Targeting the apicoplast in malaria

Malaria continues to be one of the leading causes of human mortality in the world, and the therapies available are insufficient for eradication. Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Apicomplexan parasites, including the Plasmodium spp., are descendants of photosynthetic algae, and therefore they possess an essential plastid organelle, named the apicoplast. Since humans and animals have no plastids, the apicoplast is an attractive target for drug development. Indeed, after its discovery, the apicoplast was found to host the target pathways of some known antimalarial drugs, which motivated efforts for further research into its biological functions and biogenesis. Initially, many apicoplast inhibitions were found to result in ‘delayed death’, whereby parasite killing is seen only at the end of one invasion-egress cycle. This slow action is not in line with the current standard for antimalarials, which seeded scepticism about the potential of compounds targeting apicoplast functions as good candidates for drug development. Intriguingly, recent evidence of apicoplast inhibitors causing rapid killing could put this organelle back in the spotlight. We provide an overview of drugs known to inhibit apicoplast pathways, alongside recent findings in apicoplast biology that may provide new avenues for drug development

Exploring the powerful phytoarsenal of white grape marc against bacteria and parasites causing significant diseases

Natural extracts containing high polyphenolic concentration possess antibacterial, antiparasitic and fungicidal activities. The present research characterises white grape marc, a winemaking by-product describing their physicochemical features and antimicrobial capacities. Main components of 2 different extracts generated were phenolic acids, flavan-3-ols and their gallates, and flavonols and their glycosides. As a result of this complex composition white grape marc extracts showed pronounced bioactivities with potential uses in agricultural, pharmaceutical and cosmetic industries, among others. Specifically, polyphenol compounds were extracted by using hydro-organic solvent mixtures from the by-product of Albariño white wines (Galicia, NW Spain) production. In the present work the “in vitro” antimicrobial activity of the bioactive extracts was evaluated using two different hydro-organic mixtures (HOL & HOP). The microorganisms tested included Gram positive and negative bacteria, two Apicomplexan parasite species and one Oomycota parasite. Microbial species investigated are causing agents of several human and animal diseases, such as foodborne illnesses (Bacillus cereus, Eschericha coli, Salmonella enterica), skin infections (Staphylococcus aureus), mastitis (Streptococcus uberis), parasite infections as Malaria (Plasmodium falciparum) or Toxoplasmosis (Toxoplasma gondii), and plant infections as "chestnut ink" in chestnuts or "root rot" in avocado, both diseases caused by Phytophthora cinnamomi. Both extracts verified activity against all the tested species demonstrating their potentiality to be used for the development of biocides to control a wide range of pathogenic agents; at the same time that they contribute to winemaking industry residues valorisationThis research was supported by projects GPC2017/04 (Consolidated Research Groups Program) & ED431E 2018/01 Cross-Research in Environmental Technologies (CRETUS) (Xunta de Galicia, Spain)info:eu-repo/semantics/publishedVersio

Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii.

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites

Exploring the powerful phytoarsenal of white grape marc against bacteria and parasites causing significant diseases

Natural extracts containing high polyphenolic concentration possess antibacterial, anti-parasitic and fungicidal activities. The present research characterises two extracts based on white grape marc, a winemaking by-product, describing their physicochemical features and antimicrobial capacities. The main components of these extracts are phenolic acids, flavan-3-ols and their gallates and flavonols and their glycosides. As a result of this complex composition, the extracts showed pronounced bioactivities with potential uses in agricultural, pharmaceutical and cosmetic industries. Polyphenol compounds were extracted by using hydro-organic solvent mixtures from the by-product of Albariño white wines (Galicia, NW Spain) production. The in vitro antimicrobial activity of these extracts was evaluated on Gram-positive and Gram-negative bacteria and Apicomplexan and Oomycota parasites. Microbial species investigated are causing agents of several human and animal diseases, such as foodborne illnesses (Bacillus cereus, Escherichia coli, Salmonella enterica, and Toxoplasma gondii), skin infections and/or mastitis (Staphylococcus aureus and Streptococcus uberis), malaria (Plasmodium falciparum) and plant infections as “chestnut ink” or “root rot” (Phytophthora cinnamomi). Both extracts showed activity against all the tested species, being nontoxic for the host. So, they could be used for the development of biocides to control a wide range of pathogenic agents and contribute to the enhancement of winemaking industry by-products

Isolation of a novel flavanonol and an alkylresorcinol with highly potent anti-trypanosomal activity from Libyan propolis

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4’-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction

Depletion of a Toxoplasma porin leads to defects in mitochondrial morphology and contacts with the ER

The Voltage Dependent Anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes of the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs

Chemical and antimicrobial profiling of propolis from different regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts

Metabolic phenotyping of Plasmodium falciparum mutants with impaired pyruvate dehydrogenase complex activity

