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Background: Malaria parasites establish new permeation pathways (NPPs) at the red blood cell membrane to facilitate the transport of essential nutrients from the blood plasma into the infected host cell. The NPPs are critical to parasite survival and, therefore, in the pursuit of novel therapeutics are an attractive drug target. The NPPs of the human parasite, P. falciparum, have been linked to the RhopH complex, with the monoallelic paralogues clag3.1 and clag3.2 encoding the protein RhopH1/CLAG3 that likely forms the NPP channel-forming component. Yet curiously, the combined knockout of both clag3 genes does not completely eliminate NPP function. The essentiality of the clag3 genes is, however, complicated by three additional clag paralogs (clag2, clag8 and clag9) in P. falciparum that could also be contributing to NPP formation., Methods: Here, the rodent malaria species, P. berghei, was utilised to investigate clag essentiality since it contains only two clag genes, clagX and clag9. Allelic replacement of the regions encompassing the functional components of P. berghei clagX with either P. berghei clag9 or P. falciparum clag3.1 examined the relationship between the two P. berghei clag genes as well as functional orthology across the two species. An inducible P. berghei clagX knockout was created to examine the essentiality of the clag3 ortholog to both survival and NPP functionality., Results: It was revealed P. berghei CLAGX and CLAG9, which belong to two distinct phylogenetic clades, have separate non-complementary functions, and that clagX is the functional orthologue of P. falciparum clag3. The inducible clagX knockout in conjunction with a guanidinium chloride induced-haemolysis assay to assess NPP function provided the first evidence of CLAG essentiality to Plasmodium survival and NPP function in an in vivo model of infection., Conclusions: This work provides valuable insight regarding the essentiality of the RhopH1 clag genes to the NPPs functionality and validates the continued investigation of the RhopH complex as a therapeutic target to treat malaria infections., Competing Interests: Declarations. Ethics approval and consent to participate: All animal procedures that were conducted in this study were in accordance with the relevant guidelines and was approved by the Ethics Committee of Deakin University (Project numbers G03-2020, approved 22/5/2020 and G03-2023, approved 8/5/2023). Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. Another potent tool is the CRISPR/Cas9 technology, which has enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR/Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirmed the role of CLAMP during invasion of erythrocytes., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite's biology at scale., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The inner membrane complex (IMC), a double-membrane organelle underneath the plasma membrane in apicomplexan parasites, plays a significant role in motility and invasion and confers shape to the cell. We characterized the function of PbIMC1g, a component of the IMC1 family member in Plasmodium berghei . PbIMC1g is recruited to the IMC in late schizonts, activated gametocytes, and ookinetes. Pairwise yeast two-hybrid assays demonstrate that PbIMC1g interacts with IMC1c, a component of the PHIL1 complex, and the core sub-repeat motif "EKI(V)V(I)EVP" in PbIMC1g is essential for this interaction. Localization of PbIMC1g to the IMC was dependent on its IMCp domain, while its C-terminus and palmitoylation sites were required for the full efficiency of proper IMC targeting. PbIMC1g is required for asexual stage development, and its conditional knockdown resulted in a defect in schizogony. Additionally, PbIMC1g was also important for male gametogenesis and ookinete development. As an IMC component that assists in anchoring the glideosome to the subpellicular network, PbIMC1g was also involved in ookinete motility and mosquito midgut invasion. IMC1g from the human parasite Plasmodium vivax could functionally replace PbIMC1g in P. berghei , confirming the evolutionary conservation of IMC1g proteins in Plasmodium spp. Together, this work reveals an essential role of IMC1g in the parasite life cycle and suggests that IMC1 family members likely contribute to parasite gliding and invasion., Importance: 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., Competing Interests: The authors declare no conflict of interest.

