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Lung adenocarcinoma is the most prevalent subtype of lung cancer, which is the leading cause of cancer-related mortality worldwide. Toxoplasma gondii ( T.gondii ) Rhoptry protein 16 (ROP16) has been shown to quickly enter the nucleus, and through activate host cell signaling pathways by phosphorylation STAT3 and may affect the survival of tumor cells. This study constructed recombinant lentiviral expression vector of T. gondii ROP16 I/II/III and stably transfected them into A549 cells, and the effects of ROP16 on cell proliferation, cell cycle, apoptosis, invasion, and migration of A549 cells were explored by utilizing CCK-8, flow cytometry, qPCR, Western blotting, TUNEL, Transwell assay, and cell scratch assay, and these effects were confirmed in the primary human lung adenocarcinoma cells from postoperative cancer tissues of patients. The type I and III ROP16 activate STAT3 and inhibited A549 cell proliferation, regulated the expression of p21, CDK6, CyclinD1, and induced cell cycle arrest at the G1 phase. ROP16 also regulated the Bax, Bcl-2, p53, cleaved-Caspase3, and Caspase9, inducing cell apoptosis, and reduced the invasion and migration of A549 cells, while type II ROP16 protein had no such effect. Furthermore, in the regulation of ROP16 on primary lung adenocarcinoma cells, type I and III ROP16 showed the same anticancer potential. These findings confirmed the anti-lung adenocarcinoma effect of type I and III ROP16, offering fresh perspectives on the possible application of ROP16 as a target with adjuvant therapy for lung adenocarcinoma and propelling the field of precision therapy research toward parasite treatment of tumors.

The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis, the second largest parasitic killer in the world behind malaria. A large percentage of Leishmania membrane phospholipids is phosphatidylcholine (PC), formed via the Kennedy pathway, where the enzyme CTP: phosphocholine cytidylyltransferase (CCT) catalyzes the second, rate limiting step. Leishmania major CCT was expressed in non-pathogenic Leishmania tarentolae and exhibited activity that increased 10-fold in the presence of PC:oleate lipid vesicles. Confocal microscopy of L. tarentolae expressing L. major CCT fused to a red fluorescent protein revealed the enzyme is cytoplasmic but may associate with internal membranes. A truncated mutant of L. major CCT containing the catalytic domain was expressed in Escherichia coli and in vitro analysis of the enzyme showed catalysis was divalent cation-dependent and yielded a V max of 374 nmol/min/mg and K m values of 0.0648 mM and 3.74 mM, respectively, for the substrates CTP and phosphocholine., 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 Elsevier Inc. All rights reserved.)

The malaria-causing parasite, P. falciparum, replicates through schizogony, a tightly orchestrated process where numerous daughter parasites are formed simultaneously. Proper division and segregation of one-per-cell organelles, like the mitochondrion and apicoplast, are essential, yet remain poorly understood. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during last-stage schizogony. Analysis of focused ion beam scanning electron microscopy data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation., (© 2024 Verhoef et al.)

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis., (© 2024 The Protein Society.)

Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.

Prenyltransferases act essential roles in the prenylation modification, which is significant for proteins, like small GTPases to execute various important activities in Toxoplasma gondii ( T.gondii ). The structures and partial functions of prenyltransferases (FTase, GGTase-I, and GGTase-II) in prenylation process have been dissected in T. gondii . However, the cellular effects of prenyltransferases on type 2-ME49 strain of Toxoplasma are largely unknown. To address this gap, CRISPR/Cas9-based gene-editing technology was employed to construct conditional knockdown strains of prenyltransferases in ME49 strain. Subsequent observation of ingestion ability of host cytosolic molecules (e.g, green fluorescent protein [GFP]) and status of secretory vacuolar sorting post-knockdown of prenyltransferases revealed significant findings. Our study demonstrated that degradation of FTase and GGTase-II notably affected the trafficking of endocytic GFP and vacuolar secretory trafficking to rhoptry bulb. Additionally, depletion of GGTase-II led to disordered endoplasmic reticulum and microtubules, as well as impaired gliding motility. The integrity of mitochondrion was damaged after degradation of GGTase-I. These findings underscore the critical functions of prenyltransferases in endocytosis and secretory vacuolar sorting in ME49 strain of T. gondii , thereby enhancing our understanding of prenyltransferases as potential drug targets.

Malaria is among the deadliest infectious diseases. Over 200 million annual clinical malaria cases are reported and more than half a million people, mostly children, die every year. The most advanced RTS,S/AS01 vaccine based on the P. falciparum circumsporozoite protein (CSP), targets sporozoite liver infection but achieved modest efficacy. To reduce malaria death, novel malaria vaccine development is a high priority. Most malaria vaccine candidates target three infection steps: sporozoite liver infection, merozoite red blood cell (RBC) infection, and mosquito midgut infection. However, only few malaria vaccine candidates target specific parasite-host cell interactions. Our group has implemented the phage peptide-display approach to discover new parasite ligands and host cell receptors. Here we summarize our findings and discuss their potential for the development of novel vaccines.