Malaria remains one of the most important infectious diseases in the world, counting 200 million cases and killing 300,000 people every year, mostly represented by children under the age of 5. Apicomplexan parasites of the genus Plasmodium cause malaria and P. falciparum is responsible for the most severe form of the disease. The development of drug resistance is one of the most important challenges that eradication programs face and first line therapies have started to become ineffective. A better understanding of Plasmodium biology and metabolic functions is essential for the identification of new drug targets and the interpretation of the drug resistance mechanisms to help and fight this devastating disease. The pyruvate dehydrogenase complex (PDC) is a member of the -ketoacid dehydrogenase complexes (KADH), representing essential elements of the intermediary metabolism. PDC has the important role in decarboxylating pyruvate generating NADH+H+ and acetyl-CoA, which are essential for many metabolic processes including energy and lipid metabolism. Three enzymes catalyse PDC reaction represented by pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Specific cofactors such as thiamine pyrophosphate, FAD and NAD+ are required for the activity of the enzymes and lipoic acid is essential for E2 catalysis. Usually, the main role of PDC is to link glycolysis to the tricarboxylic acid cycle and downstream oxidative phosphorylation. This dogma does not apply to Plasmodium PDC, which is exclusively found in the apicoplast and provides acetyl-CoA and NADH+H+ for fatty acid biosynthesis. Both, PDC and fatty acid biosynthesis are dispensable during the intraerythrocytic development of P. falciparum but are essential for sexual development. Therefore, the complex may be exploitable for potential transmission-blocking agents. Given that the genes encoding the PDC subunits are actively expressed during the intraerythrocytic life of P. falciparum, it was postulated that they might have additional functions independent of PDC as has been reported from other organisms. To test this hypothesis, P. falciparum mutants with a deletion of apicoplast e3 (ae3) and with impaired E2 activity, through interruption of the apicoplast located lipoic acid biosynthesis pathway by deletion of the lipoic acid protein ligase B gene (lipB), were investigated using targeted and untargeted metabolomics approaches. Metabolic fluxes in the parasite metabolism were characterised after stable isotopic labelling using 13C-U-D-glucose and 13C, 15N-U-L-glutamine. Further growth phenotypes of the mutants were detailed and quantitative western blotting was used to assess the relative expression levels of several antioxidant and redox proteins. The analysis of the growth phenotypes confirmed that disruption of lipb accelerates parasite lifecycle, while deletion of ae3 produces parasites with a persistent synchronicity. The targeted metabolomics analysis of the LipB mutants showed an up-regulation of the TCA cycle activity characterised by an increased flux of isotopic carbons from 13C-U-D-glucose to 13C-2-citrate. These results imply an incomplete cyclic activity of the TCA cycle and the transferring of citrate outside of the mitochondrion, possibly contributing to the cytosolic acetyl-CoA pool. Results obtained from the untargeted analysis sustained this hypothesis showing increased levels for CoA biosynthesis intermediates and different acetylated metabolites, suggesting adaptations in the parasite acetylome. Additionally, levels of ATP were significantly reduced in this mutant line and carbon flux into NAD(P)+ was increased suggesting the instalment of starvation and redox stress. The e3 mutants hardly showed any significant metabolic change, despite its peculiar synchronous growth phenotype. These results suggest that the two different PDC subunits may have independent roles affecting specific metabolic functions in the parasite. On the other hand, the analysis of expression levels of antioxidant and redox active proteins in both parasite lines showed that they have increased the relative expression of peroxiredoxins and glutathione S-transferase, possibly to compensate a redox-regulating role of E2 and E3. In addition, the expression levels of the mitochondrial branched-chain -ketoacid dehydrogenase were significantly up-regulated, possibly pointing towards compensatory activity providing acetyl-CoA. In a more direct approach to assess the role of P. falciparum E2, a DiCre based conditional knockout of the gene was attempted. Preliminary data suggest that conditional deletion of e2 might affect parasite growth during intraerythrocytic development. However, isolation of a clonal e2 mutant line for detailed analyses was unsuccessful due to time limitations. The data provided in this thesis suggest that the E2 and E3 subunits of PDC possibly have independent roles during P. falciparum intraerythrocytic development that affect the parasites’ energy and redox metabolisms. They also provide some evidence that the mutant parasites have adapted to the metabolic changes by up-regulating proteins involved in antioxidant and redox activities and by increasing the carbon flux from glucose into the generation on NAD(P)+ to allow for increased activities of their antioxidant machinery. In addition, it appears that mitochondrial citrate synthesis increases in response to the loss of E2 activity and that citrate may be a conduit for the transport of acetyl-CoA into the cytosol and other cellular compartments where acetylation reactions take place, in order to maintain general cellular functions and regulatory processes. The metabolic changes may affect regulation of cell cycle progression as is evidenced by the growth phenotypes of the two parasites lines