In the present work, curcuminoids-loaded lipid nanoparticles for parenteral administration were successfully prepared by a nanoemulsion technique employing high-speed homogenizer and ultrasonic ...probe. For the production of nanoparticles, trimyristin, tristerin and glyceryl monostearate were selected as solid lipids and medium chain triglyceride (MCT) as liquid lipid. Scanning electron microscopy (SEM) revealed the spherical nature of the particles with sizes ranging between 120 and 250
nm measured by photon correlation spectroscopy (PCS). The zeta potential of the particles ranged between −28 and −45
mV depending on the nature of the lipid matrix produced, which also influenced the entrapment efficiency (EE) and drug loading capacity (LC) found to be in the range of 80–94% and 1.62–3.27%, respectively. The LC increased reciprocally on increasing the amount of MCT as confirmed by differential scanning calorimetry (DSC). DSC analyses revealed that increasing imperfections within the lipid matrix allowed for increasing encapsulation parameters. Nanoparticles were further sterilized by filtration process which was found to be superior over autoclaving in preventing thermal degradation of thermo-sensitive curcuminoids. The
in vivo pharmacodynamic activity revealed 2-fold increase in antimalarial activity of curcuminoids entrapped in lipid nanoparticles when compared to free curcuminoids at the tested dosage level.
harbors two endosymbiotic organelles: a relict plastid, the apicoplast, and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually ...targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2.
Signal-dependent transport into and out of the nucleus mediated by members of the importin (IMP) superfamily of nuclear transporters is critical to the eukaryotic function and a point of therapeutic ...intervention with the potential to limit disease progression and pathogenic outcomes. Although the apicomplexan parasites
and
both retain unique IMPα genes that are essential, a detailed analysis of their properties has not been performed. As a first step to validate apicomplexan IMPα as a target, we set out to compare the properties of
and
IMPα (PfIMPα and TgIMPα, respectively) to those of mammalian IMPα, as exemplified by
IMPα (MmIMPα). Close similarities were evident, with all three showing high-affinity binding to modular nuclear localisation signals (NLSs) from apicomplexans as well as Simian virus SV40 large tumour antigen (T-ag). PfIMPα and TgIMPα were also capable of binding to mammalian IMPβ1 (MmIMPβ1) with high affinity; strikingly, NLS binding by PfIMPα and TgIMPα could be inhibited by the mammalian IMPα targeting small molecules ivermectin and GW5074 through direct binding to PfIMPα and TgIMPα to perturb the α-helical structure. Importantly, GW5074 could be shown for the first time to resemble ivermectin in being able to limit growth of
. The results confirm apicomplexan IMPα as a viable target for the development of therapeutics, with agents targeting it worthy of further consideration as an antimalarial.
In recent times, Plasmodium vivax (P. vivax) has become a serious threat to public health due to its ability to cause severe infection with fatal outcomes. Its unique biology makes it resilient to ...control measures that are otherwise effective against P. falciparum. A deeper understanding of P. vivax biology and pathogenesis is, therefore, essential for developing the right control strategies. Proteomics of P. falciparum has been helpful in studying disease biology and elucidating molecular mechanisms involved in the development of disease. However, unlike P. falciparum, proteomics data for P. vivax infection is minimal due to the absence of a continuous culture system. The dependence on clinical samples and animal models has drastically limited P. vivax research, creating critical knowledge gaps in our understanding of the disease. This study describes an in-depth proteomics analysis of P. vivax-infected human plasma and parasite isolates, to understand parasite biology, pathogenesis, and to identify new diagnostic targets for P. vivax malaria.
A mass-spectrometry- (MS) based proteomics approach (Q Exactive) was applied to analyze human plasma and parasite isolates from vivax malaria patients visiting a primary health centre in India. Additionally, a targeted proteomics assay was standardized for validating unique peptides of most recurring parasite proteins.
Thirty-eight P. vivax proteins were detected in human plasma with high confidence. Several glycolytic enzymes were found along with hypothetical, cytoskeletal, ribosomal, and nuclear proteins. Additionally, 103 highly abundant P. vivax proteins were detected in parasite isolates. This represents the highest number of parasite proteins to be reported from clinical samples so far. Interestingly, five of these; three Plasmodium exported proteins (PVX_003545, PVX_003555 and PVX_121935), a hypothetical protein (PVX_083555) and Pvstp1 (subtelomeric transmembrane protein 1, PVX_094303) were found in both plasma and parasite isolates.
