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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.

tRNAs are not only essential for decoding the genetic code, but their abundance also has a strong impact on the rate of protein production, folding, and on the stability of the translated messenger RNAs. Plasmodium expresses a unique surface protein called tRip, involved in the import of exogenous tRNAs into the parasite. Comparative proteomic analysis of the blood stage of wild-type and tRip-KO variant of P. berghei parasites revealed that downregulated proteins in the mutant parasite are distinguished by a bias in their asparagine content. Furthermore, the demonstration of the possibility of charging host tRNAs with Plasmodium aminoacyl-tRNA synthetases led us to propose that imported host tRNAs participate in parasite protein synthesis. These results also suggest a novel mechanism of translational control in which import of host tRNAs emerge as regulators of gene expression in the Plasmodium developmental cycle and pathogenesis, by enabling the synthesis of asparagine-rich regulatory proteins that efficiently and selectively control the parasite infectivity., (© 2024 International Union of Biochemistry and Molecular Biology.)

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).)

Sexual reproduction in malaria parasites is essential for their transmission to mosquitoes and offers a divergent eukaryote model to understand the evolution of sex. Through a panel of genetic screens in Plasmodium berghei, we identify 348 sex and transmission-related genes and define roles for unstudied genes as putative targets for transmission-blocking interventions. The functional data provide a deeper understanding of female metabolic reprogramming, meiosis, and the axoneme. We identify a complex of a SUN domain protein (SUN1) and a putative allantoicase (ALLC1) that is essential for male fertility by linking the microtubule organizing center to the nuclear envelope and enabling mitotic spindle formation during male gametogenesis. Both proteins have orthologs in mouse testis, and the data raise the possibility of an ancient role for atypical SUN domain proteins in coupling the nucleus and axoneme. Altogether, our data provide an unbiased picture of the molecular processes that underpin malaria parasite transmission. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The integrity of genomes of the two crucial organelles of the malaria parasite - an apicoplast and mitochondrion in each cell - must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1 leader -GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway., (Copyright © 2024 Australian Society for Parasitology. All rights reserved.)

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.)

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei , we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

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.)

Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei , which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.

Background: Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs., Methods: Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA)., Results: We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen., Conclusions: The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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.)

Malaria blood stage parasites commit to either one of two distinct cellular fates while developing within erythrocytes of their mammalian host: they either undergo another round of asexual replication or they differentiate into nonreplicative transmissible gametocytes. Depending on the state of infection, either path may support or impair the ultimate goal of human-to-human transmission via the mosquito vector. Malaria parasites therefore evolved strategies to control investments into asexual proliferation versus gametocyte formation. Recent work provided fascinating molecular insight into shared and unique mechanisms underlying the control and environmental modulation of sexual commitment in the two most widely studied malaria parasite species, Plasmodium falciparum and P. berghei. With this review, we aim at placing these findings into a comparative mechanistic context., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes., (Copyright © 2024. Published by Elsevier B.V.)

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).)

The O-fucosylation of the thrombospondin type I repeat (TSR) domain is important for TSR-containing proteins' optimal folding and stability. However, the importance of Plasmodium O-fucosyltransferase 2 (POFut2) remains unclear due to two different reports. Here, we disrupted the POFut2 gene in Plasmodium berghei and demonstrated that POFut2 KO parasites develop normally in blood and mosquito stages but show reduced infectivity in mice. We found that the reduced infectivity of POFut2 KO sporozoites was due to a diminished level of TRAP that affected the parasite gliding motility and hepatocyte infectivity. Using all-atom MD simulation, we also hypothesize that O-fucosylation impacts the TSR domain's stability more than its heparin binding capacity.

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.)