Toxoplasma gondii is a protozoan apicomplexan parasite that uses an adhesion-dependent mode of motility termed gliding to access host cells and disseminate into tissues. Previous studies on Apicomplexa motile morphotypes, including the T. gondii tachyzoite, have identified a cortical actin-myosin motor system that drives the rearward translocation of transmembrane adhesins, thus powering forward movement. However, this model is currently questioned. Here, combining micropatterning and tunable surface chemistry (to edit parasite surface ligands) with flow force and live or super-resolution imaging, we show that tachyzoites build only one apical anchoring contact with the substrate, over which it slides. Furthermore, we show that glycosaminoglycan-parasite interactions are sufficient to promote such force-productive contact and find that the apicobasal flow is set up independent of adhesin release and surface interactions. These findings should enable further characterization of the molecular functions at the T. gondii-substrate mechanosensitive interface and their comparison across apicomplexans., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The anaphase promoting complex (APC or cyclosome) is a major ubiquitin ligase that coordinates mitotic and G1 progression, acting as a major regulator of chromosome segregation. While the human APC contains fourteen subunits, it is yet to be explored in the pathogen Entamoeba histolytica. Our study reveals the existence of a single functional Apc10 homolog in E. histolytica, which acts as a processivity factor of ubiquitin ligase activity in human. A cDNA library generated from HM1:IMSS strain of E. histolytica was screened for interaction partners of EhApc10 in yeast two hybrid study. The novel interactor, a glycolytic enzyme, pyruvate phosphate dikinase (Ppdk) was found to interact with EhApc10 and further validated by in vitro assay. A comprehensive in silico study has emphasized the structural and functional aspects, encompassing physicochemical traits, predictive 3D structure modelling, validation of EhApc10-EhPpdk interaction through molecular docking and simulation. The interplay between a cell cycle protein and a glycolytic enzyme highlights the connection between cellular metabolism and the cell cycle regulatory mechanism. The study serves as the groundwork for future research on the non-mitotic role of APC beyond cell cycle., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Theileria annulata (T. annulata) is intra-erythrocytic protozoan parasite which is more prevalent in tropical and sub-tropical countries. It has a significant economic impact on the productivity of the dairy industry, and buparvaquone is used to treat infected animals in the prevalent regions of the world. Systematically, buparvaquone targets the cyto-b gene to break the electron transport chain (ETC) and Theileria annulata peptidyl-prolyl isomerase 1 (TaPIN1) gene to destabilize transcription factor JUN (c-JUN) to inhibit proliferation of infected cells, which ultimately leads to the death of T. annulata. The reported studies on drug resistance is due to inappropriate drug application, evolutionary characteristics of the cytochrome b (cyto-b) gene and oncogenic signaling pathways gene (TaPIN1) make the parasite resistant against buparvaquone. Hence, this systematic review was designed to find out non-synonymous mutation in genes (cyto-b and TaPIN1) responsible for drug resistance reported from Tunisia, Turkey, Egypt, Sudan, Iran, Pakistan, China and Germany with reference to the T. annulata Ankara strain of cyto-b (accession no. XM_949625.1) and TaPIN1 (accession no. TA18945) wild type genes. Non-synonymous point mutations were found in cyto-b (Q 01 at 130-148 and Q 02 at 253-262 regions) and TaPIN1 (A53P and A53T) genes. These point mutations are responsible for developing buparvaquone resistance against T. annulata infection. These genes can be used as biomarkers for the identification of drug resistance in any endemic area. To avoid the complication of drug resistance, development of genetically resistant cattle breeds, potent vaccines and anti-theilerial drugs (Trifloxystrobin and anti-cancerous) are currently required to control proliferating economically important T. annulata parasites., Competing Interests: Declaration of Competing Interest All 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 Elsevier B.V. All rights reserved.)

Alexandrium pacificum is a toxic dinoflagellate resulting in harmful algal blooms (HABs). ApDOT1.9 is a methyltransferase involved in the rapid growth regulation of A. pacificum, but its protein interaction information is still limited. In this study, 14 candidate interacting proteins of ApDOT1.9, which were involved in metabolism, genetic information processing, environmental information processing and cellular processes, were screened. The interaction between candidate interactor ApSNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors of Alexandrium pacificum) and ApDOT1.9 was further validated by molecular docking and GST (Glutathione S transferase) pull-down. The relevant biological functional information and gene expression of ApSNARE were also analyzed and detected. These results indicate that ApSNARE was an interactor of ApDOT1.9 and it may also participate in A. pacificum rapid growth regulation under high light or high nitrogen conditions, which will provide preliminary information on the interaction proteins of ApDOT1.9 and molecular regulation mechanisms of growth in A. pacificum., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Apicomplexa parasitic phylum rhoptry neck protein 2 (RON2) plays a key role in the process of invading host cells. Eimeria tenella, an intracellular protozoan shares a similar conserved invasion pattern. However, whether E. tenella RON2 participates in the process of invading the host intestinal epithelium is poorly understood. In this study, the sequence of EtRON2 was analyzed and expressed. The expression of the truncated extracellular N-terminal fragment of EtRON2 (403-700 aa, designated EtRON2 403-700 ) with a molecular mass of 38.3 kDa. EtRON2 in the sporozoite protein was detected at 151.4 kDa by rabbit anti-rEtRON2 403-700 antibody. Immunofluorescence results showed that EtRON2 was mainly localized to the nucleus and apex of the E. tenella sporozoite. qPCR results showed that the highest expression level of EtRON2 was detected in sporulated oocysts compared with other developmental stages of E. tenella. In vitro invasion inhibition assays showed that the capacity of sporozoites to invade DF-1 cells was significantly inhibited after pretreatment with the rabbit anti-rEtRON2 403-700 antibody. Silencing the EtRON2 gene by RNA interference (RNAi) significantly inhibited EtRON2 expression and significantly reduced the invasion of DF-1 cells by sporozoites. In vivo experiments revealed a significant decrease parasite burden and oocyst outputs in chicks after infection with EtRON2 gene-silenced sporozoites by cloacal inoculation. Recombinant EtRON2 403-700 (rEtRON2 403-700 ) immunizes chicks effectively against E. tenella infection by inducing humoral immunity and upregulating IFN-γ and CD8 + T lymphocytes. Furthermore, chicks exhibited increased relative weight gain rates, lower cecum lesion scores, and reduced oocyst outputs during the E. tenella challenge. H&E staining showed that the cecum tissue of chicks immunized with rEtRON2 403-700 showed relatively mild histopathological changes. In conclusion, the results of this study demonstrated that EtRON2 plays a key role in E. tenella invasion of the host intestinal epithelium and provides a potential target for vaccines against E. tenella infection., 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 Elsevier B.V. All rights reserved.)

Proteostasis, including protein folding mediated by molecular chaperones, protein degradation, and stress response pathways in organelles like ER (unfolded protein response: UPR), are responsible for cellular protein quality control. This is essential for cell survival as it regulates and reprograms cellular processes. Here, we underscore the role of the proteostasis pathway in Apicomplexan parasites with respect to their well-characterized roles as well as potential roles in many parasite functions, including survival, multiplication, persistence, and emerging drug resistance. In addition to the diverse physiological importance of proteostasis in Apicomplexa, we assess the potential of the pathway's components as chemotherapeutic targets., 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 Elsevier B.V. All rights reserved.)

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.)

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica, however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Repetitive elements in DNA sequences are a hallmark of Apicomplexan protozoa. A genome-wide screening for Tandem Repeats was conducted in Toxoplasma gondii and related Coccidian parasites with a novel strategy to assess compositional bias. A conserved pattern of GC skew and purine-pyrimidine bias was observed. Compositional bias was also present at the protein level. Glutamic acid was the most abundant amino acid in the purine (GA) rich cluster, while Serine prevailed in pyrimidine (CT) rich cluster. Purine rich repeats, and consequently glutamic acid abundance, correlated with high scores for intrinsically disordered protein regions/domains. Finally, variability was established for repetitive regions within a well-known rhoptry antigen (ROP1) and an uncharacterized hypothetical protein with similar features. The approach we present could be useful to identify potential antigens bearing repetitive elements., 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 Elsevier B.V. All rights reserved.)

