Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form

Yao and Wilson Parasites & Vectors (2016) 9:200 DOI 10.1186/s13071-016-1470-0 RESEARCH Open Access Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form Chaoqun Yao1* and Mary E. Wilson2,3 Abstract Background: The Leishmania spp. protozoa, the causative agents of the “neglected” tropical disease leishmaniasis, are transmitted to mammals by sand fly vectors. Within the sand fly, parasites transform from amastigotes to procyclic promastigotes, followed by development of virulent (metacyclic) promastigote forms. The latter are infectious to mammalian hosts. Biochemical components localized in the parasite plasma membrane such as proteins and sterols play a pivotal role in Leishmania pathogenesis. Leishmania spp. lack the enzymes for cholesterol synthesis, and the dynamics of sterol acquisition and biosynthesis in parasite developmental stages are not understood. We hypothesized that dynamic changes in sterol composition during metacyclogenesis contribute to the virulence of metacyclic promastigotes. Methods: Sterols were extracted from logarithmic phase or metacyclic promastigotes grown in liquid culture with or without cholesterol, and analyzed qualitatively and quantitatively by gas chromatograph-mass spectrometry (GC-MS). TriTrypDB was searched for identification of genes involved in Leishmania sterol biosynthetic pathways. Results: In total nine sterols were identified. There were dynamic changes in sterols during promastigote metacyclogenesis. Cholesterol in the culture medium affected sterol composition in different parasite stages. There were qualitative and relative quantitative differences between the sterol content of virulent versus avirulent parasite strains. A tentative sterol biosynthetic pathway in Leishmania spp. promastigotes was identified. Conclusions: Significant differences in sterol composition were observed between promastigote stages, and between parasites exposed to different extracellular cholesterol in the environment. These data lay the foundation for further investigating the role of sterols in the pathogenesis of Leishmania spp. infections. Keywords: Leishmania, Sterol, Promastigotes, Metacyclogenesis, Virulence, Sterol biosynthetic pathway Background Leishmania spp. are the etiological agents of leishmaniasis, a group of parasitic diseases that are endemic in 88 countries on four continents [1]. Over 20 Leishmania spp. are collectively responsible for varied clinic manifestations of human disease. Three major clinical forms include cutaneous leishmaniasis (CL), mucocutanous leishmaniasis and visceral leishmaniasis (VL). Data compiled by WHO show one million cases of CL in the last * Correspondence: 1 Department of Biomedical Sciences and One Health Center for Zoonoses and Tropical Veterinary Medicine, Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, St. Kitts, West Indies Full list of author information is available at the end of the article five years, and 300,000 cases of VL with 20,000 deaths annually (http://www.who.int/leishmaniasis/en/). The Leishmania spp. protozoa alternate between a flagellated promastigote in the sand fly vector and an obligate intracellular amastigote, which lacks an external flagellum, in the mammalian host. After a sand fly vector takes a blood meal, amastigotes transform in the sand fly gut to procyclic promastigotes and several other intermediate stages, multiply by binary fission, and eventually develop to the metacyclic promastigotes which are infectious for mammalian hosts. Metacyclic promastigotes are inoculated by the sand fly vector during a blood meal into a new mammalian host [2]. This process of development from the procyclic to the metacyclic promastigotes COPYRIGHT NOTICE. The article is a work of the United States Government; Title 17 U.S.C 105 provides that copyright protection is not available for any work of the United States government in the United States. Additionally, this is an open access article distributed under the terms of the Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0), which permits worldwide unrestricted use, distribution, and reproduction in any medium for any lawful purpose. Yao and Wilson Parasites & Vectors (2016) 9:200 Page 2 of 12 is termed metacyclogenesis. Due to the technical challenges in raising sand flies in laboratory, metacyclogenesis is often modeled in liquid medium by culture of promastigotes in vitro from logarithmic to stationary growth phase, from which the metacyclic promastigotes can be isolated by density for some Leishmania spp. to more than 95 % purity [3]. Metacyclic promastigotes are morphologically distinguishable from procyclic cells by their elongated flagellum (at least double the cell body length), and their smaller body size. Unlike mammalian cells but similar to fungi, Leishmania spp. are eukaryotic kinetoplastids that synthesize ergosterol [4]. They do not have the enzymes to synthesize cholesterol, although they have detectable cholesterol that they must take up from their external environment [5]. The sterols ergosterol or cholesterol are essential components of plasma membranes found in lipid rafts, membrane microdomains which are also classified as detergent resistant membranes (DRMs) due to their physicochemical properties [6, 7]. Our prior studies using the lipid-raft disrupting agent methyl-β-cyclodextrin (MβCD) suggested that lipid rafts play a pivotal role in the virulence of Leishmania spp. [8]. The anti-fungal agent amphotericin B is widely used in patients with leishmaniasis, especially in regions where parasites are resistant to standard therapy with antimony compounds [9, 10], or in patients co-infected with Leishmania sp. and human immunodeficiency virus (HIV) [11]. Amphotericin B preferentially binds to ergosterol, leading to disruption of the osmotic integrity of the membrane in target cells [12]. Given the association between sterol content and heat resistance of L. major [13], the objectives of current study were (i) to identify and quantify sterols in distinct stages of Leishmania infantum promastigotes that differ in their virulence for a mammalian host, and (ii) to determine the effects of cholesterol addition or depletion on promastigote sterol (particularly ergosterol) content. The data should provide a baseline for further study of drug- or environmentally-induced changes in parasite sterol content in the pathogenesis and control of Leishmania spp. infections. Parasites Methods Sterol identification Ethics Statement Dry derivatized trimethylsilyl derivatives were analyzed in the High Resolution Mass Spectrometry Facility at the University of Iowa using a ThermoFinnigan Voyager single quadruple mass spectrometer interfaced with a Trace2000 gas chromatograph (GC). The original software from the supplier was used for both data acquisition and processing. A DB-5ht capillary column (inn (...truncated)