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Journal of Vector Borne Diseases Apr 2024The persistent threat of drug resistant malaria demands new cures. Low prevalence of malaria in the Indian state of Kerala compared with other proximal states made us...
BACKGROUND OBJECTIVES
The persistent threat of drug resistant malaria demands new cures. Low prevalence of malaria in the Indian state of Kerala compared with other proximal states made us explore if there is any traditional practice in Kerala which may confer protection against malaria. In this context, our attention was drawn to 'Pathimugam' i.e., Ceasalpinia sappan whose heartwood is used to prepare a red aqueous extract which is a uniquely popular drink in Kerala.
METHODS
Aqueous and methanolic extracts of various organs of C. sappan were prepared and tested against Plasmodium falciparum grown in vitro culture using SYBR Green-I assay. The cytotoxicity of active extracts/fractions was studied using mammalian HeLa cell line. in vivo efficacy was determined using P. berghei ANKA infected mice.
RESULTS
The highest antiplasmodial activities in the alcoholic and aqueous extracts were observed in leaf methanolic extract (IC50 2 μg/ml) and heartwood aqueous extract (IC50 12.5 μg/ml). Ceasalpinia sappan extracts were equipotent against both chloroquine-sensitive Pf3D7 and resistant PfINDO strains and showed suppression of percentage parasitemia in P. berghei infected mice. Activity- guided chromatographic fractionation of aqueous wood extract led to the fortification of antiplasmodial activity (IC50 5 μg/ml).
INTERPRETATION CONCLUSION
Our results establish the antiplasmodial potential of C. sappan and suggest that its regular use might have prophylactic or curative actions that may assist in keeping check on malaria in the Indian state of Kerala.
PubMed: 38634464
DOI: 10.4103/JVBD.JVBD_18_24 -
MBio Apr 2024Remodeling the erythrocyte membrane and skeleton by the malarial parasite is closely associated with intraerythrocytic development. However, the mechanisms underlying...
UNLABELLED
Remodeling the erythrocyte membrane and skeleton by the malarial parasite is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not β-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of from the ring to the schizont stage. We further observed an upregulated expression of phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during development. We further revealed that inhibition of the activity of PfPI3K impaired development and infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria.
IMPORTANCE
is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after invasion, while β-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in -infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.
Topics: Humans; Animals; Mice; Plasmodium falciparum; Spectrin; Erythrocytes; Malaria, Falciparum; Ubiquitin; Phosphatidylinositol 3-Kinase; Ubiquitin-Protein Ligases
PubMed: 38470053
DOI: 10.1128/mbio.03510-23 -
MSphere Mar 2024The apicomplexans and are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the...
UNLABELLED
The apicomplexans and are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the parasites must acquire critical host resources and transport them across their PV for development. However, the mechanism by which host resources are trafficked to and across the PV remains uncertain. Here, we investigated host ADP ribosylation factors (Arfs), a class of proteins involved in vesicular trafficking that may be exploited by and for nutrient acquisition. Using overexpressed Arf proteins coupled with immunofluorescence microscopy, we found that all Arfs were internalized into the PV, with most vacuoles containing at least one punctum of Arf protein by the end of the lytic cycle. We further characterized Arf1, the most abundant Arf inside the PV, and observed that active recycling between its GDP/GTP-bound state influenced Arf1 internalization independent of host guanine nucleotide exchange factors (GEFs). In addition, Arf1 colocalized with vesicle coat complexes and exogenous sphingolipids, suggesting a role in nutrient acquisition. While Arf1 and Arf4 were not observed inside the PV during infection, our gene depletion studies showed that liver stage development and survival depended on the expression of Arf4 and the host GEF, GBF1. Collectively, these observations indicate that apicomplexans use distinct mechanisms to subvert the host vesicular trafficking network and efficiently replicate. The findings also pave the way for future studies to identify parasite proteins critical to host vesicle recruitment and the components of vesicle cargo.
IMPORTANCE
The parasites and live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by and . Our results indicate that selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For , host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.
Topics: Toxoplasma; ADP-Ribosylation Factors; Proteins; Vacuoles; Plasmodium
PubMed: 38349168
DOI: 10.1128/msphere.00770-23 -
Cellular and Molecular Life Sciences :... Nov 2023During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4...
During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.
