RESUMO
Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote.
Assuntos
Retículo Endoplasmático/enzimologia , Modelos Moleculares , Filogenia , Proteínas de Protozoários/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Biologia Computacional , Sequência Conservada , Dimerização , Deleção de Genes , Duplicação Gênica , Transferência Genética Horizontal , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/isolamento & purificação , Homologia Estrutural de ProteínaRESUMO
With the World Health Organization reporting over 30,000 deaths and 200,000 to 400,000 new cases annually, visceral leishmaniasis is a serious disease affecting some of the world's poorest people. As drug resistance continues to rise, there is a huge unmet need to improve treatment. Miltefosine remains one of the main treatments for leishmaniasis, yet its mode of action (MoA) is still unknown. Understanding the MoA of this drug and parasite response to treatment could help pave the way for new and more successful treatments for leishmaniasis. A novel method has been devised to study the metabolome and lipidome of Leishmania donovani axenic amastigotes treated with miltefosine. Miltefosine caused a dramatic decrease in many membrane phospholipids (PLs), in addition to amino acid pools, while sphingolipids (SLs) and sterols increased. Leishmania major promastigotes devoid of SL biosynthesis through loss of the serine palmitoyl transferase gene (ΔLCB2) were 3-fold less sensitive to miltefosine than wild-type (WT) parasites. Changes in the metabolome and lipidome of miltefosine-treated L. major mirrored those of L. donovani A lack of SLs in the ΔLCB2 mutant was matched by substantial alterations in sterol content. Together, these data indicate that SLs and ergosterol are important for miltefosine sensitivity and, perhaps, MoA.