Abstract
Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses.
Highlights
Leishmania spp. are trypanosomatid parasites that are transmitted as flagellated extracellular metacyclic promastigotes into the skin of the mammalian host by the bite of a female sandfly
Similar to Coxiella burnetii but in contrast to mycobacteria or Salmonella, Leishmania amastigotes thrive in a habitat designated as parasitophorous vacuole (PV) that is thought to resemble a late endosome/early lysosome based on the presence of marker proteins characteristic of these compartments such as Rab7 and LAMP-1/-2, respectively (Antoine, Prina, Jouanne, & Bongrand, 1990; Kima et al, 2010; Russell, Xu, & Chakraborty, 1992)
As demonstrated for filipin staining, we identified that the amount of NBD-cholesterol associated with the parasites is a function of the number of parasites internalized by the host cell (Figure 5c), showing a similar trend over all time points
Summary
Leishmania spp. are trypanosomatid parasites that are transmitted as flagellated extracellular metacyclic promastigotes into the skin of the mammalian host by the bite of a female sandfly. A switch toward fatty acid catabolism in amastigotes has been reported for Leishmania donovani (Rosenzweig, Smith, Myler, Olafson, & Zilberstein, 2008) This outcome has been corroborated by studies using isotope-resolved metabolomics, where amastigotes exhibited a glucose-sparing metabolism associated with increased rates of beta-oxidation (Saunders et al, 2014). This correlates with a concentration of sequestered free cellular cholesterol around the parasite, detectable as a halo. This two-way cholesterol sequestration and incorporation by the parasites amounts to one third of the host cell whole cholesterol and is associated with transcriptional upregulation of host cell genes that are involved in cholesterol biosynthesis and are induced by the ER resident, cholesterol-level-sensitive transcription factor SREBP (sterol regulatory element-binding protein)
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