Background: TgMIC1, a soluble adhesion protein that typically facilitates parasite invasion, exhibited varying expression levels among distinct virulence strains of Chinese 1 Toxoplasma. This study aims to explore its role in immunological regulation and its association with diverse postinfection outcomes in Toxoplasma infection., Methods: First, the mic1 knockout strain Wh3Δmic1 was generated and assessed for its virulence and proliferative capacity. Subsequently, the serum inflammation levels were examined in mice infected with Wh3Δmic1, Wh3, and Wh6. Furthermore, rMIC1 and rMIC1-T126A/T220A, which lack binding sites to N-glycan in TLR4, were produced for coculture with bone marrow-derived macrophages (BMDMs) to investigate their impact on pyroptosis., Results: Our data showed Wh3Δmic1 exhibited a significant reduction in invasion efficiency, limited growth, and attenuated inflammatory responses in mice. Additionally, it displayed a decreased capacity to induce pyroptosis when compared with Wh3-infected BMDMs. Moreover, rMIC1 but not rMIC1-T126A/T220A was found to be able to upregulate NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and activate GSDMD and caspase-1 in BMDMs but not in TLR4 -/- and NLRP3 -/- BMDMs., Conclusions: TgMIC1 is implicated in both parasite invasion and the modulation of macrophage pyroptosis via the TLR4/NLRP3 pathway. This investigation indicates that TgMIC1 serves diverse functions in Toxoplasma gondii infection, thereby enhancing comprehension of the immune regulatory mechanisms of the parasite., Competing Interests: Declarations. Ethics approval and consent to participate: The animal study was conducted in accordance with the National Guidelines for Animal Use in Research (China), the permission was obtained and approved by the Ethics Committee at Anhui Medical University Institute of Biomedicine (LLSC20200832). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Surveillance of drug resistance and the discovery of novel targets-key objectives in the fight against malaria-rely on identifying resistance-conferring mutations in Plasmodium parasites. Current approaches, while successful, require laborious experimentation or large sample sizes. To elucidate shared determinants of antimalarial resistance that can empower in silico inference, we examined the genomes of 724 Plasmodium falciparum clones, each selected in vitro for resistance to one of 118 compounds. We identified 1448 variants in 128 recurrently mutated genes, including drivers of antimalarial multidrug resistance. In contrast to naturally occurring variants, those selected in vitro are more likely to be missense or frameshift, involve bulky substitutions, and occur in conserved, ordered protein domains. Collectively, our dataset reveals mutation features that predict drug resistance in eukaryotic pathogens.