The successful completion of gamete fertilization is essential for malaria parasite transmission, and this process can be targeted by intervention strategies. In this study, we identified a conserved gene (PBANKA&#95;0813300) in the rodent malaria parasite Plasmodium berghei, which encodes a protein of 54 kDa (designated as Pbs54). Localization studies indicated that Pbs54 is associated with the plasma membranes of gametes and ookinetes. Functional studies by gene disruption showed that the Δpbs54 parasites had no defect in asexual proliferation, gametocyte development, or gametogenesis. However, the interactions between male and female gametes were significantly decreased compared with wild-type parasites. The Δpbs54 lines did not show a further reduction in zygote and ookinete numbers during in vitro culture, indicating that the defects were probably restricted to gamete fertilization. Consistent with this finding, mosquitoes fed on Δpbs54-infected mice showed a 30.1% reduction in infection prevalence and a 74.7% reduction in oocyst intensity. Cross-fertilization assay indicated that both male and female gametes were impaired in the Δpbs54 parasites. To evaluate its transmission-blocking potential, we obtained polyclonal antibodies from mice immunized with the recombinant Pbs54 (rPbs54) protein. In vitro assays showed that anti-rPbs54 sera inhibited ookinete formation by 42.7%. Our experiments identified Pbs54 as a fertility factor required for mosquito transmission and a novel candidate for a malaria transmission-blocking vaccine., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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).)

Long-term protection against malaria remains one of the greatest challenges of vaccination against this deadly parasitic disease. Whole-sporozoite (WSp) malaria vaccine formulations, which target the Plasmodium parasite's pre-erythrocytic stages, include radiation-attenuated sporozoites (RAS), early- and late-arresting genetically-attenuated parasites (EA-GAP and LA-GAP, respectively), and chemoprophylaxis with sporozoites (CPS). Although all these four vaccine formulations induce protective immune responses in the clinic, data on the longevity of the antimalarial protection they afford remain scarce. We employed a mouse model of malaria to assess protection conferred by immunization with P. berghei (Pb)-based surrogates of these four WSp formulations over a 36-week period. We show that EA-GAP WSp provide the lowest overall protection against an infectious Pb challenge, and that while immunization with RAS and LA-GAP WSp elicits the most durable protection, the protective efficacy of CPS WSp wanes rapidly over the 36-week period, most notably at higher immunization dosages. Analyses of liver immune cells show that CD44 hi CD8 + T cells in CPS WSp-immunized mice express increased levels of the co-inhibitory PD-1 and LAG-3 markers compared to mice immunized with the other WSp formulations. This indicates that memory CD8 + T cells elicited by CPS WSp immunization display a more exhausted phenotype, which may explain the rapid waning of protection conferred by the former. These results emphasize the need for a detailed comparison of the duration of protection of different WSp formulations in humans and suggest a more beneficial effect of RAS and LA-GAP WSp compared to EA-GAP or CSP WSp., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Diana Moita reports financial support was provided by Fundacao para a Ciencia e Tecnologia. Miguel Prudencio reports financial support was provided by la Caixa Foundation. Miguel Prudencio reports financial support was provided by GSK OpenLab Foundation., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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: The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes in most human Plasmodium strains. The aim of this study was to generate artemether-resistant (AM R ) parasites from Plasmodium berghei ANKA (AM S ), and determine their fitness cost., Methods: Artemether resistance was generated by increasing drug pressure doses gradually for 9 months. Effective doses (ED 50 and ED 90 ) were determined using the 4-day suppressive test, and the indices of resistance (I) at 50% and 90% (I 50 and I 90 ) were determined using the ratio of either ED 50 or ED 90 of AM R to AM S , respectively. The stability of the AM R parasites was evaluated by: five drug-free passages (5DFPs), 3 months of cryopreservation (CP), and drug-free serial passages (DFSPs) for 4 months. Analysis of variance was used to compare differences in growth rates between AM R and AM S with 95% confidence intervals., Results: ED 50 and ED 90 of AM S were 0.61 and 3.43 mg/kg/day respectively. I 50 and I 90 after 20 cycles of artemether selection pressure were 19.67 and 21.45, respectively; 5DFP values were 39.16 and 15.27, respectively; 3-month CP values were 29.36 and 10.79, respectively; and DFSP values were 31.34 and 12.29, respectively. The mean parasitaemia value of AM R (24.70% ± 3.60) relative to AM S (37.66% ± 3.68) at Day 7 post infection after DFSPs revealed a fitness cost of 34.41%., Conclusion: A moderately stable AM R P. berghei line was generated. Known and unknown mutations may be involved in modulating artemether resistance, and therefore molecular investigations are recommended., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

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 is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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).)