RESUMEN
BACKGROUND: Plasmodium falciparum parasitization of erythrocytes causes a substantial increase in the levels of intracellular fatty acids, notably oleic acid. How parasites acquire this monounsaturated fatty acid has remained enigmatic. Here, we report on the biochemical and enzymatic characterization of stearoyl-CoA desaturase (SCD) in P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS: Metabolic labeling experiments allowed us to demonstrate the production of oleic acid from stearic acid both in lysates of parasites incubated with [(14)C]-stearoyl-CoA and in parasite-infected erythrocytes labeled with [(14)C]-stearic acid. Optimal SCD activity was detected in schizonts, the stage of maximal membrane synthesis. This activity correlated with a late trophozoite stage-specific induction of PFE0555w transcripts. PFE0555w harbors a typical SCD signature. Similar to mammalian SCDs, this protein was found to be associated with the endoplasmic reticulum, as determined with PFE0555w-GFP tagged transgenic P. falciparum. Importantly, these parasites exhibited increased rates of stearic to oleic acid conversion, providing additional evidence that PFE0555w encodes the plasmodial SCD (PfSCD). These findings prompted us to assess the activity of sterculic acid analogues, known to be specific Delta9-desaturase inhibitors. Methyl sterculate inhibited the synthesis of oleic acid both with parasite lysates and infected erythrocytes, most likely by targeting PfSCD. This compound exhibited significant, rapid and irreversible antimalarial activity against asexual blood stages. This parasiticidal effect was antagonized by oleic acid. CONCLUSION/SIGNIFICANCE: Our study provides evidence that parasite-mediated fatty acid modification is important for blood-stage survival and provides a new strategy to develop a novel antimalarial therapeutic based on the inhibition of PfSCD.
Asunto(s)
Antimaláricos/uso terapéutico , Ácido Oléico/biosíntesis , Plasmodium falciparum/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/uso terapéutico , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Antimaláricos/química , Ciclopropanos/química , Eritrocitos/parasitología , Escherichia coli/metabolismo , Ácidos Grasos/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The antimalarial agents NAS-91 and NAS-21 were found to express potent antimycobacterial activity, NAS-91 being more active than NAS-21. They partially inhibited mycolic acid biosynthesis and profoundly altered oleic acid production. The development of a cell-free assay for Delta 9-desaturase activity allowed direct demonstration of the inhibition of oleic acid biosynthesis by these compounds.
Asunto(s)
Antituberculosos , Inhibidores Enzimáticos , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/crecimiento & desarrollo , Ácidos Micólicos/metabolismo , Ácido Oléico/biosíntesis , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Antituberculosos/química , Antituberculosos/farmacología , Sistema Libre de Células , Medios de Cultivo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Estearoil-CoA Desaturasa/metabolismoRESUMEN
The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.
Asunto(s)
Hígado/parasitología , Plasmodium berghei/patogenicidad , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Animales , Antimaláricos/farmacología , Eliminación de Gen , Malaria/parasitología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Parasitemia , Plasmodium berghei/enzimología , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Triclosán/farmacologíaRESUMEN
Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [(14)C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.