). However, the delayed PGC migration in Adamts9 KO disappeared at 48 hpf. Our study suggests a conserved function of Adamts9 in germ cell migration among invertebrates and vertebrates. In addition, our results also suggest that Adamts9 is not essential for germ cell migration as reported in C. elegans, possibly due to expansion of Adamts family members and compensatory roles from other metalloproteinases in vertebrates. Further studies are required in order to elucidate the functions and mechanisms of metalloproteinases in germ cell migration and gonad formation in vertebrates.

Development and validation of an in silico decision-tool to guide optimisation of intravenous artesunate dosing regimens for severe falciparum malaria patients

Most deaths from severe falciparum malaria occur within 24 hours of presentation to hospital. Intravenous (i.v.) artesunate is the first-line treatment for severe falciparum malaria, but its efficacy may be compromised by delayed parasitological responses. In patients with severe malaria the life-saving benefit of the artemisinin derivatives is their ability to clear circulating parasites rapidly, before they can sequester and obstruct the microcirculation. To evaluate the dosing of i.v. artesunate for the treatment of artemisinin-sensitive and reduced ring stage sensitivity to artemisinin severe falciparum malaria infections Bayesian pharmacokinetic-pharmacodynamic modelling of data from 94 patients with severe malaria (80 children from Africa and 14 adults from Southeast Asia) was performed. Assuming delayed parasite clearance reflects a loss of ring stage sensitivity to artemisinin derivatives, the median (95% credible interval) percentage of patients clearing ≥99% parasites within 24 hours (PC24≥99%) for standard (2.4 mg/kg i.v. artesunate at 0 and 12 hours) and simplified (4 mg/kg i.v. artesunate at 0 hours) regimens were 65% (52.5%-74.5%) versus 44% (25%-61.5%) for adults, 62% (51.5%-74.5%) versus 39% (20.5%-58.5%) for larger children (≥20 kg) and 60% (48.5%-70%) versus 36% (20%-53.5%) for smaller children (<20 kg). The upper limit of the credible intervals for all regimens was below a PC24≥99% of 80%, a threshold achieved on average in clinical studies of severe falciparum malaria infections. Rapid clearance of parasites, where there is loss of ring stage sensitivity to artemisinin, in patients with severe falciparum malaria is compromised with the currently recommended and proposed simplified i.v. artesunate dosing regimens.

In vitro analyses of Artemisia extracts on Plasmodium falciparum suggest a complex antimalarial effect

Dried-leaf Artemisia annua L. (DLA) antimalarial therapy was shown effective in prior animal and human studies, but little is known about its mechanism of action. Here IC50s and ring-stage assays (RSAs) were used to compare extracts of A. annua (DLAe) to artemisinin (ART) and its derivatives in their ability to inhibit and kill Plasmodium falciparum strains 3D7, MRA1252, MRA1240, Cam3.11 and Cam3.11rev in vitro. Strains were sorbitol and Percoll synchronized to enrich for ring-stage parasites that were treated with hot water, methanol and dichloromethane extracts of DLA, artemisinin, CoArtem™, and dihydroartemisinin. Extracts of A. afra SEN were also tested. There was a correlation between ART concentration and inhibition of parasite growth. Although at 6 hr drug incubation, the RSAs for Cam3.11rev showed DLA and ART were less effective than high dose CoArtem™, 8 and 24 hr incubations yielded equivalent antiparasitic results. For Cam3.11, drug incubation time had no effect. DLAe was more effective on resistant MRA-1240 than on the sensitive MRA-1252 strain. Because results were not as robust as observed in animal and human studies, a host interaction was suspected, so sera collected from adult and pediatric Kenyan malaria patients was used in RSA inhibition experiments and compared to sera from adults naïve to the disease. The sera from both age groups of malaria patients inhibited parasite growth ≥ 70% after treatment with DLAe and compared to malaria naïve subjects suggesting some host interaction with DLA. The discrepancy between these data and in-vivo reports suggested that DLA’s effects require an interaction with the host to unlock their potential as an antimalarial therapy. Although we showed there are serum-based host effects that can kill up to 95% of parasites in vitro, it remains unclear how or if they play a role in vivo. These results further our understanding of how DLAe works against the malaria parasite in vitro.

Delay in Primordial Germ Cell Migration in Adamts9 Knockout Zebrafish

Adamts9 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 9) is one of few metalloproteinases structurally conserved from C. elegans to humans and is indispensable in germ cell migration in invertebrates. However, adamts9’s roles in germ cell migration in vertebrates has not been examined. In the present study, we found zygotic expression of adamts9 started around germ ring stage and reached peak levels at 3 days post fertilization (dpf) in zebrafish. Germ cell migration completed within 24 hours in wildtype sibling, while a delay in germ cell migration was found at 15 and 24-hours post-fertilization (hpf) in the Adamts9 knockout (KO). However, this delayed effect of Adamts9 KO disappeared at 48 hpf. Our study suggests a conserved function of Adamts9 in germ cell migration among invertebrates and vertebrates. In addition, our results also suggest that Adamts9 is not essential for germ cell migration as reported in C. elegans, possibly due to expansion of Adamts family members and compensatory roles from another metalloproteinase in vertebrates. Further studies are required in order to elucidate the functions and mechanisms of metalloproteinases in germ cell migration and gonad formation in vertebrates.

PfAP2-G2 Is Associated to Production and Maturation of Gametocytes in Plasmodium falciparum via Regulating the Expression of PfMDV-1

Gametocyte is the sole form of the Plasmodium falciparum which is transmissible to the mosquito vector. Here, we report that an Apicomplexan Apetala2 (ApiAP2) family transcription factor, PfAP2-G2 (Pf3D7_1408200), plays a role in the development of gametocytes in P. falciparum by regulating the expression of PfMDV-1 (Pf3D7_1216500). Reverse transcriptase-quantitative PCR (RT-qPCR) analysis showed that PfAP2-G2 was highly expressed in the ring stage. Indirect immunofluorescence assay showed nuclear localization of PfAP2-G2 in asexual stages. The knockout of PfAP2-G2 led to a ~95% decrease in the number of mature gametocytes with a more substantial influence on the production and maturation of the male gametocytes, resulting in a higher female/male gametocyte ratio. To test the mechanism of this phenotype, RNA-seq and RT-qPCR showed that disruption of PfAP2-G2 led to the down-regulation of male development gene-1 (PfMDV-1) in asexual stages. We further found that PfAP2-G2 was enriched at the transcriptional start site (TSS) of PfMDV-1 by chromatin immunoprecipitation and qPCR assay in both ring stage and schizont stage, which demonstrated that PfMDV-1 is one of the targets of PfAP2-G2. In addition, RT-qPCR also showed that PfAP2-G (Pf3D7_1222600), the master regulator for sexual commitment, was also down-regulated in the PfAP2-G2 knockout parasites in the schizont stage, but no change in the ring stage. This phenomenon suggested that PfAP2-G2 played a role at the asexual stage for the development of parasite gametocytes and warrants further investigations in regulatory pathways of PfAP2-G2.