Search Results (40)

Obligate intracellular parasites must efficiently invade host cells to complete their life cycle and facilitate transmission. For the malaria-causing parasite Plasmodium falciparum, the invasion of an erythrocyte is a critical process, and thereby a key target for intervention strategies. In this study, we investigate the role of the ApiAP2 family transcription factor PfAP2-06B (PF3D7_0613800) in the intraerythrocytic developmental cycle of P. falciparum and focus on its regulation of genes involved in erythrocyte invasion. Conditional knockdown of PfAP2-06B resulted in a defect in asexual growth and impaired erythrocyte invasion. Bulk RNA sequencing (RNA-seq) analysis revealed that PfAP2-06B modulates the expression of invasion-related genes during the schizont stage. Single-cell RNA sequencing indicated that PfAP2-06B influences invasion gene expression and contributes to stochastic variations in expression of cell-to-cell genes. These results underscore the critical function of PfAP2-06B in the process of erythrocyte invasion and suggest its potential as a target for novel malaria control strategies. Importance: Understanding gene regulation in Plasmodium falciparum is essential for uncovering mechanisms of parasite development and pathogenicity. The research underscores the pivotal role of PfAP2-06B in regulating critical aspects of Plasmodium intraerythrocytic development and host cell invasion, demonstrating that PfAP2-06B plays a key role in orchestrating stage-specific gene expression. These findings provide new insights into the transcriptional networks of P. falciparum and highlight PfAP2-06B as a potential target for therapeutic intervention. This work advances our understanding of malaria pathogenesis and developing effective interventions. Full article

Malaria is a parasitic infectious disease that is endemic in many tropical countries. Even though several effective antimalarial agents have been implemented, treatment failure still occurs, and malaria continues to cause neurological complications and death, particularly in severe or drug-resistant cases. Hence, novel therapeutic agents with distinct mechanisms of action, as well as alternative chemical compounds that can overcome resistance, are still needed to improve malaria therapy. This study aimed to investigate the antimalarial activities of Brucea javanica, a tropical plant extracts against Plasmodium falciparum, the major species associated with severe malaria. In this study, malaria parasites were treated with plant extracts using single and co-incubation methods, along with artesunate and chloroquine, and their inhibitory effect on parasite development was determined by microscopy. The results show that all tested doses of the extracts that effectively inhibited malaria parasites did not cause hemolysis of red blood cells (RBCs). The root extract (RE) and fruit extract (FE) inhibited parasite growth at IC₅₀ values of 0.41 ± 1.14 µg/mL and 0.26 ± 1.15 µg/mL, respectively. These plant extracts significantly interrupted malaria development at the ring stage, as presented by a reduction in the conversion rate to trophozoites and schizonts. The defective parasites treated with plant extracts were characterized by nuclear clumping, leading to pyknotic cell death. Moreover, RE and FW extracts elicited an additive effect with artesunate and chloroquine, significantly reducing IC₉₀ levels for the inhibition of parasite development. In conclusion, B. javanica extracts inhibited the asexual blood-stage development of malaria parasites. They distinctively show the additive effects of ATS and CRQ, elucidating their potential for further studies on novel formulas of antimalarial drug regimens. Full article

Background: Malaria remains a global health crisis, with the World Health Organization (WHO) reporting 241 million cases and 627,000 deaths worldwide in 2020, predominantly affecting Sub-Saharan Africa. The region accounted for 95% of cases and 96% of deaths, reflecting the immense challenges in malaria prevention and treatment. Plasmodium falciparum Schizont Egress Antigen-1 (PfSEA-1) is crucial in facilitating immune evasion and promoting the sequestration of infected red blood cells (RBCs), contributing to severe malaria symptoms, including cerebral malaria, and necessitates the urgent identification of novel or repurposed drugs targeting PfSEA1. Methods: The protein structure of PfSEA-1 (UniProt ID: A0A143ZXM2) was modelled in three dimensions, prepared, and subjected to a 50 ns molecular dynamics (MD) simulation to achieve a stable structure. The equilibrated structure was minimised for molecular docking against the DrugBank compound library. Docking analysis identified potential inhibitors, including Alparabinos, Dihycid, Ambenzyne, Amiflupipquamine, Ametchomine, and Chlobenethyzenol, with docking scores ranging from −8.107 to −4.481 kcal/mol. Advanced analyses such as interaction fingerprints, density functional theory (DFT), and pharmacokinetics evaluations were conducted. Finally, a 100 ns MD simulation in the NPT ensemble was performed to assess the stability of protein–ligand complexes, with binding free energy and total energy calculations derived from the simulation trajectories. Results and Discussion: The identified compounds exhibited satisfactory pharmacokinetic profiles and binding interactions with PfSEA-1. The MD simulations demonstrated overall stability, with minor fluctuations in some instances. Key intermolecular interactions were observed, supporting the binding stability of the identified compounds. Binding free energy calculations confirmed favourable interactions, underscoring their potential as therapeutic agents against Plasmodium falciparum. While the in silico results are promising, experimental validation is essential to confirm their efficacy and safety for clinical use. Conclusion: These findings highlight PfSEA-1 as a promising antimalarial target and identify potential inhibitors with strong binding affinities and favourable pharmacokinetics. While the computational results are encouraging, further in vitro and in vivo validation is necessary to confirm their therapeutic potential and facilitate future drug development. Full article

Malaria remains a global health concern, with 249 million cases and 608,000 deaths being reported by the WHO in 2022. Traditional diagnostic methods often struggle with inconsistent stain quality, lighting variations, and limited resources in endemic regions, making manual detection time-intensive and error-prone. This study introduces an automated system for analyzing Romanowsky-stained thick blood smears, focusing on image quality evaluation, leukocyte detection, and malaria parasite classification. Using a dataset of 1000 clinically diagnosed images, we applied feature extraction techniques, including histogram bins and texture analysis with the gray level co-occurrence matrix (GLCM), alongside support vector machines (SVMs), for image quality assessment. Leukocyte detection employed optimal thresholding segmentation utility (OTSU) thresholding, binary masking, and erosion, followed by the connected components algorithm. Parasite detection used high-intensity region selection and adaptive bounding boxes, followed by a custom convolutional neural network (CNN) for candidate identification. A second CNN classified parasites into trophozoites, schizonts, and gametocytes. The system achieved an F1-score of 95% for image quality evaluation, 88.92% for leukocyte detection, and 82.10% for parasite detection. The F1-score—a metric balancing precision (correctly identified positives) and recall (correctly detected instances out of actual positives)—is especially valuable for assessing models on imbalanced datasets. In parasite stage classification, CNN achieved F1-scores of 85% for trophozoites, 88% for schizonts, and 83% for gametocytes. This study introduces a robust and scalable automated system that addresses critical challenges in malaria diagnosis by integrating advanced image quality assessment and deep learning techniques for parasite detection and classification. This system’s adaptability to low-resource settings underscores its potential to improve malaria diagnostics globally. Full article

Malaria is a leading cause of morbidity and mortality in tropical and sub-tropical regions. This research proposed a malaria diagnosis system based on the you only look once algorithm for malaria parasite detection and the convolutional neural network algorithm for malaria parasite life stage classification. Two public datasets are utilized: MBB and MP-IDB. The MBB dataset includes human blood smears infected with Plasmodium vivax (P. vivax). While the MP-IDB dataset comprises 4 species of malaria parasites: P. vivax, P. ovale, P. malariae, and P. falciparum. Four distinct stages of life exist in every species, including ring, trophozoite, schizont, and gametocyte. For the MBB dataset, detection and classification accuracies of 0.92 and 0.93, respectively, were achieved. For the MP-IDB dataset, the proposed algorithms yielded the accuracies for detection and classification as follows: 0.84 and 0.94 for P. vivax; 0.82 and 0.93 for P. ovale; 0.79 and 0.93 for P. malariae; and 0.92 and 0.96 for P. falciparum. The detection results showed the models trained by P. vivax alone provide good detection capabilities also for other species of malaria parasites. The classification performance showed the proposed algorithms yielded good malaria parasite life stage classification performance. The future directions include collecting more data and exploring more sophisticated algorithms. Full article

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of P. berghei NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in P. berghei and could assist in validating its protein constituents. Full article

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control. Full article

Malaria is caused by apicomplexan parasites of the Plasmodium genus. Plasmodium chabaudi is an excellent animal model for the study of human malaria caused by P. falciparum. Merozoites invade erythrocytes but are also found in other host cells including macrophages from the spleen and liver. Methodologies for obtaining merozoites usually involve treatment with protease inhibitors. However, merozoites obtained in this way may have their enzymatic profile altered and, therefore, are not ideal for cell-interaction assays. We report the obtainment of P. chabaudi merozoites naturally egressed from a synchronous erythrocyte population infected with schizonts forms. Merozoites had their infectivity and ultrastructure analyzed. Interaction assays were performed with mice erythrocytes and classically activated mice peritoneal macrophages, a very well-established classic model. Obtained merozoites were able to kill mice and efficiently infect erythrocytes. Interestingly, a lower merozoite:erythrocyte ratio resulted in a higher percentage of infected erythrocytes. We describe a simpler method for obtaining viable and infective merozoites. Classically activated macrophages killed merozoites, suggesting that these host cells may not serve as reservoirs for these parasites. These findings have implications for our understanding of P. chabaudi merozoite biology and may improve the comprehension of their host–parasite relationship. Full article

