Background and Objectives: Malaria airs a life-threatening risk in Tropical African countries, stemming from infection by Plasmodium species. This region is richly endowed by nature with a wealth of diverse and largely unexplored plants that hold the potential for managing this protozoan parasite. The currently accessible over-the-counter drugs for disease management often present affordability challenges for the average person, exacerbated by the parasite\'s increasing resistance to them. This study investigated the phytoconstituents present in the ethyl acetate fraction of Spilanthes filicaulis (EFSF) and explored the antimalarial effects of EFSF on mice infected with Plasmodium berghei. Methods: Standard methods and gas chromatography-mass spectrometry (GC-MS) were used to identify phytoconstituents. Chloroquine phosphate-sensitive P. berghei (NK-65) was intraperitoneally inoculated into Swiss mice. The in vivo antimalarial activity of EFSF was assessed at dose levels of 250, 500, and 750 mg/kg, using 4-day suppressive and curative antimalarial models. Parameters evaluated in the inoculated mice included rectal temperature (RT), body weight (BW), packed cell volume (PCV), level of parasitemia, and mean survival time (MST). Results: Steroids, alkaloids, flavonoids, tannins, saponins, terpenoids, and cardiac glycosides were the identified phytochemicals present in EFSF, and GC-MS alongside reveals the presence of 20 bioactive compounds predominantly fatty acids and alcohol esters. Significant prevention of reductions in RT, BW, and PCV was observed in the EFSF-treated groups dose dependently relative to the untreated group. In addition, EFSF-treated groups significantly (p < 0.05) suppressed parasitemia and exhibited chemosuppression of 79.46% and 77.38% in 4-day suppressive, whereas suppression of 59.74% and 58.66% in curative treatment, respectively, at 500 and 750 mg/kg thus consequently extending the MST of infected treated mice compared with the untreated group. Interpretation and Conclusion: Put together, the EFSF exhibited enhanced antimalarial efficacy against mice infected with P. berghei thus affirming that plants still maintain lead way as a potential source of novel antimalarial remedies.

Upon infecting its vertebrate host, the malaria parasite initially invades the liver where it undergoes massive replication, whilst remaining clinically silent. The coordination of host responses across the complex liver tissue during malaria infection remains unexplored. Here, we perform spatial transcriptomics in combination with single-nuclei RNA sequencing over multiple time points to delineate host-pathogen interactions across Plasmodium berghei-infected liver tissues. Our data reveals significant changes in spatial gene expression in the malaria-infected tissues. These include changes related to lipid metabolism in the proximity to sites of Plasmodium infection, distinct inflammation programs between lobular zones, and regions with enrichment of different inflammatory cells, which we term \'inflammatory hotspots\'. We also observe significant upregulation of genes involved in inflammation in the control liver tissues of mice injected with mosquito salivary gland components. However, this response is considerably delayed compared to that observed in P. berghei-infected mice. Our study establishes a benchmark for investigating transcriptome changes during host-parasite interactions in tissues, it provides informative insights regarding in vivo study design linked to infection and offers a useful tool for the discovery and validation of de novo intervention strategies aimed at malaria liver stage infection.

BACKGROUND: Studying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites.
METHODS: Differences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte-ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development.
RESULTS: The phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte-ookinete conversion and transmission to mosquitoes.
CONCLUSIONS: Seven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect\'s innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways.

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.

Oxidative stress is involved in the pathogenesis of malaria, causing anemia, respiratory complications, and cerebral malaria. To mitigate oxidative stress, we investigated the effect of nutritional supplementation whit lycopene (LYC) on the evolution of parasitemia and survival rate in mice infected with Plasmodium berghei ANKA (Pb), comparing to the effects promoted by N-acetylcysteine (NAC). Therefore, 175 mice were randomly distributed into 4 groups; Sham: untreated and uninfected animals; Pb: animals infected with Pb; LYC+Pb: animals treated with LYC and infected with Pb; NAC+Pb: animals treated with NAC and infected with Pb. The animals were followed for 12 days after infection, and survival and parasitemia rates were evaluated. There was a 40.1% increase in parasitemia in the animals of the Pb group on the 12th day, and a survival rate of 45%. LYC supplementation slowed the development of parasitemia to 19% and promoted a significative increase in the survival rate of 80% on the 12th day after infection, compared to the Pb group, effects superior to those promoted by NAC, providing strong evidence of the beneficial effect of LYC on in vivo malaria and stressing the importance of antioxidant supplementation in the treatment of this disease.

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

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

BACKGROUND: Malaria continues to wreak havoc on the well-being of the community. Resistant parasites are jeopardizing the treatment. This is a wake-up call for better medications. Folk plants are the key starting point for antimalarial drug discovery. After crushing and mixing the leaves of Coriandrum sativum with water, one cup of tea is drunk daily for a duration of three to five days as a remedy for malaria by local folks in Ethiopia. Additionally, in vitro experiments conducted on the plant leaf extract elsewhere have also demonstrated the plant\'s malaria parasite inhibitory effect. There has been no pharmacologic research to assert this endowment in animals, though. This experiment was aimed at evaluating the antimalarial efficacy of C. sativum in Plasmodium berghei infected mice.
METHODS: The plant\'s leaf was extracted using maceration with distilled water. The extract was examined for potential acute toxicity. An evaluation of secondary phytoconstituents was done. Standard antimalarial screening models (prophylactic, chemosuppressive, curative tests) were utilized to assess the antiplasmodial effect. In each test, thirty mice were organized into groups of five. To the three categories, the test substance was given at doses of 100, 200 and 400 mg/kg/day before or after the commencement of P. berghei infection. Positive and negative control mice were provided Chloroquine and distilled water, respectively. Rectal temperature, parasitemia, body weight, survival time and packed cell volume were ultimately assessed. Analysis of the data was performed using Statistical Package for Social Sciences.
RESULTS: No toxicity was manifested in mice. The extract demonstrated a significant inhibition of parasitemia (p < 0.05) in all the models. The inhibition of parasite load was highest with the upper dose in the suppressive test (82.74%) followed by the curative procedure (78.49%). Likewise, inhibition of hypothermia, weight loss hampering, improved survival and protection against hemolysis were elicited by the extract.
CONCLUSIONS: The results of our experimental study revealed that the aqueous crude leaf extract of C. sativum exhibits significant antimalarial efficacy in multiple in vivo models involving mice infected with P. berghei. Given this promising therapeutic attribute, in depth investigation on the plant is recommended.