Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei Guiomar Pérez-Moreno a , Marco Sealey-Cardona a , Carlos Rodrigues-Poveda a , Michael H. Gelb b , Luis Miguel Ruiz-Pérez a , Víctor Castillo-Acosta a , Julio A. Urbina c , Dolores González-Pacanowska a,⇑ a Instituto de Parasitología y Biomedicina ‘‘López-Neyra’’, Consejo Superior de Investigaciones Científicas, Avda. del Conocimiento s/n, 18100 Armilla, Granada, Spain b Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA c Instituto Venezolano de Investigaciones Científicas, Centro de Bioquímica y Biofísica, Caracas, Venezuela article info Article history: Received 25 June 2012 Received in revised form 19 July 2012 Accepted 22 July 2012 Available online 29 August 2012 Keywords: Sterols Trypanosoma brucei RNA interference Squalene synthase Squalene epoxidase Sterol methyl transferase abstract Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we ana- lyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosyn- thesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epox- idase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial struc- ture and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24- alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Protozoan parasites are responsible for a significant portion of global morbidity, mortality and economic hardship, and in most countries current control and treatment regimes are either failing or under serious threat (Barrett et al., 2003). As the lipid metabo- lism of trypanosomes differs in many respects from that of its mammalian host, it offers potential and promising targets for the development of urgently needed and new chemotherapeutic drugs to combat these parasites (Lorente et al., 2004; van Hellemond and Tielens, 2006; Lepesheva et al., 2007, 2008; Urbina, 2009). African trypanosomes do not synthesize cholesterol de novo, but instead synthesize ergosterol and other 24-alkylated sterols, similar to plants and fungi (Roberts et al., 2003). However, when cholesterol is present in their environment it is incorporated by the parasite without further metabolism from the host via receptor-mediated endocytosis of low density lipoproteins (LDLs) (Coppens and Courtoy, 2000) followed by lysosomal degradation (Coppens and Courtoy, 1995; Coppens et al., 1995). While bloodstream forms are highly dependent on this exogenous supply of sterols for ade- quate growth, procyclic trypanosomes are flexible with respect to the source of sterols and adjust their de novo ergosterol biosynthe- sis to the external supply of cholesterol (Coppens and Courtoy, 1995). The close similarities to fungi in relation to sterol composition and sterol biosynthesis have offered a unique opportunity for the development of chemotherapy by targeting the sterol biosynthetic pathway using the types of drugs already successfully employed against fungal pathogens (Roberts et al., 2003). In fungi, sterols are responsible for membrane stability and modulate morphogen- esis, intracellular trafficking, membrane permeability and the activities of membrane-bound enzymes (Heese-Peck et al., 2002; Sharma, 2006). Indeed, sterol biosynthesis inhibitors have been successfully tested as anti-trypanosomals in the case of Trypano- soma cruzi and Leishmania spp. (Magaraci et al., 2003; Lorente et al., 2004; Urbina et al., 2004; Hucke et al., 2005). Curiously, certain studies have demonstrated that inhibitors of the enzymes, sterol methyl transferase and sterol 14a-demethylase, are active against bloodstream forms of Trypanosoma brucei (Cammerer 0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.07.012 ⇑ Corresponding author. Tel.: +34 958181631; fax: +34 958181632. E-mail address: dgonzalez@ipb.csic.es (D. González-Pacanowska). International Journal for Parasitology 42 (2012) 975–989 Contents lists available at SciVerse ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara