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Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC 50 32 nM), Ugandan field isolates (mean ex vivo IC 50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED 90 0.34 and 0.57 mg kg −1 , respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3 , which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target. Benzoxaboroles have been shown to be active against different pathogens. Here, the authors show that the benzoxaborole AN3661 inhibits Plasmodium falciparum in vitro and in mouse models, and identify a homologue of a mammalian cleavage and polyadenylation specificity factor as a drug target.

Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3' untranslated region motif is implicated in this process.

Malaria infection involves an obligatory, yet clinically silent liver stage 1 , 2 . Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis 3 , but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing 4 and single-molecule transcript imaging 5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These ‘abortive hepatocytes’, which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte. Single-cell RNA sequencing and single-molecule RNA transcript imaging have been used to characterize spatially and temporally resolved mouse liver and parasite expression programmes during infection with the rodent malaria parasite Plasmodium berghei ANKA.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

The life cycle of Plasmodium parasites involves intricate, multistage processes that are tightly regulated by stage-specific transcription factors. These factors bind to regulatory regions within gene promoters, enabling the precise expression of genes required for each developmental stage. Despite the importance of these transcriptional mechanisms, our understanding remains limited, particularly in the rodent model organism P. berghei. To address this, we conducted a genome-wide analysis of RNA-Seq data from different developmental stages of P. berghei by initially integrating data from human malaria parasites P. falciparum and P. vivax . We identified unique transcriptional signatures across Plasmodium species. Our analysis of P. berghei revealed stage-specific gene sets clustered by expression profiles and predicted regulatory motifs involved in their control. We interpreted these motifs using known binding sites for eukaryotic transcription factors including ApiAP2 proteins. Additionally, we expanded the annotation of the AGGTAA motif which resembles a de novo motif linked to erythrocytic development in P. falciparum , and identified its potential interacting proteins including members of the PfMORC and GCN5 complexes. This study enhances our understanding of gene regulation in P. berghei and provides new insights into the transcriptional dynamics underlying Plasmodium development.

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na + regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na + homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na + homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes. Novel antimalarial drugs are urgently needed to combat parasite drug resistance. Here, Vaidya et al . describe a new chemical class of potent antimalarial compounds that act by disrupting the parasite's sodium homeostasis.

The malaria parasite’s inner membrane complex is critical to maintain its structural integrity and motility. Here, we identified the function of the IMC1g protein, a member of the IMC1 family, in invasive and proliferative stages of P. berghei . We found that the IMCp domain of PbIMC1g is critical for proper IMC targeting, and PbIMC1g interacts with PbIMC1c. Conditional knockdown of PbIMC1g expression affects schizogony, gametogenesis, and ookinete conversion. PbIMC1g interacts with IMC1c to firmly anchor the glideosome to the subpellicular network. Additionally, we confirmed that IMC1g is functionally conserved in Plasmodium spp. These data reveal the function of IMC1g protein in anchoring the glideosome, providing further insight into the mechanism of the glideosome function.

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp., the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within 8 min followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified 4 Nek genes, of which 2, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM), and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation, and kinetochore attachment. These data suggest that P . berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation.

The circumsporozoite protein (CSP), an essential protein that covers the surface of the Plasmodium sporozoite, is a key player in multiple stages of the parasite development within the mosquito and during interactions between sporozoites and mammalian hepatocytes. Here, we identify a novel function of Plasmodium berghei CSP: preventing parasite elimination during the early stages of hepatic infection, through its ubiquitylation at two lysine (K) residues, K252 and K258, located in the C-terminal domain. A Plasmodium berghei transgenic line lacking these lysine residues exhibited a significant decrease in hepatic infectivity, with parasites being eliminated 4 h after infection. The reduced infectivity correlated with an increased association of host autophagy markers, LC3 and LAMP1, to the parasitophorous vacuole membrane of the liver stage parasite. Notably, inhibiting the host autophagy pathway fully rescued the mutant parasites from elimination. Collectively, we reveal a strategy employed by Plasmodium to evade early clearance during hepatic infection, which relies on the ubiquitylation of specific CSP lysine residues, that results in reduced parasite elimination via host autophagic and lysosomal activity.

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium . However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium . RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development. Guan et al. identify a male gametocyte-specific RNA-binding protein RBPm1 in the malaria parasite. RBPm1 controls the intron splicing of axonemal genes. RBPm1- deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission of Plasmodium.