Malaria caused by Plasmodium parasites remains a large health burden. One approach to combat this disease involves vaccinating individuals with whole sporozoites that have been genetically modified to arrest their development at a specific stage in the liver by targeted gene deletion, resulting in a genetically attenuated parasite (GAP). Through a comprehensive phenotyping screen, we identified the hscb gene, encoding a putative iron-sulfur protein assembly chaperone, as crucial for liver stage development, making it a suitable candidate gene for GAP generation. Parasites lacking Plasmodium berghei HscB (PbHscB) exhibited normal sporozoite production in mosquitoes, but their liver stage development was severely impaired, characterized by slow growth and delayed expression of merozoite surface protein 1 (MSP1). In vivo experiments demonstrated that PbHscB-deficient parasites exhibited a delay in prepatency of 2-4 days, emphasizing the significance of PbHscB for exo-erythrocytic development. Although knockout of PbHscB alone allowed breakthrough infections, it is a potent candidate for a dual gene deletion strategy. PlasMei2, an RNA-binding protein, was previously found to be crucial for the completion of liver stage development. We generated a PbHscB-PbMei2-double attenuated parasite line, serving as a late liver stage-arresting replication-competent (LARC) GAP, providing a solid block of liver-to-blood stage transition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schmid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plasmodium parasites, the causative agents of malaria, undergo closed mitosis without breakdown of the nuclear envelope. Unlike closed mitosis in yeast, Plasmodium berghei parasites undergo multiple rounds of asynchronous nuclear divisions in a shared cytoplasm. This results in a multinucleated organism prior to the formation of daughter cells within an infected red blood cell. During this replication process, intact nuclear pore complexes (NPCs) and their component nucleoporins play critical roles in parasite growth, facilitating selective bi-directional nucleocytoplasmic transport and genome organization. Here, we utilize ultrastructure expansion microscopy to investigate P. berghei nucleoporins at the single nucleus level throughout the 24-hour blood-stage replication cycle. Our findings reveal that these nucleoporins are distributed around the nuclei and organized in a rosette structure previously undescribed around the centriolar plaque, responsible for intranuclear microtubule nucleation during mitosis. By adapting the recombination-induced tag exchange system to P. berghei through a single plasmid tagging system, which includes the tagging plasmid as well as the Cre recombinase, we provide evidence of NPC formation dynamics, demonstrating Nup221 turnover during parasite asexual replication. Our data shed light on the distribution of NPCs and their homeostasis during the blood-stage replication of P. berghei parasites., Importance: Malaria, caused by Plasmodium species, remains a critical global health challenge, with an estimated 249 million cases and over 600,000 deaths in 2022, primarily affecting children under five. Understanding the nuclear dynamics of Plasmodium parasites, particularly during their unique mitotic processes, is crucial for developing novel therapeutic strategies. Our study leverages advanced microscopy techniques, such as ultrastructure expansion microscopy, to reveal the organization and turnover of nuclear pore complexes (NPCs) during the parasite's asexual replication. By elucidating these previously unknown aspects of NPC distribution and homeostasis, we provide valuable insights into the molecular mechanisms governing parasite mitosis. These findings deepen our understanding of parasite biology and may inform future research aimed at identifying new targets for anti-malarial drug development., Competing Interests: The authors declare no conflict of interest.

Background: Studying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites., Methods: Differences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte-ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development., Results: The phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte-ookinete conversion and transmission to mosquitoes., Conclusions: Seven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening., Competing Interests: Declaration of competing interest Declarations of interest: none., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plasmodium, the causative agent of malaria, infects hepatocytes prior to establishing a symptomatic blood stage infection. During this liver stage development, parasites reside in a parasitophorous vacuole (PV), whose membrane acts as the critical interface between the parasite and the host cell. It is well-established that host cell autophagy-related processes significantly impact the development of Plasmodium liver stages. Expression of genes related to autophagy and lysosomal biogenesis is orchestrated by transcription factor EB (TFEB). In this study, we explored the activation of host cell TFEB in Plasmodium berghei-infected cells during the liver stage of the parasite. Our results unveiled a critical role of proteins belonging to the Gamma-aminobutyric acid receptor-associated protein subfamily (GABARAP) of ATG8 proteins (GABARAP/L1/L2 and LC3A/B/C) in recruiting the TFEB-blocking FLCN-FNIP (Folliculin-Folliculin-interacting protein) complex to the PVM. Remarkably, the sequestration of FLCN-FNIP resulted in a robust activation of TFEB, reliant on conjugation of ATG8 proteins to single membranes (CASM) and GABARAP proteins. Our findings provide novel mechanistic insights into host cell signaling occurring at the PVM, shedding light on the complex interplay between Plasmodium parasites and the host cell during the liver stage of infection., Competing Interests: Competing interests: The authors declare the following competing interests: L.O.M. and T.L. are employees and shareholders of Casma Therapeutics. A.B. is a co-founder and shareholder of CASMA Therapeutics, Inc, and Advisory board member of Avilar Therapeutics and Amplify Therapeutics. L.O.M. is an author on a patent related to TFEB modulation, in addition. M.L. is a co-founder and member of the scientific advisory board of Automera. Other authors declare no competing interests., (© 2024. The Author(s).)

