PCR cycling conditions were performed using the default conditions of the ABI Prism 7300 SDS Software. Quantification of gene expression was done using the Relative Standard Curve Method (Applied Biosystems bulletin). with each invasive cycle (Cowman and Crabb, 2006) whereas one-time replication in the infected hepatocyte produces up to 40 000 merozoites (Shortt parasites were initially assumed to lack the ability to synthesize their own fatty acids and thus rely on their hosts for lipid scavenging (Vial and Ancelin, 1992). However, this model came into question with the discovery of the apicoplast, a relict plastid organelle of (Kohler genome (Gardner pathway by which can synthesize fatty acids from derivatives of acetate and malonate. The fatty acid chain extension step of FAS II is catalysed by four key enzymes C FabB/F, FabG, FabI and FabZ and the substrate/product of each Rabbit Polyclonal to OR10D4 reaction is covalently bound to the acyl carrier protein (ACP) cofactor (Fig. 1A). Conversely, the mammalian FAS I pathway utilizes a single enzyme complex and is not present in based on genome sequence analysis (Bahl encodes both FAS I and FAS II enzymes, has FAS I enzyme whereas does not harbour either FAS I or FAS II pathways (Mazumdar and Striepen, 2007). Deletion of ACP from has demonstrated that apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in this parasite (Mazumdar PlasmoDB identifier PY04452). -Ketoacyl-ACP is reduced by -ketoacyl-ACP reductase (FabG, PY02416) to form -hydroxyacyl-ACP, dehydrated by -hydroxyacyl-ACP dehydratase (FabZ, PY01586) to form life cycle, reverse transcribed and used for quantitative PCR. Expression levels were measured in salivary gland sporozoites (SG SPZ), mixed blood stages (BS), blood stage schizonts (SCH), and in liver stages 12, 24, 40 and 50 h after salivary gland sporozoite infection (LS-12, LS-24, LS-40 and LS-50 respectively). The expression profile for (B) FabB/F, (C) FabI, (D) FabZ and (E) FabG are shown. Note that for all four genes, expression is highly upregulated in liver stages. The four FAS II enzymes are promising drug targets because they are of bacterial origin. The enzymes have been expressed and used to reconstitute the elongation Ezutromid module of FAS II (Sharma system mimicked the machinery and known inhibitors of the enzymes of the elongation module caused the expected accumulation of intermediates. Thus, possesses a functional FAS II pathway. An early study identified a FabI and showed that the FabI inhibitor triclosan kills blood stage parasites (Surolia and Surolia, 2001) and subsequently a significant effort has been undertaken to develop blood stage FAS II inhibitors to treat malaria (Gornicki, 2003; Sato and Wilson, 2005; Wiesner and Seeber, 2005). Although the data suggested that FAS II is necessary for intra-erythrocytic replication, the expression of FAS II enzymes has not been studied throughout the complex infection cycle of the parasite and their importance in parasite progression throughout the life cycle remains unknown. We recently carried out a liver stage transcriptome and proteome analysis in the model rodent malaria parasite and observed that (i) the transcription of FAS II genes was increased in liver stages when compared with blood stages; (ii) FAS II enzymes were present in the liver stage proteome; Ezutromid and (iii) hexachlorophene, an inhibitor of FabG, was able to inhibit liver stage development (Tarun and blood stage replication. Results The transcript abundance of FAS II genes is highly upregulated in late liver stage development We used quantitative RT-PCR (qPCR) to show upregulation of FAS II genes in liver stages because our previous microarray analysis had indicated preferential expression in liver stages of (Tarun life cycle stages for the four FAS II genes encoding the enzymes involved in fatty acid extension (Fig. 1A). For all four genes, the level of transcription was highly increased in pre-erythrocytic stages but the most consistent and substantial expression was seen in late liver stages when compared with Ezutromid blood stages (Fig. 1B to E). The qPCR data suggest that FAS II is induced in liver stages and might thus play an important role for liver stage development. FAS II enzymes are expressed in sporozoites and liver stages and localize to the apicoplast To further investigate the expression of FAS II during the parasite life cycle we generated a transgenic line expressing a myc epitope-tagged FabI under the control of the endogenous promoter (PyFabI-myc) (Fig. S1). The quadruple myc tag was fused to the carboxyl (C)-terminus of FabI and was followed by the 3 UTR from dihydrofolate reductase/thymidylate synthase (subtilase 1 (Yeoh FAS II enzymes including FabI possess a bipartite leader sequence that predicts import into the apicoplast (data not shown). As FAS II enzymes localize to the apicoplast in the apicomplexan (Waller was localized to the apicoplast (Ferguson sporozoite apicoplast. To analyse FabI expression during liver stage development, HepG2:CD81 hepatoma cells (Silvie blood stages with reference.