Ubiquitin C-terminal hydrolase 37 (UCH37 also known as UCHL5) is a conserved deubiquitinating enzyme (DUB) with dual roles in proteasomal degradation and chromatin remodeling in humans. Its Plasmodium falciparum ortholog, PfUCH37, is unusual in that it possesses both DUB and deneddylating activities. While PfUCH37 is enriched in proteasome preparations, its direct interaction and broader functions in Plasmodium remain unclear, particularly given the absence of the chromatin remodeling complex INO80 homologs. This study utilizes transgenic parasites and proteomics to identify PfUCH37-associating proteins. We confirm a direct interaction with the proteasome and demonstrate that the interaction mechanism is evolutionarily conserved. Notably, we discover a divergence in localization compared to the human enzyme and identify novel interacting partners, suggesting alternative functions for PfUCH37 in Plasmodium. These findings provide insights into the unique biology of this enzyme in malaria parasites, potentially opening avenues for targeted therapeutic interventions., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The PIWI-interacting RNA (piRNA) pathway is crucial for transposon repression and the maintenance of genomic integrity. Gametocyte-specific factor 1 (GTSF1), a PIWI-associated protein indispensable for transposon repression, has been recently shown to potentiate the catalytic activity of PIWI in many metazoans. Whether the requirement of GTSF1 extends to PIWI proteins beyond metazoans is unknown. In this study, we identified a homolog of GTSF1 in the unicellular eukaryote Paramecium tetraurelia (PtGtsf1) and found that its role as a PIWI-cofactor is conserved. PtGtsf1 interacts with PIWI (Ptiwi09) and Polycomb Repressive Complex 2 and is essential for PIWI-dependent DNA elimination of transposons during sexual development. PtGtsf1 is crucial for the degradation of PIWI-bound small RNAs that recognize the organism's own genomic sequences. Without PtGtsf1, self-matching small RNAs are not degraded and results in an accumulation of H3K9me3 and H3K27me3, which may disturb transposon recognition. Our results demonstrate that the PIWI-GTSF1 interaction also exists in unicellular eukaryotes with a role in transposon silencing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Giardia duodenalis (G. duodenalis) is a globally distributed zoonotic protozoan that parasitizes the small intestines of humans and various mammals, such as goats and sheep. The objective of this study was to establish a convenient, accurate, and specific method based on restriction fragment length polymorphism (RFLP) for genotyping assemblages A, B and E of G. duodenalis in goats. The β-giardin gene was amplified using primer pairs bgF1, bgR1, bgF2 and bgR2 by nested PCR. The PCR products were digested with the restriction enzymes Hinf I and Bgl I. The established PCR-RFLP method was used to detect and analyze the genetic subtypes of G. duodenalis in 130 fecal samples from goats and compared simultaneously with microscopic examination and nucleic acid sequencing for G. duodenalis., Results: Genetic sequencing confirmed that the PCR-RFLP method accurately distinguished G. duodenalis assemblages A, B and E, as well as different combinations of mixed infections of these three assemblages. Among the 130 samples tested by PCR-RFLP, a total of 26 samples (20.00%) tested positive for G. duodenalis, a higher sensitivity than microscopic examination at 13.85% (18/130). Sequence alignment analysis revealed that among the 26 PCR-positive samples, two were identified as assemblage AI, while the remaining 24 were identified as assemblage E or E12., Conclusions: This study established an accurate, efficient and rapid PCR-RFLP genotyping method using the bg sequence of G. duodenalis, enabling accurate identification and effective differentiation of goat-derived G. duodenalis assemblages without requiring sequencing., Competing Interests: Declarations. Ethics approval and consent to participate: This study was conducted under the approval and instructions of the ethics committee of Foshan University and the animal ethics requirements of the People’s Republic of China [No. FS2022107]. Permissions was obtained from animal owners before stool sampling, and none of the animals were injured during the specimen collection. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Plasmodium vivax transmission occurs throughout the tropics and is an emerging threat in areas of Plasmodium falciparum decline, causing relapse infections that complicate treatment and control. Targeted sequencing for P. falciparum has been widely deployed to detect population structure and the geographic spread of antimalarial and diagnostic resistance. However, there are fewer such tools for P. vivax. Leveraging global variation data, we designed four molecular inversion probe (MIP) genotyping panels targeting geographically differentiating SNPs, neutral SNPs, putative antimalarial resistance genes, and vaccine candidate genes. We deployed these MIP panels on 866 infections from the Peruvian Amazon and identified transmission networks with clonality (IBD[identity by descent]>0.99), copy number variation in Pvdbp and multiple Pvrbps, mutations in antimalarial resistance orthologs, and balancing selection in 13 vaccine candidate genes. Our MIP panels are the broadest genotyping panel currently available and are poised for successful deployment in other regions of P. vivax transmission., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Acrasids are amoebae with the capacity to form multicellular fruiting bodies in a process known as aggregative multicellularity (AGM). This makes acrasids the only known example of multicellularity among the earliest branches of eukaryotes (the former Excavata). Here, we report the Acrasis kona genome sequence plus transcriptomes from pre-, mid- and post-developmental stages. The genome is rich in novelty and genes with strong signatures of horizontal transfer, and multigene families encode nearly half of the amoeba's predicted proteome. Development in A. kona appears molecularly simple relative to the AGM model, Dictyostelium discoideum. However, the acrasid also differs from the dictyostelid in that it does not appear to be starving during development. Instead, developing A. kona appears to be very metabolically active, does not induce autophagy and does not up-regulate its proteasomal genes. Together, these observations strongly suggest that starvation is not essential for AGM development. Nonetheless, development in the two amoebae appears to employ remarkably similar pathways for signaling, motility and, potentially, construction of an extracellular matrix surrounding the developing cell mass. Much of this similarity is also shared with animal development, suggesting that much of the basic tool kit for multicellular development arose early in eukaryote evolution., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: The genetic diversity of malaria parasites contributes to their ability to adapt to environmental changes, develop drug resistance and circumvent the host immune system. This study aimed to analyse the genetic diversity of the Pfmsp1 and Pfmsp2 genes in Plasmodium falciparum and the Pvmsp-3α gene in Plasmodium vivax isolates from District Nowshera in Pakistan., Methods: Blood samples from 124 consenting patients with uncomplicated malaria presenting to different hospitals from the Nowshera district were collected between March and August 2019, representing 28 P. falciparum and 96 P. vivax isolates. The genomic DNA extracted from the isolates were subjected to nested PCR and allele-specific analysis. Pvmsp-3α amplified fragments were further treated with restriction fragment length polymorphism (RFLP)-based Hha1 restriction enzyme., Results: Of the analyzed P. falciparum, 21 distinct alleles were detected, including 14 alleles for Pfmsp-1 and 7 alleles for Pfmsp-2. The sub-allelic families MAD20 (50%) of Pfmsp-1and FC27 (75%) of Pfmsp-2 were predominant. The multiplicity of infection (MOI) was calculated as 1.4 and 1.2 for Pfmsp-1 and Pfmsp-2, respectively, with an overall mean MOI of 1.34. In P. vivax, 4 allelic variants, Pvmsp-3α types A, B, C and D, were detected, while RFLP digestion of amplicons, detected 9 sub-allelic variants (A1-A4, B1, B2, C1, C2 and D1) at the Pvmsp-3α locus., Conclusion: This first ever report of molecular characterization of P. falciparum and P. vivax genotypes from District Nowshera, Pakistan reveals moderate to high allelic diversity in parasite population from District Nowshera, Pakistan., Competing Interests: Declarations. Ethics approval and consent to participate: The study protocol was approved by the Ethical Review Committee of Abdul Wali Khan University Mardan under the letter of AWKUM/ERC/578. Consent was obtained from all participants before conducting the study. Consent for publication: All authors have given their consent for publication of this manuscript. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Introduction: Malaria molecular surveillance has the potential to generate information on biological threats that compromise the effectiveness of antimalarial interventions. This study aims to streamline surveillance activities to inform the new strategic plan of the Mozambican National Malaria Control Programme (2023-2030) for malaria control and elimination., Methods and Analyses: This prospective genomic surveillance study aims to generate Plasmodium falciparum genetic data to monitor diagnostic failures due to pfhrp2/3 deletions and molecular markers of antimalarial drug resistance, to characterise transmission sources and to inform the implementation of new antimalarial approaches to be introduced in Mozambique (chemoprevention and child malaria vaccination). The study, to be conducted between 2024 and 2026, will use three sampling schemes: a multicluster probabilistic health facility survey in the 10 provinces of the country to detect pfhrp2/3 deletions and markers of antimalarial drug resistance; dense sampling of all clinical cases in representative districts in the south targeted for elimination to characterise malaria importation and identify sources of transmission; and testing of pregnant women for malaria at their first antenatal care visit to assess malaria burden and molecular trends. Using a multiplex amplicon-based sequencing approach, the study will target microhaplotypes informative of genomic diversity and relatedness, as well as key drug resistance-associated genes, pfhrp2/3 deletion and malaria vaccine targets. Key genomic information will be visualised in a dashboard integrated into the District Health Information System V.2-based Malaria Information Storage System for programmatic use., Ethics and Dissemination: The protocol was reviewed and approved by the national ethics committee of Mozambique (Comité Nacional de Bioética para Saúde, Ref: 680/CNBS/23). Project results will be presented to all stakeholders using study-specific brochures and published in open-access journals., Trial Registration Number: NCT06529237., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study., Methods: By meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing., Results: The entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 10 4 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples., Conclusions: This study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site., Competing Interests: Declarations. Ethics approval and consent to participate: The studies involving humans were approved by the medical ethics committee of the Affiliated Hospital of Youjiang Medical University for Nationalities. The studies were conducted in accordance with local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Benznidazole (BZ) is the trypanocidal compound of choice for Chagas disease, a neglected tropical disease in the Americas. However, this drug often fails to cure the infection. The regulation of gene expression in Trypanosoma cruzi, the causative agent of Chagas disease, is based on post-transcriptional mechanisms. When environmental changes cause translational arrest, RNA-binding proteins, and their target mRNAs assemble into cytoplasmic bodies, known as RNA granules, which act as RNA sorting centers. We have characterized the T. cruzi RNA-binding protein DRBD3, which has two RRMs domains, and a C-terminal low-complexity sequence rich in proline and glutamines. Using a tagged form of TcDRBD3 (rTcDRBD3), we showed that this protein resides in the cytoplasm, but localizes into perinuclear cytoplasmic foci after BZ exposure. RNA staining after BZ also showed that this molecule accumulates into perinuclear cytoplasmic foci. Moreover, BZ and puromycin treatment enhanced the colocalization of rTcDRBD3 and RNA, suggesting that TcDRBD3 granules repertoire harbors RNAs released from polysomes. Under starvation, rTcDRBD3 granules localized throughout the cytoplasm and also increased in number in the presence of puromycin. Our results suggest that TcDRBD3 accumulates into perinuclear granules that harbor RNA and also that its localization varies according to the type of stress.

The actin cytoskeleton is a ubiquitous feature of eukaryotic cells, yet its complexity varies across different taxa. In the parasitic protist Trypanosoma brucei , a rudimentary actomyosin system consisting of one actin gene and two myosin genes has been retained despite significant investment in the microtubule cytoskeleton. The functions of this highly simplified actomyosin system remain unclear, but appear to centre on the endomembrane system. Here, advanced light and electron microscopy imaging techniques, together with biochemical and biophysical assays, were used to explore the relationship between the actomyosin and endomembrane systems. The class I myosin (TbMyo1) had a large cytosolic pool and its ability to translocate actin filaments in vitro was shown here for the first time. TbMyo1 exhibited strong association with the endosomal system and was additionally found on glycosomes. At the endosomal membranes, TbMyo1 colocalised with markers for early and late endosomes (TbRab5A and TbRab7, respectively), but not with the marker associated with recycling endosomes (TbRab11). Actin and myosin were simultaneously visualised for the first time in trypanosomes using an anti-actin chromobody. Disruption of the actomyosin system using the actin-depolymerising drug latrunculin A resulted in a delocalisation of both the actin chromobody signal and an endosomal marker, and was accompanied by a specific loss of endosomal structure. This suggests that the actomyosin system is required for maintaining endosomal integrity in T. brucei ., Competing Interests: FL, SJ, XM, FZ, AK, AB, CB, MW, MP, AN, JK, CV, ME, BM No competing interests declared, (© 2024, Link et al.)

The Tyrosine Kinase-Like (TKL) family of proteins are a set of poorly studied kinases that have garnered attention in recent years for their role in Toxoplasma biology. The Toxoplasma genome contains eight TKL kinases, of which six have been predicted to be important for parasite propagation. We have previously shown that TgTKL1 is a nuclear kinase that is critical for the parasite lytic cycle and is essential for acute virulence in the animal model. However, the contribution of the kinase domain to the functioning of TgTKL1 was not known. Hence to determine the significance of its catalytic function, we first validated that TgTKL1 is a true kinase using purified recombinant protein. Furthermore, we successfully generated a TgTKL1 kinase mutant strain of Toxoplasma via CRISPR-Cas9 gene editing. Our studies revealed that the kinase mutant of TgTKL1 displays defects in parasite growth and host-cell invasion. Additionally, loss of kinase function alters the transcriptomic profile of the parasite, including downregulation of the invasion-related gene, TgSUB1. Importantly, this dysregulation of TgSUB1 expression leads to defects in post-exocytosis processing of micronemal proteins, an event critical for normal host-cell invasion. Furthermore, the TgTKL1 kinase mutant is completely avirulent in the mouse model of acute toxoplasmosis. Since the loss of kinase function leads to phenotypic manifestations seen previously with TgTKL1 knockout parasites, we conclude that kinase activity is important for TgTKL1 function in Toxoplasma propagation and virulence., Importance: Toxoplasma gondii is a protozoan parasite that can cause life-threatening disease in humans. Hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. We have previously shown that a member of the TKL protein kinase family, TgTKL1, is a plant-like kinase that is required for effective Toxoplasma growth in vitro and essential for virulence in vivo . Herein, we show that the TgTKL1 is, indeed, a bona fide kinase, and loss of its kinase function in the Toxoplasma leads to similar defects seen in parasites with complete loss of TgTKL1. More specifically, the TgTKL1 kinase mutant exhibits defects in parasite growth, host-cell invasion, gene expression profile, and virulence in the animal model. Together, these findings suggest that TgTKL1 is a true kinase, and loss of its kinase activity leads to disruption of TgTKL1 function in Toxoplasma ., Competing Interests: The authors declare no conflict of interest.

cAMP plays an important role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP, and cAMP-response element binding protein transcription factors, to initiate signal transduction in humans. The Giardia genome only contains two adenylate cyclases (ACs), a single phosphodiesterase (PDE) and a single known PKA effector, and the specific functions of these components are not fully understood. In our previous research, we demonstrated the important role of AC2-dependent cAMP signaling in promoting the encystation program. Using the NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of the PKA regulatory domain (PKAr) and PKA catalytic domain (PKAc). In this study, our objectives are twofold: first, we used the newly developed Split-Halo to examine subcellular interactions of Gl PKAr and Gl PKAc in Giardia ; and second, we investigated whether PKAc regulates encystation-specific proteins. Our findings revealed distinct subcellular locations where Gl PKAr and Gl PKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytoplasm. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Knockdown of Gl PKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in the transcriptional regulation of encystation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its important role in governing Giardia 's encystation process., Importance: The precise timing of interactions and subcellular compartmentation play crucial roles in signal transduction. The co-immunoprecipitation assay (CO-IP) has long been utilized to validate protein-protein interactions; however, CO-IPs lack spatial and temporal resolutions. Our recent study used the NanoBit assay, which showcased the reversible protein-protein interaction between PKAr and PKAc in response to cAMP analogs and encystation stimuli. Expanding on this groundwork, this study employs the Split-Halo assay to uncover the subcellular compartments where the PKAr and PKAc protein-protein interactions take place and respond to encystation stimuli. Taken together, these molecular tools provide spatiotemporal information on the protein-protein interaction, which will be useful in the field., Competing Interests: The authors declare no conflict of interest.

The lifecycle progression of the malaria parasite Plasmodium falciparum requires precise tuning of gene expression including histone methylation. The histone methyltransferase Pf SET10 was previously described as an H3K4 methyltransferase involved in var gene regulation, making it a prominent antimalarial target. In this study, we investigated the role of Pf SET10 in the blood stages of P. falciparum in more detail, using tagged Pf SET10-knockout (KO) and -knockdown (KD) lines. We demonstrate a nuclear localization of Pf SET10 with peak protein levels in schizonts. Pf SET10 deficiency reduces intraerythrocytic growth but has no effect on gametocyte commitment and maturation. Screening of the Pf SET10-KO line for histone methylation variations reveals that lack of Pf SET10 renders the parasites unable to mark H3K18me1, while no reduction in the H3K4 methylation status could be observed. Comparative transcriptomic profiling of Pf SET10-KO schizonts shows an upregulation of transcripts particularly encoding proteins linked to red blood cell remodeling and antigenic variation, suggesting a repressive function of the histone methylation mark. TurboID coupled with mass spectrometry further highlights an extensive nuclear Pf SET10 interaction network with roles in transcriptional regulation and mRNA processing, DNA replication and repair, and chromatin remodeling. The main interactors of Pf SET10 include ApiAP2 transcription factors, epigenetic regulators like Pf HDAC1, chromatin modulators like Pf MORC and Pf ISWI, mediators of RNA polymerase II, and DNA replication licensing factors. The combined data pinpoint Pf SET10 as a histone methyltransferase essential for H3K18 methylation that regulates nucleic acid metabolic processes in the P. falciparum blood stages as part of a comprehensive chromatin modulation network.IMPORTANCEThe fine-tuned regulation of DNA replication and transcription is particularly crucial for the rapidly multiplying blood stages of malaria parasites and proteins involved in these processes represent important drug targets. This study demonstrates that contrary to previous reports the histone methyltransferase Pf SET10 of the malaria parasite Plasmodium falciparum promotes the methylation of histone 3 at lysine K18, a histone mark to date not well understood. Deficiency of Pf SET10 due to genetic knockout affects genes involved in intraerythrocytic development. Furthermore, in the nuclei of blood-stage parasites, Pf SET10 interacts with various protein complexes crucial for DNA replication, remodeling, and repair, as well as for transcriptional regulation and mRNA processing. In summary, this study highlights Pf SET10 as a methyltransferase affecting H3K18 methylation with critical functions in chromatin maintenance during the development of P. falciparum in red blood cells., Competing Interests: The authors declare no conflict of interest.

The malaria parasite Plasmodium falciparum employs antigenic variation of the virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) to escape adaptive immune responses during blood infection. Antigenic variation of PfEMP1 occurs through epigenetic switches in the mutually exclusive expression of individual members of the multi-copy var gene family. var genes are located in perinuclear clusters of transcriptionally inactive heterochromatin. Singular var gene activation is linked to locus repositioning into a dedicated zone at the nuclear periphery and deposition of histone 3 lysine 4 di-/trimethylation (H3K4me2/3) and H3K9 acetylation marks in the promoter region. While previous work identified the putative H3K4-specific methyltransferase PfSET10 as an essential enzyme and positive regulator of var gene expression, a recent study reported conflicting data. Here, we used iterative genome editing to engineer a conditional PfSET10 knockout line tailored to study the function of PfSET10 in var gene regulation. We demonstrate that PfSET10 is not required for mutually exclusive var gene expression and switching. We also show that PfSET10 is dispensable not only for asexual parasite proliferation but also for sexual conversion and gametocyte differentiation. Furthermore, comparative RNA-seq experiments revealed that PfSET10 plays no obvious role in regulating gene expression during asexual parasite development and gametocytogenesis. Interestingly, however, PfSET10 shows different subnuclear localization patterns in asexual and sexual stage parasites and female-specific expression in mature gametocytes. In summary, our work confirms in detail that PfSET10 is not involved in regulating var gene expression and is not required for blood-stage parasite viability, indicating PfSET10 may be important for life cycle progression in the mosquito vector or during liver stage development.IMPORTANCEThe malaria parasite Plasmodium falciparum infects hundreds of millions of people every year. To survive and proliferate in the human bloodstream, the parasites need to escape recognition by the host's immune system. To achieve this, P. falciparum can change the expression of surface antigens via a process called antigenic variation. This fascinating survival strategy is based on infrequent switches in the expression of single members of the var multigene family. Previous research reported conflicting results on the role of the epigenetic regulator PfSET10 in controlling mutually exclusive var gene expression and switching. Here, we unequivocally demonstrate that PfSET10 is neither required for antigenic variation nor the expression of any other proteins during blood-stage infection. This information is critical in directing our attention toward exploring alternative molecular mechanisms underlying the control of antigenic variation and investigating the function of PfSET10 in other life cycle stages., Competing Interests: The authors declare no conflict of interest.

Malaria remains a global health burden, killing over half a million people each year. Decreased therapeutic efficacy to artemisinin, the most efficacious antimalarial, has been detected in sub-Saharan Africa, a worrying fact given that over 90% of deaths occur on this continent. Mutations in Kelch13 are the most well-established molecular marker for artemisinin resistance, but these do not explain all artemisinin-resistant isolates. Understanding the biological underpinnings of drug resistance is key to curbing the emergence and spread of artemisinin resistance. Artemisinin-mediated non-specific alkylation leads to the accumulation of misfolded and damaged proteins and activation of the parasite unfolded protein response (UPR). In addition, the parasite proteasome is vital to artemisinin resistance, as we have previously shown that chemical inhibition of the proteasome or mutations in the β2 proteasome subunit increase parasite susceptibility to dihydroartemisinin (DHA), the active metabolite of artemisinins. Here, we investigate parasites with mutations at the Kelch13 and/or 19S and 20S proteasome subunits with regard to UPR regulation and proteasome activity in the context of artemisinin resistance. Our data show that perturbing parasite proteostasis kills parasites, early parasite UPR signaling dictates DHA survival outcomes, and DHA susceptibility correlates with impairment of proteasome-mediated protein degradation. Importantly, we show that functional proteasomes are required for artemisinin resistance in a Kelch13-independent manner, and compound-selective proteasome inhibition demonstrates why artemisinin-resistant Kelch13 mutants remain susceptible to the related antimalarial peroxide OZ439. These data provide further evidence for targeting the parasite proteasome and UPR to overcome existing artemisinin resistance.IMPORTANCEDecreased therapeutic efficacy represents a major barrier to malaria treatment control strategies. The malaria proteasome and accompanying unfolded protein response are crucial to artemisinin resistance, revealing novel antimalarial therapeutic strategies., Competing Interests: The authors declare no conflict of interest.

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.)

Entamoeba nuttalli is genetically the closest to Entamoeba histolytica, the causative agent of human amebiasis, and its natural host is Macaca species. A unique E. nuttalli specific surface protein (PTORS) containing 42 repeats of octapeptide was identified by comparative genomic analysis of Entamoeba species. We aimed to elucidate the function of this protein. When trophozoites from various E. nuttalli strains were examined by immunofluorescence microscopy and flow cytometry using a PTORS-specific monoclonal antibody, only a limited proportion of trophozoites were stained, indicating that the protein was not commonly expressed in all E. nuttalli trophozoite. The proportion of trophozoites expressing PTORS increased after passage in hamster livers, suggesting that the protein functions in the virulence of trophozoites in the liver tissue. Single-cell analysis revealed that in the cluster including trophozoites with PTORS gene expression, genes of virulence-related proteins were also upregulated. Trophozoites of E. histolytica transfected with PTORS showed enhanced adherence and subsequent phagocytic activity towards human Jurkat cells, independent of the lectin. E. histolytica trophozoites expressing PTORS formed larger liver abscesses in hamsters. These results demonstrate that PTORS is a novel virulence factor in Entamoeba species., 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 Inc. All rights reserved.)

Entamoeba histolytica causes amebiasis, a significant global health issue, with millions affected annually, especially in developing countries. EhDUF2419, an important protein involved in E. histolytica 's queuine salvage pathway and its interaction network, remains unclear. To explore this, we transfected E. histolytica trophozoites with a plasmid encoding Myc-tagged EhDUF2419 and achieved successful overexpression. Through immunoprecipitation with the Myc antibody followed by mass spectrometry, we identified 335 proteins interacting with Myc-tagged EhDUF2419, including over 100 ribosomal proteins, along with translation initiation and elongation factors, and aminoacyl-tRNA synthetases. Ribosome purification revealed the presence of EhDUF2419 in ribosomal protein-enriched fractions. Treatment with queuosine (Q) significantly reduced the EhDUF2419 protein levels and decreased the Q-modified tRNA in Myc-tagged EhDUF2419 overexpressing trophozoites. This effect, which was Q-dependent, was not observed in strains carrying an empty vector control or overexpressing a truncated form of EhDUF2419 lacking catalytic activity. The reduction in the EhDUF2419 protein levels was regulated by proteasome-mediated degradation, as evidenced by the reduced degradation in the presence of MG132, a proteasome inhibitor. Our study uncovers the novel interaction of EhDUF2419 with ribosomal proteins and its regulation by the proteasome machinery, providing new insights into its role in E. histolytica and potential therapeutic strategies.

Background: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H 2 O 2 ., Methods: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding LiΔchrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages., Results: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in LiΔchrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (Sb III ) and isoniazid, reduced tolerance to H 2 O 2 , and unchanged macrophage infectivity compared with LiWT. In contrast, LiΔchrAPX::LbAPX parasites were more sensitive to Sb III and isoniazid, exhibited greater susceptibility to H 2 O 2 -induced oxidative stress, and 72 h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites., Conclusions: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), secreted by the dense granule, play significant roles in modifying the parasitophorous vacuole, maintenance of morphology, and regulating host-cell interactions. However, their precise involvement in tachyzoite egress remains inadequately characterized. In this study, we identified a homologous gene, Ncgra41, corresponding to the dense granule protein 41 (GRA41) of Toxoplasma gondii, which is associated with egress, utilizing NCBI and ToxoDB databases. NcGRA41 is localized extracellularly within dense granules and intracellularly within parasitic vacuoles. Deletion of NcGRA41 did not affect tachyzoites invasion or proliferation but significantly reduced egress capacity and pathogenicity in mice. The phenotypic characteristics were restored in a complementary strain. Further investigation revealed that the absence of NcGRA41 reduced gliding motility and the transcription level of the subtilisin-like protein (SUB1). A microneme secretion assay demonstrated a significant decrease in NcMIC1 secretion, along with reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings demonstrate that NcGRA41, a novel dense granule protein in N. caninum, modulates tachyzoites egress and influences pathogenicity by regulating microneme secretion., Competing Interests: Declarations Ethics approval The mice used in this study were handled in compliance with the recommendations outlined in the “Guidelines for the Care and Use of Laboratory Animals” of the Ministry of Science and Technology of China. Additionally, our research protocol received approval from the Laboratory Animal Welfare and Animal Experimental Ethical Committee of China Agricultural University (Certificate No.: CAU-AW31901202-2–1). Consent to participate Done and confirmed. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmodium knowlesi is a zoonotic form of human malaria, the pathology of which is poorly understood. While the J domain protein (JDP) family has been extensively studied in Plasmodium falciparum , and shown to contribute to malaria pathology, there is currently very limited information on the P. knowlesi JDPs (PkJDPs). This review provides a critical analysis of the literature and publicly available data on PkJDPs. Interestingly, the P. knowlesi genome encodes at least 31 PkJDPs, with well over half belonging to the most diverse types which contain only the signature J domain (type IIIs, 19) or a corrupted version of the J domain (type IVs, 2) as evidence of their membership. The more typical PkJDPs containing other domains typical of JDPs in addition to the J domain are much fewer in number (type IIs, 8; type Is, 2). This study indentifies PkJDPs that are potentially involved in: folding of newly synthesized or misfolded proteins within the P. knowlesi cytosol (a canonical type I and certain typical type IIs); protein translocation (a type III) and folding (a type II) in the ER; and protein import into mitochondria (a type III). Interestingly, a type II PkJDP is potentially exported to the host cell cytosol where it may recruit human HSP70 for the trafficking and folding of other exported P. knowlesi proteins. Experimental studies are required on this fascinating family of proteins, not only to validate their role in the pathology of knowlesi malaria, but also because they represent potential anti-malarial drug targets.

Faithful chromosome segregation in eukaryotes requires the assembly of a bipolar spindle and the faithful attachment of kinetochores to spindle microtubules, which are regulated by various spindle-associated proteins (SAPs) that play distinct functions in regulating spindle dynamics and microtubule-kinetochore attachment. The protozoan parasite Trypanosoma brucei employs evolutionarily conserved and kinetoplastid-specific proteins, including some kinetoplastid-specific nucleus- and spindle-associated proteins (NuSAPs), to regulate chromosome segregation. Here, we characterized NuSAP4 and its functional interplay with diverse SAPs in promoting chromosome segregation in T. brucei. NuSAP4 associates with the spindle during mitosis and concentrates at spindle poles where it interacts with SPB1 and MAP103. Knockdown of NuSAP4 impairs chromosome segregation by disrupting bipolar spindle assembly and spindle pole protein localization. These results uncover the mechanistic role of NuSAP4 in regulating chromosome segregation by promoting bipolar spindle assembly, and highlight the unusual features of mitotic regulation by spindle-associated proteins in this early divergent microbial eukaryote., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The Babesia divergens/B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. Currently, diagnostic antigen of the group has not been established. In this study, we investigated the ortholog of Bd37, a glycosylphosphatidylinositol (GPI)-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the group. From two genomic isolates from sporozoite/sporoblast stages, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3-64.1% amino acid sequence identity. Discriminative blood direct PCR revealed that Bd37 JP-A was encoded in all parasites infecting wild sika deer examined (n=22). While Bd37 JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Sequencing of all JP-A variants revealed that the gene was polymorphic, with a low ratio of non-synonymous to synonymous substitutions (dN/dS) and that a highly polymorphic region was not related to predicted B-cell epitopes. A recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in sika deer in Japan from north (Hokkaido) to south (Kyushu island) across 24 prefectures (n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.6%). The geographical trends in infection rates were consistent. This study demonstrated that direct examination was informative for revealing genetic background and selecting antigen candidates. Bd37 orthologs may serve diagnostic purposes in combination with indirect fluorescence assay, which requires biological isolates.

Current understanding of genetic polymorphisms and natural selection in Plasmodium falciparum circumsporozoite (PfCSP), the leading malaria vaccine, is crucial for the development of next-generation vaccines, and such data is lacking in Africa. Blood samples were collected among Plasmodium-infected individuals living in four Cameroonian areas (Douala, Maroua, Mayo-Oulo, Pette). DNA samples were amplified using nested PCR protocols, sequenced, and BLASTed. Single nucleotide polymorphisms (SNPs) were analysed in each PfCSP region, and their impact on PfCSP function/structure was predicted in silico. The N-terminal region showed a limited polymorphism with four haplotypes, and three novel SNPs (N68Y, R87W, K93E) were found. Thirty-five haplotypes were identified in the central region, with several variants (e.g., NVNP and KANP). The C-terminal region was also highly diverse, with 25 haplotypes and eight novel SNPs (N290D, N308I, S312G, K317A, V344I, D356E, E357L, D359Y). Most polymorphic codon sites were mainly observed in the Th2R subregion in isolates from Douala and Pette. The codon site 321 was under episodic positive selection. One novel (E357L) and three known (K322I, G349D, D359Y) SNPs show an impact on function/structure. This study showed extensive genetic diversity with geographical patterns and evidence of the selection of Cameroonian PfCSP central and C-terminal regions., 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 Elsevier B.V. All rights reserved.)

This study aimed to analyze polymorphisms in Pfcrt, Pfmdr1, and Pfk13 genes' markers of resistance to Artemisinin-based combination therapy (ACT), in Plasmodium falciparum isolates from southern Brazzaville, 15 years after the adoption of ACT in the Republic of Congo. A total of 369 microscopy-confirmed malaria-infected individuals were enrolled from March to October 2021 in the community and in health facilities during a cross-sectional study. The K76T mutation in the Pfcrt gene, N86Y and Y184F mutations in the Pfmdr1 gene were investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) while the codons region (1005-1300) of the Pfmdr1gene, and Pfk13 gene were sequenced. The prevalences of K76T, N86Y, Y184F mutations were 26.0%, 6.8%, and 27.7%, respectively. However, no mutations were detected in codons 1034, 1042, and 1246 of the Pfmdr1 gene. None of the mutations previously associated with artemisinin-based resistance were detected in the Pfk13 gene. The results reveal a significant decrease in the prevalence of K76T, N86Y, Y184F mutations, in Plasmodium falciparum isolates following the change of therapeutic policy. As artemisinin resistance is emerging throughout Africa, continued surveillance for early detection of these mutations and relevant partner markers of drug resistance are recommended in the Republic of Congo., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica, the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E. histolytica. Through gene silencing of highly transcribed Ehrho/Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM's multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E. histolytica, highlighting the significance of EhRacM in both macropinocytosis and cellular migration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shimoyama 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.)

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb -/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4 + T cells and most predominantly CD8 + T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma , the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb -/- and IL-10 -/- mice. Furthermore, resistance against OMP was restored in Tcrb -/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4 + and CD8 + T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii . Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma ., Competing Interests: The authors declare no conflict of interest.

We previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high-confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase that interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signaling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasizes a role in calcium signaling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development., Importance: Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology., Competing Interests: The authors declare no conflict of interest.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles, which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here, we identify a Golgi-localizing protein (ULP1), which is structurally similar to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness that is the result of defects in microneme secretion, invasion, replication, and egress. Using ULP1 as bait for TurboID proximity labeling and immunoprecipitation, we identify 11 more Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii -conserved oligomeric Golgi (COG) complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these 11 proteins on parasite fitness. Together, this work reveals a diverse set of T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites., Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite fitness and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labeling to identify 11 additional Golgi-associated proteins, which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention., Competing Interests: The authors declare no conflict of interest.

Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA for several decades. Here, we demonstrate that 5hmU contents vary among different dinoflagellates and are generated through thymidine hydroxylation. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homolog, catalyzing 5hmU production using both in vivo and in vitro biochemical assays. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae , we depicted a comprehensive 5hmU landscape and found that 5hmU loci are significantly enriched in repeat elements. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements, implying that 5hmU appears to serve as an epigenetic mark for silencing transposon. Together, our results revealed the biogenesis, genome-wide landscape, and molecular function of dinoflagellate 5hmU, providing mechanistic insight into the function of this enigmatic DNA mark., Competing Interests: Competing interests statement:The authors declare no competing interest.

TBVs are suggested to inhibit parasite transmission from humans to Anopheles mosquitoes. For the transmission of Plasmodium parasite, a variety of factors are included in gametes fusion phase. In this step, conserved male-specific generative cell specific 1 antigen is necessary for fusion of cytoplasmic membranes of micro- and macro-gametocytes and zygot formation. The partial blocking activities of elicited antibodies against either the HAP2-GCS1 domain or the cd loop of this antigen have been recorded to hinder the transmission of Plasmodium species in Anopheles mid-gut. Thus, the objective of the present study was to investigate if the cd loop-fusion can enhance the quantity and quality of humoral and cellular immune responses against Plasmodium falciparum GCS1 in comparison to non-fusion antigen (without cd loop), in the adjuvanted and non-adjuvanted mouse groups. The immunogenicity of two constructs of P. falciparum generative cell specific 1 antigen, a fusion protein composed of cd loop and HAP2-GCS1 domain (cd-HAP) and another recombinant PfGCS1 containing solo HAP2-GCS1 domain (HAP2) were assessed to impede Plasmodium gametocytes integration before zygote formation. The antibodies profiling, titer, and avidity of induced antibodies were measured by the immunized mice sera, and the released cytokines (IL-5, TNF, and INF-γ) were analyzed in the supernatants of stimulated splenocytes. Furthermore, the inhibitory potency of the elicited antibodies against HAP2 and cd-HAP was measured during oocyst development by Standard Membrane Feeding Assay (SMFA). The comparative results in the present study showed the higher titer of IgG antibodies and IgG2a subclass, avidity, and transmission-reducing activity (TRA = 72.5 %) when mice were immunized by cd-HAP rather than HAP2. Moreover, our findings confirmed intensified Th1-directed immune responses in group 4 received cd-HAP/Poly(I:C). These findings declared the potential ability of cd loop fusion (cd-HAP) to upsurge humoral and cellular immune responses. However, the immune responses may switch to stronger Th1-type using alternative formulations. Explicitly, the cd-HAP-based vaccine may enhance the overall efficiency of immune responses and present a promising implementation in aiming malaria transmission., Competing Interests: Declaration of competing interest The article entitled “Cd loop fusion enhances the immunogenicity and the potential transmission blocking activity of Plasmodium falciparum Generative Cell Specific 1 (GCS1) antigen” has not been previously published elsewhere in any language and is not currently under consideration by any other publication. The authors also declare that they contributed significantly to the review, i.e. drafting, critical revision of the article, and also approval of the version to be published., (Copyright © 2024 Elsevier Inc. All rights reserved.)