A parasite proteomics investigation is essential to understand disease pathobiology and design novel interventions. Control strategies against P. vivax also depend on early diagnosis. This work provides deeper insights into the biology of P. vivax by identifying proteins expressed by the parasite during its complex life-cycle within the human host. The study also reports antigens that may be explored as diagnostic candidates.
Although common in plants, very few proteins are currently known to be localized to both the plastid and the mitochondrion in Plasmodium falciparum. One such protein is P. falciparum glutathione ...peroxidase‐like thioredoxin peroxidase (PfTPxGl) which we show, by immunofluorescence imaging and bioinformatics predictions, is localized to the apicoplast, the mitochondrion and the cytosol. The distribution of PfTPxGl was random in the population, with the protein localizing to any one organelle in some parasites and to both in others. It has been proposed that targeting to each organelle occurs via independent pathways that do not proceed via the Golgi. However, for PfTPxGl, both incubation at low temperature (15 °C) and Brefeldin A treatment reversibly blocked targeting to these organelles, suggesting the involvement of a novel trafficking route, most probably via the endoplasmic reticulum and Golgi. This idea is further supported by the lack of cleavage of the putative N‐terminal signal sequence of PfTPxGl, and this N‐terminal extension did not compromise PfTPxGl enzyme activity. In the context of evolution, a common pathway for the dual localization of a single gene product, such as the primitive endoplasmic reticulum–Golgi route, may have been retained as opposed to optimization for individual organellar import pathways.
P. falciparum glutathione peroxidase‐like thioredoxin peroxidase (PfTPxGl), we show, is localized to the apicoplast, the mitochondrion and the cytosol by a non‐canonical pathway involving the ER and Golgi. The localization of PfTPxGl was heterogeneous in the population. In the context of evolution, the primitive ER‐Golgi route may have been retained as opposed to optimization for individual organellar import pathways
The World Health Organization (WHO) has set forth a global call for eradicating malaria, caused majorly by the protozoan parasites
and
. The lack of diagnostic biomarkers for
, especially those that ...differentiate the parasite from
, significantly hinders
elimination. Here, we show that
tryptophan-rich antigen (PvTRAg) can be a diagnostic biomarker for diagnosing
in malaria patients. We report that polyclonal antibodies against purified PvTRAg protein show interactions with purified PvTRAg and native PvTRAg using Western blots and indirect enzyme-linked immunosorbent assay (ELISA). We also developed an antibody-antigen-based qualitative assay using biolayer interferometry (BLI) to detect vivax infection using plasma samples from patients with different febrile diseases and healthy controls. The polyclonal anti-PvTRAg antibodies were used to capture free native PvTRAg from the patient plasma samples using BLI, providing a new expansion range to make the assay quick, accurate, sensitive, and high-throughput. The data presented in this report provides a proof of concept for PvTRAg, a new antigen, for developing a diagnostic assay for
identification and differentiation from the rest of the
species and, at a later stage, translating the BLI assay into affordable, point-of-care formats to make it more accessible.
Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium ...falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC50 of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.
Plasmodium species are causative agents of malaria, a disease that is a serious global health concern. FDA-approved HIV-1 protease inhibitors (HIV-1 PIs) have been reported to be effective in ...reducing the infection by Plasmodium parasites in the population co-infected with both HIV-1 and malaria. However, the mechanism of HIV-1 PIs in mitigating Plasmodium pathogenesis during malaria/HIV-1 co-infection is not fully understood. In this study we demonstrate that HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum. Crystal structures of the complexes of PMII with both drugs have been determined. The inhibitors interact with PMII via multiple hydrogen bonding and hydrophobic interactions. The P4 moiety of RTV forms additional interactions compared to LPV and exhibits conformational flexibility in a large S4 pocket of PMII. Our study is also the first to report inhibition of P. falciparum PMX by RTV and the mode of binding of the drug to the PMX active site. Analysis of the crystal structures implies that PMs can accommodate bulkier groups of these inhibitors in their S4 binding pockets. Structurally similar active sites of different vacuolar and non-vacuolar PMs suggest the potential of HIV-1 PIs in targeting these enzymes with differential affinities. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing HIV-1 PIs as antimalarial drugs.
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•HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) inhibit activity of plasmepsin II (PMII) and plasmepsin X (PMX), pepsin-like aspartic proteases of P. falciparum, a parasite causing the deadliest forms of malaria.•First report of crystal structures of a plasmepsin (PMII) complexed with antiretroviral drugs RTV and LPV.•Molecular insights into the binding of HIV-1 drugs to the active site of vacuolar and non-vacuolar plasmepsins.•Repurposing of HIV-1 drugs as antimalarials.
This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about ...disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates the potential of this analytical approach for the detection of malaria as well as other human diseases.
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