High mobility group box (HMGB) proteins belong to the high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression, and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation, and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Site-directed mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-α release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses shows prominent germinal centers leading to effective humoral responses and enhanced T follicular helper cells. There is also complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible to cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated blood-stage vaccines for malaria., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium . Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include W D-repeat protein i mportant for g ametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCE Plasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium . Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zeeshan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Malaria vaccination approaches using live Plasmodium parasites are currently explored, with either attenuated mosquito-derived sporozoites or attenuated blood-stage parasites. Both approaches would profit from the availability of attenuated and avirulent parasites with a reduced blood-stage multiplication rate. Here we screened gene-deletion mutants of the rodent parasite P. berghei and the human parasite P. falciparum for slow growth. Furthermore, we tested the P. berghei mutants for avirulence and resolving blood-stage infections, while preserving sporozoite formation and liver infection. Targeting 51 genes yielded 18 P. berghei gene-deletion mutants with several mutants causing mild infections. Infections with the two most attenuated mutants either by blood stages or by sporozoites were cleared by the immune response. Immunization of mice led to protection from disease after challenge with wild-type sporozoites. Two of six generated P. falciparum gene-deletion mutants showed a slow growth rate. Slow-growing, avirulent P. falciparum mutants will constitute valuable tools to inform on the induction of immune responses and will aid in developing new as well as safeguarding existing attenuated parasite vaccines., (© 2024. The Author(s).)

Background: Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax . Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax ., Methods and Findings: The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution., Conclusions: Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zheng, Cheng, Liu, Yu, Zhao, Yang, Thongpoon, Roobsoong, Sattabongkot, Luo, Cui and Cao.)

Background: Malaria, a global health concern, is caused by parasites of the Plasmodium genus, which undergo gametogenesis in the midgut of mosquitoes after ingestion of an infected blood meal. The resulting male and female gametes fuse to form a zygote, which differentiates into a motile ookinete. After traversing the midgut epithelium, the ookinete differentiates into an oocyst on the basal side of the epithelium., Methods: Membrane proteins with increased gene expression levels from the gamete to oocyst stages in P. berghei were investigated utilizing PlasmoDB, the functional genomic database for Plasmodium spp. Based on this analysis, we selected the 184-kDa membrane protein, Pb184, for further study. The expression of Pb184 was further confirmed through immunofluorescence staining, following which we examined whether Pb184 is involved in fertilization using antibodies targeting the C-terminal region of Pb184 and biotin-labeled C-terminal region peptides of Pb184., Results: Pb184 is expressed on the surface of male and female gametes. The antibody inhibited zygote and ookinete formation in vitro. When mosquitoes were fed on parasite-infected blood containing the antibody, oocyst formation decreased on the second day after feeding. Synthesized biotin-labeled peptides matching the C-terminal region of Pb184 bound to the female gamete and the residual body of male gametes, and inhibited differentiation into ookinetes in the in vitro culture system., Conclusions: These results may be useful for the further studying the fertilization mechanism of Plasmodium protozoa. There is also the potential for their application as future tools to prevent malaria transmission., (© 2024. The Author(s).)

