Abstract
Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.
Highlights
Upon invasion of a host cell, the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment, the parasitophorous vacuole (PV).1 The PV is unique because it is predominantly derived from the host cell plasma membrane [1] but is devoid of host cell transmembrane proteins [2, 3] and does not fuse with endocytic or exocytotic vesicles of the host cell (4 –7)
Absence or Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) Activity Decreases Parasite Replication—To determine whether T. gondii replication is affected by alteration in cholesterol ester formation, tachyzoites were used to infect either murine embryonic fibroblasts (MEF) deficient for acyl-CoA:cholesterol acyltransferase 1 (ACAT-1) or HFF treated with ACAT inhibitors SaH 58-035 and CI 976 [34] (Fig. 1A)
In ACAT-1Ϫ/Ϫ MEF, parasite replication was reduced by 60% compared with that in ACAT-1ϩ/ϩ MEF
Summary
Upon invasion of a host cell, the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment, the parasitophorous vacuole (PV).1 The PV is unique because it is predominantly derived from the host cell plasma membrane [1] but is devoid of host cell transmembrane proteins [2, 3] and does not fuse with endocytic or exocytotic vesicles of the host cell (4 –7). For ACAT activity assay in T. gondii, tachyzoites were harvested from ACAT-1Ϫ/Ϫ MEF after 2 lysis cycles, washed in PBS to remove cellular debris, and aliquots of 107 parasites tested for incorporation of [14C]oleate into cholesterol esters as described above.
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