Plants, animals, and yeast have the capacity to produce and accumulate neutral lipids, sterol esters and triacylglycerol (TAG). However, their accumulation and the genes underlying their synthesis in intracellular microorganisms are poorly understood. Plasmodium falciparum is a parasitic protozoan that causes the most virulent form of human malaria. During erythrocytic infection, parasite-induced fatty acid and phospholipid synthesis have been of interest as drug targets. Cholesterol is also important during infection of erythrocytes. However, the accumulation of cholesterol esters (CE) and TAG in P. falciparum-infected erythrocytes has not been reported. Here we show by mass and incorporation of radiolabeled fatty acids, that TAG accumulates during intracellular parasite growth in a stage-dependent manner, with high levels present at the later stages of infection. We fail to detect parasite-induced accumulation or synthesis of CE suggesting that TAG is the major neutral lipid resident in lipid droplets within the parasite. Further, we have identified a plasmodial homologue for the gene acyl CoA:diacylglycerol acyltransferase (DGAT), which is expressed in a stage-dependent manner and may provide the enzyme that catalyzes the last step of TAG synthesis. Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by Salmonella pathogenicity island 2 (SPI-2). High levels of cholesterol are detected at the Salmonella-containing vacuole (SCV). Here we show that the SPI-2 effector SseJ esterifies cholesterol in vitro, in cells and during infection. Intracellular infections with wild type as compared to sseJ bacteria led to higher levels of CE production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets. Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in cultured macrophages and epithelial cells have reported no significant change in viability of sseJ relative to wild type bacteria at 16 h post-infection. We also see this at 2 and 10 h, but sseJ mutants reliably display larger vacuoles and a detectable increase in total GFP-positive bacteria that may reflect higher levels of non-viable Salmonella.

Last modified

• 09/08/2018

Creator

• Parwez Nawabi

DOI

• https://doi.org/10.21985/N2T724

Subject

• Integrated Graduate Program in the Life Sciences

Keyword

• Plasmodium
• Neutral Lipids
• Cholestrol Esters
• Salmonella
• Triacylglycerol
• SseJ

Date created

• 2008-03-26

Resource type

• Dissertation

Rights statement

• In Copyright