Increasing reports of insecticide resistance continue to hamper the gains of vector control strategies in curbing malaria transmission. This makes identifying new insecticide targets or alternative vector control strategies necessary. CLassifier of Essentiality AcRoss EukaRyote (CLEARER), a leave-one-organism-out cross-validation machine learning classifier for essential genes, was used to predict essential genes in Anopheles gambiae and selected predicted genes experimentally validated. The CLEARER algorithm was trained on six model organisms: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and employed to identify essential genes in An. gambiae. Of the 10,426 genes in An. gambiae, 1,946 genes (18.7%) were predicted to be Cellular Essential Genes (CEGs), 1716 (16.5%) to be Organism Essential Genes (OEGs), and 852 genes (8.2%) to be essential as both OEGs and CEGs. RNA interference (RNAi) was used to validate the top three highly expressed non-ribosomal predictions as probable vector control targets, by determining the effect of these genes on the survival of An. gambiae G3 mosquitoes. In addition, the effect of knockdown of arginase (AGAP008783) on Plasmodium berghei infection in mosquitoes was evaluated, an enzyme we computationally inferred earlier to be essential based on chokepoint analysis. Arginase and the top three genes, AGAP007406 (Elongation factor 1-alpha, Elf1), AGAP002076 (Heat shock 70kDa protein 1/8, HSP), AGAP009441 (Elongation factor 2, Elf2), had knockdown efficiencies of 91%, 75%, 63%, and 61%, respectively. While knockdown of HSP or Elf2 significantly reduced longevity of the mosquitoes (p<0.0001) compared to control groups, Elf1 or arginase knockdown had no effect on survival. However, arginase knockdown significantly reduced P. berghei oocytes counts in the midgut of mosquitoes when compared to LacZ-injected controls. The study reveals HSP and Elf2 as important contributors to mosquito survival and arginase as important for parasite development, hence placing them as possible targets for vector control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Adedeji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Malaria transmission-blocking vaccines (TBVs) aim to inhibit malaria parasite development in mosquitoes and prevent further transmission to the human host. The putative-secreted ookinete protein 25 (PSOP25), highly conserved in Plasmodium spp., is a promising TBV target. Here, we investigated PvPSOP25 from P. vivax as a TBV candidate using transgenic murine parasite P. berghei and clinical P. vivax isolates., Methods and Findings: A transgenic P. berghei line expressing PvPSOP25 (TrPvPSOP25Pb) was generated. Full-length PvPSOP25 was expressed in the yeast Pichia pastoris and used to immunize mice to obtain anti-rPvPSOP25 sera. The transmission-blocking activity of the anti-rPvPSOP25 sera was evaluated through in vitro assays and mosquito-feeding experiments. The antisera generated by immunization with rPvPSOP25 specifically recognized the native PvPSOP25 antigen expressed in TrPvPSOP25Pb ookinetes. In vitro assays showed that the immune sera significantly inhibited exflagellation and ookinete formation of the TrPvPSOP25Pb parasite. Mosquitoes feeding on mice infected with the transgenic parasite and passively transferred with the anti-rPvPSOP25 sera showed a 70.7% reduction in oocyst density compared to the control group. In a direct membrane feeding assay conducted with five clinical P. vivax isolates, the mouse anti-rPvPSOP25 antibodies significantly reduced the oocyst density while showing a negligible influence on mosquito infection prevalence., Conclusions: This study supported the feasibility of transgenic murine malaria parasites expressing P. vivax antigens as a useful tool for evaluating P. vivax TBV candidates. Meanwhile, the moderate transmission-reducing activity of the generated anti-rPvPSOP25 sera necessitates further research to optimize its efficacy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of PbARID, a putative subunit of a SWI/SNF chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. PbARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of gametocytes with detectable male features and the production of abnormal female gametocytes. ChIP-seq analysis of PbARID showed that it forms a complex with gSNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element, TGTCT. Further ChIP-seq of PbARID in gsnf2-knockout parasites revealed an association of PbARID with another cis-regulatory element, TGCACA. RIME and DNA-binding assays suggested that HDP1 is the transcription factor that recruits PbARID to the TGCACA motif. Our results indicated that PbARID could function in two chromatin remodeling events and paly essential roles in both male and female gametocyte development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., (© 2024. The Author(s).)

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP., (© 2024 John Wiley & Sons Ltd.)

The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed., (© 2024. The Author(s).)

Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8 to target proteins at lysine residues. However, the role of neddylation in malaria parasites has not been determined. Here, for the first time, we showed that neddylation plays an essential role in malaria transmission in Plasmodium berghei . We found that disruption of Nedd8 did not affect blood-stage propagation, gametocyte development, gamete formation, or zygote formation while abolishing the formation of ookinetes and further transmission of the parasites in mosquitoes. These phenotypic defects in Nedd8 knockout parasites were complemented by reintroducing the gene that restored mosquito transmission to wild-type levels. Our data establish the role of P. berghei Nedd8 in malaria parasite transmission.IMPORTANCENeddylation is a process by which Nedd8 is covalently attached to target proteins through three-step enzymatic cascades. The attachment of Nedd8 residues results in a range of diverse functions, such as cell cycle regulation, metabolism, immunity, and tumorigenesis. The potential neddylation substrates are cullin (CUL) family members, which are implicated in controlling the cell cycle. Cullin neddylation leads to the activation of cullin-RING ubiquitin ligases, which regulate a myriad of biological processes through target-specific ubiquitylation. Neddylation possibly regulates meiosis in zygotes, which subsequently develop into ookinetes. Our findings point to an essential function of this neddylation pathway and highlight its possible importance in designing novel intervention strategies., Competing Interests: The authors declare no conflict of interest.

Plasmodium sporozoite development in and egress from oocysts in the Anopheles mosquito remains largely enigmatic. In a previously performed high-throughput knockout screen, the putative subunit 5 of the prefoldin complex (PbPCS5, PBANKA&#95;0920100) was identified as essential for parasite development during mosquito and liver stage development. Here we generated and analyzed a PbPCS5 knockout parasite line during its development in the mosquito. Interestingly, PbPCS5 deletion does not significantly affect oocyst formation but leads to a growth defect resulting in aberrantly shaped sporozoites. Sporozoites produced in the absence of PbPCS5 were thinner, markedly elongated, and did, in most cases, not contain a nucleus. Sporozoites contained fewer subpellicular microtubules, which reached deep into the sporoblast during sporogony where they contacted and indented nuclei. These aberrantly shaped sporozoites did not reach the salivary glands, and we, therefore, conclude that PbPCS5 is essential for sporogony and the life cycle progression of the parasite during its mosquito stage., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function., (© 2023 John Wiley & Sons Ltd.)

Background: Plasmodium lacks an mRNA export receptor ortholog, such as yeast Mex67. Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain, suggesting that NTF2-like domain-containing proteins might be associated with mRNA export in Plasmodium. In this study, the relationship between mRNA export and an NTF2-like domain-containing protein, PBANKA&#95;1019700, was investigated using the ANKA strain of rodent malaria parasite Plasmodium berghei., Methods: The deletion mutant Δ1019700 was generated by introducing gene-targeting vectors into the P. berghei ANKA genome, and parasite growth and virulence were examined. To investigate whether PBANKA&#95;1019700 is involved in mRNA export, live-cell fluorescence imaging and immunoprecipitation coupled to mass spectrometry (IP-MS) were performed using transgenic parasites expressing fusion proteins (1019700::mCherry)., Results: Deletion of PBANKA&#95;1019700 affected the sexual phase but not the asexual phase of malaria parasites. Live-cell fluorescence imaging showed that PBANKA&#95;1019700 localizes to the cytoplasm. Moreover, IP-MS analysis of 1019700::mCherry indicated that PBANKA&#95;1019700 interacts with ubiquitin-related proteins but not nuclear proteins., Conclusions: PBANKA&#95;1019700 is a noncanonical NTF2-like superfamily protein., (© 2024. The Author(s).)

Importance: Cerebral malaria (CM) is the deadliest complication of malaria infection with an estimated 15%-25% mortality. Even with timely and effective treatment with antimalarial drugs such as quinine and artemisinin derivatives, survivors of CM may suffer long-term cognitive and neurological impairment. Here, we show that human apolipoprotein E variant 2 (hApoE2) protects mice from experimental CM (ECM) via suppression of CD8 + T cell activation and infiltration to the brain, enhanced cholesterol metabolism, and increased IFN-γ production, leading to reduced endothelial cell apoptosis, BBB disruption, and ECM symptoms. Our results suggest that hApoE can be an important factor for risk assessment and treatment of CM in humans., Competing Interests: The authors declare no conflict of interest.

Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite., (© 2023. The Author(s).)

Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum . The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin -based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive., Competing Interests: EG, EJ, DK, RP, AG, SB, EM No competing interests declared, (© 2023, Green et al.)

Background: Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays., Methods: IC 50 values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 10 3 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH., Results: The IC 50 values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC 50 of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification., Conclusions: Results from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody IC 50 values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system., (© 2023. The Author(s).)

Background: Plasmodium berghei has been used as a preferred model for studying human malaria, but only a limited number of disease-associated genes of P. berghei have been reported to date. Identification of new disease-related genes as many as possible will provide a landscape for better understanding the pathogenesis of P. berghei., Methods: Network module analysis method was developed and applied to identify disease-related genes in P. berghei genome. Sequence feature identification, gene ontology annotation, and T-cell epitope analysis were performed on these genes to illustrate their functions in the pathogenesis of P. berghei., Results: 33,314 genes were classified into 4,693 clusters. 4,127 genes shared by six malaria parasites were identified and are involved in many aspects of biological processes. Most of the known essential genes belong to shared genes. A total of 63 clusters consisting of 405 P. berghei genes were enriched in rodent malaria parasites. These genes participate in various stages of parasites such as liver stage development and immune evasion. Combination of these genes might be responsible for P. berghei infecting mice. Comparing with P. chabaudi, none of the clusters were specific to P. berghei. P. berghei lacks some proteins belonging to P. chabaudi and possesses some specific T-cell epitopes binding by class-I MHC, which might together contribute to the occurrence of experimental cerebral malaria (ECM)., Conclusions: We successfully identified disease-associated P. berghei genes by network module analysis. These results will deepen understanding of the pathogenesis of P. berghei and provide candidate parasite genes for further ECM investigation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, the complex regulation of gene expression and genotype-phenotype associations in the mosquito vector, along with sexual reproduction, have hindered the development of screens in this critical part of the parasite life cycle. To address this, we developed a genetic approach in the rodent parasite Plasmodium berghei that, in combination with barcode sequencing, circumvents the fertilization roadblock and enables screening for gametocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii. Our results confirm previous findings, validating our approach for scaling up, and identify genes necessary for mosquito midgut infection, oocyst development, and salivary gland infection. These findings can aid efforts to study malaria transmission biology and to develop interventions for controlling disease transmission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite., (© 2023. Springer Nature Limited.)

Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H + from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its in vivo druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 × IC 50 (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their in vivo activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials., Competing Interests: The authors declare no conflict of interest.

Background: Malaria caused by Plasmodium species is a prominent public health concern worldwide, and the infection of a malarial parasite is transmitted to humans through the saliva of female Anopheles mosquitoes. Plasmodium invasion is a rapid and complex process. A critical step in the blood-stage infection of malarial parasites is the adhesion of merozoites to red blood cells (RBCs), which involves interactions between parasite ligands and receptors. The present study aimed to investigate a previously uncharacterized protein, PbMAP1 (encoded by PBANKA&#95;1425900), which facilitates Plasmodium berghei ANKA (PbANKA) merozoite attachment and invasion via the heparan sulfate receptor., Methods: PbMAP1 protein expression was investigated at the asexual blood stage, and its specific binding activity to both heparan sulfate and RBCs was analyzed using western blotting, immunofluorescence, and flow cytometry. Furthermore, a PbMAP1-knockout parasitic strain was established using the double-crossover method to investigate its pathogenicity in mice., Results: The PbMAP1 protein, primarily localized to the P. berghei membrane at the merozoite stage, is involved in binding to heparan sulfate-like receptor on RBC surface of during merozoite invasion. Furthermore, mice immunized with the PbMAP1 protein or passively immunized with sera from PbMAP1-immunized mice exhibited increased immunity against lethal challenge. The PbMAP1-knockout parasite exhibited reduced pathogenicity., Conclusions: PbMAP1 is involved in the binding of P. berghei to heparan sulfate-like receptors on RBC surface during merozoite invasion., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The control of malaria parasite transmission from mosquitoes to humans is hampered by decreasing efficacies of insecticides, development of drug resistance against the last-resort antimalarials, and the absence of effective vaccines. Herein, the anti-plasmodial transmission blocking activity of a recombinant Aspergillus oryzae (A. oryzae-R) fungus strain, which is used in human food industry, was investigated in laboratory-reared Anopheles stephensi mosquitoes. The recombinant fungus strain was genetically modified to secrete two anti-plasmodial effector peptides, MP2 (midgut peptide 2) and EPIP (enolase-plasminogen interaction peptide) peptides. The transstadial transmission of the fungus from larvae to adult mosquitoes was confirmed following inoculation of A. oryzae-R in the water trays used for larval rearing. Secretion of the anti-plasmodial effector peptides inside the mosquito midguts inhibited oocyst formation of P. berghei parasites. These results indicate that A. oryzae can be used as a paratransgenesis model carrying effector proteins to inhibit malaria parasite development in An. stephensi. Further studies are needed to determine if this recombinant fungus can be adapted under natural conditions, with a minimal or no impact on the environment, to target mosquito-borne infectious disease agents inside their vectors., (© 2023. The Author(s).)

Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Chemical genetic approaches have had a transformative impact on discovery of drug targets for malaria but have primarily been used for parasite targets. To identify human pathways required for intrahepatic development of parasite, we implemented multiplex cytological profiling of malaria infected hepatocytes treated with liver stage active compounds. Some compounds, including MMV1088447 and MMV1346624, exhibited profiles similar to cells treated with nuclear hormone receptor (NHR) agonist/antagonists. siRNAs targeting human NHRs, or their signaling partners identified eight genes that were critical for Plasmodium berghei infection. Knockdown of NR1D2, a host NHR, significantly impaired parasite growth by downregulation of host lipid metabolism. Importantly, treatment with MMV1088447 and MMV1346624 but not other antimalarials, phenocopied the lipid metabolism defect of NR1D2 knockdown. Our data underlines the use of high-content imaging for host-cellular pathway deconvolution, highlights host lipid metabolism as a drug-able human pathway and provides new chemical biology tools for studying host-parasite interactions., Competing Interests: Declaration of interests All authors affiliated with Recursion Pharmaceuticals, Inc have real or potential ownership of public equities in the company and thereby declare a potential conflict., (Copyright © 2023. Published by Elsevier Ltd.)

Plasmodium sporozoites associated with the midgut and in the hemolymph of mosquitoes differ from sporozoites in the secretory cavities and ducts of the insects' salivary glands in their transcriptome, proteome, motility, and infectivity. Using an ex vivo Anopheles stephensi salivary gland culture system incorporating simple microfluidics and transgenic Plasmodium berghei with the fluorescent protein gene mCherry under the transcriptional control of the Pbuis4 promoter whose expression served as a proxy for parasite maturation, we observed rapid parasite maturation in the absence of salivary gland invasion. While in vivo Pbuis4::mCherry expression was only detectable in sporozoites within the salivary glands (mature parasites) as expected, the simple exposure of P. berghei sporozoites to dissected salivary glands led to rapid parasite maturation as indicated by mCherry expression. These results suggest that previous efforts to develop ex vivo and in vitro systems for investigating sporozoite interactions with mosquito salivary glands have likely been unsuccessful in part because the maturation of sporozoites leads to a loss in the ability to invade salivary glands.

All protozoan parasites are lacking the pathway to synthesize purines de novo and therefore they depend on their host cells to provide purines. A number of highly conserved nucleoside transporter (NT) proteins are encoded in malaria parasite genomes, of which NT1 is characterized in Plasmodium falciparum and P. yoelii as a plasma membrane protein that is responsible for salvage of purines from the host, and NT2 is an endoplasmic membrane NT protein. Whereas NT3 is only present in primate malaria parasites, little is known about NT4, which is conserved in all malaria parasite species. Herein, we targeted NT4 gene for deletion in P. berghei . NT4 knockout parasites developed normally as blood stages, ookinetes and formed oocysts with sporozoites compared with wild-type (WT) P. berghei ANKA parasites. However, nt4(-) sporozoites showed significantly decreased egress from oocysts to hemolymph, significant reduction of colonization of the salivary glands, and complete abolishment of infection of the mammalian host by salivary gland and hemolymph sporozoites. Therefore, we identify NT4 as a NT that is important, not for replication and growth, but for sporozoite infectivity functions.

Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCF FBXO1 ) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCF FBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts., (© 2023. The Author(s).)

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In the acidic lysosome-like digestive vacuole, Plasmodium parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the residual hemozoin is phagocytized by macrophages principally in the liver and spleen where the heme crystals can persist for months to years, as heme oxygenase does not readily degrade the crystal. Previous studies demonstrated hemozoin modulation of monocytes and macrophages. Hemozoin modulates immune function activity of monocytes/macrophages. Here, we used purified/washed hemozoin (W-Hz) isolated from murine Plasmodium berghei infections and intravenously (i.v.) injected it back into naive mice. We characterized the modulating effect of W-Hz on liver-stage replication. Purified washed hemozoin decreases P. berghei liver levels both at 1 week and 1 month after i.v. injection in a dose and time dependent fashion. The injected hemozoin fully protected in nine out of 10 mice given a 50 sporozoite inoculum, and in 10 out of 10 mice against 2,000 sporozoites when they were infected an hour or a day after hemozoin inoculation. DNase treatment at the hemozoin reversed the observed liver load reduction. The liver load reduction was similar in mature B- and T-cell-deficient RAG-1 knockout (KO) mice suggesting an innate immune protection mechanism. This work indicates a role for residual hemozoin in down modulation of Plasmodium liver stages.

During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3' untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2- glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.

The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in human; therefore, these can be explored as potential drug targets. We deleted the MCA-2 gene from Plasmodium berghei genome using a gene knockout strategy to decipher its precise function. This study has identified that MCA-2 plays an important role in parasite transmission since it is critical for the formation of gametocytes and for maintaining an appropriate number of infectious sporozoites required for sporogony. It is noticeable that a significant reduction in gametocyte, oocysts, ookinete and sporozoites load along with a delay in hepatocytes invasion were observed in the MCA-2 knockout parasite. Furthermore, a study found the two MCA-2 inhibitory molecules known as C-532 and C-533, which remarkably inhibited the MCA-2 activity, abolished the in vitro parasite growth, and also impaired the transmission cycle of P. falciparum and P. berghei in An. stephensi . Our findings indicate that the deletion of MCA-2 hampers the Plasmodium development during erythrocytic and exo-erythrocytic stages, and its inhibition by C-532 and C-533 critically affects the malaria transmission biology.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups - Nup176, 269, 335, 390, and 434 - are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position. IMPORTANCE The nuclear pore complex (NPC) is a platform for constant evolution and has been used to study the evolutionary patterns of early-branching eukaryotes. The Plasmodium NPC is poorly defined due to its evolutionary divergent nature making it impossible to characterize it via homology searches. Although 2 decades have passed since the publication of the Plasmodium genome, 30% of the genes still lack functional annotation. Our study demonstrates the ability of proximity labeling using TurboID to assign function to orphan proteins in the malaria parasite. We have identified a total of 10 Nups that will allow further study of NPC dynamics, structural elements, involvement in nucleocytoplasmic transport, and unique non-transport functions of nucleoporins that provide adaptability to this malaria parasite.

Malaria is among the deadliest infectious diseases, and Plasmodium , the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to occur. Two promising strategies to curb transmission are transgenesis, consisting of genetically engineering mosquitoes to express antimalarial effector molecules, and paratransgenesis, consisting of introducing into the mosquito commensal bacteria engineered to express antimalarial effector molecules. Although both approaches restrict parasite development in the mosquito, it is not known how their effectiveness compares. Here we provide an in-depth assessment of transgenesis and paratransgenesis and evaluate the combination of the two approaches. Using the Q-system to drive gene expression, we engineered mosquitoes to produce and secrete two effectors - scorpine and the MP2 peptide - into the mosquito gut and salivary glands. We also engineered Serratia , a commensal bacterium capable of spreading through mosquito populations to secrete effectors into the mosquito gut. Whereas both mosquito-based and bacteria-based approaches strongly reduced the oocyst and sporozoite intensity, a substantially stronger reduction of Plasmodium falciparum development was achieved when transgenesis and paratransgenesis were combined. Most importantly, transmission of Plasmodium berghei from infected to naïve mice was maximally inhibited by the combination of the two approaches. Combining these two strategies promises to become a powerful approach to combat malaria., Competing Interests: WH, JV, CK, SC, MJ No competing interests declared

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei . It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.