The apicoplast link to fever-survival and artemisinin-resistance in the malaria parasite.

The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite's algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite's genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors

Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis

Malaria is caused by eukaryotic Plasmodium spp. parasites that classically infect red blood cells. These are difficult organisms to investigate genetically because of their AT-rich genomes. Zhang et al. have exploited this peculiarity by using piggyBac transposon insertion sites to achieve saturation-level mutagenesis for identifying and ranking essential genes and drug targets (see the Perspective by White and Rathod). Genes that are current candidates for drug targets were identified as essential, in contrast to many vaccine target genes. Notably, the proteasome degradation pathway was confirmed as a target for developing therapeutic interventions because of the several essential genes involved and the link to the mechanism of action of the current frontline drug, artemisinin

A novel Modulator of Ring Stage Translation (MRST) gene alters artemisinin sensitivity in Plasmodium falciparum

The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum. In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7_1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite’s ART drug response

Chemogenomic profiling of a Plasmodium falciparum transposon mutant library reveals shared effects of dihydroartemisinin and bortezomib on lipid metabolism and exported proteins

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance

Phenotypic screens identify genetic factors associated with gametocyte development in the human malaria parasite Plasmodium falciparum

Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi

Essential genes of the parasitic apicomplexa

Genome-scale mutagenesis screens for genes essential for apicomplexan parasite survival have been completed in three species: Plasmodium falciparum, the major human malaria parasite, Plasmodium berghei, a model rodent malaria parasite, and the more distantly related Toxoplasma gondii, the causative agent of toxoplasmosis. These three species share 2606 single-copy orthologs, 1500 of which have essentiality data in all three screens. In this review, we explore the overlap between these datasets to define the core essential genes of the phylum Apicomplexa. We further discuss the implications of these groundbreaking studies for understanding apicomplexan parasite biology, and we identify promising areas of focus for developing new pan-apicomplexan parasite interventions

Uncovering Host-microbiome Interactions in Global Systems with Collaborative Programming: A Novel Approach Integrating Social and Data Sciences [version 1; peer review: awaiting peer review]

Microbiome data are undergoing exponential growth powered by rapid technological advancement. As the scope and depth of microbiome research increases, cross-disciplinary research is urgently needed for interpreting and harnessing the unprecedented data output. However, conventional research settings pose challenges to much-needed interdisciplinary research efforts due to barriers in scientific terminologies, methodology and research-culture. To breach these barriers, our University of South Florida OneHealth Codeathon was designed to be an interactive, hands-on event that solves real-world data problems. The format brought together students, postdocs, faculty, researchers, and clinicians in a uniquely cross-disciplinary, team-focused setting. Teams were formed to encourage equitable distribution of diverse domain-experts and proficient programmers, with beginners to experts on each team. To unify the intellectual framework, we set the focus on the topics of microbiome interactions at different scales from clinical to environmental sciences, leveraging local expertise in the fields of genetics, genomics, clinical data, and social and geospatial sciences. As a result, teams developed working methods and pipelines to face major challenges in current microbiome research, including data integration, experimental power calculations, geospatial mapping, and machine-learning classifiers. This broad, transdisciplinary and efficient workflow will be an example for future workshops to deliver useful data-science products

Kernbach_WGCNA_blue

Genes belonging to the blue module