The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Preliminary characterization of Plasmodium vivax sporozoite antigens as pre-erythrocytic vaccine candidates.

Plasmodium vivax pre-erythrocytic (PE) vaccine research has lagged far behind efforts to develop Plasmodium falciparum vaccines. There is a critical gap in our knowledge of PE antigen targets that can induce functionally inhibitory neutralizing antibody responses. To overcome this gap and guide the selection of potential PE vaccine candidates, we considered key characteristics such as surface exposure, essentiality to infectivity and liver stage development, expression as recombinant proteins, and functional immunogenicity. Selected P. vivax sporozoite antigens were surface sporozoite protein 3 (SSP3), sporozoite microneme protein essential for cell traversal (SPECT1), sporozoite surface protein essential for liver-stage development (SPELD), and M2 domain of MAEBL. Sequence analysis revealed little variation occurred in putative B-cell and T-cell epitopes of the PE candidates. Each antigen was tested for expression as refolded recombinant proteins using an established bacterial expression platform and only SPELD failed. The successfully expressed antigens were immunogenic in vaccinated laboratory mice and were positively reactive with serum antibodies of P. vivax-exposed residents living in an endemic region in Thailand. Vaccine immune antisera were tested for reactivity to native sporozoite proteins and for their potential vaccine efficacy using an in vitro inhibition of liver stage development assay in primary human hepatocytes quantified on day 6 post-infection by high content imaging analysis. The anti-PE sera produced significant inhibition of P. vivax sporozoite invasion and liver stage development. This report provides an initial characterization of potential new PE candidates for a future P. vivax vaccine.

Population genomics in neglected malaria parasites.

Malaria elimination includes neglected human malaria parasites Plasmodium vivax, Plasmodium ovale spp., and Plasmodium malariae. Biological features such as association with low-density infection and the formation of hypnozoites responsible for relapse make their elimination challenging. Studies on these parasites rely primarily on clinical samples due to the lack of long-term culture techniques. With improved methods to enrich parasite DNA from clinical samples, whole-genome sequencing of the neglected malaria parasites has gained increasing popularity. Population genomics of more than 2200 P. vivax global isolates has improved our knowledge of parasite biology and host-parasite interactions, identified vaccine targets and potential drug resistance markers, and provided a new way to track parasite migration and introduction and monitor the evolutionary response of local populations to elimination efforts. Here, we review advances in population genomics for neglected malaria parasites, discuss how the rich genomic information is being used to understand parasite biology and epidemiology, and explore opportunities for the applications of malaria genomic data in malaria elimination practice.

New Plasmodium vivax Genomes From the China-Myanmar Border.

Plasmodium vivax is increasingly the dominant species of malaria in the Greater Mekong Subregion (GMS), which is pursuing regional malaria elimination. P. vivax lineages in the GMS are poorly characterized. Currently, P. vivax reference genomes are scarce due to difficulties in culturing the parasite and lack of high-quality samples. In addition, P. vivax is incredibly diverse, necessitating the procurement of reference genomes from different geographical regions. Here we present four new P. vivax draft genomes assembled de novo from clinical samples collected in the China-Myanmar border area. We demonstrate comparable length and content to existing genomes, with the majority of structural variation occurring around subtelomeric regions and exported proteins, which we corroborated with detection of copy number variations in these regions. We predicted peptides from all PIR gene subfamilies, except for PIR D. We confirmed that proteins classically labeled as PIR D family members are not identifiable by PIR motifs, and actually bear stronger resemblance to DUF (domain of unknown function) family DUF3671, potentially pointing to a new, closely related gene family. Further, phylogenetic analyses of MSP7 genes showed high variability within the MSP7-B family compared to MSP7-A and -C families, and the result was comparable to that from whole genome analyses. The new genome assemblies serve as a resource for studying P. vivax within the GMS.

Population genomics identifies a distinct Plasmodium vivax population on the China-Myanmar border of Southeast Asia.

Plasmodium vivax has become the predominant malaria parasite and a major challenge for malaria elimination in the Greater Mekong Subregion (GMS). Yet, our knowledge about the evolution of P. vivax populations in the GMS is fragmental. We performed whole genome sequencing on 23 P. vivax samples from the China-Myanmar border (CMB) and used 21 high-coverage samples to compare to over 200 samples from the rest of the GMS. Using genome-wide single nucleotide polymorphisms (SNPs), we analyzed population differentiation, genetic structure, migration and potential selection using an array of methods. The CMB parasites displayed a higher proportion of monoclonal infections, and 52% shared over 90% of their genomes in identity-by-descent segments with at least one other sample from the CMB, suggesting preferential expansion of certain parasite strains in this region, likely resulting from the P. vivax outbreaks occurring during this study period. Principal component, admixture, fixation index and phylogenetic analyses all identified that parasites from the CMB were genetically distinct from parasites from eastern parts of the GMS (Cambodia, Laos, Vietnam, and Thailand), whereas the eastern GMS parasite populations were largely undifferentiated. Such a genetic differentiation pattern of the P. vivax populations from the GMS parasite was largely explainable through geographic distance. Using the genome-wide SNPs, we narrowed down to a set of 36 SNPs for differentiating parasites from different areas of the GMS. Genome-wide scans to determine selection in the genome with two statistical methods identified genes potentially under drug selection, including genes associated with antifolate resistance and genes linked to chloroquine resistance in Plasmodium falciparum.

A glance of the blood stage transcriptome of a Southeast Asian Plasmodium ovale isolate.

Plasmodium ovale accounts for a disproportionate number of travel-related malaria cases. This parasite is understudied since there is a reliance on clinical samples. We collected a P. ovale curtisi parasite isolate from a clinical case in western Thailand and performed RNA-seq analysis on the blood stage transcriptomes. Using both de novo assembly and alignment-based methods, we detected the transcripts for 6628 out of 7280 annotated genes. For those lacking evidence of expression, the vast majority belonged to the PIR and STP1 gene families. We identified new splicing patterns for over 2500 genes, and mapped at least one untranslated region for over half of all annotated genes. Our analysis also detected a notable presence of anti-sense transcripts for over 10% of P. ovale curtisi genes. This transcriptomic analysis provides new insights into the blood-stage biology of this neglected parasite.