Coccidiosis poses a significant challenge in poultry production and is typically managed with ionophores and chemical anticoccidials. However, the emergence of drug resistance and limitations on their use have encouraged the exploration of alternative solutions, including botanical compounds and improvements in in vitro screening methods. Prior research focused only on the impact of these alternatives on Eimeria invasion, with intracellular development in cell cultures receiving limited attention. This study assessed the impact of thyme (Thymus vulgaris), oregano (Origanum vulgare), and garlic (Allium sativum) essential oils, as well as their bioactive compounds, on the initial phase of schizogony in Madin–Darby bovine kidney cells, comparing their effectiveness to two commercially used anticoccidial drugs. Using image analysis and quantitative PCR, the study confirmed the efficacy of commercial anticoccidials in reducing invasion and schizont formation, and it found that essential oils were equally effective. Notably, thymol and carvacrol exhibited mild inhibition of intracellular replication of the parasite but significantly reduced schizont numbers, implying a potential reduction in pathogenicity. In conclusion, this research highlights the promise of essential oils and their bioactive components as viable alternatives to traditional anticoccidial drugs for mitigating coccidiosis in poultry, particularly by disrupting the intracellular development of the parasites. Full article

Innovative strategies to control malaria are urgently needed. Exploring the interplay between Plasmodium sp. parasites and host red blood cells (RBCs) offers opportunities for novel antimalarial interventions. Pyruvate kinase deficiency (PKD), characterized by heightened 2,3-diphosphoglycerate (2,3-DPG) concentration, has been associated with protection against malaria. Elevated levels of 2,3-DPG, a specific mammalian metabolite, may hinder glycolysis, prompting us to hypothesize its potential contribution to PKD-mediated protection. We investigated the impact of the extracellular supplementation of 2,3-DPG on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. The results showed an inhibition of parasite growth, resulting from significantly fewer progeny from 2,3-DPG-treated parasites. We analyzed differential gene expression and the transcriptomic profile of P. falciparum trophozoites, from in vitro cultures subjected or not subjected to the action of 2,3-DPG, using Nanopore Sequencing Technology. The presence of 2,3-DPG in the culture medium was associated with the significant differential expression of 71 genes, mostly associated with the GO terms nucleic acid binding, transcription or monoatomic anion channel. Further, several genes related to cell cycle control were downregulated in treated parasites. These findings suggest that the presence of this RBC-specific glycolytic metabolite impacts the expression of genes transcribed during the parasite trophozoite stage and the number of merozoites released from individual schizonts, which supports the potential role of 2,3-DPG in the mechanism of protection against malaria by PKD. Full article

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen’s kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata. Full article

Background: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). Methods: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. Results: The liposome–replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. Conclusion: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines. Full article

Medicinal plants have historically been a source of drugs in multiple applications, including the treatment of malaria infections. The Cabo Verde archipelago harbors a rich diversity of native plants, most of which are used for medicinal purposes. The present study investigated the in vitro antiplasmodial activities of four native plants from Cabo Verde (i.e., Artemisia gorgonum, Lavandula rotundifolia, Sideroxylon marginatum, and Tamarix senegalensis). Traditional preparations of these medicinal plants, namely aqueous extracts (infusions) and ethanolic extracts, were tested against both chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains using the SYBR Green detection method. The in vitro cytotoxicity was evaluated in Caco-2 and PLP2 cells using a sulforhodamine B colorimetric assay. An ethanolic extract of A. gorgonum and infusions of T. senegalensis exhibited high antiplasmodial activities (EC₅₀ < 5 μg/mL) without cytotoxicity (GI₅₀ > 400 μg/mL). Extracts of L. rotundifolia and S. marginatum exhibited moderate activities, with EC₅₀ values ranging from 10–30 μg/mL. The A. gorgonum ethanolic extract showed activity toward early ring stages, and parasites treated with the T. senegalensis infusions progressed to the early trophozoite stage, although did not develop further to the late trophozoite or schizont stages. Antimalarial activities and the lack of cytotoxicity of the extracts are reported in the present study and support previous claims by traditional practitioners for the use of these plants against malaria while suggesting their ethnopharmacological usefulness as future antimalarials. Full article

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies. Full article

Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1·× 10⁻⁸ mol/L) and porcine CPB (Ki = 8.15·× 10⁻⁸ mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC₅₀ = 5.5 μmol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite’s intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite’s life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies. Full article