Ancient Plasmodium genomes shed light on the history of human malaria.

Malaria-causing protozoa of the genus Plasmodium have exerted one of the strongest selective pressures on the human genome, and resistance alleles provide biomolecular footprints that outline the historical reach of these species1. Nevertheless, debate persists over when and how malaria parasites emerged as human pathogens and spread around the globe1,2. To address these questions, we generated high-coverage ancient mitochondrial and nuclear genome-wide data from P. falciparum, P. vivax and P. malariae from 16 countries spanning around 5,500 years of human history. We identified P. vivax and P. falciparum across geographically disparate regions of Eurasia from as early as the fourth and first millennia BCE, respectively; for P. vivax, this evidence pre-dates textual references by several millennia3. Genomic analysis supports distinct disease histories for P. falciparum and P. vivax in the Americas: similarities between now-eliminated European and peri-contact South American strains indicate that European colonizers were the source of American P. vivax, whereas the trans-Atlantic slave trade probably introduced P. falciparum into the Americas. Our data underscore the role of cross-cultural contacts in the dissemination of malaria, laying the biomolecular foundation for future palaeo-epidemiological research into the impact of Plasmodium parasites on human history. Finally, our unexpected discovery of P. falciparum in the high-altitude Himalayas provides a rare case study in which individual mobility can be inferred from infection status, adding to our knowledge of cross-cultural connectivity in the region nearly three millennia ago.

Plasmodium Egress Across the Parasite Life Cycle.

Human malaria, caused by infection with Plasmodium parasites, remains one of the most important global public health problems, with the World Health Organization reporting more than 240 million cases and 600,000 deaths annually as of 2020 (World malaria report 2021). Our understanding of the biology of these parasites is critical for development of effective therapeutics and prophylactics, including both antimalarials and vaccines. Plasmodium is a protozoan organism that is intracellular for most of its life cycle. However, to complete its complex life cycle and to allow for both amplification and transmission, the parasite must egress out of the host cell in a highly regulated manner. This review discusses the major pathways and proteins involved in the egress events during the Plasmodium life cycle-merozoite and gametocyte egress out of red blood cells, sporozoite egress out of the oocyst, and merozoite egress out of the hepatocyte. The similarities, as well as the differences, between the various egress pathways of the parasite highlight both novel cell biology and potential therapeutic targets to arrest its life cycle.

The parasite intraerythrocytic cycle and human circadian cycle are coupled during malaria infection.

During infections with the malaria parasites Plasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al., Science 368, 746-753 (2020); Smith et al., Science 368, 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

Differentiation of Plasmodium male gametocytes is initiated by the recruitment of a chromatin remodeler to a male-specific cis-element.

Plasmodium parasites, the causative agents of malaria, possess a complex lifecycle; however, the mechanisms of gene regulation involved in the cell-type changes remain unknown. Here, we report that gametocyte sucrose nonfermentable 2 (gSNF2), an SNF2-like chromatin remodeling ATPase, plays an essential role in the differentiation of male gametocytes. Upon disruption of gSNF2, male gametocytes lost the capacity to develop into gametes. ChIP-seq analyses revealed that gSNF2 is widely recruited upstream of male-specific genes through a five-base, male-specific cis-acting element. In gSNF2-disrupted parasites, expression of over a hundred target genes was significantly decreased. ATAC-seq analysis demonstrated that decreased expression of these genes correlated with a decrease of the nucleosome-free region upstream of these genes. These results suggest that global changes induced in the chromatin landscape by gSNF2 are the initial step in male differentiation from early gametocytes. This study provides the possibility that chromatin remodeling is responsible for cell-type changes in the Plasmodium lifecycle.

Ape Origins of Human Malaria.