Topics: Animals; Mice; Cysteine Proteases; Parasites; Plasmodium berghei; Autophagy-Related Protein 8 Family; Autophagy; Malaria; Protozoan Proteins
PubMed: 37910326
DOI: 10.1007/s00018-023-05004-2 -
Journal of Parasitology Research 2023Reduction of oxidative stress during malaria infection is considered as being of great benefit so long as treatment and drug development approaches are concerned. This...
BACKGROUND
Reduction of oxidative stress during malaria infection is considered as being of great benefit so long as treatment and drug development approaches are concerned. This study had the aim of evaluating the antimalarial and antioxidant activities of the ethanolic extract of in Swiss albino mice infected with the NK65 strain.
METHODS
the antiplasmodial activity of the plant ethanolic extract was tested in a four-day suppressive and curative assay using in Swiss albino mice. The extract was administered to the mice at doses of 125, 250, and 500 mg/kg per day. Then, parameters, such as parasite suppression and survival time of the mice, were evaluated. Furthermore, the effect of plant extract on liver damage, oxidative stress indicators, and lipid profile changes in -infected mice were studied.
RESULTS
Administration of significantly suppressed infection by 55.17%, 70.69%, and 71.10% at doses of 125, 250, and 500 mg/kg, respectively, whereas chloroquine had 84.64% suppression relative to the untreated group 1% Dimethyl sulfoxide (1% DMSO) at day 4 (post-infection) in the four-day suppressive test. This suppression activity rate was dose-dependent. The curative test also presented a significant reduction in parasitemia and an extension of the survival time of the treated groups. Treatment of infected parasitized mice with the extract of had a significant ( < 0.05) reduction in parameters, such as total protein, aspartate aminotransferase, and alanine aminotransferase. Infection may also lead to a significant increase in the enzymatic activity of liver catalase and superoxide dismutase compared with the normal control group. The non-enzymatic antioxidant activity in parasitized mice was significantly reduced in malondialdehyde and increased in glutathione and nitric oxide when compared with the normal control group.
CONCLUSIONS
These findings support the ethnobotanical use of stem bark as an antimalarial remedy coupled with antioxidant activity. However, further toxicity tests are required to ascertain its safety.
PubMed: 37435530
DOI: 10.1155/2023/3350293 -
Experimental Parasitology Nov 2023Cerebral malaria (CM) is a severe manifestation of malaria that commonly occurs in children and is hallmarked by neurologic symptoms and significant Plasmodium...
Cerebral malaria (CM) is a severe manifestation of malaria that commonly occurs in children and is hallmarked by neurologic symptoms and significant Plasmodium falciparum parasitemia. It is currently hypothesized that cerebral hypoperfusion from impaired microvascular oxygen transport secondary to parasitic occlusion of the microvasculature is responsible for cerebral ischemia and thus disease severity. Animal models to study CM, are known as experimental cerebral malaria (ECM), and include the C57BL/6J infected with Plasmodium berghei ANKA (PbA), which is ECM-susceptible, and BALB/c infected with PbA, which is ECM-resistant. Here we sought to investigate whether changes in oxygen (O) delivery, O flux, and O utilization are altered in both these models of ECM using phosphorescence quenching microscopy (PQM) and direct measurement of microvascular hemodynamics using the cranial window preparation. Animal groups used for investigation consisted of ECM-susceptible C57BL/6 (Infected, n = 14) and ECM-resistant BALB/c (Infected, n = 9) mice. Uninfected C57BL/6 (n = 6) and BALB/c (n = 6) mice were included as uninfected controls. Control animals were manipulated in the exact same way as the infected mice (except for the infection itself). C57BL/6 ECM animals at day 6 of infection were divided into two cohorts: Early-stage ECM, presenting mild to moderate drops in body temperature (>34 < 36 °C) and Late-stage ECM, showing marked drops in body temperature (<33 °C). Data taken from new experiments conducted with these animal models were analyzed using a general linear mixed model. We constructed three general linear mixed models, one for total O content, another for total O delivery, and the third for total O content as a function of convective flow. We found that in both the ECM-susceptible C57BL/6J model and ECM-resistant BALB/c model of CM, convective and diffusive O flux along with pial hemodynamics are impaired. We further show that concomitant changes in p50 (oxygen partial pressure for 50% hemoglobin saturation), only 5 mmHg in the case of late-stage CM C57BL/6J mice, and O diffusion result in insufficient O transport by the pial microcirculation, and that both these changes are required for late-stage disease. In summary, we found impaired O transport and O affinity in late-stage ECM, but only the former in either early-stage ECM and ECM-resistant strains.
PubMed: 37673369
DOI: 10.1016/j.exppara.2023.108608 -
Life Sciences Oct 2023Malaria is a deadly parasitic disease caused a by protozoan parasite of the genus plasmodium. The challenges facing by chemotherapy and vector control couple with the...
Prophylactic effects of probiotic bacterium Latilactobacillus sakei on haematological parameters and cytokine profile of mice infected with Plasmodium berghei ANKA during early malaria infection.
Malaria is a deadly parasitic disease caused a by protozoan parasite of the genus plasmodium. The challenges facing by chemotherapy and vector control couple with the lack of vaccine against malaria necessitate an urgent need for the development of alternative treatment regimens to combat this disease. One possible antimalarial treatment regimen is the use of probiotic bacteria as dietary supplements. Traditionally fermented milk is a rich source of probiotic bacteria that up to date, very few studies have been carried out on their immunoprotective effects against early malaria infection in mice. This study sought to assess the prophylactic activities of a probiotic bacterium Latilactobacillus sakei on malaria and inflammation in Plasmodium berghei infected mice. The probiotic bacterium was isolated from the Fulani's traditionally fermented milk and identified using the sequencing of the 16S r RNA gene. The repository activity of L. sakei on malaria was assessed using the method described by Peters with slight modification. Eighty-four BALB/c mice were randomly divided into two sets of seven groups of six mice each. One set received orally different doses of L. sakei Chloroquine and Sulfadoxine/Pyrimethamine for seven days before infection while the other set received for fourteen days before infection with 0.1 mL of 10Plasmodium berghei. Parasitaemia density, haematological parameters and inflammatory cytokines profile were evaluated. Data were presented as Mean ± SEM and analysed using SPSS version 20.0. The results of this study revealed that L. sakei significantly (p < 0.05) reduced in dose dependent manner parasite load, body weight loss and reduction of body temperature in all the treated mice when compare to untreated mice. Leukocytopenia, thrombocytosis and inflammation were also found to be significantly (p < 0.05) prevented in treated mice as compared to untreated mice. This study suggested that L sakei possesses immunomodulation and protective effects on early malaria infection in Plasmodium berghei mice.
Topics: Animals; Mice; Plasmodium berghei; Latilactobacillus sakei; Malaria; Probiotics; Bacteria; Cytokines
PubMed: 37652156
DOI: 10.1016/j.lfs.2023.122056 -
Antimicrobial Agents and Chemotherapy Jul 2023The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new...
The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC concentration. Using 72-h exposure of phebestin at concentrations of 1 μM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.
Topics: Humans; Animals; Mice; Antimalarials; Aminopeptidases; Parasitemia; Chloroquine; Malaria; Malaria, Falciparum; Plasmodium falciparum; Plasmodium berghei
PubMed: 37314349
DOI: 10.1128/aac.01606-22 -
BioRxiv : the Preprint Server For... Jul 2023parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug...
parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypetide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual liver stage parasites and host cells simultaneously. After validation, we demonstrate specific, concentration-dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds, and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between direct and indirect translation inhibitors. We identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in liver stages and confirm this potential mode of action in asexual blood stages.
PubMed: 37461595
DOI: 10.1101/2023.07.05.547872 -
Natural Product Research Oct 2023S Walp., () is used traditionally in West Africa for the treatment of malaria. However, no scientific reports validating these effects and its active constituents are on...
S Walp., () is used traditionally in West Africa for the treatment of malaria. However, no scientific reports validating these effects and its active constituents are on record. Therefore, this study is aimed at evaluating the antimalarial effects, of its ethanolic extract and isolated compounds against 3D7 and ANKA strains. Using chromatographic, spectrometric and spectroscopic techniques three compounds were isolated and characterised. The extract of was active against 3D7, in an assay with IC of 12.0 ± 0.32 µg/ml. The three isolated compounds, namely 1,10-dihydroxy-6-benzo[]chromen-6-one (), 8- hydroxy-3,4-dimethoxydibenzo[,]furan-1-carboxylic acid () and benzyl-2-methoxybenzoate (), also showed antimalarial activity against ANKA strain in curative and suppressive assays. The ethanolic extract and isolated compounds of possess antimalarial effects. The isolated compounds may be responsible, at least in part, for the observed activities of the extract.
PubMed: 37867307
DOI: 10.1080/14786419.2023.2272288