African apes harbor at least twelve Plasmodium species, some of which have been a source of human infection. It is now well established that Plasmodium falciparum emerged following the transmission of a gorilla parasite, perhaps within the last 10,000 years, while Plasmodium vivax emerged earlier from a parasite lineage that infected humans and apes in Africa before the Duffy-negative mutation eliminated the parasite from humans there. Compared to their ape relatives, both human parasites have greatly reduced genetic diversity and an excess of nonsynonymous mutations, consistent with severe genetic bottlenecks followed by rapid population expansion. A putative new Plasmodium species widespread in chimpanzees, gorillas, and bonobos places the origin of Plasmodium malariae in Africa. Here, we review what is known about the origins and evolutionary history of all human-infective Plasmodium species, the time and circumstances of their emergence, and the diversity, host specificity, and zoonotic potential of their ape counterparts.

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle.

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of Plasmodium in Asymptomatic Malaria.

Asymptomatic infections of Plasmodium parasites are major obstacles to malaria control and elimination. A sensitive, specific, and user-friendly method is urgently needed for point-of-care (POC) Plasmodium diagnostics in asymptomatic malaria, especially in resource-limited settings. In this work, we present a POC method (termed Cas13a-SDT) based on the cascade sequence recognition and signal amplification of dual Cas13a trans-cleavage and strand displacement-triggered transcription (SDT). Cas13a-SDT not only achieves exceptional specificity in discriminating the target RNA from nontarget RNAs with any cross-interaction but also meets the sensitivity criterion set by the World Health Organization (WHO) for effective malaria detection. Remarkably, this novel method was successfully applied to screen malaria in asymptomatic infections from clinical samples. The proposed method provides a user-friendly and visually interpretable output mode while maintaining high accuracy and reliability comparable to RT-PCR. These excellent features demonstrate the significant potential of Cas13a-SDT for POC diagnosis of Plasmodium infections, laying a vital foundation for advancing malaria control and elimination efforts.

Emerging biology of noncoding RNAs in malaria parasites.

In eukaryotic organisms, noncoding RNAs (ncRNAs) have been implicated as important regulators of multifaceted biological processes, including transcriptional, posttranscriptional, and epigenetic regulation of gene expression. In recent years, it is becoming clear that protozoan parasites encode diverse ncRNA transcripts; however, little is known about their cellular functions. Recent advances in high-throughput "omic" studies identified many novel long ncRNAs (lncRNAs) in apicomplexan parasites, some of which undergo splicing, polyadenylation, and encode small proteins. To date, only a few of them are characterized, leaving a big gap in our understanding regarding their origin, mode of action, and functions in parasite biology. In this review, we focus on lncRNAs of the human malaria parasite Plasmodium falciparum and highlight their cellular functions and possible mechanisms of action.

Inhibitors of ApiAP2 protein DNA binding exhibit multistage activity against Plasmodium parasites.

Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified putative AP2-EXP interacting compounds. Four compounds were found to block DNA binding by AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that interact with AP2 DNA binding domains. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.

RNA-seq analysis of parasitism by Intoshia linei (Orthonectida) reveals protein effectors of defence, communication, feeding and growth.

Orthonectida is a group of multicellular endoparasites of a wide range of marine invertebrates. Their parasitic stage is a multinuclear shapeless plasmodium infiltrating host tissues. The development of the following worm-like sexual generation takes place within the cytoplasm of the plasmodium. The existence of the plasmodial stage and the development of a sexual stage within the plasmodium are unique features to Bilateria. However, the molecular mechanisms that maintain this peculiar organism, and hence enable parasitism in orthonectids, are unknown. Here, we present the first-ever RNA-seq analysis of the plasmodium, aimed at the identification and characterization of the plasmodium-specific protein-coding genes and corresponding hypothetical proteins that distinguish the parasitic plasmodium stage from the sexual stage of the orthonectid Intoshia linei Giard, 1877, parasite of nemertean Lineus ruber Müller, 1774. We discovered 119 plasmodium-specific proteins, 82 of which have inferred functions based on known domains. Thirty-five of the detected proteins are orphans, at least part of which may reflect the unique evolutionary adaptations of orthonectids to parasitism. Some of the identified proteins are known effector molecules of other endoparasites suggesting convergence. Our data indicate that the plasmodium-specific proteins might be involved in the plasmodium defense against the host, host-parasite communication, feeding and nutrient uptake, growth within the host, and support of the sexual stage development. These molecular processes in orthonectids have not been described before, and the particular protein effectors remained unknown until now.

Genotype-environment associations reveal genes potentially linked to avian malaria infection in populations of an endemic island bird.

Patterns of pathogen prevalence are, at least partially, the result of coevolutionary host-pathogen interactions. Thus, exploring the distribution of host genetic variation in relation to infection by a pathogen within and across populations can provide important insights into mechanisms of host defence and adaptation. Here, we use a landscape genomics approach (Bayenv) in conjunction with genome-wide data (ddRADseq) to test for associations between avian malaria (Plasmodium) prevalence and host genetic variation across 13 populations of the island endemic Berthelot's pipit (Anthus berthelotii). Considerable and consistent spatial heterogeneity in malaria prevalence was observed among populations over a period of 15 years. The prevalence of malaria infection was also strongly positively correlated with pox (Avipoxvirus) prevalence. Multiple host loci showed significant associations with malaria prevalence after controlling for genome-wide neutral genetic structure. These sites were located near to or within genes linked to metabolism, stress response, transcriptional regulation, complement activity and the inflammatory response, many previously implicated in vertebrate responses to malarial infection. Our findings identify diverse genes - not just limited to the immune system - that may be involved in host protection against malaria and suggest that spatially variable pathogen pressure may be an important evolutionary driver of genetic divergence among wild animal populations, such as Berthelot's pipit. Furthermore, our data indicate that spatio-temporal variation in multiple different pathogens (e.g. malaria and pox in this case) may have to be studied together to develop a more holistic understanding of host pathogen-mediated evolution.

Review of MrsFreqPhase methods: methods designed to estimate statistically malaria parasite multiplicity of infection, relatedness, frequency and phase.

Malaria parasites are haploid within humans, but infections often contain genetically distinct groups of clonal parasites. When the per-infection number of genetically distinct clones (i.e., the multiplicity of infection, MOI) exceeds one, and per-infection genetic data are generated in bulk, important information are obfuscated. For example, the MOI, the phases of the haploid genotypes of genetically distinct clones (i.e., how the alleles concatenate into sequences), and their frequencies. This complicates many downstream analyses, including relatedness estimation. MOIs, parasite sequences, their frequencies, and degrees of relatedness are used ubiquitously in malaria studies: for example, to monitor anti-malarial drug resistance and to track changes in transmission. In this article, MrsFreqPhase methods designed to estimate statistically malaria parasite MOI, relatedness, frequency and phase are reviewed. An overview, a historical account of the literature, and a statistical description of contemporary software is provided for each method class. The article ends with a look towards future method development, needed to make best use of new data types generated by cutting-edge malaria studies reliant on MrsFreqPhase methods.

Genomic variation in Plasmodium relictum (lineage SGS1) and its implications for avian malaria infection outcomes: insights from experimental infections and genome-wide analysis.

BACKGROUND: The globally transmitted avian malaria parasite Plasmodium relictum (lineage SGS1) has been found to infect hundreds of different bird species with differences in infection outcomes ranging from more or less latent to potentially mortal. However, to date basic knowledge about the links between genetic differentiation and variation in infection outcome within this single malaria parasite species is lacking. METHODS: In this study, two different isolates of SGS1, obtained in the wild from two different host species, were used to investigate differences in their development in the blood and virulence in the experimentally infected canaries. Simultaneously, 258 kb of the parasite genome was screened for genetic differences using parasite mRNA and compared between experimental groups. RESULTS: The two isolates showed differences in development and caused mortality as well as effects on the blood parameters of their hosts. Although previous studies using single genes have shown very limited within lineage genetic diversity in the European population of SGS1, 226 SNPs were found across 322 genes, which separated the two experimental groups with a total of 23 SNPs that were fixed in either of the experimental groups. Moreover, genetic variation was found within each experimental group, hinting that each avian malaria infection harbours standing genetic variation that might be selected during each individual infection episode. CONCLUSION: These results highlight extensive genetic variation within the SGS1 population that is transferred into individual infections, thus adding to the complexity of the infection dynamics seen in these host-parasite interactions. Simultaneously, the results open up the possibility of understanding how genetic variation within the parasite populations is linked to the commonly observed differences in infection outcomes, both in experimental settings and in the wild.

RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

From eradication to reemergence: the growing threat of malaria in Turkey.

According to WHO, between 2000 and 2021, there were approximately 247 million malaria cases and 627,000 deaths globally, with the majority of cases occurring in sub-Saharan Africa. In Turkey, indigenous P. vivax malaria was a major public health problem until its eradication was achieved in 2010. Although indigenous malaria transmission has been significantly reduced since 2010, the country is challenged with imported malaria due to increasing global travel and migration from endemic regions. In this study, all imported malaria cases admitted to Dr. Sadi Konuk Research and Training Hospital, Istanbul, between 2018 and 2023 were included. DNA extraction was performed using archived slides and EDTA blood samples. Real-time PCR was performed to identify samples at the species level using previously reported primers and probes. In addition, all available patient demographics are presented. During the six years between 2018 and 2023, 157 patients were diagnosed with imported malaria. According to the real-time PCR results, 149 cases were P. falciparum (94.9%), five cases were P. vivax (3.2%), two cases were P. ovale (1.3%), and one case was P. malariae (0.6%). The male/female ratio among diagnosed patients was 2.34 (110♂/47♀) among diagnosed patients. Plasmodium falciparum was detected in patients from all African regions, whereas P. vivax was detected only in patients from Liberia and Djibouti. Although malaria cases in Turkey have significantly decreased due to elimination efforts and effective public health interventions, the recent increase in both imported and indigenous cases, as well as the presence of suitable vector species in the country, indicates that malaria still remains a serious public health problem for Turkey.

Avian haemosporidians of the genera Plasmodium and Haemoproteus from resident and Neotropical migrant birds in Colombia.

Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus. Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo. Seventeen haemosporidian lineages were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager (neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes.

Post-Translational Modifications of Proteins of Malaria Parasites during the Life Cycle.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host.

A toolbox for conditional control of gene expression in apicomplexan parasites.

Apicomplexan parasites encompass diverse pathogens for humans and animals, including the causative agents of malaria and toxoplasmosis, Plasmodium spp. and Toxoplasma gondii. Genetic manipulation of these parasites has become central to explore parasite biology, unravel gene function and identify new targets for therapeutic strategies. Tremendous progress has been achieved over the past years with the advent of next generation sequencing and powerful genome editing methods. In particular, various methods for conditional gene expression have been developed in both Plasmodium and Toxoplasma to knockout or knockdown essential genes, or for inducible expression of master developmental regulators or mutant versions of proteins. Conditional gene expression can be achieved at three distinct levels. At the DNA level, inducible site-specific recombinases allow conditional genome editing. At the RNA level, regulation can be achieved during transcription, using stage-specific or regulatable promoters, or post-transcriptionally through alteration of mRNA stability or translation. At the protein level, several systems have been developed for inducible degradation or displacement of a protein of interest. In this review, we provide an overview of current systems for conditional control of gene expression in Plasmodium and Toxoplasma parasites, highlighting the advantages and limitations of each approach.

Microtubule number and length determine cellular shape and function in Plasmodium.

Microtubules are cytoskeletal filaments essential for many cellular processes, including establishment and maintenance of polarity, intracellular transport, division and migration. In most metazoan cells, the number and length of microtubules are highly variable, while they can be precisely defined in some protozoan organisms. However, in either case the significance of these two key parameters for cells is not known. Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two α-tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that α-tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit.